UNIPROT:P56851 - FACTA Search

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Query: UNIPROT:P56851 (epididymal)
11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We previously identified an insoluble 50-kDa acrosomal matrix protein (AM50) localized to the ventral region of the guinea pig sperm apical segment. AM50 is converted to a 42-kDa polypeptide and released during matrix dispersion in acrosome-reacting sperm. This study examines the sorting pathways and assembly processes which generate the domain-specific distribution of AM50 in the acrosome. AM50 was expressed during early acrosome development and localized to the matrix of proacrosomal granules and the acrosomal vesicle. Initial sorting of AM50 occurred in Golgi phase spermatids, where it became concentrated in the matrix surrounding the acrosomal granule. AM50 remained restricted to the apical segment of acrosome phase spermatids and was finally sorted to the ventral matrix of the apical segment in maturation phase spermatids. By reducing SDS-PAGE testicular AM50 exhibited a slightly higher M(r) of 52 kDa than the 50-kDa form of cauda epididymal spermatozoa. Nonreducing SDS-PAGE demonstrated that testicular and epididymal AM50 were assembled into homomeric complexes of 480 and 450 kDa respectively. Cauda epididymal sperm apical segments contained a second disulfide-cross-linked homomeric complex of 520 kDa composed of a 68-kDa subunit. These studies indicate that AM50 is first assembled into a disulfide-cross-linked complex and subsequently processed into mature AM50. These data also suggest that disulfide-linked complexes of different structural proteins may assemble into distinct acrosomal matrix compartments.
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PMID:Sorting of the domain-specific acrosomal matrix protein AM50 during spermiogenesis in the guinea pig. 785 54

1. Earlier studies have shown that exposure of fat-cells to insulin results in the rapid increased phosphorylation of an acid-soluble protein which migrates as a doublet on SDS/PAGE with an apparent molecular mass of close to 22 kDa; agents such as isoprenaline, which increase cell concentrations of cyclic AMP, also increase phosphorylation, but to a lesser extent [Belsham, Brownsey, Hughes and Denton (1980) Diabetologia 18, 307-312; Diggle and Denton (1992) Biochem. J. 282, 729-736]. 2. The protein has been purified from rat epididymal adipose tissue, and the sequences of six tryptic peptides were determined. All six peptides are present in the deduced sequence of a protein of similar properties, designated PHAS-I by Hu, Pang, Kong, Velleca and Lawrence [(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3730-3734]. Hence the proteins are the same or extremely similar. 3. A rabbit anti-peptide antibody has been raised against one of the peptides (AGGDESQFEMD). The antibody was found to be highly specific for the phosphorylated and non-phosphorylated forms of the acid-soluble 22 kDa protein in Western blots and by immunoprecipitation. Studies with the antibody preparation have shown that both phosphorylated and non-phosphorylated forms of the protein appear to be exclusively located in the cytoplasm, and that exposure of cells to isoprenaline causes increased phosphorylation of the same acid-soluble 22 kDa protein as does insulin treatment. 4. Western blots carried out with the antibody preparation indicate that the protein is also present in other insulin-sensitive tissues, including liver, skeletal muscle, heart and brown adipose tissue. The protein was also detected in lung and spleen, but not brain and kidney. It is concluded that the protein may play an important role in some of the actions of insulin.
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PMID:Further characterization of the acid-soluble phosphoprotein (SDS/PAGE apparent molecular mass of 22 kDa) in rat fat-cells by peptide sequencing and immuno-analysis: effects of insulin and isoprenaline. 786

