Partial purification and characterization of a phosphoprotein phosphatase from sperm plasma membrane

1999 ◽  
Vol 11 (6) ◽  
pp. 379 ◽  
Author(s):  
Madhabi Barua ◽  
Anil K. Ghosh ◽  
Gopal C. Majumder
Keyword(s):  
Plasma Membrane ◽  
Concanavalin A ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Tween 20 ◽  
Partial Purification ◽  
Appreciable Effect ◽  
Phosphoprotein Phosphatase ◽  
Sperm Plasma Membrane

A phosphoprotein phosphatase (PPase M-I) that dephosphorylates serine and threonine residues of histones was isolated from the goat cauda-epididymal sperm plasma membrane and partially characterized. The PPase was solubilized from the sperm membrane by treating it with 0.1 N NaOH at pH 11.4 and the solubilized enzyme was partially purified by concanavalin A-sepharose affinity chromatography and high-performance liquid chromatography (HPLC), revealing it to be a 520-kDa protein. The PPase gave a single protein band in native polyacrylamide gel electrophoresis (PAGE), but in the presence of SDS it resolved into multiple proteins (35–170 kDa) showing that the isolated enzyme contained a few contaminating proteins. The enzyme is a glycoprotein because it binds with high affinity to concanavalin A. It was maximally active at pH 8.0 and its activity was not dependent on bivalent metal ions. The enzyme is a specific phosphatase as it displayed higher affinity for dephosphorylation of large molecular weight phosphate esters. The PPase showed broad substrate specificity for the dephosphorylation of a variety of proteins. The membrane-associated PPase was strongly (70–80%) inhibited by detergents (0.5%) such as Nonidet P-40, Lubrol PX, Triton X-100 and Tween-20. Pyrophosphate (5 mM) and orthovanadate (400 M) had no significant effect on the activity of the isolated PPase whereas polyamines such as spermine (10 mM) nd spermidine (10 mM) slightly inhibited (20%) the enzymatic activity. Inorganic phosphate (10 mM) and NaF (10 mM), the well-known inhibitors of the cytosolic PPases, had no appreciable effect on the activity of PPase M-I, indicating that the membrane-bound PPase is distinct from the cytosolic PPases. The enzyme was radiolabelled when the intact spermatozoa were subjected to lactoperoxidase-mediated radioiodination reaction. The results show that the PPase M-I is an ecto-enzyme that may play an important role in sperm physiology by causing the dephosphorylation of the sperm outer surface phosphoproteins.

Phosphorylation of liver plasma membrane-bound calmodulin

1988 ◽  
Vol 66 (8) ◽  
pp. 922-927 ◽  
Author(s):  
Shobha Ghosh ◽  
Jon G. Church ◽  
Basil D. Roufogalis ◽  
Antonio Villalobo
Keyword(s):  
Plasma Membrane ◽  
Calcium Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Band ◽  
Bovine Brain ◽  
Antibody Treatment ◽  
Liver Plasma Membrane ◽  
Phosphate Donor ◽  
Membrane Bound ◽  
Stimulation Of

In highly purified rat liver plasma membrane preparations, membrane-bound calmodulin was phosphorylated by a membrane-bound protein kinase using [γ-32P] ATP as phosphate donor. Maximum phosphorylation of calmodulin occurred in the absence of calcium ion, but was significantly decreased in its presence. Plasma membrane-bound calmodulin was identified by the following criteria: (i) extraction from the membrane by EGTA, (ii) stimulation of the activity of the Ca2+-calmodulin-dependent enzyme, (3′:5′ AMP)-phosphodiesterase, by the EGTA extract, and (iii) electrophoretic comigration of EGTA-extracted protein with standard bovine brain calmodulin, both in the presence and the absence of Ca2+. Phosphorylation of the plasma membrane-bound calmodulin was shown by electrophoretic comigration of the 32P-labelled molecule with bovine brain calmodulin, the absence of phosphorylation of this protein band in calmodulin-depleted membranes, and a Western blot of the phosphorylated band using a calmodulin antibody. Treatment of plasma membrane preparations with sheep anticalmodulin serum prevented the phosphorylation of the calmodulin band. Phosphocalmodulin, which could be partially extracted from the membrane by EGTA, comigrated with bovine brain calmodulin in polyacrylamide gel electrophoresis.

