Article

The Cleavage of Prolyl Peptides by Kidney Peptidases

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Abstract

Carboxypeptidase activity was detected in swine kidney microsomes. The enzyme was solubilised by treatment with trypsin and toluene. Approximately 330-fold purification was achieved by subsequent chromatography on DEAE-cellulose and hydroxylapatite. As shown by polyacrylamide gel investigation, the carboxypeptidase preparation thus obtained was practically pure. It still contains trace impurities of aminopeptidase M which is quite similar in physical properties. The molecular weight of the carboxypeptidase was 240000 as determined by Sephadex gel filtration. It is activated by manganese ions and exhibits a pH-optimum at 7.75. The carboxypeptidase preferentially attacks substrates with a prolyl residue in the last but one position. No chain length dependence was found. Peptides free of proline were also cleaved, but normally at a rather low rate. The designation carboxypeptidase P (indicating its preference of proline peptide bonds) is therefore suggested. The physiological role of the new peptidase is discussed.

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... Analysis of the isolates demonstrated that they had mutations in the pepQ gene, which encodes a putative Xaa-Pro aminopeptidase (Bloemberg et al. 2015;Harris et al. 2003). Such proteins catalyze the release of an Nterminal amino acid linked to a proline and are highly conserved across all three domains of life (Dehm and Nordwig 1970;Cottrell et al. 2000). However, the mechanism by which pepQ mutations result in increased resistance to bedaquiline is unknown. ...
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Article
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Article
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Article
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Article
A method for the preparation of brush border from rabbit kidneys is described. Contamination by other organelles was checked by electron microscopy and by the assay of marker enzymes and was low. Seven enzymes, all hydrolases, were substantially enriched in the brush border preparation and are considered to be primarily located in this structure. They are: alkaline phosphatase, maltase, trehalase, aminopeptidase A, aminopeptidase M, γ glutamyl transpeptidase and a neutral peptidase assayed by its ability to hydrolyse [125I]iodoinsulin B chain. Adenosine triphosphatases were also present in the preparation, but showed lower enrichments. Alkaline phosphatase was the most active phosphatase present in the preparation. The weak hydrolysis of AMP may well have been due to this enzyme rather than a specific 5' nucleotidase. The two disaccharidases in brush border were distinguished by the relative heat stability of trehalase compared with that of maltase. The individuality of the four peptidases was established by several means. The neutral peptidase and aminopeptidase M, both of which can attack insulin B chain, differed not only in response to inhibitors and activators but also in the inhibitory effect of a guinea pig antiserum raised to rabbit aminopeptidase M. This antiserum inhibited both the purified and the brush border activities of aminopeptidase M. The neutral peptidase and γ glutamyl transpeptidase were unaffected but aminopeptidase A was weakly inhibited. The characteristic responses to Ca2+ and serine with borate served to distinguish aminopeptidase A and γ glutamyl transpeptidase from other peptidases. No dipeptidases, tripeptidases or carboxypeptidases were identified as brush border enzymes. Incubation of brush border with papain released almost all the aminopeptidase M activity but only about half the activities of maltase, γ glutamyl transpeptidase and aminopeptidase A. No release of alkaline phosphatase, trehalase or the neutral peptidase was observed.
Article
Peptides of the X-Pro-Y type were found to be readily cleaved by crude swine kidney homo-genates. Using Gly-Pro-Hyp as a substrate, a peptidase releasing glycine was isolated and partially purified from the microsomal fraction of the homogenate. The purification steps used included treatment of the kidney microsomes with n-butanol, and chromatography of the protein fraction thus released on DEAE-cellulose as well as gel filtration on Sephadex G-200. The increase in specific activity was approximately 250-fold.The purified preparation consisted of two enzymatically active components exhibiting molecular weights of one million and two millions, respectively. There is evidence that these components are lipoproteins, i.e. membrane fragments of different size.The activity of the enzyme was optimal at pH 8 and was greatly increased in the presence of manganese ions and albumin. Since the peptidase required a prolyl residue in the second position of the substrate, the designation “X-prolyl-aminopeptidase” is suggested. Although the enzyme acted on substrates of different chain length, the reaction rates were greatest with peptides of the structure Gly-Pro-Y. Therefore, a role of X-prolyl-aminopeptidase in the degradation of collagen appears plausible.
