Article

RESOLUTION OF GRANULES FROM RABBIT HETEROPHIL LEUKOCYTES INTO DISTINCT POPULATIONS BY ZONAL SEDIMENTATION

Authors:
To read the full-text of this research, you can request a copy directly from the author.

Abstract

Postnuclear supernates from homogenates of essentially pure rabbit heterophil leukocytes were fractionated by means of zonal differential centrifugation through a discontinuous sucrose gradient at various speeds. Three distinct groups of granules were characterized biochemically and morphologically. They were, in order of decreasing sedimentation coefficient: (a) Large, relatively dense granules, identified morphologically as the azurophil or primary granules, and containing essentially all of the myeloperoxidase activity of the preparations, about one-third of their lysozyme activity, and between 50 and 80% of their content in five acid hydrolases typically associated with lysosomes in other cells; (b) smaller, less dense granules, with the morphological appearance of the specific or secondary granules, and carrying most of the alkaline phosphatase and the remainder of the lysozyme activity of the preparations; (c) a second group of lysosome-like particles, associated with a morphologically heterogeneous fraction, and containing the remainder of the acid hydrolases, but little or no myeloperoxidase. When p-nitrophenyl phosphate was used instead of ß-glycerophosphate for the assay of acid phosphatase, only small proportions of the total activity accompanied the two main lysosomal bands, and considerable activity was found in a zone slightly retarded with respect to the slowly moving band of acid hydrolases.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the author.

... Similar distributions and RSA were found for the lysosomal marker activities of a-galactosidase and acid P-glycerophosphatase. The sonication conditions used in these studies will, of course, give substantial percent of intact leukocytes that appear in the N (7) y-Glutamyl transferase acceptor: Glycylglycine (7) I.-Cystine (7) L-Glutamine (3) /3-Galactosidase (6) Acid P-Glycerophosphatase (3) Lactic dehydrogenase (3) fraction. This was borne out by the distribution of lactate dehvdrogenase in the fractions. ...
... However, the in these granule preparations of a heterogeneous population of particles was suggested by morphologic differences and by the inclusion of membrane-bound enzymes, such as alkaline phosphatase, not normally found in lysosomes (12,44). In subsequent studies on rabbit heterophil leukocytes by zonal sedimentation and isopycnic centrifugation, four types of particles were identified (2,3,14). Four sedimenting bands and a nonsedimenting band that collected at the junction of the gradient and sample volumes were also visible after isopycnic centrifugation of PN fractions of human leukocytes. ...
Article
Evidence has been obtained in three different cell types, by a combination of biochemical and histologic approaches, that some y-glutamyl transferase (EC 2.3.2.2) activity is associated with lysosomes. The distribution of y-glutamyl transferase in subcellular fractions of human leukocytes and its enrichment in a postnuclear granule fraction were similar to the corresponding findings for lysosomal marker enzymes. Isopycnic centrifugation of the postnuclear fraction showed that although the bulk of the y-glutamyl transferase activity was associated with nonlysosomal particles containing alkaline phosphatase, a clearly separated fraction (approximately 20%) comigrated with lysosomal marker enzymes. L-cystine was the most potent of the amino acids tested as acceptors of the γ-glutamyl moiety of L-γ-glutamyl-P-nitroanilide. Apparent Km for L-cystine was 1.3 mM compared to 8.7 mM for L-glutamine, the second best amino acid acceptor. Optimum pH for the transferase was 9.0; there was no activity below pH 6. Further evidence that leukocyte γ-glutamyl transpeptidase includes a lysosomal component was obtained by demonstrating histochemically that the transpeptidase appears in cytoplasmic granules in rabbit neutrophile bone marrow precursors at a time when the synthesis of azurophile (lysosomal) granules is predominant.
Chapter
Cells observed in cytological specimens can generally be classified by origin into one of four tissue groups: hemic, epithelial‐glandular, connective, or nervous. Mesenchymal cells tend to exfoliate poorly and provide poorly cellular cytological specimens. Plant material is frequently observed in fecal cytology specimens from many species as variably sized, pleomorphic plant fragments, which are consistent with normal cytologic findings. Cerebrospinal fluid analysis is a critical tool in the evaluation of central nervous system disease in mammals with signs of neurologic disease. The inflammatory response of mammals can be classified as either neutrophilic/heterophilic, eosinophilic, mixed cell, or macrophagic depending upon the predominant cell type. Fine‐needle aspiration cytology is a rapid, efficient, cost‐effective, relatively painless procedure and produces a speedy result with high diagnostic accuracy. Tissue hyperplasia is a proliferative process of tissues responding to cellular injury or chronic stimulation. Neoplasia of the lungs of animals occurs with either primary or metastatic lesions.
Article
Extracts from the granule fraction obtained from leukaemic myeloid cells contained several basic proteins with electrophoretic mobilities against the cathod faster than that for lysozyme. Similar proteins have previously been demonstrated in rabbit polymorphonuclear granules. By chromatography on sulphoethyl Sephadex and on a weak dipolar ion adsorbant the most basic proteins were isolated. The amino acid analysis of the protein gave a preponderance of basic amino acids (29 %) and electrophoresis after treatment with sodium dodecyl sulphate revealed components with the molecular weight in the range 10, 000–25, 000. These proteins seem to be specific for granulocytes and probably have antibacterial properties.
Article
A neutral α-glucosidase (EC 3.2.1.20) activity was shown to be associated with granules which are sedimentable at 10 000 g after differential centrifugation of mouse peritoneal macrophage homogenates. When the post-nuclear supernatant was centrifuged in a sucrose density gradient, high activities for neutral α-glucosidase and β-glucuronidase (EC 3.2.1.31) were detected in the bottom fractions because of aggregation of the granules. Neutral α-glucosidase-containing granules were completely disaggregated by the addition of 20 units/ml of heparin and 10 mM Tris-HCl (pH 7.2), which caused only a partial disaggregation of β-glucuronidase-containing granules. The addition of a high concentration of heparin, Tris buffer, or KCl to the gradient gave the same patterns of disaggregation of the granules. Under the condition in which about 50% of the total β-glucuronidase activity was released into the medium, depending on phagocytosis, very little α-glucosidase was released. These observations suggested that neutral α-glucosidase may localize in non-lysosomal granules.
Article
1.1. Human polymorphonuclear leukocyte cytoplasmic extracts were preincubated at pH 7.2 in 0.34 M sucrose with exogenous phospholipase C or with deoxycholate, and immediately free and soluble activities were measured for several primary and specific granule enzymes.2.2. Both treatments caused almost complete activation of latent lysosomal enzymes.3.3. Specific granule membrane was strongly resistant to phospholipase C and deoxycholate treatments, because free and soluble alkaline phosphatase activity, used as marker enzyme for these granules, did not increase significantly.4.4. The above results are evidence for a biochemical heterogeneity between primary and specific granule membranes.