Comparison of plasma membrane FABP and mitochondrial isoform of aspartate aminotransferase from rat liver

1993 ◽  
Vol 265 (5) ◽  
pp. G894-G902 ◽  
Author(s):  
D. D. Stump ◽  
S. L. Zhou ◽  
P. D. Berk
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Amino Acid ◽  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fatty Acid Binding ◽  
Sds Page

A relationship between plasma membrane fatty acid binding protein (FABPpm), a putative membrane transporter for long-chain fatty acids, and the mitochondrial isoform of aspartate aminotransferase (m-AspAT) has been reported. Accordingly, we have compared the chemical and immunological properties of rat liver m-AspAT with those of rat liver FABPpm isolated by two procedures: 1) detergent solubilization of the membranes followed by purification via fatty acid affinity chromatography (FABP-1) or 2) salt extraction of the membranes and subsequent purification by high-performance liquid chromatography (HPLC; FABP-2). Comparison of the three protein preparations revealed no differences with respect to NH2-terminal amino acid sequence, amino acid composition, peptides from tryptic digests, AspAT enzymatic activity, isoelectric point, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), retention on five different HPLC columns, and immunoprecipitation and immunoblotting of SDS-PAGE separated proteins with polyclonal antisera. Examination of the proteins by nondenaturing PAGE showed a consistent second band in FABP-1 and FABP-2 not always present in m-AspAT. However, whenever present, this band was immunoreactive with antibodies to both m-AspAT and FABP-1. Hence, FABP-1 and FABP-2 are indistinguishable from one another. They are also at least closely related, if not identical, to m-AspAT.

D-Glucose uptake and insulin binding by the human adipose cell plasma membrane as a function of its polypeptide composition

1977 ◽  
Vol 55 (2) ◽  
pp. 126-133 ◽  
Author(s):  
Bluma G. Brenner ◽  
Shiro Ozaki ◽  
Norman Kalant ◽  
Arthur Kahlenberg
Keyword(s):  
Plasma Membrane ◽  
Glucose Uptake ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insulin Binding ◽  
Partial Purification ◽  
Scatchard Analysis ◽  
Molecular Weight Range

A preparation of plasma membranes isolated from human omental lipocytes is composed of about 15 major polypeptide components including three major glycoproteins with an apparent molecular weight range from 100 000 to 23 000, as determined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Extraction of this membrane preparation with sodium iodide or 2,3-dimethylmaleic anhydride solubilized 50 and 70% of the membrane protein, respectively, resulting from the extensive extraction of protein from all but two of the major membrane polypeptide components. This removal of protein did not affect the membrane's stereospecific D-glucose-uptake activity but did reduce its total specific [l25I]insulin-binding activity by 46–67%. The binding of [125I]insulin to its specific receptor on lipocyte plasma membranes was detected at physiologic concentrations of the hormone and could be competitively displaced by increasing concentrations of native insulin. The kinetic behaviour of this reaction was approximated by Scatchard analysis, and both the affinity and binding capacity of the plasma membrane for insulin were increased at lower temperatures.These results suggest that D-glucose transport in human adipose tissue is mediated by an intrinsic component of the hydrophobic structure of the lipocyte plasma membrane, and represent a partial purification of this component. In addition, these studies demonstrate and characterize the binding of insulin to the plasma membrane isolated from human lipocytes. A quantitative study of this binding reaction may provide further understanding of the mechanisms underlying the decreased insulin responsiveness characteristic of human diabetes.

