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ABSTRACT: Isolation of glycosylated 26 kDa rat prolactin and subsequent proper carbohydrate characterization has so far not been reported. In the present work the hormone isoform was isolated to 95% homogeneity by preparative electrophoretic separation on Mini Prep Cell of rat pituitary homogenate. The isoform was then investigated by 2-mercaptoethanol gradient electrophoresis, Cleveland's sequential SDS-PAGE, digestion with endoproteinase Asp-N and N-glycanase. The glycosidic part of the isoform was examined in O-profiling and its monosaccharide composition obtained by FACE and HPAE-PAD analysis. The outcome of the experimental data is: 1) in contrast to unglycosylated 23 kDa rat prolactin, intra-chain S-S bridging is not affected in 26kDa rat prolactin, neither by transiting through a thiol gradient nor in sequential nonreducing/reducing SDS-PAGE; 2) the conformational availability of Asp residues involved in the endoproteinase Asp-N attack is the same in 23- and 26 kDa rat prolactin; the glycan moiety apparently does not cause steric hindrance at this level; 3) no glycosidic N-linkage could be detected, only O-linkage(s); 4) 26 kDa rat prolactin is no glycosyl-phosphaditylinositol-anchored protein; 5) in O-profiling an oligosaccharide chain of Mr +/- 1.4 kDa was recorded; 6) the monosaccharide composition obtained in FACE is peculiar in the sense that next to Fuc, Man, GalNac, GlcNac and NeuAc also Rib was determined; 7) HPAE-PAD analysis identified NeuAc subtypes; 8) in vitro, glycosylation of rat prolactin modulates immune recognition through steric hindrance of the access to the epitope sites.
Archives of Physiology and Biochemistry 05/2001; 109(2):180-90.
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ABSTRACT: To gain an insight in the routing, processing and export of rat prolactin, rat pituitary cells were cultured in serum-free medium in the presence of cycloheximide, carbonyl cyanide m-chlorophenylhydrazone, Brefeldin A and monensin. The potential influence of these perturbants, whose well documented effects are the altering of protein synthesis and transport, was studied on rat prolactin molecular size isoforms appearing in cellular extracts and in culture medium. The outcome of the culture experiments as recorded in vertical SDS-PAGE, thiol gradient electrophoresis and sequential SDS-PAGE followed by prolactin specific immunoblotting and densitometry, was as follows: (1) at the cellular level we were able to characterize a novel 36 kDa protein as a disulphide-bridged oligomeric precursor prolactin, which is presumably rapidly transformed in the cis/medial Golgi; to designate monomeric rat prolactin as an early Golgi protein and t o advance evidence that the main processing of the glycosylated rat prolactin is a cis/medial Golgi event; (2) in release none of the perturbants disturbed the relative distribution of monomeric and glycosylated rat prolactin, the main molecular size isoforms currently secreted by untreated pituitary cells, or induced the appearance of transformed molecular size isoforms; (3) the secretion mode indicates that rat prolactin is released via the regulated pathway in the presence of the perturbants used.
Archives of Physiology and Biochemistry 11/1999; 107(4):312-22.
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ABSTRACT: The modulation of both the molecular size heterogeneity and the relative distribution of rat prolactin variants, synthesized and secreted in vitro by rat pituitary cells in the course of postnatal ontogeny and in gestation, lactation and weaning was investigated by SDS-PAGE, immunoblotting, radioimmunological techniques and O-sialoendopeptidase digestion. The outcome of the experiments is as follows: 1) from day 1 of postnatal life 20-, 23-, 26-, 40-44 kDa and oligomeric rat prolactin isoforms were stored and secreted; 2) perinatal life is characterized by a high degree of variability of prolactin size isoforms and their respective repartition in storage and release; in addition to the major variants, transient ones of M, 25-, 28-, 33- and 36 kDa were secreted and/or stored; 3) O-sialoglycoprotease digestion of pituitary cell lysate gave good evidence for 25 kDa prolactin being a glycoform; 4) at 1 month of age 16 kDa rat prolactin appeared and persisted over the whole postnatal span (1 day-->1 year) but only in stored form; 5) the physiology of gestation was essentially characterized by the M(r)-modulation of the glycoform (26 kDa-->26.3 kDa) and the virtual absence of stored 26 kDa rat prolactin at week 1 of pregnancy; 6) in lactation and weaning uncommon multiple banding was observed in secreted oligomeric prolactin; 7) in pregnancy, lactation and weaning the differential distribution of released and stored prolactin isoforms displayed a considerable intra- and intervariability; 8) in the vast array of size isoforms observed in all our experiments monomeric 23 kDa prolactin was always the dominating variant. In conclusion, the molecular size heterogeneity and the differential distribution of secreted and stored rat pituitary prolactin is considerably influenced by age and physiological stimuli. The nature of polymeric prolactin and of the transient variants is presently unclear, and the exact physiological role of molecular heterogeneity modulation is unknown, both in humans and rat, but the patterns of change we observed in definite stages of life, suggest that this phenomenon is important in the maturation of the hypothalamus-pituitary axis and in the metabolic and hormonal changes accompanying gestation.