Previous studies from this laboratory have identified a novel alpha-D-mannosidase on the sperm plasma membranes of several species, including man, which may have a role in fertilization. The polyclonal antibody raised against an isoform of the enzyme purified from rat epididymal fluid was found to cross-react with the alpha-D-mannosidase activity present in the detergent-solubilized spermatozoa and sperm plasma membranes. In the present study, we have used affinity-purified as well as monospecific anti-mannosidase IgG to demonstrate that the sperm mannosidase is an integral plasma membrane component of the rat sperm and is localized on the periacrosomal region of the sperm head. In addition, we demonstrate proteolytic processing of the membrane-bound alpha-D-mannosidase during maturation of spermatozoa. The membrane fractions prepared from testis, and spermatozoa from the caput, corpus, and cauda regions of the epididymis, were solubilized in SDS and resolved by SDS-PAGE. The resolved polypeptides, when subjected to Western blot analysis using affinity-purified anti-mannosidase IgG as the primary antibody, revealed the presence of three specific immunoreactive bands (apparent M(r), 135, 125, and 115 kDa) in the membranes from testis, caput, and corpus spermatozoa. However, the cauda sperm plasma membranes showed only one immunoreactive band of apparent M(r) 115 kDa. The disappearance of the 135-and 125-kDa forms and the appearance of a sharp 115-kDa band on cauda spermatozoa suggests a precursor-product relationship between various molecular forms of the enzyme. Trypsin treatment of testicular and caput sperm membranes largely converted the precursor forms to the mature (115-kDa) form. The in vitro proteolysis resulted in an elevated level of the alpha-D-mannosidase activity in the caput (but not cauda) sperm plasma membrane. Inclusion of trypsin inhibitors (benzamidine and aprotinin) largely prevented the conversion of precursor form to the mature form. These data are consistent with the observed increase in the levels of sperm enzyme activity as spermatozoa move from the caput to the cauda region and suggest that the increase is due to the conversion of enzymatically inactive/less active high molecular weight precursor forms (135 and 125 kDa) into enzymatically active mature form (115 kDa) during sperm maturation.
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PMID:Rat sperm plasma membrane mannosidase: localization and evidence for proteolytic processing during epididymal maturation. 787 80

3-Quinuclidinyl benzilate (QNB), a potent antagonist of muscarinic acetylcholine receptors, has been demonstrated to inhibit specifically the zona pellucida (ZP)-induced acrosome reaction (AR) in mouse sperm (Florman and Storey, 1982; Dev Biol 91:121-130). In this study we describe the solubilization and partial purification of the mouse sperm QNB binding activity which may represent a component of the putative receptor complex for ZP on the sperm plasma membrane. Sperm membranes were isolated from cell homogenates of washed, capacitated, epididymal mouse sperm. Scatchard plots of QNB binding to these membranes indicated a single class of binding sites with KD = 7.2 nM and Bmax = 8700 sites/cell. These binding characteristics are similar to those seen with QNB binding to whole cells (Florman and Storey, 1982, J Androl 3:157-164). Sperm membranes were solubilized using 1% digitonin/0.2% cholate, and the resultant detergent-soluble fraction possessed QNB binding activity similar to that of intact membranes. The detergent-soluble fraction maintained intact ZP receptor(s)-G protein coupling in that treatment of this fraction with either ZP or mastoparan resulted in a 35% or 65% increase in specific GTP gamma S binding, respectively. The solubilized membrane preparation was fractionated by gel permeation HPLC. A majority of specific QNB binding activity was confined to one HPLC fraction. Analysis of this fraction by SDS-PAGE revealed a complex of approximately 5 proteins unique to this fraction. The most prominent protein had a M(r) of 72 kDa, which is within the M(r) range for muscarinic receptors. A protein with M(r) = 41 kDa was also present within this fraction. Subsequent pertussis toxin (PTX)-catalyzed ADP-ribosylation of this fraction revealed this protein to be the alpha subunit of the G(i) class of G proteins. Although the QNB binding activity could not be positively identified, we propose that it is contained in one or more of the proteins unique to this fraction and that these proteins, including G(i), may act as part of a sperm receptor complex for the ZP.
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PMID:Solubilization and partial purification from mouse sperm membranes of the specific binding activity for 3-quinuclidinyl benzilate, a potent inhibitor of the zona pellucida-induced acrosome reaction. 789 91