Binding of porcine sperm plasma membrane proteins to sheep, hamster and mouse oocyte plasma membrane

Zygote ◽  
2000 ◽  
Vol 8 (2) ◽  
pp. 181-187 ◽  
Author(s):  
Elizabeth Ward ◽  
Trish Berger
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Vesicles ◽  
Protein Band ◽  
Negative Control ◽  
Plasma Membrane Vesicles ◽  
Plasma Membrane Proteins ◽  
Mouse Oocytes ◽  
Sperm Plasma Membrane ◽  
Porcine Oocyte

Four porcine sperm plasma membrane proteins were previously identified as putative ligands for the oocyte plasma membrane. The present study examined the binding of these proteins and two additional porcine sperm membrane proteins to oocytes from sheep, mice and hamsters as a first step in assessing potential conservation of these putative sperm ligands across species and across mammalian orders. Plasma membrane vesicles were isolated from porcine sperm, solubilised, and the proteins separated by one-dimensional gel electrophoresis. The 7, 27, 39 and 62 kDa porcine sperm protein bands demonstrating predominant binding of the porcine oocyte plasma membrane on ligand blots, a 90 kDa protein band demonstrating minor binding, and a 97 kDa protein band that did not bind the oocyte plasma membrane probe were electroeluted. Proteins were biotinylated, and incubated with zona-free oocytes. Bound biotinylated protein was labelled with fluorescent avidin and the oocytes examined with a confocal microscope. The 7 kDa, 27 kDa and the 39 kDa proteins bound to the sheep oocytes but not to a majority of the hamster or mouse oocytes. The 62 kDa protein bound to sheep oocytes and mouse oocytes but not to a majority of the hamster oocytes. The 90 kDa protein bound to oocytes from all three species. The 97 kDa protein, which did not recognise the porcine oocyte probe on a Western ligand blot, did not bind to oocytes from any species and served as a negative control. These observations are consistent with significant conservation of molecule and function among species within the same mammalian order. Hence, one species may be a good model for other species from the same order. Only limited conservation of binding activity of porcine sperm plasma membrane proteins to rodent oocytes was observed, suggesting a greater divergence either in molecular structure or in function among species from different orders.

scholarly journals Isolation and electrophoretic characterization of the plasma membrane of sea-urchin sperm

1983 ◽  
Vol 59 (1) ◽  
pp. 13-25
Author(s):  
N.L. Cross
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Coomassie Blue Staining ◽  
Electrophoretic Patterns ◽  
Sperm Plasma Membrane

A subcellular fraction containing plasma membranes was isolated from flagella of the sperm of Strongylocentrotus purpuratus by differential centrifugation, and analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Coomassie Blue staining revealed nine major bands and 14 minor species. Five bands of apparent molecular weights approximately 200 X 10(3), 149 X 10(3), 120 X 10(3), 75 X 10(3) and 59 X 10(3) also stained with periodic acid-Schiff's reagent and so are probably glycoproteins. These five components are externally exposed, as determined by lactoperoxidase-catalysed radio-iodination. Isolation of membranes from radio-iodinated sperm results in an enrichment of about tenfold in the specific activity of 125I. Comparison of the electrophoretic patterns of labelled sperm and of the membranes isolated from 125I-labelled sperm suggests that no major labelled proteins are lost during the isolation procedure, and so to this extent the membrane fraction is representative of the entire sperm plasma membrane.

Identification of spermatogenic cell plasma membrane glycoproteins by two-dimensional electrophoresis and lectin blotting

1984 ◽  
Vol 65 (1) ◽  
pp. 233-248
Author(s):  
C.F. Millette ◽  
B.K. Scott
Keyword(s):  
Plasma Membrane ◽  
Concanavalin A ◽  
Polyacrylamide Gel Electrophoresis ◽  
Spermatogenic Cell ◽  
Two Dimensional ◽  
Spermatogenic Cells ◽  
Membrane Glycoproteins ◽  
Differentiation Markers ◽  
Pachytene Spermatocytes