Journal of Neuroendocrinology 10/1996; 8(9):721-30. · 3.14 Impact Factor
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ABSTRACT: The secretion of 23 kDa prolactin by rat pituitary cells has been thoroughly investigated, but secretion of glycosylated rat prolactin is not currently known. This is mainly due to the lack of an antiserum which is solely specific for glycosylated rat prolactin and therefore we studied the basal secretion of this variant by an indirect method. Rat pituitary cells were cultured in total culture medium and three different serum-free media (DMEM, keratinocyte-serum-free medium, protein-free hybridoma medium) and secretion of 23 kDa and glycosylated rat prolactin was recorded by radioactive techniques and immunoblotting. The pituitary cell quality was monitored by electron microscopy, cell activation-and cell death assessment. In short-range culture (2 days) the pituitary cell quality and behaviour was very good and comparable in total culture medium, DMEM and keratinocyteserum-free medium, i.e. numerous secretory granules, moderate amount of ER, cristae well in place in the mitochondriae. In medium-range culture (8 days) only cells cultured in total culture medium and DMEM presented a parallel behaviour: migration of cells toward each other, marked degranulation, massive array of ER. The inner membrane of the mitochondria was no longer folded into cristae leaving an unoccupied central space. At day 2 of the culture span secretion of 23 kDa rat prolactin was very comparable in all media used; hereafter, secretion of 23 kDa rat prolactin in total culture medium and DMEM assumed the well known pattern of peaking and slowing down, whereas in the other serumfree media it steadily decreased over the culture span. Pertaining to the important novel point of glycosylated rat prolactin secretion, it was low in comparison to the one of 23 kDa rat prolactin and it assumed a near steady pattern in all media used. 26 kDa rat prolactin was identified as the preferentially secreted glycoform, and the 23 kDa isoform as the major secretory product of rat pituitary lactotroph cells.
Endocrine 01/1995; 3(1):61-8. · 1.42 Impact Factor
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ABSTRACT: Rat pituitary homogenates were submitted to differential and density gradient centrifugation. Subcellular fractions as well as the purified secretory granules were examined in electron microscopy, radioimmunological techniques, protease digestion, alkaline treatment and immunoblotting. The global outcome of these experiments was that: 1) the glycosylated rPRL was foremost recorded in the crude secretory granular fraction, also in the microsomal fraction and the cytosol, but virtually not in the plasma membrane fraction; 2) in purified secretory granules glycosylated rPRL appeared as an array of near Mr, such as was formerly obtained by enzymatic deglycosylation; 3) protease digestion and ice-cold alkaline treatment of the secretory granules showed that 23,000 rPRL appears in three different physicochemical states in these organelles: unsequestered within a closed system, membrane-bounded and bound state; 4) likewise treatment of microsomal vesicles showed that 23,000 and glycosylated rPRL are sequestered in these bodies, but apparently 23,000 rPRL appears as both integral membrane-bound and released from the lumen, whereas glycosylated rPRL is chiefly retained as an integral membrane protein. 5) dopamine alters the pattern of glycosylation as well in Mr as in relative percentages of the molecular variants. The systematical occurrence of the array of near Mr glycosylated rPRL is biosynthesized as a pool of proteins with a different degree of glycosylation. On the basis of our data, we speculate that selection of definite molecular variants from this pool could play an important role in the biological function of 23,000 rPRL and that oligosaccharides could perhaps target the glycosylated forms of rPRL to specific sites of action.