Article
Prolylleucylglycinamide (MIF) at 1.0 mM concentration and pH 7.0 was hydrolyzed by mouse brain homogenate at a rate of 140 nmol/mg protein/hr. Nearly all of this activity can be accounted for by the action of two enzymes, both of which cleave Pro and Leu sequentially from the N-terminus of MIF. At pH 7.0 the predominant enzyme is arylamidase, inhibited by puromycin (1 mM) and Mn2+ (2.5 mM). At pH 8.5, in the presence of Mn2+, a second enzyme with a higher potential activity (570 nmol/mg protein/hr) was observed. While the arylamidase is primarily localized in the cytosol, the Mn2+-stimulated enzyme is equally divided between soluble and particulate fractions. Because of its ability to cleave leucinamide, its high pH optimum, and its Mn2+ dependence, it can be classified as a leucine aminopeptidase (LAP). In its substrate specifically and its preference for Mn2+ over Mg2+ it resembles the LAP from connective tissue more than that from other sources.
Article
The cytosol peptidase activities hydrolyzing glycl-L-leucine and L-leucyl-glycine as well as the activities of the brush border peptidases (oligoaminopeptidase, aminopeptidase A, gamma-glutamyl-transpeptidase, dipeptidyl-aminopeptidase IV, and carboxypeptidase) are present in rat fetuses during the early differentiation of the intestine (17th to 19th days of fetal life); they increase then at a different rate, reaching a maximum at various times, in the second and third wk after birth, and then decrease to the adult values during the first month of postnatal life. Only the oligoaminopeptidase activity increases steadily after birth, reaching maximal activity in the last decade of the first month. In human fetuses aged between 8 and 22 wk, the gamma-glutamyltranspeptidase was the only brush border peptidase found to be higher than in adults and children. On the other hand, the aminopeptidase A is remarkably reduced. The dipeptidylaminopeptidase IV and the carboxypeptidase are already at adult level in the youngest fetuses, and the oligoaminopeptidase increases during the period of fetal life studied; at the end of this period, the enzyme activity does not differ from the values found in children and adults. The small intestine of the term and preterm newborn should, therefore be able, with some possible exceptions, to efficiently digest peptides.
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A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.
Article
Peptidases which are specific for proline residues have been described and include endopeptidases (post-proline cleaving enzyme and proline specific endopeptidase), N-terminal exopeptidases (post-proline dipeptidyl aminopeptidase, proline iminopeptidase, aminopeptidase P), C-terminal exopeptidases (prolylcarboxypeptidase, and carboxypeptidase P) and dipeptidases (prolyl dipeptidase and proline dipeptidase). The properties, distinguishing charcteristics, and possible significance of these proline specific endo- and exopeptidases are discussed. In addition, reference is made to a series of enzymes which can hydrolyze proline containing peptide bonds, but which are not specific for proline.
Article
Biochemical studies on human prolidase (EC 3.4.13.9) and prolidase deficiency are described. The urine sample from a 32-year-old female with prolidase deficiency was examined. Diagnosis was based on clinical features and defects of prolidase in her erythrocytes. She excreted massive amounts of iminopeptides, where three major peptides were identified; aspartyl-proline, glutamyl-proline and glycyl-proline. The prolidase was purified approximately 10,000-fold from the normal human erythrocytes through an eight step procedure. The purified enzyme consisted of two identical subunits of which the molecular weight was calculated to be 55,000. The relative cleavage rates of the enzyme for glycyl-L-proline, L-alanyl-L-proline, L-leucyl-L-proline, L-prolyl-L-proline, and glycyl-hydroxy-L-proline were 100%, 53%, 27%, 31% and 2%, respectively. The relative substrate specificity of the enzyme offers a reasonable explantation for the presence of a higher level of urinary imidodipeptides in a patient with prolidase deficiency. An attempt at erythrocyte transfusion was performed, aimed at enzyme replacement therapy. After the transfusion (erythrocytes from 800 ml of whole blood), the prolidase activity of the peripheral erythrocyte was elevated to approximately 35% of the normal values and gradually decreased (half-life, 41 days). During this period urinary peptide-bound proline was monitored, but no significant change was observed.