Plasma membrane glycoproteins present in purified mouse spermatogenic cells have been identified by two-dimensional polyacrylamide gel electrophoresis and lectin blotting techniques. Four membrane glycoproteins labelled with Bandeiraea simplicifolia lectin (I) have been detected, ranging in Mr from 55 000 to 76 000 and in pI from 6.0 to 6.3. Only one of these proteins, p76/6.3, is synthesized by short-term in vitro cultures of spermatogenic cells, as determined by the incorporation of [35S]methionine. Approximately 20 surface glycoproteins labelled with concanavalin A have been identified, ranging in Mr from 50 000 to 151 000 and in pI from 5.7 to 7.0. None of the components detected with B. simplicifolia lectin (I) are labelled significantly with concanavalin A. A major concanavalin A-binding protein in the membranes of both pachytene spermatocytes and round spermatids is p151/6.0. This glycoprotein has been previously shown to be exposed on the outer surface of spermatogenic cell membranes and may represent a mediator of germ cell-Sertoli cell interactions. Furthermore, two constituents identified in the present study represent stage-specific markers. Component p73/5.7 is detected with concanavalin A only in the membranes of pachytene spermatocytes. Conversely, p84/6.3 is found only in round spermatid membranes. These results, then have: (a) provided a map of membrane glycoproteins in developing mouse male germ cells; (b) identified p151/6.0 as a membrane constituent of possible functional significance; and (c) identified the first reported glycoprotein surface differentiation markers for mouse spermatogenesis.

scholarly journals Increase in the concentration of major boar sperm surface proteins during maturation in the epididymis

1986 ◽  
Vol 82 (1) ◽  
pp. 295-308
Author(s):  
N.K. Saxena ◽  
N. Saxena ◽  
W. Hunt ◽  
R.N. Peterson ◽  
L. Henry ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Protein ◽  
Surface Proteins ◽  
Secretory Cells ◽  
Boar Sperm ◽  
Sperm Plasma Membrane ◽  
Antigen Localization ◽  
Polyclonal Antisera ◽  
Narrow Area

High-resolution two-dimensional polyacrylamide gel electrophoresis analyses have indicated that several major boar sperm plasma membrane polypeptides (PMPs) increased in concentration during maturation in the epididymis. To investigate this further, monoclonal antibodies (MAbs) to two of these PMPs/glycoproteins referenced as 4.85 and 5.0 and polyclonal antisera (PCA) raised against PMP 5.0 were used to quantify these changes. ELISA assays for both PMPs, using solubilized plasma membrane (PM) and intact PM as antigens, indicated that both PMPs were present in greater concentration in cauda PM than in caput PM. ELISA assay using PCA against 5.0 and whole sperm from the cauda and caput epididymis also showed an increase in the concentration of this protein during transit through the epididymis. Indirect FITC fluorescence microscopy showed that MAbs reacted specifically with the luminal aspect of secretory cells of the corpus epididymis and, with greater intensity, with secretory cells of the cauda segment. When MAb tags were used the fluorescence technique indicated that PMP 5.0 was restricted to a narrow area of PM overlying the principal segment of the head of caput sperm but fluorescence extended further into the principal segment and into the flagellum of cauda PM. Since MAb binding sites appear to be partially masked, PCA were also used to localize antigenic sites. PCA to PMP 5.0 showed antigen localization in both head and flagellar PM of cauda and caput sperm. The fluorescence of cauda sperm PM, however, was significantly more intense than that of caput sperm PM. MAb to PMP 4.85 also bound to antigen more extensively on cauda sperm. The sum of these analyses suggests that major sperm PMPs are secreted by the epididymis and absorbed by sperm. The magnitude of surface protein changes occurring during epididymal transit in boar sperm appears to be greater than heretofore recognized.