The sperm acrosome contains a variety of hydrolytic enzymes that exhibit differential release during the acrosome reaction. The interaction between hydrolases and the acrosomal matrix, and their compartmentalization into chemically unique matrix domains may represent mechanisms by which this ordered release is achieved. This study characterizes an acrosomal matrix protein that is restricted to the ventral-most region of the apical segment of guinea pig cauda epididymal spermatozoa. Polyclonal antiserum, prepared against an SDS-PAGE-purified apical segment protein, recognized a 50-kDa band, termed AM50, on Western blots. Native AM50 is resistant to solubilization. However, during the acrosome reaction, AM50 is converted into a 42-43-kDa doublet protein (AM50AR) and released into the incubation medium. AM50 and AM50AR are not recognized by antiserum to guinea pig proacrosin and do not exhibit protease activity in a gelatinolytic SDS-PAGE assay. However, AM50AR does bind to p-aminobenzamidine affinity columns, suggesting that it may remain associated with proteases after the acrosome reaction. Light and electron microscopic immunocytochemistry established that AM50 was exclusively localized to the ventral aspect of the apical segment of the acrosome.
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PMID:A domain-specific 50-kilodalton structural protein of the acrosomal matrix is processed and released during the acrosome reaction in the guinea pig. 791 64

In vivo microperifusion and micropuncture were used to study tubule protein synthesis and proluminal secretion by the male reproductive tract in vivo. Seminiferous and caput and cauda epididymal tubules were perifused for 3 h. with [35S]-methionine. Perifused interstitial fluid (IF), lumen fluid (LF), and tubule extract (TE) were collected. Proteins were separated by SDS-PAGE, and autoradiograms were developed. Trichloroacetic acid precipitable proteins in each fluid were determined and a protein synthesis index (PSI) was calculated. PSI values demonstrated that the cauda epididymis synthesized less protein in vivo than did either seminiferous or caput tubules. Seminiferous tubules synthesized and secreted into the tubule lumen a relatively constant panel of proteins. Epididymal tubules synthesized and secreted proteins in a region-specific manner. In the caput epididymis the most prominent secreted bands were consistent with the heavy and light chains of epididymal clusterin. In the cauda epididymis, the most prominent synthesized and secreted protein was a 25 kDa protein consistent with the protein D. The above approach to studying protein synthesis and secretion will allow direct study of the physiological and pathophysiological effects on this important epithelial function in vivo.
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PMID:Assessment of protein synthesis and secretion by rat seminiferous and epididymal tubules in vivo. 799 57

The organization of sperm chromatin in the dasyurid marsupial, Sminthopsis crassicaudata, was investigated using various morphological techniques. Transmission electron microscopy indicates two quite distinct chromatin regions became evident late in spermiogenesis with an outer globular region containing blocks of very electron-dense chromatin. Fluorescent light microscopical studies after staining with DNA dyes and 7-amino actinomycin D of testicular, caput, and cauda epididymal spermatozoa showed that this region fluoresced less brightly than the rest of the nucleus, indicating the presence of fewer DNA binding sites. Freeze fracture showed that the chromatin in most of the nucleus had randomly arranged particles of various sizes, but that of the outer region was composed entirely of small particles. This outer region was more resistant to low concentrations of the ionic detergent, SDS, whereas both guanidine hydrochloride and urea together with sodium chloride generally dispersed all the chromatin except that in the outer globular region and in a localized area of the nucleus beneath the acrosome. This study has thus revealed that the outer globular chromatin of these spermatozoa responds differently to ionic detergents and protein denaturing agents and has a different chromatin organization than most of the rest of the nucleus. The significance of these differences remains, however, to be determined.
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PMID:Unusual nuclear structure of the spermatozoon in a marsupial, Sminthopsis crassicaudata. 812 34