Article
The liquid centrifuge using a density-gradient method has certainly played an essential role in almost every advance in molecular and cellular biology. For the velocity-sedimentation method, when a rotor reaches its full speed, the density-gradient solution establishes its stable profile and particles of the sample begin to separate and to form their respective bands, and to sediment toward the wall of rotor. While particles move outward in bands, some of the bands separate into more bands during sedimentation and sometimes the separated bands overtake and cross each other depending on local properties such as density, dispersion coefficient, particle size, etc. Then each band starts to separate and to sediment outward until reaching its respective isopycnic zone. The locations where the separated bands cross each other or combine together may be termed bifurcation point(s) in density-gradient centrifugation. The method of Poincare's bifurcation analysis together with numerical simulation are used to analyze the effect of various local properties on bifurcation point(s). It is found that a major factor in having bifurcation(s) is the steepness of the density-gradient profile. The other properties are minor.
Article
Riassunto In tutte le fasi dell’infiammazione (vasodilatazione, suppurazione e cicatrizzazione) intervengono numerosi fattori non tutti perfettamente conosciuti biochimicamente. Essi derivano principalmente dai fattori del complemento, dai fattori della coagulazione e dal sistema delle chinine. Tra i fattori responsabili delle reazioni infiammatorie generali (febbre, leucocitosi, modificazioni della concentrazione delle plasmaproteine), quelli che causano la febbre sono ben caratterizzati. Le conoscenze sulla struttura dei pirogeni sono ancora all’inizio, sebbene sia ormai accertata la loro origine leucocitaria. L’inizio dei fenomeni infiammatori può essere attribuito alia degranulazione delle mastcellule, alla formazione delle chinine o all’attivazione del complement». Quest’ultimo processo, come è stato ricordato, può avvenire anche in assenza di reazioni immunologiche. L’andamento del processo infiammatorio è regolato da enzimi quali la istaminasi, la carbossipeptidasi e da varie proteine plasmatiche ad attività anti-proteasica come l’α1-antitripsina, l’α1-macroglobulina e l’inattivatore del C1. Lo sviluppo dei granulomi, formazioni caratteristiche dell’infiammazione cronica, è probabilmente dovuto all’attivazione del fattore di Hageman con conseguente liberazione di chinine.
Article
Ein Überblick über neuere Entwicklungen auf dem Gebiet der Gewebefraktionierung wird angegeben. Die Trennverfahren konnten wesentlich verbessert werden, hauptsächlich durch die Konstruktion verschiedener automatischer Rotoren zum Zentrifugieren mit Dichtegradient. Auch die Analyse der Fraktionen wurde durch Automation biochemischer Verfahren und besonders durch den Einsatz quantitativer morphologischer Methoden weitgehend verbessert.
Article
Die erfolgreiche Abwehr von Infektionen durch Mikroorganismen hängt von Resistenz und Immunität ab. Resistenz ist nicht antigenspezifisch; sie wird in ihrer Ausprägung durch genetische Faktoren und umweltbedingte Einflüsse wie Ernährung, Überanstren-gung und Krankheit bestimmt. Hingegen bedeutet Immunität die Abwehrleistung, welche auf dem Vorhandensein von Prodiikten des Immunsystems beruht. Immunität ist er-worben, da sie erst nach Kontakt mit Antigenen des Mikroorganismus entsteht und im Gegensatz zur Resistenz spezifisch ist.
Article
Dispersion coefficients of a polystyrene latex bead (diameter: 0.091 ± 0.0058 μ) and bovine serum albumin in sucrose and Ficoll (polysucrose by Pharmacia Fine Chemicals, Sweden) gradient solutions were measured in an Oak Ridge B-XV zonal centrifuge rotor. Measurements were analyzed by the moment method. Dispersion coefficients were then correlated in a reduced form in terms of Schmidt number as a function of reduced centrifugal force field strength, Taylor number, reduced sedimentation coefficient, density ratio between particle and gradient medium, and reduced time. The correlation is also given in a formulawhich fits all the experimental data points with an average deviation of 2.5% and a maximum deviation of 6.3%. Some information on the eddy and molecular components of the dispersion coefficients is also presented.
Article
1Large amounts of granulocytes can be isolated from bovine blood by differential centrifugation and hypotonic lysis of erythrocytes, followed by separation of neutrophils and eosinophils by centrifugation through a gradient of colloidal silica.2Careful homogenization of the purified neutrophils and subfractionation of the postnuclear supernatant by centrifugation through a discontinuous sucrose gradient provides a membrane fraction (at the 20/32%, w/w, sucrose interface), which collects about 20–35% of the activity of plasma membrane marker enzymes.3Treatment of the plasma membrane fraction with 0.5 M KCl removes some protein and activity of granule enzymes, leading to an about 20–35-fold enrichment in specific activity of plasma membrane marker enzymes. In particular, there is a 25-fold enrichment in a Ca2+-dependent ATPase, whose half-maximal reaction velocity is reached at 2.2 × 10-7 M Ca2+.4High-resolution sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals a complex composition of the neutrophil plasma membrane, with about 40 polypeptides stained by Coomassie blue. Ten of these peptides are more intensely stained by the dye; their apparent molecular weight ranges from 81000 to 34000. All these ten polypeptides but one (which is likely to be actin) are markedly enriched in the plasma membrane fraction over the original homogenate.5Since bovine blood can be obtained in unlimited amounts, the procedures here described can be applied to a large-scale purification of the neutrophil plasma membrane, suitable for biochemical studies.
Article
Inflammatory bowel diseases are common chronic inflammatory disorders. The majority are idiopathic and can be broadly divided into Crohn's disease and ulcerative colitis. Their cause is unknown, but most hypotheses focus on a primary role for T-cell dysfunction. Conversely, there is a collection of congenital disorders of phagocyte function that result not only in immunodeficiency but also in noninfectious inflammatory bowel disease. In all cases, the latter is strikingly reminiscent of the clinical and pathological features of Crohn's disease. This coincides with recent work demonstrating that despite previous emphasis on adaptive immune dysfunction, patients with Crohn's disease actually possess an unusually weak acute innate inflammatory response. This review consolidates the literature on inflammatory bowel disease in congenital immunodeficiencies and considers the role of phagocyte dysfunction in Crohn's disease. Concepts about pathogenesis and treatment that can be carried across these disorders are also discussed.(Inflamm Bowel Dis 2008)
Article
1. The distribution of lysosomal enzymes obtained by density gradient centrifugation of cell-free extracts of Xenopus tail tissue was compared during growth and involution. The particles containing lysosomal enzymes were also characterized by means of electron microscopy. 2. Cytochromeoxidase is recovered to more than 90% in bound form (mitochondria), whereas lysosomal enzymes are characterized by a high proportion of soluble activity (up to 67%). The particle-bound activity of-glucuronidase, cathepsin, and acid RNase show two peaks after centrifugation, in contrast to the activity of acid phosphatase, which is distributed over the whole gradient. During tail involution an increasing proportion of soluble activity is observed, especially for cathepsin and acid RNase. 3. Electronmicroscopical examination reveals a rather heterogeneous composition of the particle populations obtained by density gradient centrifugation. In fractions with highest activity of lysosomal enzymes various types of electron dense granules are found, thus precluding characterization of a specific lysosomal structure. 4. The distribution of enzymes after induction of tail involution with thyroxine was almost similar to the results obtained from spontaneously metamorphosing animals. Since actinomycin D inhibits the increase in lysosomal enzymes and tissue involution it is concluded that the synthesis of soluble acid hydrolases is a prerequisite of hormonal induced tissue involution.