Journal of Neuroendocrinology 01/1994; 5(6):669-76. · 3.14 Impact Factor
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ABSTRACT: Abstract In the rat two major molecular variants of prolactin are recorded i.e. 23,000 M(r) and glycosylated 26,000 M(r). In order to further characterize the glycosylated 26,000 rat prolactin molecular variant, rat pituitary cell lysates were digested with several glycoen-zymes and the digestion products submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis and subsequent immunoblotting. The results were as follows: treatment with 1) neuraminidase, specific for sialic acid, yielded an M(r) decrease of the glycosidic variant from 26,000 to 24,500, 23,800, 23,000 and 22,000; 2) endo-alpha-N-acetylgalactosaminidase, which releases the disaccharide Gal (beta 1-3) GalNac from O-glycans, split 26,000 rat prolactin into a doublet of M(r) 26,000 to 25,500; and 3) mixed exoglycosidases from Turbo cornutus caused a gradual M(r) shift from 26,000 to 23,000. Affinity chromatography on wheat germ agglutinin Sepharose 6MB and soybean agglutinin agarose of rat pituitary homogenates and competitive inhibition tests showed that glycosylated rat prolactin has distinct affinity for these lectins. From the experimental data it is proposed that glycosylated rat prolactin is O-linked through threonine by the disaccharide Gal (beta 1-3) GalNac and possesses at least GalNac, and/or Gal and sialyl residues.
Journal of Neuroendocrinology 09/1991; 3(4):375-81. · 3.14 Impact Factor
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ABSTRACT: Abstract Prolactin cells derived from the anterior pituitaries of female rats were cultured in the presence of tunicamycin, swainsonine, castanospermine, beta-hydroxynorvaline and monensin in order to study their effect on the post-translational processing of the M(r) 17,000, 23,000 and 26,000 prolactin molecular forms. Sodium-dodecyl-sulphate polyacrylamide electrophoresis and subsequent immunoblotting revealed that: 1) tunicamycin, swainsonine and castanospermine, compounds that are essentially known as inhibitors of the N-glycosylation processus, had no effect on M(r) 17,000, 23,000 and 26,000 rat prolactin; 2) betahydroxynorvaline, which has been assumed to inhibit processing of pre-prolactin to mature 23,000 prolactin, did not increase the synthesis of 26,000 rat prolactin. In case of inhibition of the processing of a pre-prolactin to mature prolactin, one would expect an increase of the pre-prolactin; consequently, we could not establish the 26,000 rat prolactin, we revealed in immunoblotting, as a pre-prolactin; 3) monensin affected the post-translational processing of 17,000 and 26,000 rat prolactin, but left the 23,000 mature form intact. This is an important finding for the following reasons: monensin blocks the transport of secretory and membrane proteins, and this blockade prevents the cleavage of these molecules; indeed, production of 17,000 rat prolactin, a form of cleaved prolactin, was inhibited. Monensin also affects glycosylation and 26,000 rat prolactin has been identified as a presumably O-iinked glycosylated variant. The fact that its synthesis is inhibited by monensin treatment, but not by inhibitors of the N-linked process, particularly tunicamycin, and that 26,000 rat prolactin is susceptible to mild alkali and decomposition via beta-elimination are decisive arguments in favour of the O-linked glycosidic linkage.
Journal of Neuroendocrinology 01/1990; 1(6):427-31. · 3.14 Impact Factor
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ABSTRACT: Recently we reported the isolation and partial biochemical characterization of the novel polypeptide h3 from human brain and liver. In this report, the physicochemical characterization is further established by the use of several analytical methods. The following results were obtained: the ultraviolet absorption spectrum is not influenced by pH, and the circular dichroism (CD) spectrum reveals that this protein has no alpha-helices, whereas approximately 25% of the polypeptide chain is found to be folded as a beta-pleated sheet structure. Neither the conformation of h3 as assessed by CD nor the titration kinetics of sulfhydryl groups with Ellman's reagent are affected by the presence of the ions K+, Na+, Ca2+, and Mg2+. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a beta-mercaptoethanol gradient and Cleveland sequential SDS-PAGE showed that the frequent formation of h3 polymers and doublets, as observed earlier, is almost exclusively due to disulfide bonding.
