Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We prepared a monoclonal antibody (MAb) against N-acetylglucosaminide beta 1----4 galactosyltransferase purified from F9 embryonal carcinoma cells. The MAb recognized the protein portion of the enzyme, since it inhibited galactosyltransferase activity, reacted with the enzyme both from F9 cells and from bovine milk, and did not exhibit anti-carbohydrate activity. Using this MAb, we studied the subcellular localization of the enzyme by immunoelectron microscopy. Intense staining was observed in trans-Golgi stacks within testicular interstitial cells and mucous neck cells, confirming the specificity of the immunological reaction. Cell surface galactosyltransferase was detected in the following regions: cultured cells such as F9 embryonal carcinoma cells, testicular interstitial cells, seminiferous tubule epithelial cells, Sertoli cells, the head of the epididymal sperm, epididymal epithelial cells, and apical surfaces of epithelial cells in the fundic gland and of intestinal goblet cells. The use of Triton X-100 intensified the cell surface immunoreactivity, and in certain cases the mode of distribution of the cell surface enzyme was different from that described in previous reports. In addition, nuclear envelopes of cultured cells were distinctly stained. The possible significance of the latter finding is discussed in relation to recent advances in nuclear localization of glycoproteins.
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PMID:Subcellular localization of N-acetylglucosaminide beta 1----4 galactosyltransferase revealed by immunoelectron microscopy. 194 Mar 15

Previous studies from this laboratory and others have identified several enzymes on the surface of mammalian spermatozoa. Some of these enzymes, namely a galactosyltransferase and a novel alpha-D-mannosidase, are believed to play a ligand-like role in recognizing and binding to the complementary moiety(ies) present on zona pellucida glycoconjugates. However, little or no information is available about the occurrence of these enzymes in human spermatozoa. In the present report, we show that a very small amount of the total galactosyltransferase activity present in human semen is associated with spermatozoa. Moreover, our failure to find a significant amount of the enzyme on sperm plasma membranes suggests that the enzyme is not associated with the sperm surface. Therefore, it is unlikely that galactosyltransferase in humans has the same ligand-like role in zona binding that is demonstrated in mouse sperm. In contrast, nearly 5% of alpha-D-mannosidase activity was repeatedly found in the salt-washed plasma membrane fraction. The recovery and enrichment of the alpha-D-mannosidase was nearly one-half that observed for adenylate cyclase and nearly one-third that for phosphodiesterase I, the two sperm plasma membrane marker enzymes. The differential enrichment and recovery of the sperm surface alpha-D-mannosidase is consistant with our previous studies in rat spermatozoa, and suggests that alpha-D-mannosidase may be localized on morphologically distinct region(s) of the sperm plasma membranes. The properties of human sperm surface alpha-D-mannosidase are quite similar to those reported by us for rat sperm plasma membrane mannosidase, but quite different from human sperm acid alpha-D-mannosidase. In addition, whereas anti-rat epididymal alpha-D-mannosidase antibody (IgG-fraction) cross-reacted with the human sperm acid alpha-D-mannosidase, no cross-reactivity was observed with the sperm surface mannosidase. A small amount of fucosyltransferase (less than 1% of the enzyme originally present on spermatozoa) was found in the salt-washed plasma membrane, but the enrichment of the enzyme was only one-tenth of that observed for adenylate cyclase. The potential ligand-like role of human sperm surface alpha-D-mannosidase and other sperm surface enzymes during fertilization is discussed.
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PMID:Human sperm plasma membranes possess alpha-D-mannosidase activity but no galactosyltransferase activity. 211 23

The development and evaluation of a method for the determination of galactosyltransferase and alpha-lactalbumin activities using the addition of Dowex resin to the sample to separate substrate from products are described. For both assays galactosyltransferase activity was optimized by the addition of detergent, and relevant control incubations were included. The assay conditions were optimized for epididymal tissue and standards, and the assays were validated for accuracy and specificity with authentic bovine proteins and lactating rat mammary gland homogenates. Galactosyltransferase and alpha-lactalbumin activities in tissues were dependent on the extraction procedure used. Epididymal and testicular homogenates reduced the slopes of internal standards of galactosyltransferase but only testicular homogenates depressed slopes of internal standards of alpha-lactalbumin, necessitating the use of internal standards in the validation of the assays.
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PMID:Improved assays of alpha-lactalbumin and galactosyltransferase. 212 Oct 61