Article
The segregation of human lysosomal arylsulfatase A (ARS-A) has been evaluated in 50 primary hybrid clones derived from four separate fusions involving WBCs from two unrelated individuals and three hamster cell lines. ARS-A was expressed in the hybrids as a dimeric molecule of very similar or identical subunits. The expression of this enzyme was concordant with that of mitochondrial aconitase (ACON-M), an isozyme assigned to chromosome 22, in all 50 clones and with chromosome 22 segregation in all but one of the 29 karyotyped hybrids. No other human chromosome cosegregated with 22 in these clones, suggesting that this enzyme is specified in hybrid cells by a locus (or loci) on a single chromosome. ß-Galactosidase (B-GAL) expression was analyzed with two different electrophoresis systems and with a number of cell extract preparation methods in 39 of the primary hybrid clones. The B-GAL isozyme expressed in these hybrid cells was concordant with the expression of glutathione peroxidase-1 (GPX-1), an isozyme assigned to chromosome 3, in all 39 clones and with the segregation of this chromosome in 97% of the 29 karyotyped hybrids. These observations substantiate the prior tentative assignments of an ARS-A locus to chromosome 22 and a B-GAL locus to chromosome 3 (Bruns et al., 1978a, b). The implications of the chromosome assignments of loci for 12 human lysosomal enzymes for the cellular assembly of these organelles are discussed.
Article
Sequential changes in the kidney during the generalized Shwartzman reaction were studied electron microscopically. The first anatomical change was infiltration of neutrophils into the glomerular capillaries. Endothelial damage was not noticeable until the capillaries were filled with fibrin deposits. Fibrin appeared in the mesangium at almost the same time as in the capillary lumina, traversing through the endothelial fenestrae. Endothelial damage was more common in the mesangial portion than in the peripheral portion of the capillaries. Severe mesangiolysis developed after loss of endothelial cells had been followed by massive penetration of intracapillary contents. Later, signs of repair were evident in some parts of the damaged endothelium. The development of cortical necrosis coincided with the appearance of mesangiolysis and arteriolar thrombotic lesions.
Article
Human subjects exhibiting the anomaly described byAlius andGrignaschi lack myeloperoxidase in their neutrophil polymorphonuclear leukocytes (PMN). Chicken heterophile granulocytes naturally lack myeloperoxidase as well. Therefore they must be incapable for killing ingested microorganisms by aid of H2O2, an oxidizable cofactor and myeloperoxidase, a mechanism proposed for human PMN. To get closer insight into alternative bactericidal mechanisms we chose chicken PMN as a model. We checked the antimicrobial potencies of these cells in vitro, isolated their different classes of granules, determined their enzyme content and localized an antimicrobial mechanism within these granules. The following results could be established: 1. Vital chicken PMN are not inferior to human PMN in respect to their antimicrobial potencies against four strains of microorganisms. 2. An antibacterial mechanism was located in the large granules of chicken heterophile PMN. 3. Evidently this antibacterial mechanism consists of five or more basic proteins. One of these is lysozyme. Up to now the other proteins could not be shown to be enzymes. These results prove that there exists an alternative antimicrobial mechanism within PMN. Basic proteins appear to be of central importance for this mechanism. Our results might shed some light upon the controversial observations concerning the antimicrobial potencies of patients exhibiting the anomaly ofAlius andGrignaschi.
Article
The ultrastructural localization of peroxidase activity has been studied in the cells of normal human bone marrow using the diaminobenzidine peroxidase technique. Peroxidase activity has been localized within the primary (azurophil) granules of the neutrophilic series as well as in the cytoplasmic granules of eosinophils, basophils and monocytes. Peroxidase activity appears within the cisternal system (nuclear envelope, Golgi complex and rough endoplasmic reticulum) of these cells during the period of peroxidase-containing lysosome production. With the cessation of granulogenesis, peroxidase activity disappears from the cisternal system and does not reappear in subsequent developmental stages. In cells incubated in peroxide-free media, staining of granular components, but not of cisternae, is reduced. The inclusion of catalase in peroxide-free media eliminates all staining. This indicates that an endogenous peroxide is present within the cisternae and granules of these cell types.
Article
Full-text available
The influence on diaminobenzidine staining of four variables: prefixation in aldehyde, temperature and pH of incubation, and H2O2 concentration, was investigated in catalase-, as well as in peroxydase-containing material. Catalase from five different sources and five types of peroxidase were examine. It is conclude: (a) when cells are incubated without prior fixation, in a DAB medium at room temperature and pH 7.3 with 0.003% H2O2, peroxidases produce a visible cytochemical stain, while catalases do not; (b) the cytochemical reaction elicited by catalases is stimulated by prior aldehyde fixation in specified conditions, and incubation at 45 C and pH 9.7 with 0.06% H2O2; (c) under the latter circumstances several peroxidases also stain. Ultrastructural preservation is satisfactory in tissues incubated prior toxation.
Article
The enzyme components of secretory granules in mast cells and basophils together with biogenic amines and negatively charged proteoglycan impart qualitatively and quantitatively distinct molecular compositions to these cell types. Acute pathologic reactions elicited by mast cells and basophils depend upon the release of mediators. The preformed secretory granule components of mast cells include histamine, heparin proteoglycan, chemotactic peptides, enzymes with neutral protease, oxidative and acid hydrolase activities, and in rodent mast cells only serotonin.X Also included are the newly generated mediators, most notably, prostaglandin D2 (PGD2) in rat serosal 2"3 and human pulmonary 2"4 mast cells, slow reacting substances (SRSs) in human mast cells 4 and mouse mastocytoma, 5 and platelet activating factor (PAF) in human mast cells.6 The preformed mediators of basophils, like mast cells, include histamine and chemotactic activities, 7 but in contrast to mast cells probably include chondroitin sulfate rather than heparin proteoglycan s and different neutral protease activities. Human basophils apparently do not generate appreciable amounts of SRS, PAF, 9 and PGD2, even though activated rabbit and human leukemic basophils can generate PAF 1°-12 and both rat and human leukemic basophils apparently can generate SRS activity. 13'14 This review describes the enzyme components localized to secretory granules of highly purified rodent mast cells and partially purified human mast cells and basophils (Table 1), the latter two cells are most relevant for understanding human allergic disease. Mast cell and basophil enzymes have been assigned to cytoplasmic granules based upon histochemical techniques, subcellular fractionation, and noncytotoxic immunologic secretion parallel to histamine. Only the latter technique discriminates between components of secretory and nonsecretory granules and permits quantification of mediator levels in each granule subtype. Nevertheless, histochemical studies of mast cell mediators have provided several interesting insights in terms of vertebrate evolution. Detectable levels of heparin-like proteoglycan are present in mast cells of fish, amphibia, reptiles, birds, and mammals; histamine appears only in the latter three species; the acid exoglycosidase termed ~-hexosaminidase is detected in amphibia, birds,
Article
The effects of nonsteroidal anti-inflammatory agents on superoxide production and granule enzyme release by human polymorphonuclear leukocytes stimulated with either formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe)) or immune complexes were investigated. Cytochrome c reduction and the release of lysozyme, β-glucuronidase, myeloperoxidase and gelatinase were measured. Auranofin, phenylbutazone, sulfasalazine and the phospholipase A2 inhibitor, 4-bromophenacyl bromide, strongly inhibited these responses in fMet-Leu-Phe stimulated cells, at concentrations below 50 μM. Indomethacin, piroxicam, mefenamic acid, primaquine and quinacrine at 50–250 μM were inhibitory. Up to 1 mM ibuprofen and chloroquine inhibited superoxide production but had little effect on degranulation. With cells stimulated by IgG aggregates (immune complexes), up to 1 mM ibuprofen, mefenamic acid and piroxicam did not inhibit either response. Indomethacin, phenylbutazone, sulfasalazine and primaquine inhibited, but considerably higher concentrations were required than with fMet-Leu-Phe. Quinacrine inhibited superoxide production equally well with both stimuli but inhibited enzyme release only with fMet-Leu-Phe. Only auranofin, 4-bromophenacyl bromide, and the weakly effective chloroquine exerted approximately the same effect with both stimuli. d-Penicillamine did not affect enzyme release with either stimulus and interfered in the superoxide assay. Gelatinase release induced by fMet-Leu-Phe was affected to the same extent, or slightly more, than release of the other granule enzymes. With immune complexes, there was only modest inhibition of gelatinase release by any of the drugs at 250–1000 μM. Our results reinforce previous observations that many anti-inflammatory drugs affect neutrophil functions, but their effects vary with stimulus. The relative insensitivity of immune complex-induced responses to most of the drugs must be taken into account when considering their mode of action.