Journal of Neurochemistry 05/1989; 52(4):1123-6. · 4.06 Impact Factor
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ABSTRACT: Prolactin and GH cells from rat pituitary glands were separated into three main fractions on discontinuous Percoll gradient layers. SDS-PAGE and subsequent immunoblotting of these fractions revealed that: (1) multiple rat prolactin (rPRL) molecular variants were present in total culture, Percoll layer 1 and 2; four variants were clear-cut: Mr approximately 23,000, Mr doublet approximately 25,000-26,000, Mr approximately 40,000 and Mr approximately 42,000; (2) cell cytosol from Percoll gradient layer 1 was particularly enriched in prolactin; (3) cells from gradient layer 1 secreted into the culture medium only prolactin in detectable amounts; (4) three distinct molecular forms of rat growth hormone (rGH) were recorded in layer 3: Mr approximately 36,000, 24,000 and 20,000; the 20,000 variant was paramount; and (5) cells from layer 3 secreted both rPRL and rGH into the culture medium. Reduction experiments showed that, on the one hand, 42,000 and 40,000 rPRL variants and, on the other hand, 36,000 rGH variants are disulphide-bridged dimers. An important finding was the presence of glycosylated rPRL and rGH: indeed Concanavalin A-Sepharose 4B affinity chromatography indicated that 26,000 rPRL and 24,000 rGH display a very strong affinity for lectin. Competitive inhibition tests showed that this affinity is specific and not due to hydrophobic binding. When rPRL was submitted to deglycosylation in conditions specific for O-linked glycoproteins, the 26,000 rPRL variant disappeared. The biological role of glycosylated rPRL is as yet unknown.
Journal of Endocrinology 03/1989; 120(2):201-6. · 3.55 Impact Factor
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ABSTRACT: Recently we reported the isolation and partial biochemical characterization of a novel polypeptide, h3, from the human brain and liver. Thin-layer isoelectric focusing showed that the polypeptide was ubiquitously distributed throughout the human brain. Immunophosphatase transfer electrophoresis showed that this protein was localized in several mammalian species and different tissues. In addition, h3 or h3-like protein was demonstrated in subsets of tissues from one avian species. Protein h3 was present in epithelial and muscular tissue, as well as in nervous tissue; however, for all species investigated, it was most abundant in CNS and muscle.
Journal of Neurochemistry 05/1988; 50(4):1210-4. · 4.06 Impact Factor
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ABSTRACT: A patient affected with multiple myeloma displayed in the serum, urine, and cerebrospinal fluid a paraprotein with identical electrophoretic mobility. The paraprotein, which was polymeric, appeared in the serum and cerebrospinal fluid mainly as the dimer and tetramer, whereas in the urine the tetramer was predominant. The myeloma protein, identified as an IgA1 kappa, was isolated from the serum and urine and submitted to structural analysis. For reasons of scarcity of material, it was decided to approach the structure of the cerebrospinal fluid paraprotein by means of antiidiotypic antiserum. Complete idiotypic identity between, on the one hand, cerebrospinal fluid and, on the other hand, IgA1 isolated from serum and urine and F(ab)2 alpha derived from serum IgA1 was observed. Adsorption experiments confirmed the idiotypic identity among the three biological fluids. Although the blood-brain barrier of the patient was only slightly disturbed, IgA polymers of MW varying from approximately 280,000 to approximately 840,000 appeared in the cerebrospinal fluid. Consequently the results are good evidence for synthesis within the central nervous system by subsequent generations of a malignant B-cell line which invaded the central nervous system.
Journal of Clinical Immunology 08/1986; 6(4):319-25. · 3.08 Impact Factor
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ABSTRACT: Total protein content, alpha 1-antitrypsin, alpha 2-macroglobulin and plasminogen levels and measles antibody titers were determined in serum and plasma from patients affected with multiple sclerosis and patients affected with non-neurological diseases. The results were compared with those from a control group of healthy donors. Both multiple sclerosis patients and patients affected with non-neurological diseases differed from controls for the following parameters: total protein, plasminogen and measles antibody activity. However, when studied longitudinally the different parameters were not altered to the same degree in multiple sclerosis and non-neurological diseases, a fact which is translated in the difference of significance levels. Individual plasminogen values were very often higher in non-neurological diseases than in multiple sclerosis, whereas for increased measles antibody titers it was the reverse. Also, there were no notable changes in alpha 1-antitrypsin and alpha 2-macroglobulin values in multiple sclerosis, whereas in some non-neurological disease patients particularly high alpha 1-antitrypsin and alpha 2-macroglobulin values were observed. In the multiple sclerosis patients, no correlations existed between the duration of the disease and disturbed biochemical parameters, or between the disturbed parameters themselves.
