Immotile bovine caput epididymal sperm contain levels of protein phosphatase activity twofold higher than do mature motile caudal sperm. Comparison of the inhibition profiles of endogenous phosphatase activities detected by okadaic acid (OA) and calyculin A (CA) revealed a pattern consistent with the predominance of a type 1 protein phosphatase (PP1). Immunoblot analysis identified PP1 gamma 2 (the testis-specific isoform of PP1) as the only PP1 isoform in sperm and showed little protein phosphatase 2A (PP2A). In addition, of the known PP1 inhibitors, i.e., DARPP-32, inhibitor 1 (I1), and inhibitor 2 (I2), only I2-like activity was detected in sperm. Inhibition of PP1 by the heat-stable I2-like activity purified from sperm could be reversed with purified glycogen synthase kinase-3 (GSK-3). Furthermore, sperm extracts contain an inactive complex of PP1 and I2 (termed PP1I) that could also be activated by purified GSK-3. The presence of GSK-3 in sperm was demonstrated by activation of purified PP1I, and quantitation revealed that immotile caput sperm contained sixfold higher GSK-3 activity than motile caudal sperm. Immunoblot analysis confirmed the expression of GSK-3 in sperm and revealed the occurrence of both the alpha and beta isoforms. Our findings suggest that the higher PP1 activity measured in immotile sperm, presumably due to higher GSK-3 activity, is responsible for holding motility in check. This conclusion was supported by the observation that the phosphatase inhibitors OA and CA, at micromolar and nanomolar levels, respectively, were able to induce motility in completely immotile bovine caput epididymal sperm and to stimulate the kinetic activity of mature caudal sperm. The intrasperm levels of cAMP, pH, and calcium were unaltered by treatment with these inhibitors. The results suggest a biochemical basis for the development and regulation of sperm motility and a possible physiological role for the PP1/I2/GSK-3 system.
Reactions catalyzed by human milk N-acetyl lactosamine synthetase in the presence and absence of a-lactalbumins have been investigated by steady-state kinetics. N-Acetyl lactosamine synthesis and lactose synthesis in the absence of a-lactalbumin appear to proceed by an ordered sequential reaction, with substrates attaching in the order : Mn2+, UDP-galactose and monosaccharide. Under the conditions used (pH 7.4, 37 "C) the attachment of Mn2+ is not a t thermodynamic equilibrium and it appears that the enzyme can accept either free UDP-galactose or its Mn2+ complex as substrate. Evidence is presented which suggests that the Mn2+ complex of UDP may be the final product released from the enzyme. Reactions in the presence of a-lactalbumin proceed by a similar ordered mechanism. Kinetic effects observed in the presence of human a-lactalbumin with three different monosaccharide acceptors, and in the presence of bovine a-lactalbumin with glucose, can be reasonably explained only by assuming that a-lactalbumin attaches to the enzyme immediately before monosaccharides, contrary to suggestions by other workers. It is proposed that a-lactalbumin attaches to an enzyme . Mn2+ . UDPgalactose complex a t thermodynamic equilibrium, producing a new enzyme form with increased affinity for monosaccharides. The inhibitory effects of a-lactalbumin on N-acetyl lactosamine synthesis are attributed to inhibition resulting from attachment of the protein to a central complex in an alternative pathway in the reaction scheme. The kinetic effects of four a-lactalbumins with human galactosyl transferase are characterized and intrinsic differences shown to be independent of the source of galactosyl transferase. The function deduced for a-lactalbumin in the lactose synthetase system is discussed in relation to its structure and a procedure is indicated for quantitatively measuring activity differences in a-lactalbumins.Lactose synthetase consists of two protein components [l]. One is a widely-distributed galactosyl transferase which in isolation catalyzes the synthesis of N-acetyl lactosamine : Abbreviations. GlcNAc, N-acetyl glucosamine; MnUDP, the Mn2+ complex of UDP; MnUDP-galactose, the Mn2+ complex of UDP-galactose.Enzyme. Lactose synthetase or UDP-galactose : D-glucose 8-4-galactosyl transferase (EC 2.4.1.22). a-Lactalbumin, which has been designated the "specifier protein" of lactose synthetase as it controls the organ-specific nature of lactose production [2], thus exerts a unique form of regulation in the enzyme system. It is therefore particularly impartant to determine the mechanism of action of the lactose synthetase system to help in understanding one aspect of the biosynthasis of the components of milk, in particular the nature of the regulation of lactose synthesis by or-lactalbumin [7]. Furthermore, a-lactalbumin is homologous with chicken egg-white lysozyme [8] and it is hoped that a better understanding of its mechanism of action will help to reveal the nature of any functional relationship with lysozyme.As part of an investig...