Article
Proline residues confer unique structural constraints on peptide chains and markedly influence the susceptibility of proximal peptide bonds to protease activity. This review presents a critical analysis of peptidases involved in the cleavage of proline-containing peptide bonds, with particular attention to the role of proline peptidases in the regulation of the lifetime of biologically active peptides. Peptidases discussed include aminopeptidase P, prolidase, dipeptidyl peptidase IV, prolyl endopeptidase, and prolyl iminopeptidase. Attention is also given to HIV-1 protease, because this key enzyme processes an Xaa-Pro peptide bond. Analysis of the above enzymes reveals that they may function as key pacemakers in the control of the activity of many peptide hormones and that they are involved in a variety of immunological processes, including T-cell-mediated immune response. The novel occurrence of cis-trans isomerization about Xaa-Pro bonds and the biological function of peptidyl-prolyl cis-trans isomerases (immunophilins) are reviewed.
Article
A proline-specific dipeptidylcarboxypeptidase, which removes diproline from the C-terminus of the proline-containing peptides, such as Boc-Pro-Pro-Pro-Pro and Leu-Pro-Pro-Pro-Pro-Pro, has recently been purified from a Streptomyces sp. The specificity of the enzyme for various imino acid-containing synthetic peptide substrates was further studied. The peptides with proline, hydroxyproline, or dehydroproline at the P2' position were found to be good substrates, while those with pipecolic acid, D-proline or other usual amino acids at the P2' position were scarcely hydrolyzed. The peptides with proline, dehydroproline, pipecolic acid, or N-methyl-alanine at the P1' position were well-hydrolyzed, while those with hydroxyproline or D-proline at the P1' position were not hydrolyzed. Utilizing this high specificity for imino acids, Boc-Pro-Pro-Pro-Pro was synthesized by the enzyme using Boc-Pro-Pro as the acidic component and Pro-Pro as the basic component.
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CD26/DPPIV can be considered a moonlighting protein because it is a multifunctional protein that exerts its different functions depending on cell type and intra- or extracellular conditions in which it is expressed. In the present review, we summarize all its known functions in relation to physiological and pathophysiological conditions. The protein is a proteolytic enzyme, receptor, costimulatory protein, and is involved in adhesion and apoptosis. The CD26/DPPIV protein plays a major role in immune response. Abnormal expression is found in the case of autoimmune diseases, HIV-related diseases and cancer. Natural substrates for CD26/DPPIV are involved in immunomodulation, psycho/neuronal modulation and physiological processes in general. Therefore, targeting of CD26/ DPPIV and especially its proteolytic activity has many therapeutic potentials. On the other hand, there are homologous proteins with overlapping proteolytic activity, which thus may prevent specific modulation of CD26/DPPIV. In conclusion, CD26/DPPIV is a protein present both in various cellular compartments and extracellularly where it exerts different functions and thus is a true moonlighting protein.
Article
Carboxypeptidases from animal, plant, fungal, and bacterial sources were tested for their ability to bind to the carboxypeptidase inhibitor from Russet Burbank potatoes. Enzymes which participate in the degradation of dietary protein were partially purified from animal species as diverse as the cow and the limpet, and all were potently affected by the inhibitor. However, several zymogens of the enzymes in this group were tested and shown not to bind immobilized inhibitor. With the exception of an enzyme from mast cells and a novel carboxypeptidase A-like enzyme from bovine placenta, all animal carboxypeptidases which were not of digestive tract origin were not affected by the inhibitor. The inhibitor had no effect on the enzymic activities of all plant and most microbial carboxypeptidases. However, a strong association between the inhibitor and Streptomyces griseus carboxypeptidase has been noted previously and a low affinity (K(i) about 10 micromolar) for a carboxypeptidase G(1) from an acinetobacterium was found in this study.
Article
Full-text available
X-prolyl aminopeptidases catalyze the removal of a penultimate prolyl residue from the N termini of peptides. Mammalian X-prolyl aminopeptidases are shown to be responsible for the degradation of bradykinin, a blood pressure regulator peptide, and have been linked to myocardial infarction. The x-ray crystal structure of human cytosolic X-prolyl aminopeptidase (XPN-PEP1) was solved at a resolution of 1.6Å. The structure reveals a dimer with a unique three-domain organization in each subunit, rather than the two domains common to all other known structures of X-prolyl aminopeptidase and prolidases. The C-terminal catalytic domain of XPNPEP1 coordinates two metal ions and shares a similar fold with other prolyl aminopeptidases. Metal content analysis and activity assays confirm that the enzyme is double Mn(II) dependent for its activity, which contrasts with the previous notion that each XPNPEP1 subunit contains only one Mn(II) ion. Activity assays on an E41A mutant demonstrate that the acidic residue, which was considered as a stabilizing factor in the protonation of catalytic residue His498, plays only a marginal role in catalysis. Further mutagenesis reveals the significance of the N-terminal domain and dimerization for the activity of XPNPEP1, and we provide putative structural explanations for their functional roles. Structural comparisons further suggest mechanisms for substrate selectivity in different X-prolyl peptidases.