Article
Human neutrophils were disrupted by brief sonication under conditions which preserve the hormone sensitivity of adenylate cyclase and yield minimal granule lysis. Fractions enriched in adenylate cyclase were analysed for hormonal and guanine nucleotide regulation of the enzyme as well as structural proteins. Adenylate cyclase was activated by PGE1 and isoproterenol in a GTP-dependent fashion, while f-met-leu-phe and C5a gave no stimulation. Cholera toxin treatment, which specifically modifies cyclase-related GTP-binding proteins, caused a dose-dependent enhancement of GTP activation, in which GTP alone activated maximally and PGE1 was without further effect. The following proteins were detected in the cyclase-containing vesicles: (a) a 42 K mol. wt protein labeled selectively by cholera toxin; (b) protein subunits observed in SDS gels at 214, 165, 105 and 47 K, of which the 47 K band was the most prominent and comigrated with actin; (c) prominent lectin-binding activities at 165 K (concanavalin A and wheat germ agglutinin) as well as at 100 K (wheat germ agglutinin); and (d) a set of proteins and lectin-binding activities in fractions containing β-glucuronidase activity distinct from adenylate cyclase containing vesicles. The identification of receptor-controlled cyclase, GTP-binding regulatory proteins, cytoskeletal elements and unique lectin-binding activities in a single vesicle preparation should contribute to an understanding of receptor-mediated control of neutrophil function.
Article
SummaryPhagocytes represent the last vestige in higher species of the ancient cellular function for which they are named. Human genetic diseases indicate that phagocytosis and oxidant production are critical to the defense of the host against pathogenic bacteria and fungi, which undoubtedly served to select and maintain these functions over evolutionary time. Phagocytes may also be deleterious to the host, contributing to acute and chronic inflammation. Many chronic inflammatory conditions occur in adulthood after reproduction has occurred, and therefore do not impose selective pressure upon the germ line. Moreover, mechanisms for regulating phagocyte functions would by necessity evolve more slowly than the micro-organisms they are designed to control. Thus, inflammation may be an intrinsic property of a system that only crudely responds over evolutionary time to hypermutable microbial targets, while awaiting establishment of a finely tuned, specific immune response.
Article
—Rat brain d-amino acid oxidase was found to be confined to the hindbrain, distributed more or less equally between cerebellum and medulla. Histochemical staining showed the enzyme to be localized largely in the molecular layer of the cerebellum. After fractionation by means of several distinct density gradient centrifugation procedures exploiting differences in sedimentation coefficient or in density or in both, the enzyme was found to be entirely or almost entirely associated with cytoplasmic particles with a median diameter of the order of 0·2 μm, and a median equilibrium density in aqueous sucrose of 1·18. Comparison with the behavior of cytochrome oxidase and of N-acetyl-β-glucosaminidase indicates that these particles are not mitochondria and are unlikely to be lysosomes. They also differ significantly from the bulk of the catalase-containing particles, which on an average appear to be somewhat smaller. The possibility that they might contain some catalase activity, and thereby qualify as ‘peroxisomes’, can however not be excluded. In any case, they differ profoundly from the peroxisomes of liver or kidney.
Article
Es wird über 4 Fälle unreifzelliger Monozytenleukämien berichtet, die durch folgende Eigenschaften charakterisiert sind: Die leukämischen Zellpopulationen zeigen einen geringen zytologischen Ausreifungsgrad. Zytochemisch läßt sich in diesen Zellen eine mäßig starke, durch NaF hemmbare N-AS-E nachweisen, sowie eine mäßig starke α-N-E und eine ebenfalls mäßige saure Phosphataseaktivität. Die Anfärbung mit Sudanschwarz B bzw. beim Nachweis der N-AS-D-Cl-E und der Peroxydase ist gering oder fehlt (4 Fälle untersucht). Die leukämischen Zellen wandern reichlich in Hautfensterexsudate ein und wandeln sich in Makrophagen um (3 Fälle untersucht). Bei der Kultivierung in vitro wandeln sich die leukämischen Zellen in Makrophagen um (1 Fall untersucht). Der Nachweis eines Rezeptors für IgG an den leukämischen Zellen ergibt ein positives Resultat (1 Fall untersucht). Die Lysozymausscheidung im Harn ist kaum oder nur mäßiggradig erhöht; in den leukämischen Zellen ist immunzytologisch kein Lysozym nachweisbar oder nur unsicher nachweisbar (3 Fälle untersucht). Elektronenmikroskopisch bieten die leukämischen Zellen ein überwiegend undifferenziertes Bild mit runden bis ovalen Kernen, einem reichlichen Gehalt an Ribosomen bei geringer Ausprägung der übrigen Zellorganellen (2 Fälle untersucht).
Chapter
Rabbits have become increasingly popular as companion animals. With this popularity, pet owners demand the same level of diagnostic investigation and medical treatment as they would for cats and dogs. As prey species, rabbits instinctively hide signs of illness. Therefore, clinical pathology is especially important in diagnosing disease. Collection of diagnostic samples and interpretation of results differ in rabbits as compared to other companion species. This chapter offers complete information on obtaining samples, performing tests, and interpreting laboratory results in domestic rabbits. It emphasizes details on clinical biochemistries, urinalysis, and common laboratory diagnostic tests. Bacterial respiratory disease is extremely common in rabbits, and Pasteurella multocida is frequently implicated. Serologic testing for Pasteurella multocida is widely available. However, testing is of limited usefulness in clinical practice. Demonstration of a fourfold rise in titer within a two‐ to four‐week interval is suggestive of active infection.
