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.

Serogroups of the beer spoilage bacterium Megasphaera cerevisiae correlate with the molecular weight of the major EDTA-extractable surface protein

2000 ◽  
Vol 46 (2) ◽  
pp. 95-100 ◽  
Author(s):  
Barry Ziola ◽  
Lori Gee ◽  
Nancy N Berg ◽  
Sun Y Lee
Keyword(s):  
Ethylenediaminetetraacetic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Cross Reactivity ◽  
Surface Reactivity ◽  
Surface Proteins ◽  
Bacterial Surface ◽  
Spoilage Bacteria ◽  
Beer Spoilage

Megasphaera cerevisiae is a Gram-negative obligate anaerobe that causes turbidity and off-flavour and aroma in beer. Seven isolates of M. cerevisiae were obtained worldwide, and their extractable surface antigens were focused upon to determine if there is more than one serogroup of this bacterium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of ethylenediaminetetraacetic acid (EDTA) bacterial extracts revealed a predominant protein with apparent molecular weights of 46 000, 45 000, and 43 000 for three, two, and two isolates, respectively. When mouse antiserum generated against any of the EDTA extracts was reacted with denatured bacterial proteins in immunoblots, all bacterial isolates exhibited extensive cross-reactivity involving three antigens, one being the major EDTA-extractable protein. In contrast, when the sera were tested for surface reactivity with intact bacteria, three cross-reactivity groups were observed, with the groups individually comprised of bacteria having the same size major EDTA-extractable surface protein. When BALB/c mice immunized with a bacterium from each of the three serogroups were used for monoclonal antibody (Mab) hybridoma production, bacterial surface-reactive Mabs were obtained whose reactivities parallel the three polyclonal antibody-defined serogroups. Through combining these surface-reactive Mabs, it will be possible to rapidly detect and identify beer contamination by M. cerevisiae belonging to any serogroup. Key words: beer spoilage bacteria, Megasphaera cerevisiae, monoclonal antibodies, surface proteins, serogroups.

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.

Capacitation Induces Tyrosine Phosphorylation of Proteins in the Boar Sperm Plasma Membrane

1999 ◽  
Vol 262 (3) ◽  
pp. 787-792 ◽  
Author(s):  
Frits M. Flesch ◽  
Ben Colenbrander ◽  
Lambert M.G. van Golde ◽  
Barend M. Gadella
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Phosphorylation ◽  
Phosphorylation Of Proteins ◽  
Boar Sperm ◽  
Sperm Plasma Membrane

scholarly journals Electrophoretic Map of Boar Sperm Plasma Membrane Polypeptides and Localization and Fractionation of Specific Polypeptide Subclasses

1983 ◽  
Vol 28 (2) ◽  
pp. 393-413 ◽  
Author(s):  
Lonnie D. Russell ◽  
R. N. Peterson ◽  
Terri A. Russell ◽  
Wally Hunt
Keyword(s):  
Plasma Membrane ◽  
Boar Sperm ◽  
Sperm Plasma Membrane

Effect of CB1 Receptors on Boar Sperm Plasma Membrane

2007 ◽  
Vol 31 (S1) ◽  
pp. 189-191
Author(s):  
M. G. Pistilli ◽  
N. Bernabò ◽  
A. Gloria ◽  
M. Mattioli ◽  
B. Barboni
Keyword(s):  
Plasma Membrane ◽  
Cb1 Receptors ◽  
Boar Sperm ◽  
Sperm Plasma Membrane

The interaction of living boar sperm and sperm plasma membrane vesicles with the porcine zona pellucida

1981 ◽  
Vol 84 (1) ◽  
pp. 144-156 ◽  
Author(s):  
R.N. Peterson ◽  
L.D. Russell ◽  
D. Bundman ◽  
M. Conway ◽  
M. Freund
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Boar Sperm ◽  
Sperm Plasma Membrane

scholarly journals Surface proteins of thymus-derived lymphocytes and bone-marrow-derived lymphocytes. Selective isolation of immunoglobulin and the θ-antigen by non-ionic detergents

1974 ◽  
Vol 140 (3) ◽  
pp. 345-354 ◽  
Author(s):  
Robert E. Cone ◽  
John J. Marchalonis
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
B Cells ◽  
T Lymphocytes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Immunoglobulin A ◽  
Surface Protein ◽  
Surface Proteins ◽  
Ionic Detergent