Article
DURING the past few decades the mechanism of hydrolysis of peptides has been intensively investigated. An important outcome of these studies has been the establishment of the fact that the formation of amino-acids by the splitting of dipeptides does not occur in the cavity of the small intestine, but during the transport of peptides through the intestinal wall. The conclusion has been drawn, based on the analysis of correlation between the peptidase activity of the mucous liquid and homogenate of the intestine, that hydrolysis occurs intracellularly, that is, the peptides are passively transported through the membrane of the brush border into the cells of the small intestine, where they are split up into amino-acids1,2. Data exist which suggest that this process occurs in the apical parts of the cytoplasm3. On the other hand, investigations of the effect of liquid convection relative to the surface of mucosa on the rate of hydrolysis of dipeptides4 have revealed that this process is more likely to be localized on the external surface of the cell membranes (membrane digestion) than within the cells.
Article
The effects of amino acids and peptides on Chinard's (2) method of estimating proline were investigated.Of the 18 α-amino acids tested, all except aspartic acid interfered with the method. Of the 17 di- and tripeptides tested, only prolyglycine and N-glycyl peptides interfered. The interference by peptides was found to be secondary to their hydrolysis to amino acids during the estimation.By adding glycine to the solution to be analyzed, the interference by 10 amino acids and by all the peptides except glycylproline could be eliminated and the sensitivity of the method increased by 50%.
Article
Peptides of the X-Pro-Y type were found to be readily cleaved by crude swine kidney homo-genates. Using Gly-Pro-Hyp as a substrate, a peptidase releasing glycine was isolated and partially purified from the microsomal fraction of the homogenate. The purification steps used included treatment of the kidney microsomes with n-butanol, and chromatography of the protein fraction thus released on DEAE-cellulose as well as gel filtration on Sephadex G-200. The increase in specific activity was approximately 250-fold.The purified preparation consisted of two enzymatically active components exhibiting molecular weights of one million and two millions, respectively. There is evidence that these components are lipoproteins, i.e. membrane fragments of different size.The activity of the enzyme was optimal at pH 8 and was greatly increased in the presence of manganese ions and albumin. Since the peptidase required a prolyl residue in the second position of the substrate, the designation “X-prolyl-aminopeptidase” is suggested. Although the enzyme acted on substrates of different chain length, the reaction rates were greatest with peptides of the structure Gly-Pro-Y. Therefore, a role of X-prolyl-aminopeptidase in the degradation of collagen appears plausible.
Article
A method of peptidase assay based upon a differential effect of cupric ion on the reaction of trinitrobenzenesulfonate (TNBS) with amino acids and peptides has facilitated the separation and characterization of renal peptidases. Three such peptidases, all active in the hydrolysis of leucylglycine, were separated and further characterized; these peptidases were the leucine aminopeptidase of the soluble fraction and the leucyl-β-naphtyhlamidase and the “solubilized aminopeptidase” of the endoplasmic membranes of renal tissue. The solubilized aminopeptidase appears to be identical with the renal iminodipeptidase (prolinase).
Article
An improved method for the liberation and purification of the particle-bound aminopeptidase of pig kidney is presented. Large quantities of enzyme may be prepared by separating the so-called "microsomal" particles by pH aggregation rather than by ultracentrifugation. The enzyme is distinguished from other aminopeptidases by its relative stability to heat and extremes of pH. It is denatured by alcohols, especially by long-chain alcohols, and by other organic solvents. The purest preparations were homogeneous and had molecular weights of 280,000. The most useful pH for the hydrolysis of peptides appears to be between 7 and 7.3, where the enzyme has maximum affinity for its substrate. The true maximum rate of hydrolysis is at pH 9, but here the affinity of the enzyme for its substrate is at its lowest. The turnover number is 10,450 moles of leucine p-nitroanilide/mole of aminopeptidase at 37°.