Chapter
Der biochemische Mechanismus der Zellschädigung im ischämischen Myokard ist nicht vollständig geklärt. Untersuchungen im Lebergewebe zeigten, daß saure Hydrolasen während ischämischer Schädigung über mehrere Stunden freigesetzt werden [1, 2]. Ähnliche Untersuchungen wurden am Skelettmuskel nach Unterbindung der arteriellen Blutzufuhr beschrieben [3]. Wenn lysosomale Enzyme wesentlich zur Entwicklung eines Myokardinfarktes aus einer anfänglichen re versiblen hypoxischen Schädigung beitragen, müssen Veränderungen der Enzym aktivität in einem frühen Stadium des ischämischen Prozesses zu finden sein. Deshalb untersuchten wir ischämisches und Kontrollgewebe nach einer einstündigen ischämischen Schädigung in 5 subzellulären Fraktionen.
Article
Polymorphonuclear leukocytes were obtained from rabbit peritoneal exudates. Granule preparations from these cells were subjected to zonal fractionation through a discontinuous sucrose gradient. Azurophilic and specific granules were enzymatically characterized by peroxidase and alkaline phosphatase, respectively. Collagenase was consistently associated with the alkaline phosphatase-containing specific granules.
Chapter
In this article we will review selected aspects of the physiology of granulocytes and macrophages as related to the formation of cytoplasmic organelles, endocytic activity and the process of intracellular digestion. Both cell types, which are richly endowed in digestive enzymes, have as their primary role the uptake and degradation of exogenous molecules. Although the overall picture of the function of these cells was described by Metchnikoff it is only in the past decade that the detailed mechanisms have been examined.
Article
Symptomatic infection by Neisseria gonorrhoeae (Gc) produces a potent inflammatory response, resulting in a neutrophil-rich exudate. A population of Gc can survive the killing activities of neutrophils for reasons not completely understood. Unlike other Gram-negative bacteria, Gc releases monomeric peptidoglycan (PG) extracellularly, dependent on two nonessential, nonredundant lytic transglycosylases (LTs), LtgA and LtgD. PG released by LtgA and LtgD can stimulate host immune responses. We report that ΔltgAΔltgD Gc were decreased in survival in the presence of primary human neutrophils but otherwise grew equally to wild-type Gc. Adding PG monomer failed to alter ΔltgAΔltgD Gc survival. Thus, LTs protect Gc from neutrophils independently of monomer release. We found two reasons to explain decreased survival of the double LT mutant. First, ΔltgAΔltgD Gc was more sensitive to the neutrophil antimicrobial proteins lysozyme and neutrophil elastase, but not others. Sensitivity to lysozyme correlated with decreased Gc envelope integrity. Second, exposure of neutrophils to ΔltgAΔltgD Gc increased the release of neutrophil granule contents extracellularly and into Gc phagosomes. We conclude that LtgA and LtgD protect Gc from neutrophils by contributing to envelope integrity and limiting bacterial exposure to select granule-localized antimicrobial proteins. These observations are the first to link bacterial degradation by lysozyme to increased neutrophil activation.
Chapter
The lysosomes obtained by subcellular fractionation may be defined as cytoplasmic organelles containing acid hydrolases and surrounded by a semipermeable membrane. In the living cell, however, the lysosomes are not single entities; they are part of the vacuolar apparatus, a membrane-bound system performing mainly digestive functions (de Duve, 1969). The components of this system are in continuous exchange with each other through selective processes of membrane fusion and fission. The enzyme-carrying vesicles (primary lysosomes) are formed in the Golgi apparatus. They subsequently merge with vesicles containing cellular or extracellular substrates (auto- or heterophagic vacuoles) to form secondary lysosomes, where digestion takes place. Secondary lysosomes may enlarge by fusion with each other, with primary lysosomes, or with vacuoles containing additional substrates, or may split into smaller bodies.
Chapter
Various organelles in a cell work together to accomplish cell functions. For example, elements of the cell surface, the endoplasmic reticulum, the Golgi apparatus, and the lysosomes work in concert forming the intracellular digestive tract. Although the intracellular digestive tract may have developed to serve as a food-gathering function in unicellular organisms, the basic components of it have been modified during evolution to subserve a variety of complex and diverse functions in the multicellular organism. Some of these diverse functions are discussed in this chapter.
Chapter
Schon bald nach Abgrenzung der akuten Leukämie als eigenständiges Krankheitsbild setzten vielfältige Versuche ein, aufgrund des cytomorphologischen Befundes weitere Unterteilungen dieser Erkrankung zu treffen. Die Vielzahl der bei akuten Leukämien zu beobachtenden Zellformen führte zu mannigfachen Einteilungsvorschlägen und zu einer Vielzahl von Bezeichnungen für die leukämischen Blasten, die meist im wesentlichen von den subjektiven Vorstellungen der jeweiligen Autoren geprägt waren und sich nicht ohne weiteres auf die Befunde anderer Untersucher übertragen ließen [23, 32, 59, 86, 113, 143, 154, 183,188]. Aufgrund der bei manchen akuten Leukämien sichtbaren Differenzierungstendenzen der Blasten zu Promyelocyten wurde eine myeloische, granulocytäre Form der akuten Leukämie allgemein anerkannt. Weitreichende Meinungsverschiedenheiten herrschten jedoch bezüglich der übrigen Fälle, wobei neben akuten lymphatischen auch akute undifferenzierte Leukämien sowie akute Monocytenleukämien, hier wiederum myeloische (Typ Naegeli) und histiocytäre (Typ Schilling) Formen, beschrieben wurden.
Chapter
Der höher entwickelte Organismus verfügt über Abwehrmechanismen, die an die weißen Blutkörperchen und an ein weitgehend stationäres Zellsystem, das retikuloendotheliale System (RES), gebunden sind. Hierzu gehört die Phagozytose eingedrungener Fremdkörper, wie Bakterien und Viren, die neben dem retikuloendothelialen System, den Granulozyten und den Monozyten obliegt. Das stationäre Abwehrsystem der Makrophagen (RES) kann hier nicht weiter besprochen werden.
Chapter
The release of hydrolytic enzymes by cells present at sites of inflammation is now well recognized. These enzymes are released following either the death of cells after interaction with cytotoxic agents or in a selective manner, where hydrolase release occurs by secretion from viable cells. The latter process has attracted much attention in recent years since it constitutes a mechanism by which hydrolytic enzymes can be released in a directed manner towards an extracellular stimulus. Also viable cells under certain conditions, can be induced to release newly synthesized hydrolases over a long period of time, illustrating a possible mechanism by which tissue damage, degradation and remodelling seen during chronic inflammatory processes can occur.