Accessible surface proteins of thymus-derived lymphocytes (T-cells) of normal CBA mice and bone-marrow-derived lymphocytes (B-cells) of congenitally athymic nu/nu mice were analysed. The surfaces of lymphocytes were radioiodinated by using the enzyme lactoperoxidase (EC 1.11.1.7), then solubilized either in acid–urea or in the non-ionic detergent Nonidet P-40. These lysates were then precipitated with antisera specific to either immunoglobulin or the θ-alloantigen in order to assess the presence of these surface markers. Comparable amounts of radioactivity in proteins specifically precipitable as immunoglobulin were obtained from T-lymphocytes and B-lymphocytes when the cells were disrupted by acid–urea. This immunoglobulin had mol. wt. approx. 180000 and was composed of light chains and μ-type heavy chains. When radioiodinated lymphocytes were solubilized with Nonidet P-40, 3–4% of radioiodinated high-molecular-weight protein of B-cells consisted of immunoglobulin, a result similar to that found with acid–urea extraction. However, with the detergent extraction, only 0.1% of T-cell surface protein was precipitable by anti-globulin reagents. The θ-alloantigen was isolated from CBA T-cells both by acid–urea and by detergent lysis. This protein possessed a mobility on polyacrylamide-gel electrophoresis in sodium dodecyl sulphate which was consistent with a mol. wt. of 60000. An identical component was isolated from the θ-positive thymoma WEHI 105. The θ-antigen was not isolated from B-cells by either of the extraction procedures used. These results provide further evidence that the surface membranes of normal T-cells and B-cells differ in physicochemical properties. In particular, various surface components possess differential solubilities in non-ionic or organic solvents. This observation provides an explanation for discrepant results that have appeared in the literature concerning the isolation of immunoglobulin from T-lymphocytes.

scholarly journals A mouse sperm decapacitation factor receptor is phosphatidylethanolamine-binding protein 1

Reproduction ◽  
2005 ◽  
Vol 130 (4) ◽  
pp. 497-508 ◽  
Author(s):  
Rachel Gibbons ◽  
Susan A Adeoya-Osiguwa ◽  
Lynn R Fraser
Keyword(s):  
Plasma Membrane ◽  
Binding Protein ◽  
Human Sperm ◽  
Surface Proteins ◽  
Mouse Sperm ◽  
Fertility Treatments ◽  
State Dependent ◽  
Sperm Plasma Membrane ◽  
Associated Proteins ◽  
Functional Consequences

Capacitation is a pivotal event for mammalian spermatozoa, involving the loss of surface proteins known as decapacitation factors (DF) and consequent acquisition of fertilizing ability. Earlier studies showed that a mouse sperm DF binds to a receptor, DF-R, whose attachment to the sperm plasma membrane appears to involve a glycosylphosphatidylinositol (GPI) anchor. In the present study, purification and subsequent sequencing of DF-R has identified this ~23 kDa protein as phosphatidyletha-nolamine-binding protein 1 (PEBP 1). To obtain functional evidence that supports sequence homology data, purified recombinant PEBP 1 and PEBP 2 were evaluated for biological activity. While PEBP 1 was able to remove DF activity in solution at concentrations above ~1 nmol/l, PEBP 2 was ineffective, even at 600 nmol/l; this confirmed that DF-R is PEBP 1. Anti-PEBP 1 antiserum recognized recombinant PEBP 1 and a ~23 kDa protein in both mouse and human sperm lysates. Immunolocalization studies revealed that DF-R/PEBP 1 is located on the acrosomal cap, the post-acrosomal region and the flagellum of both mouse and human spermatozoa, with epitope accessibility being capacitation state-dependent and reversible. Treatment of cells with a phospholipase able to cleave GPI anchors essentially abolished immunostaining, thus confirming the extracellular location of DF-R/PEBP 1. We suggest that DF-R/PEBP 1 plays its fundamental role in capacitation by causing alterations in the sperm plasma membrane in both head and flagellum, with functional consequences for membrane-associated proteins. Obtaining more detail about DF ↔ DF-R interactions could lead to useful applications in both fertility treatments and new contraceptive approaches.

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.

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