Article
Methods for preparation of stable columns of calcium phosphate (hydroxylapatite) suitable for protein chromatography and conditions for specific elution of proteins on such columns have been worked out. Elution is brought about by stepwise or continuous increase in the concentration of the eluant (phosphate buffer of constant pH), or by displacement. A number of proteins of molecular weights from about 10,000 to several millions have been chromatographed. In several cases multiple components were observed, indicating inhomogeneity. Some characteristic features of the procedure have been discussed, particularly the narrow elution ranges and the marked mutual displacement effects shown by many proteins. Low-molecular-weight material was found to show little or no adsorption on these columns. A description is given of photographic and television methods of observing directly colorless protein zones on the columns, making use of their light absorption in the short-wave ultraviolet region.
Article
A colorimetric method for the quantitative analysis of pure amino acids is described. It is a modification of one reported by Yemm and Cocking, which employs cyanide and ninhydrin. The present method avoids the necessity for the preparation of solutions of reduced ninhydrin, which is an unstable reagent difficult to prepare and impracticable to store.The method is applicable to amino acid mixtures when allowances are made for slight variability of color yields, and the presence of interfering compounds.
Article
IT has been found that a Carboxypeptidase (or a mixture of various carboxypeptidases) occurs in the peel of citrus fruit, for example, of the orange (Citrus sinensis), lemon (Citrus medica), and grapefruit (Citrus maxima). This peptidase differs in specificity and in other properties from the known carboxypeptidases A (ref. 1) and B (ref. 2) of the pancreas and from the catheptic Carboxypeptidase of brain3. This citrus peptidase or enzyme mixture, referred to for short as Carboxypeptidase C, is the first Carboxypeptidase to be demonstrated in vegetable tissue. CarboxypeptidaseC belongs to the catheptic peptidases-a group which, if we extend and re-define the term `cathep-sin', can also be said to include the glycyl-glycine-dipeptidase4 and leucinaminopeptidase5 found in leaves.
Article
The digestive cycle following reabsorption of hemoglobin by cells of the proximal convoluted tubules in mouse kidney and the uptake of ferritin by glomerular mesangial cells in the kidney of normal and nephrotic rats were investigated by electron microscopical histochemical procedures. Mouse kidneys, sampled at closely spaced time points between 1 to 48 hours after intraperitoneal injection of hemoglobin, and rat (normal and nephrotic) kidneys, sampled at 30 minutes, 2 hours, and 48 hours after intravenous injection of ferritin, were fixed in glutaraldehyde, cut at 50 µ on a freezing microtome, incubated for acid phosphatase and thiolacetate-esterase, and postfixed in OsO4. Satisfactory preservation of fine structure permitted the localization of the enzymatic reaction products on cell structures involved in uptake and digestion of exogenous proteins. The latter were identified either by their density (hemoglobin) or their molecular structure (ferritin). It was found that lysosomal enzymic activities and incorporated exogenous proteins occur together in the same membrane-bounded structures. In the cells of the proximal convolution, lytic activities become demonstrable within 1 hour after hemoglobin injection, appear first in apical vacuoles filled with hemoglobin, and persist in fully formed protein absorption droplets. At the end of the lytic cycle (∼48 hours post injection), the cells have an increased population of polymorphic bodies which exhibit lytic activities. In smaller numbers, identical bodies occur in controls. It is concluded that they represent remnants of previous digestive events. The means by which the resorptive vacuoles acquire hydrolytic activities remain unknown. Fusion of newly formed vacuoles with residual bodies was not seen, and hemoglobin incorporation into such bodies was only occasionally encountered. Acid phosphatase activity was found sometimes in the Golgi complex, but enzyme transport from the complex to the resorbing vacuoles could not be established. Autolytic vacuoles containing mitochondria or mitochondrial remnants were frequently found during the early stages of hemoglobin resorption, but no definite conclusions about the mechanism involved in the segregation of endogenous material were obtained. In nephrotic rats ferritin was segregated in membrane-bounded bodies mainly in the mesangial cells and to a lesser extent in epithelial and endothelial cells. Most of these sites were marked by the reaction products of acid phosphatase and organophosphorus-resistant esterase and therefore identified as lysosomes connected with the digestion of incorporated exogenous proteins.
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.
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