Journal of clinical chemistry and clinical biochemistry. Zeitschrift für klinische Chemie und klinische Biochemie 11/1984; 22(10):653-9.
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ABSTRACT: Recently (1) we reported the isolation and partial biochemical characterization of a novel polypeptide from human brain. Now we report the isolation of an almost identical polypeptide from human liver tissue and further biochemical characterization of both polypeptides. Thorough analytical investigation with several methods, fully described hereafter, of both polypeptides, leads on the one hand to a more complete picture of the polypeptide and on the other hand to the correction of earlier assumptions. The monomeric polypeptide, now identified as a glycopolypeptide, is indeed an independent chain of approximately 220 amino-acid residues and possesses 2 free sulphydryl groups. From the almost complete identity between the CNS and liver polypeptide it is concluded that they are most probably one and the same polypeptide and consequently, that h3 is not brain specific.
Neuropeptides 05/1983; 3(4):243-54. · 1.55 Impact Factor
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ABSTRACT: The serum, cerebrospinal (CSF) and brain of a patient (NAG) affected with multiple sclerosis (MS) were examined for measles antibodies with CF and HI techniques, and the kappa-lambda light chain ratios of all samples available were evaluated, kappa-lambda populations of the matched serum, CSF and brain specimens were all lambda-predominant and in agreement with each other; the light chain distribution f the brain specimens confirmed previous findings [3]. Only the serum immunoglobulins showed significant measles antibody titers, but slightly increased measles antibody titers were also observed in ventricular plaques. The amount of immunoglobulin G (IgG) synthesized per day by the central nervous system (CNS) was estimated. The IgG synthesis in CNS NAG (11.6 mg/day) was above the upper limit of the normal range (3.3 mg/day), but apparently there was no positive correlation between the intracerebral IgG synthesis and specific anti-measles IgG.
Journal of Neurology 02/1981; 225(2):135-43. · 3.47 Impact Factor
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ABSTRACT: The presence of measles antibodies in serum immunoglobulin G fractions from seven patients affected with multiple sclerosis was investigated with HI technic. The kappa-lambda light chain ratios of all samples under investigation were evaluated. Three multiple sclerosis patients, who displayed either fractionation or a tendency towards fractionation in their serum, had slightly elevated measles antibody titers associated to increased kappa/lambda ratios.
Journal of Neurology 04/1979; 220(2):105-12. · 3.47 Impact Factor
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ABSTRACT: The presence of measles antibodies in white and grey brain material and in 8 demyelination plaques from 6 patients affected with multiple sclerosis was investigated with the hemagglutination inhibition (HI) and complement fixation (CF) techniques. White and grey matter of 5 controls were run in parallel. No measles antibodies could be detected, except for one plaque, where the answer could be considered as doubtful. The immunoglobulin G (IgG) content and the kappa/lambda light chain ratios of all the samples were evaluated. No unique monoclonal immunoglobulin population could be detected, but kappa or lambda predominant oligoclonal populations appeared in controls as well as in MS IgG.
Brain Research 09/1978; 152(1):133-44. · 2.73 Impact Factor
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ABSTRACT: In this report the structure of the novel polypeptide h3, isolated from subsets of tissues of a single species, and the structural similarity between interspecies h3 were investigated by peptide mapping of enzymatic and chemical cleavage fragments of h3 in one-dimensional SDS-PAGE; the peptide maps were commented on in comparison with the known sequence of 21 kDa protein, a h3-like ox brain protein. The following results were obtained: peptides generated by chymotrypsin, protease XX, BNPS-skatole and CNBr cleavage of different tissues in a single species were strikingly identical, whereas peptide maps obtained from analogue tissues in different species revealed slight structural differences. Possible ligand-h3 binding was studied by comparing the c.d. spectra of native h3, and h3 incubated with several phospholipids. Given the presence of h3 or h3-like protein in rat and human platelets, h3 was also assessed in platelet aggregation in the presence of h3 and specific anti-h3 antiserum. So far, the results emphasize the unique intra- and interspecies molecular form of h3, allow us to assign to known amino acid sequence of 21 kDa to a large extent to human h3, but do not identify h3 as a phospholipid binding protein.
Archives internationales de physiologie, de biochimie et de biophysique 101(1):63-9.