Purification and characterization of a protein kinase from goat sperm plasma membrane

1994 ◽  
Vol 72 (5-6) ◽  
pp. 218-226 ◽  
Author(s):  
Sutapa Mitra ◽  
Debjani Nath ◽  
Gopal C. Majumder
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Membrane Proteins ◽  
Kinase Activity ◽  
Muscle Protein ◽  
Diazonium Salt ◽  
Deae Cellulose ◽  
Outer Cell ◽  
Appreciable Effect ◽  
Sperm Plasma Membrane

A protein kinase that causes phosphorylation of serine and threonine residues of casein has been partially purified from goat cauda-epididymal sperm plasma membrane and characterized. The kinase, solubilized from the membrane with 1.0% Triton X-100, was purified to 480-fold by using DEAE-cellulose and casein-Sepharose affinity chromatographic techniques. The kinase is a strongly basic protein with pI of 9.5. The enzyme has a molecular mass of 310 kilodaltons as estimated by Sephacryl S-300 gel exclusion. The kinase showed affinity for protein substrates in the order membrane proteins > casein > phosvitin > histone > protamine. The apparent Km values of the kinase for casein and membrane proteins were 1 and 0.15 mg/mL, respectively. The synthetic peptides Kemptide and poly(Glu80Tyr20) did not serve as substrates of the enzyme. ATP, rather than GTP or PP, is the donor of phosphate for the phosphorylation reaction. Cyclic AMP and GMP, NaCl (0.25 M), KCl (0.25 M), Ca2+, calmodulin, phosphatidylserine, and muscle protein kinase inhibitor had no appreciable effect on the kinase activity. Heparin (0.5 μg/mL) showed high affinity for inhibiting only 40% of the kinase activity, whereas polyamines at a relatively high concentration (5 mM) inhibited 40–50% of the enzymic activity. The kinase appears to be distinct from other protein kinases including casein kinases. The activity of the kinase derived from the purified sperm plasma membrane was markedly (~ 90%) lost when the intact spermatozoa were pretreated with diazonium salt of sulfanilic acid, a membrane nonpenetrating surface probe. The data are consistent with the view that the isolated enzyme is an ecto-protein kinase whose catalytic site is oriented primarily to the surface of viable sperm cell to cause phosphorylation of the endogenous outer cell-surface phosphoproteins.Key words: spermatozoa, plasma membrane, protein kinase, phosphoproteins, ectoenzyme.

Effects of pronase and concanavalin A upon the freeze-etch morphology of cell membranes of intact human erythrocytes

1978 ◽  
Vol 30 (1) ◽  
pp. 63-76
Author(s):  
G.E. Wise ◽  
F.L. Shienvold ◽  
R.W. Rubin
Keyword(s):  
Plasma Membrane ◽  
Concanavalin A ◽  
Gel Electrophoresis ◽  
Particle Distribution ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Erythrocytes ◽  
Sodium Dodecylsulphate ◽  
Con A ◽  
Erythrocyte Plasma Membrane ◽  
Pronase Treatment

Freeze-etch studies reveal that mild pronase treatment with subsequent incubation in concanavalin A induces aggregation of intramembranous particles (IMP) in intact human erythrocytes. This alteration in particle distribution is accompanied by a change in the distribution of the Con A molecules such that they also become clustered on the extracellular etch face. If divalent succinyl Con A is used after pronase instead of tetravalent Con A the IMPs still become clustered. Pronase only, Con A only, or succinyl Con A only does not cause the IMPs to become aggregated. Most surprising is the finding that pronase followed by Con A causes partial haemolysis of the cells whereas pronase only, Con A only, or pronase+succinyl Con A do not cause this haemoglobin loss. These perturbations of the erythrocyte plasma membrane appear to be a result of the pronase+Con A exerting a transmembrane effect on the spectrin. This conclusion is supported by sodium dodecylsulphate polyacrylamide gel electrophoresis of material crosslinked with dimethyl adipimidate dihydrochloride, which indicates that spectrin is more susceptible to being cross-linked after pronase+Con A; i.e. the spectrin is probably aggregated by the enzyme and lectin incubation.

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