Galactosyltransferase and alphalactalbumin-like activities have been reported to be present in the post-testicular fluids of the male reproductive tract. In the lactating mammary gland, these activities constitute the lactose synthetase complex. Kinetic parameters and acceptor specificities previously reported, along with recent amino acid sequence analysis argue against the mammary gland and epididymal activities being products of the same gene. In this paper we present cell-free translation of rat epididymal mRNA and Northern blot analysis of epididymal mRNA hybridized with authentic rat alpha-lactalbumin cDNA supporting this lack of identity and describe the differential synthesis and secretion of the androgen-regulated 18 kDa component of the so-called rat epididymal alphalactalbumin-like complex along the length of the epididymis. We conclude that although the 18 kDa component of the so-called epididymal alphalactalbumin moiety (E alpha LA) is capable, in common with a number of unrelated molecules, of modifying galactosyltransferase acceptor specificity in vitro, there is no primary structural similarity between it and authentic rat mammary alphalactalbumin. In view of the fact that the activity of E alpha LA is 1/100th that of authentic milk alphalactalbumin, we suggest that it may not be of physiological importance and that modification of galactosyltransferase activity may not be the function of the 18 kDa molecule.
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PMID:An 18-kDa androgen-regulated protein that modifies galactosyltransferase activity is synthesized by the rat caput epididymidis, but has no structural similarity to rat milk alphalactalbumin. 212 11

Using an assay for alpha-lactalbumin in which galactosyltransferase activity was stabilized and a tissue phosphatase inhibitor was present, no evidence was found for alpha-lactalbumin-like activity in rat epididymal tissue, epididymal fluids or medium from cultured epididymal epithelial cells with either glucose or N-acetylglucosamine as acceptor. However, when assay conditions were suboptimal, apparent transfer of radioactivity to both acceptors could be demonstrated in the epididymis and other tissues. In these assays the amount of alpha-lactalbumin registered was linearly correlated to the extent of stimulation of alpha-lactalbumin added exogenously to tissue extracts as internal standards. When rete testis fluid from rats was used as source of galactosyltransferase under suboptimal conditions, no transfer to glucose was demonstrable in epididymal fluid and an apparent decreased transfer to N-acetylglucosamine could be explained by increases in (pyro)phosphatase activity. Putative alpha-lactalbumin activity in the epididymis may be an artefact of unoptimized assays.
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PMID:Re-examination of the presence of alpha-lactalbumin in the epididymis of the rat. 225 Feb 49

Immature sperm from the caput epididymis are immotile and infertile. It is thought that caput epididymal sperm are infertile due to their immotility, as well as to an inability to bind to the zona pellucida, suggesting the absence of a functional receptor for the zona. However, the sperm receptor for the zona pellucida has been identified previously as the enzyme galactosyltransferase (GalTase) (L. C. Lopez et al. (1985) J. Cell Biol. 101, 1501-1510) and is present on the surface of caput as well as cauda epididymal sperm (N. F. Scully et al., (1987) Dev. Biol. 124, 111-124.). In this paper we examine this apparent conflict and show that immotile caput epididymal sperm are able to bind to the zona pellucida if they are first washed free of caput epididymal secretions, which contain factors that inhibit sperm-zona binding. Consistent with this finding are results that show that caput epididymal fluid is capable of inhibiting the binding of mature, cauda epididymal sperm to the zona pellucida. Caput epididymal fluid contains, among many other components, a soluble GalTase and an alpha-lactalbumin-like protein, both of which are capable of inhibiting mouse sperm-zona binding. Thus, caput epididymal sperm have the appropriate receptor, i.e., GalTase, for the zona pellucida, to which they can bind if removed from the inhibitory factors that mask their zona-binding ability.
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PMID:Binding of caput epididymal mouse sperm to the zona pellucida. 282 54

We have previously shown that sperm-egg recognition in the mouse is mediated by the binding of galactosyltransferase (GalTase) on the sperm surface to its appropriate glycoside substrate in the egg zona pellucida [L. C. Lopez, E. M. Bayna, D. Litoff, N. L. Shaper, J. H. Shaper, and B. D. Shur (1985) J. Cell Biol. 101, 1501-1510]. In the present study, we have defined the spatial and temporal expression of surface GalTase during spermatogenesis and epididymal maturation. Purified populations of spermatogenic cells were isolated by unit gravity sedimentation, and surface GalTase expression was determined by indirect immunofluorescence and by direct enzymatic assay. GalTase is present on the surface of all spermatogenic cells assayed. During differentiation, there is a progressive redistribution of GalTase from an initially diffuse and uniform localization on the surface of primary spermatocytes to a restricted plasma membrane domain overlying the dorsal aspect of the mature acrosome. This apparent redistribution of surface GalTase was confirmed by direct enzymatic assays, which show that surface GalTase activity, normalized per cell, remains relatively constant throughout spermatogenesis, despite a drastic reduction in cell surface area. When normalized to the relevant cell surface area, the GalTase concentration per square micrometer increases 77-fold from pachytene spermatocytes to cauda epididymal sperm. Cell surface GalTase is thought to be a cytoskeletally associated transmembrane protein [N. L. Shaper, P. L. Mann, and J. H. Shaper (1985) J. Cell Biochem. 28, 229-239]; consequently we examined whether cytoskeletal components may be involved in the redistribution of GalTase during spermatogenesis. beta-Tubulin, monomeric actin, and filamentous actin were found to be present during spermatogenesis, as assayed by indirect immunofluorescence and by Western immunoblotting. alpha-Actinin and vinculin were not detectable under these conditions and served as negative controls. During spermatogenesis, the distribution of tubulin coincides with the appearance of the mitotic spindle, flagellum, and manchette. On the other hand, the distribution of filamentous actin coincides with surface GalTase, suggesting that actin-containing microfilaments may participate in the redistribution of surface GalTase during spermatogenesis.
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PMID:Spatial and temporal expression of cell surface galactosyltransferase during mouse spermatogenesis and epididymal maturation. 311 4