Article
Full-text available
Long-term catheterization inevitably leads to a catheter-associated bacteriuria caused by multispecies bacterial biofilms growing on and in the catheters. The overall goal of the presented study was (I) to unravel bacterial community structure and function of such a uropathogenic biofilm and (II) to elucidate the interplay between bacterial virulence and the human immune system within the urine. To this end, a metaproteomics approach combined with in vitro proteomics analyses was employed to investigate both, the pro- and eukaryotic protein inventory. Our proteome analyses demonstrated that the biofilm of the investigated catheter is dominated by three bacterial species, i.e. Pseudomonas aeruginosa, Morganella morganii and Bacteroides sp., and identified iron limitation as one of the major challenges in the bladder environment. In vitro proteome analysis of P. aeruginosa and M. morganii isolated from the biofilm revealed that these opportunistic pathogens are able to overcome iron restriction via the production of siderophores and high expression of corresponding receptors. Notably, a comparison of in vivo and in vitro protein profiles of P. aeruginosa and M. morganii also indicated that the bacteria employ different strategies to adapt to the urinary tract. Whilst P. aeruginosa seems to express secreted and surface-exposed proteases to escape the human innate immune system and metabolizes amino acids, M. morganii is able to take up sugars and to degrade urea. Most interestingly, a comparison of urine protein profiles of three long-term catheterized patients and three healthy control persons demonstrated the elevated level of proteins associated with neutrophils, macrophages and the complement system in the patient 's urine, which might point to a specific activation of the innate immune system in response to biofilm-associated urinary tract infections. We thus hypothesize that the often asymptomatic nature of CAUTIs might be based on a fine-tuned balance between the expression of bacterial virulence factors and the human immune system. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
Chapter
We feel that our main function at this symposium is to act as devil’s advocates. Several of the speakers this week have discussed protein degradation in many different tissues. However, I would like to gently remind the audience that about half of the total weight of the body, or 40% of the total protein of the body is muscle, and of this protein, 60% is represented by contractile protein (1). The average turnover time of the contractile proteins has been estimated to be about 6 days, varying from 4 days for actin to about 11 days for myosin (2). It is obvious that the protein reservoir is not only large, but highly mobile in the musculature of the body.
Chapter
The accumulation of phagocytic cells at the site of infection represents one of the most important defense mechanisms for protecting the host from noxious microorganisms, viruses, and parasites. The defense cells, also known as professional phagocytes, include neutrophils, eosinophils, monocytes, and a variety of macrophage phenotypes.
Article
Myeloperoxidase (MPO) belongs to the class of oxidoreductases (EC 1.11.1.7). The electron acceptor substrate for this enzyme is solely hydrogen peroxide, while the electron donor substrates are different reductants including halides (with the exception of fluoride).
Article
While sialic acid exists in free form, or as a part of relatively small heterosaccharide molecules, the major portion of naturally occurring sialic acid is present as a constituent of either glycoproteins or gangliosides.* Distribution of these sialic-acid-containing compounds in nature and their biosynthesis, are dealt with in Chapters 2 and 4. As for any other chemical constituents of biological systems, sialic acids exist in a metabolically dynamic state which, naturally, is the net result of constant biosynthesis and degradation.
Article
Myeloperoxidase (MPO), H2O2, and a halide form a powerful antimicrobial system which is effective against a variety of microorganisms. This system is also toxic to certain mammalian cells, namely, spermatozoa, erythrocytes, leukocytes, platelets, and tumor cells. This review (see also Klebanoff, 1975a; Klebanoff and Clark, 1978) will consider the properties of the MPO-mediated antimicrobial system and its role in the intracellular and extracellular toxicity of the polymorphonuclear leukocyte (PMN).
Article
A number of acute allergic responses e.g. the Arthus reaction, experimental nephrotoxic nephritis etc involve the chemotaxis, lysosomal enzyme secretion and phagocytosis of neutrophils. In these reactions, chemotactic factors liberated at the site causes infiltration of neutrophils and the secretion of lysosomal enzymes from these cells induced by actual or frustrated phagocytosis of the antigen-antibody complexes initiating the reaction induces the tissue damage (1). These functions of the neutrophil are not completely independent and unrelated. There are a number of similarities and differences in the biochemical requirements for each of these functions (2). In addition all the chemotactic factors tested, C5a, pronase sensitive and pronase insensitive fractions from E. coli culture filtrates and synthetic peptides are capable of inducing not only chemotaxis but lysosomal enzyme release (3, 4) and if these same factors are presented to the neutrophil at the same time as the particle, EAC423 they also will inhibit erythrophago- cytosis (5).
Article
The most dramatic changes induced by phagocytosis in PMN leucocytes are: 1) increase of oxygen uptake, insensitive to cyanide, rotenone, Antimicin A and amytal; 2) increase of H2O2 production; 3) increase of glucose oxidation through the HMP pathway (1–8). Similar changes are induced by treatment of leucocytes with surfactants (9,10) or antileucocyte antibodies (11).
Article
Acute inflammatory reactions, regardless of etiology, are recognized by the presence of polymorphonuclear leukocytes at sites of tissue damage. During the past two decades, it has become appreciated that these cells are not merely innocent bystanders to the events which occur at an inflammatory site, but play an active role in the mediation of tissue injury and suppuration. Central to this role are the inflammatory substances contained within these cells, particularly those sequestered within their cytoplasmic granules, or lysosomes. The nature and mode of action of these substances are the subjects of this chapter. Particular attention will be directed toward human polymorphonuclear leukocytes and relationships between these cells and other inflammatory mediator systems in the production of human disease.
Chapter
Proteinases of NeutrophilsProteinases of MacrophagesMechanisms of Enzyme ReleaseDegradation of Connective Tissue Material by Neutrophils: Possible Role of Elastase, Cathepsin G and Plasminogen ActivatorReferenceDiscussionReference
Article
Disintegration of rabbit polymorphonuclear leucocytes in suspension is achieved by extrusion under controlled pressure. The operating pressure is the key factor influencing the degree of disintegration. The number of cells disintegrated increases with higher pressure. At 35 kp/cm2, 80–90 per cent of the cells are disintegrated. The disintegrated material was subsequently analysed by following the subcellular distribution of leucocyte A and B granule marker enzymes. The analysis reveals that although 80–90 per cent of the cells are disintegrated, a high degree of subcellular integrity remains. Leucocyte A and B granules, abundantly present in the disintegrated material, were separated from soluble components by zonal centrifugation. Extrusion under controlled pressure as a cell disintegration method is discussed, particularly with reference to the study of polymorphonuclear leucocyte structure and function.
Article
A method is presented whereby inflammatory mediators may be detected and quantified in individual follicular casts. Lysozyme, lactoferrin, IgG, IgM, C3 and material reacting with antiserum to polymorphonuclear leukocytes (PMN) were assayed by functional and immunologic methods. By these techniques, lysozyme, IgG and anti-PMN reactive material were detected in clinically uninflamed follicular casts from acne subjects.
Article
The cationic antibacterial proteins of rabbit PMN lysosomes have been resolved into at least five subfractions. Each of these showed substantial selectivity in its antibacterial action against several pathogenic bacteria, including two smooth and two rough Escherichia coli strains, three Staphylococcus aureus strains, one S. albus, three proteus species and four different cultures of streptococcus. Each of the subfractions possesses a different electrophoretic mobility. Amino acid analyses of the three most cationic components revealed high contents of arginine consistent with their relative electrophoretic mobilities and very high arginine to lysine ratios. Aromatic amino acids were present in very low concentrations in these proteins and their light absorption at 2800 A was correspondingly weak. The evidence of antibacterial specificity, along with marked differences in the arginine-lysine ratios, shows that the cationic antibacterial components of rabbit PMN lysosomes are biologically and chemically heterogeneous.