The long-term goal of our work is to understand biochemical mechanisms underlying sperm motility and fertility. In a recent study we showed that tyrosine phosphorylation of a 55-kDa protein varied in direct proportion to motility. Tyrosine phosphorylation of the protein was low in immotile compared to motile epididymal sperm. Inhibition or stimulation of motility by high calcium levels or cAMP, respectively, results in a corresponding decrease or increase in tyrosine phosphorylation of the 55-kDa protein. Here we report purification and identification of this motility-associated protein. Soluble extracts from bovine caudal epididymal sperm were subjected to DEAE-cellulose, Affi-Gel blue, and cellulose phosphate chromatography. Tyrosine phosphate immunoreactive fractions contained glycogen synthase kinase-3 (GSK-3) activity, suggesting a possible correspondence between these proteins. This suggestion was verified by Western blot analyses following one-dimensional and two-dimensional gel electrophoresis of the purified protein using monoclonal and affinity-purified polyclonal antibodies against the catalytic amino-terminus and carboxy-terminus regions of GSK-3. Further confirmation of the identity of these proteins came from Western blot analysis using antibodies specific to the tyrosine phosphorylated GSK-3. Using this antibody, we also showed that GSK-3 tyrosine phosphorylation was high in motile compared to immotile sperm. Immunocytochemistry revealed that GSK-3 is present in the flagellum and the anterior portion of the sperm head. These data suggest that GSK-3, regulated by phosphorylation, could be a key element underlying motility initiation in the epididymis and regulation of mature sperm function.
Serine/threonine phosphatase PP1gamma2 is a testis-specific protein phosphatase isoform in spermatozoa. This enzyme appears to play a key role in motility initiation and stimulation. Catalytic activity of PP1gamma2 is higher in immotile compared with motile spermatozoa. Inhibition of PP1gamma2 activity causes both motility initiation and motility stimulation. Protein phosphatases, in general, are regulated by their binding proteins. The objective of this article is to understand the mechanisms by which PP1gamma2 is regulated, first by identifying its regulatory proteins. We had previously shown that a portion of bovine sperm PP1gamma2 is present in the cytosolic fraction of sperm sonicates. We purified PP1gamma2 from soluble bovine sperm extracts by immunoaffinity chromatography. Gel electrophoresis of the purified enzyme showed that it was complexed to a protein 43 M(r) x 10(-3) in size. Microsequencing revealed that this protein is a mammalian homologue of sds22, which is a yeast PP1 binding protein. Phosphatase activity measurements showed that PP1gamma2 complexed to sds22 is catalytically inactive. The complex cannot be activated by limited proteolysis. The complex is unable to bind to microcystin sepharose. This suggests that sds22 may block the microcystin binding site in PP1gamma2. A proportion of PP1gamma2 in sperm extracts, which is presumably not complexed to sds22, is catalytically active. Fluorescence immunocytochemistry was used to determine the intrasperm localization of PP1gamma2 and sds22. Both proteins are present in the tail. They are also present in distinct locations in the head. Our data suggest that PP1gamma2 binding to sds22 inhibits its catalytic activity. Mechanisms regulating sds22 binding to PP1gamma2 are likely to be important in understanding the biochemical basis underlying development and regulation of sperm function.
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