We have used perifusion organ culture of proximal and distal caput epididymal tubules of the rat to study the secretion of proteins by epididymal epithelium and uptake of the luminal radioactive proteins by sperm. The amount of incorporation of L-[35S]methionine into luminal fluid proteins was time dependent and completely inhibited by cycloheximide. The association of labeled proteins with cultured sperm was also dependent on time and continuous, with sperm still acquiring labeled luminal proteins after protein synthesis was arrested. A Mr = 46,000 molecule was found to be heavily labeled in luminal fluid and sperm extracts. Fluorograms of all L-[35S]methionine extracts immunoprecipitated using an antiepididymal alpha-lactalbumin antibody (Klinefelter and Hamilton, 1984) showed labeling of an Mr = 18,000 molecule and, in addition, the Mr = 46,000 molecule, but immunostaining was specific only for the Mr = 18,000 molecule and the heavy chain of the immunoglobulin. We suggest that the Mr = 46,000 molecule may be galactosyltransferase. Galactose oxidase-NaB[3H]4 labeling of the cultured caput sperm cell surface revealed a Mr = 23,000 molecule that was able to be immunoprecipitated with antiepididymal alpha-lactalbumin antibody. Our data suggest that this cell surface molecule is similar to one component of the fluid epididymal alpha-lactalbumin-like complex and, in addition, show that glycosylation of the sperm surface can occur in the caput epididymidis.
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PMID:Synthesis and secretion of proteins by perifused caput epididymal tubules, and association of secreted proteins with spermatozoa. 408 28

alpha-Lactalbumin, a modifier protein that changes the substrate specificity of galactosyltransferase, to promote the synthesis of lactose, is found in the mammary glands of lactating mammals and in milk. Molecules similar to mammary gland alpha-lactalbumin but distinct in their modifier activity have been found in rat epididymal fluid. We report here, using a rat mammary gland alpha-lactalbumin cDNA clone as a hybridization probe, RNA sequences homologous to alpha-lactalbumin mRNA were detected in total RNA from the rat epididymis. This finding suggests that alpha-lactalbumin or similar molecules, in addition to regulating lactose synthesis in the mammary gland, may have other important functions in mammalian reproduction.
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PMID:The presence of the milk protein, alpha-lactalbumin and its mRNA in the rat epididymis. 641 37

Studies using genetic and biochemical probes have suggested that mouse sperm surface galactosyltransferases may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the egg zona pellucida. In light of these results, we examined sperm surface galactosyltransferase activity during in vitro capacitation to determine whether changes in enzymatic activity correlated with fertilizing ability. Results show that surface galactosyltransferases on uncapacitated sperm was preferentially loaded with poly N-acetyllactosamine substrates. As a consequence of capacitation in Ca(++)-containing medium, these polylactosaminyl substrates are spontaneously released from the sperm surface, thereby exposing the sperm galactosyltransferase for binding to the zona pellucida. Sperm capacitation can be mimicked, in the absence of Ca(++), either by washing sperm in Ca(++)-free medium, or by pretreating sperm with antiserum that reacts with the galactosyltransferase substrate. In both instances, sperm galgactosylation of endogenous polylactosaminyl substrates is reduced, coincident with increased galactosylation of exogenous GlcNAc, and increased binding to the zona pellucida. Binding of capacitated sperm to the egg can be inhibited by pronase-digested high molecular weight polyactosaminyl glycoside extracted from epidymal fluids or from undifferentiated F9 embryonal carninoma cells. Thus, these glycosides function as "decapacitation factors" when added back to in vitro fertilization assays. These glycoside "decapacitation factors" inhibit sperm-egg binding by competeing for the sperm surface galactosyltransferase, since (a) they are galactosylated by sperm in the presence of UDP[(3)H]galactose, and (b) enzymatic removal of terminal GlcNAc residues reduces "decapacitation factio" competition. On the other hand "conventional" low molecular weight glycosides, isolated from either epididymal fluid or differentiated F9 cells, fail to inhibit capacitated sperm binding to the zona pellucida. These results define a molecular mechanism for one aspect of sperm capacitation, and help explain why removal of "decapacitation factos" is a necessary prerequisite for sperm binding to the zona pellucida.
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PMID:Sperm surface galactosyltransferase activities during in vitro capacitation. 681 11


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