Article
Biochemical and morphological evidence of a green particulate isolated for the first time from the leucocyte of normal human blood indicates that the particulate fulfills sufficient criteria to suggest that it is a lysosome. The presence of peroxidase, β-glucuronidase, and acid phosphatase, and the absence of enzymes of the electron transport system, support this. The human leucocytic lysosome is heavier than that reported for the guinea pig and lighter than that of the horse. Its distribution in a number of animal species as measured by the peroxidase content of the neutrophile suggests that its content per cell is remarkably constant in nature.
Article
The contents of selected hydrolytic enzymes of oil-induced peritoneal, normal alveolar, and BCG-induced alveolar macrophages have been studied. On a per cell or nitrogen basis the normal alveolar cells contained considerably more acid phosphatase, cathepsin, acid ribonuclease, lysozyme, and lipase than peritoneal cells. The BCG-induced alveolar macrophage exhibited increased levels of acid phosphatase, lysozyme, and lipase as compared to alveolar macrophages from unstimulated rabbits. The morphological differences between these cells was discussed and electron micrographs of the BCG-induced macrophage presented. Fractionation of the BCG-induced macrophage by differential centrifugation showed that 60 to 80 per cent of the total cell content of acid phosphatase, cathepsin, beta glucuronidase, acid ribonuclease, acid deoxyribonuclease, aryl sulfatase, lysozyme, and lipase were localized in a postnuclear fraction which sedimented at 15,000 g. This fraction also contained the majority of the mitochondria as evidenced by its content of cytochrome oxidase. Non-specific esterase was not localized to this fraction. A separation of the hydrolase-containing particles and mitochondria was achieved by isopycnic sucrose gradient centrifugation. Under the conditions employed, the mitochondria distributed at densities of 1.19 to 1.20, whereas the hydrolase particles sedimented to a density of 1.26 to 1.27. Each of the hydrolases including acid phosphatase, beta glucuronidase, cathepsin, lysozyme, and acid ribonuclease exhibited maximum activities in the same gradient fraction. The isolated granules exhibited enzymatic latency, and activation could be achieved by cycles of freezing and thawing or surface active agents. The majority of each of the hydrolytic enzymes could be liberated in a non-particulate form by mechanical trauma. Macrophages which had been stained supravitally with neutral red were fractionated by differential and gradient centrifugation. More than 70 per cent of the dye could be recovered in the particulate hydrolase fraction. The isolated, stained granules resembled those seen in the intact cell.
Article
In rabbit heterophils, acid phosphatase activity occurs in primary (azurophil) granules which predominate in early cells and persist in mature cells and in tertiary granules which are seen only in mature cells. Alkaline phosphatase activity occurs in secondary granules which appear in intermediate heterophils and later predominate in mature cells. Acid phosphatase activity in heterophil Golgi zones coincides developmentally with the genesis of primary and, later, tertiary granules, whereas alkaline phosphatase in the Golgi complex coincides with secondary granulogenesis. In developing eosinophils, acid phosphatase reaction product occurs in Golgi elements, rims the spherical precursors of angular, mature granules and appears inconsistently within mature granules. Basophil myelocytes show acid phosphatase in Golgi elements but not in specific granules. Additional acid phosphatase reactive structures include: granules of mononuclear cells; phagocytic vacuoles in macrophages; autophagic vacuoles in maturing erythroid cells; small dense granules of platelets; dense bodies in lipocytes; and Golgi elements of mononuclear cells, macrophages, nucleated red cells, megakaryocytes and lipocytes. Localized deposits were absent in control specimens except for enzyme-independent nuclear staining in alkaline phosphatase preparations.
Article
Human leukocytes in suspension or in monolayer cultures have been processed for electron microscopy by fixation in a freshly made cold mixture of glutaraldehyde and osmium tetroxide and by "postfixation" in uranyl acetate. Simultaneous exposure to glutaraldehyde and osmium tetroxide eliminates many of the shortcomings seen when either of these agents is used alone as the initial fixative. Specimens are processed to the stage of dehydration as single cell suspensions or as very small clumps to assure rapid penetration of fixatives and efficient washing. The technique is rapid and reproducible. Electron micrographs presented in this report illustrate the ultrastructural features of human white cells prepared by this method.
Article
The origin, nature, and distribution of polymorphonuclear leukocyte (PMN) granules were investigated by examining developing granulocytes from normal rabbit bone marrow which had been fixed in glutaraldehyde and postfixed in OsO(4). Two distinct types of granules, azurophil and specific, were distinguished on the basis of their differences in size, density, and time and mode of origin. Both types are produced by the Golgi complex, but they are formed at different stages of maturation and originate from different faces of the Golgi complex. Azurophil granules are larger ( approximately 800 mmicro) and more dense. They are formed only during the progranulocyte stage and arise from the proximal or concave face of the Golgi complex by budding and subsequent aggregation of vacuoles with a dense core. Smaller ( approximately 500 mmicro), less dense specific granules are formed during the myelocyte stage; they arise from the distal or convex face of the Golgi complex by pinching-off and confluence of vesicles which have a finely granular content. Only azurophil granules are found in progranulocytes, but in mature PMN relatively few (10 to 20%) azurophils are seen and most (80 to 90%) of the granules present are of the specific type. The results indicate that inversion of the azurophil/specific granule ratio occurs during the myelocyte stage and is due to: (a) reduction of azurophil granules by multiple mitoses; (b) lack of new azurophil granule formation after the progranulocyte stage; and (c) continuing specific granule production. The findings demonstrate the existence of two distinct granule types in normal rabbit PMN and their separate origins from the Golgi complex. The implications of the observations are discussed in relationship to previous morphological and cytochemical studies on PMN granules and to such questions as the source of primary lysosomes and the concept of polarity within the Golgi complex.
Article
Methods have been developed for the quantitative assay of cytochrome oxidase, esterase, and 11 acid hydrolases in rat-spleen homogenates. These methods seem to be applicable also to other lymphoid tissues. Preliminary studies, extended to nine of the acid hydrolases, indicate that these enzymes occur in partly latent and sedimentable form and that they can be unmasked and rendered soluble by some of the treatments that liberate the enzymes from rat-liver lysosomes. The spleen particles appear to be very sensitive to mechanical injury, a property which necessitates special precautions in homogenizing the tissue. Agglutination of spleen particles takes place to a larger extent in 0.25 M sucrose than in 0.15 M KCl.