The genetically obese Zucker rat is a well-characterized model of early-onset human obesity. The 120 kDa protein was recently found in the liver cytosol of obese Zucker rats at levels higher than that in lean Zucker rats. We isolated this protein using precipitation with ammonium sulfate, DEAE-Sephacel chromatography, and preparative polyacrylamide gel electrophoresis; the product showed a single band on SDS-polyacrylamide gel electrophoresis. Immunoblotting analysis revealed that the 120 kDa protein was predominantly localized in the liver cytosol of obese Zucker rats. The amount of this protein in lean Zucker rats was less than one-fifth of that found in obese Zucker rats. Further, there were only trace amounts of this protein in the lung tissues, and no detectable amount in other tissues, such as kidney, epididymal adipose tissue, brain, spleen, skeletal muscle, or serum, in either strain of rat. These data suggest that the 120 kDa protein contributes to the abnormal lipid metabolism in obese Zucker rats.
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PMID:Isolation and localization of the 120 kDa protein in the liver of genetically obese Zucker rats. 818 33

alpha-L-Fucosidase (alpha-fucosidase) is the most active of a number of glycosidases measured in rat epididymal sperm through use of artificial 4-methylumbelliferyl substrates. In addition, enzyme activity can be detected in purified populations of testicular germ cells; in comparison to round spermatids, caput epididymal sperm show at least a 1.8-fold increase in alpha-fucosidase activity per cell. Metabolic labeling of cultured testicular germ cells, followed by immunoprecipitation and SDS-PAGE, reveals synthesis of alpha-fucosidase as a polypeptide of 54 kDa in pachytene spermatocytes and round spermatids but no synthesis in condensing spermatids. This polypeptide is N-glycosylated but does not undergo further processing as determined by pulse/chase labeling and treatment with endoglycosidase H. In contrast, the alpha-fucosidase synthesized by cultured clone 9 cells (a rat liver-derived cell line) undergoes processing from a 54-kDa precursor to a slightly larger intermediate centered at 56 kDa on SDS-PAGE (via carbohydrate modification) and finally to a 52-kDa mature polypeptide. Immunoblotting confirms the presence of the 54-kDa form of alpha-fucosidase in testicular germ cells and shows the existence of a 52-kDa mature polypeptide in epididymal sperm. In addition, immunoreactive polypeptide is more prominent in caput epididymal sperm preparations; this is consistent with the increase in enzyme activity. In the absence of alpha-fucosidase synthesis beyond the round spermatid stage of development, it appears that alpha-fucosidase may be acquired from the luminal fluid by sperm during transit through the excurrent duct system. This hypothesis is supported by evidence that metabolically labeled cultured epididymal epithelial cells synthesize and secrete into culture medium an immunoprecipitable 58-kDa alpha-fucosidase polypeptide. Analysis of sperm isolated from the first part of the caput epididymis indicates that both acquisition and processing of sperm-associated alpha-fucosidase takes place prior to or concomitant with arrival of sperm in the epididymis.
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PMID:Synthesis and processing of rat sperm-associated alpha-L-fucosidase. 831 78

We examine here the biochemical properties and epididymal localization of a maturation dependent ram sperm surface antigen. A monoclonal antibody, ESA152, identifies an antigen that is present on the surface of ejaculated sperm, but is absent from testicular sperm. Crosslinking of the ESA152 antigen with bivalent antibodies induces the acrosome reaction, redistributing the antigen into the anterior region of the sperm head where it associates with the fusion product of the plasma membrane and the outer acrosomal membrane. The ESA152 antigen appears as a polypeptide of 18 kDa on immunoblots of SDS-polyacrylamide gels. The ESA152 epitope includes the sialic acid termini of N-linked oligosaccharides, as shown by its sensitivity to neuraminidase and endoglycosidase F. The ESA152 antigen is a highly hydrophobic integral membrane protein that resists aqueous extraction, partitions into the detergent phase of Triton-X-114, and solubilizes in chloroform-methanol mixtures. The anchoring of ESA152 is unaffected by phosphtidylinositol specific phospholipase C. The antigen is absent from extracts of caput and corpus epididymidis but appears abruptly in the first segment of the cauda. Immunofluorescence reveals that the ESA152 epitope first appears in clusters of cells in the luminal epithelium of the proximal cauda, prior to or concurrent with its appearance on sperm.
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PMID:Biochemical characterization and epididymal localization of the maturation-dependent ram sperm surface antigen ESA152. 835 35

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