Article
A technique has been developed for collecting large numbers of polymorphonuclear leucocytes from peritoneal exudates in rabbits. These cells are obtained essentially free from other cell types and from debris. When microphages so procured are disrupted by physical methods and extracted with aqueous salt solutions, the soluble fraction manifests striking bactericidal activity, especially on Gram-negative enteric bacilli. The susceptible microorganisms are not lysed. This bactericidal substance, which has been called phagocytin, appears to be limited in distribution mainly to the polymorphonuclear leucocyte. No phagocytin is present in extracts of rabbit heart, kidney, or skeletal muscle, and rabbit liver and spleen contain much less than do packed leucocytes. Extracts of human and of guinea pig microphages show less bactericidal activity than rabbit cell preparations. Similar extracts of rat and mouse polymorphonuclear leucocytes contain no demonstrable phagocytin. As indicated by its behavior on dialysis, on exposure to proteolytic enzymes, and on salt fractionation, phagocytin appears to be a protein with general properties characteristic of a globulin. It is clearly different from lysozyme and from properdin. Although phagocytin is reasonably stable at temperatures of 65°C. and lower for several hours, solutions of it gradually lose bactericidal activity on standing for prolonged periods at 4°C. This instability, and also the ease with which phagocytin is inactivated, presumably by adsorption, on exposure to a variety of materials, have thus far rendered fruitless efforts to isolate it.
Article
1. The activities of lysosomal enzymes in the cortexes and medullas and the principal subcellular fractions of rat kidney were measured. 2. A method is described for the isolation of rat-kidney lysosomes and a detailed analysis of the enzymic composition of the lysosomes is reported. Enzyme analysis of the other principal subcellular fractions is included for comparison. 3. Studies of the distribution of alpha-glucosidase showed that the lysosomal fraction contained only 10% of the total enzyme activity. The microsomal fraction contained most of the particulate alpha-glucosidase. Lysozyme was concentrated mainly in the lysosomal fraction with only small amounts present in the microsomal fraction. Lysosomal alpha-glucosidase had optimum pH5 whereas the microsomal form had optimum pH6. Both lysosomal and microsomal lysozyme had optimum pH6.2. 4. The stability of lysosomal suspensions was studied. Incubation at 37 degrees and pH7 resulted in first an increased availability of enzymes without parallel release of enzyme. This was followed by a second stage during which the availability of enzymes was closely related to the release of enzymes. These changes were closely paralleled by changes in light-scattering properties of lysosomes. 5. The latent nature of the alpha-glucosidase and lysozyme of intact kidney lysosomes was demonstrated by their graded and parallel release with other typical lysosomal enzymes. 6. Isolated lysosomes were unstable at pH values lower than 5, most stable at pH6-7 and less stable at pH 8-9. Lysosomes were not disrupted when the osmolarity of the suspending medium was decreased from 0.6m to 0.25m. 7. The discussion compares the properties and composition of kidney lysosomes, liver lysosomes and the granules of macrophages. 8. The possible origin of the lysozyme in kidney lysosomes by reabsorption of the lysozyme in blood is discussed.
  • D A Waters
  • W D Fisher
  • G B Cline
  • C E Nunley
  • L H Elrod
  • C T Rankin
ANDERSON, N. G., D. A. WATERS, W. D. FISHER, G. B. CLINE, C. E. NUNLEY, L. H. ELROD, and C. T. RANKIN, JR. 1967. Anal. Biochem. 21:235.
  • D F Bainton
  • M G Farquhar
BAINTON, D. F., and M. G. FARQUHAR. 1968a. J. Cell Biol. 39:286.
  • C De Duve
BEAUFAY, H., and C. DE DUvE. 1954. Bull. Soc. Chim. Biol. 36:1525.
  • M Bessrs
  • J Thiery
BEssrs, M., and J. THIERY. 1961. Int. Rev. Cytol. 12:199.
  • W E Bowers
  • J T Finkenstaedt
  • C De Duve
BOWERS, W. E., J. T. FINKENSTAEDT, and C. DE DUvE. 1967. J. Cell Biol. 32:325.
  • T Y Toribara
  • H Warner
CHEN, JR., P. S., T. Y. TORIBARA, and H. WARNER. 1956. Anal. Chem. 28:1756.
  • J G Hirsch
COHN, Z. A., and J. G. HIRSCH. 1960. J. Exp. Med. 112:983.
  • E Wiener
COHN, Z. A., and E. Wiener. 1963. J. Exp. Med. 118:991.
  • De Duve
DE DUVE, C., and R. WATTIAUX. 1966. Annu. Rev. Physiol. 28:435.
  • Z A Cohn
HIRSCH, J. G., and Z. A. COHN. 1960. J. Exp. Med. 112:1005.
  • M E Fedorko
HIRSCH, J. G., and M. E. FEDORKO. 1968. J. Cell Biol. 38:615.
  • N Berger
  • M J Bonner
  • J Schultz
  • S J Klebanoff
  • S J S Fowler
JOHN, S., N. BERGER, M. J. BONNER, and J. SCHULTZ. 1967. Nature. 215:1483. 20. KLEBANOFF, S. J. 1967. J. Exp. Med. 126:1063. 21. KLEBANOFF, S. J. 1968. J. Bacteriol. 95:2131. 22. LEIGHTON, F., B. POOLE, H. BEAUFAY, P. BAUDHUIN, J. W. COFFEY, S. FOWLER, and C.
  • De Duve
DE DuVE. 1968. J. Cell Biol. 37:482.
  • R Corlin
  • F Oddi
  • K Kaminker
  • W Jones
SCHULTZ, J., R. CORLIN, F. ODDI, K. KAMINKER, and W. JONES. 1965. Arch. Biochem. Biophys. 111:73.
  • A L Tappel
SHIBKO, S., and A. L. TAPPEL. 1965. Biochem. J. 95:731.
  • R G Horn
  • S S Spicer
WETZEL, B. K., R. G. HORN, and S. S. SPICER. 1967. Lab. Invest. 16:349.
  • J K Spitznagel
ZEYA, H. I., and J. K. SPITZNAGEL. 1968. J. Exp. Med. 127:927.
  • H Beaufay
  • C De Duve
BEAUFAY, H., and C. DE DUvE. 1954. Bull. Soc. Chim. Biol. 36:1525.
  • J R Chen
  • T Y Toribara
CHEN, JR., P. S., T. Y. TORIBARA, and H. WARNER. 1956. Anal. Chem. 28:1756.
  • Z A Cohn
  • J G Hirsch
COHN, Z. A., and J. G. HIRSCH. 1960. J. Exp. Med. 112:1015.
  • J G Hirsch
HIRSCH, J. G. 1962. J. Exp. Med. 116:827.
  • S John
  • N Berger
  • M J Bonner
  • J Schultz
JOHN, S., N. BERGER, M. J. BONNER, and J. SCHULTZ. 1967. Nature. 215:1483.
  • H Ohta
OHTA, H. 1964. Acta Haematol. Jap. 27:555. 24. PAESE, D. C. 1956. Blood. 11:501.
  • R E Vercauteren
VERCAUTEREN, R. E. 1964. Enzymol. 27:369.
  • Y Watanabe
WATANABE, Y. 1957. J. Electronmicroscopy. 5:46.
  • Zucker-Franki
  • D In
  • J G Hirsch
ZUCKER-FRANKI.IN, D., and J. G. HIRSCH. 1964. J. Exp. Med. 120:569.