Lectin binding to guinea-pig sperm zipper particles

1983 ◽  
Vol 60 (1) ◽  
pp. 303-329
Author(s):  
G.C. Enders ◽  
Z. Werb ◽  
D.S. Friend
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Lectin Binding ◽  
Mammalian Species ◽  
Sodium Dodecyl ◽  
Freeze Fracture ◽  
Binding Study ◽  
Detergent Solubilization ◽  
Triton X

Zipper particles are morphologically distinct transmembrane specializations of sperm tails. In freeze-fracture replicas of the guinea-pig sperm, they appear as an interdigitating double row of intramembranous particles running longitudinally within the plasma membrane of the principal piece. In thin section, zipper particles appear as an increase in electron density both above and below the bilayer. Zipper particles have been observed on a variety of both mammalian and non-mammalian species, suggesting that they have been conserved to serve an essential sperm function. As a first step towards biochemically characterizing guinea-pig zipper particles and towards developing a zipper isolation procedure, we performed an in situ lectin-binding study. Examination of nine gold- or ferritin-conjugated lectins revealed that three lectins, concanavalin A, Ricinus communis agglutinin I and wheatgerm agglutinin, bound to zipper particles. These lectin binding results suggest the presence of N-linked oligosaccharides within zipper particles. The results of the lectin binding study were then used in conjunction with a detergent solubilization procedure to identify potential zipper components. Detergent solubilization involved two non-ionic detergents: digitonin, which solubilized most of the plasma membrane, but left approximately two thirds of the zipper particles attached to the cytoskeleton, and Triton X-100, which solubilized the remaining zipper particles while leaving most other sperm structures intact. Within sodium dodecyl sulphate/polyacrylamide gels of the Triton-X-100-soluble fraction potential zipper particle components with the same lectin-binding characteristics as in situ zipper particles were identified.

scholarly journals Calpain modulates capacitation and acrosome reaction through cleavage of the spectrin cytoskeleton

Reproduction ◽  
2010 ◽  
Vol 140 (5) ◽  
pp. 673-684 ◽  
Author(s):  
Yadira Bastián ◽  
Ana L Roa-Espitia ◽  
Adela Mújica ◽  
Enrique O Hernández-González
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
Mammalian Species ◽  
Physiological Changes ◽  
Signal Transducer ◽  
Mammalian Sperm ◽  
Important Player ◽  
Calpain 2 ◽  
Spectrin Cytoskeleton

Research on fertilization in mammalian species has revealed that Ca2+is an important player in biochemical and physiological events enabling the sperm to penetrate the oocyte. Ca2+is a signal transducer that particularly mediates capacitation and acrosome reaction (AR). Before becoming fertilization competent, sperm must experience several molecular, biochemical, and physiological changes where Ca2+plays a pivotal role. Calpain-1 and calpain-2 are Ca2+-dependent proteases widely studied in mammalian sperm; they have been involved in capacitation and AR but little is known about their mechanism. In this work, we establish the association of calpastatin with calpain-1 and the changes undergone by this complex during capacitation in guinea pig sperm. We found that calpain-1 is relocated and translocated from cytoplasm to plasma membrane (PM) during capacitation, where it could cleave spectrin, one of the proteins of the PM-associated cytoskeleton, and facilitates AR. The aforementioned results were dependent on the calpastatin phosphorylation and the presence of extracellular Ca2+. Our findings underline the contribution of the sperm cytoskeleton in the regulation of both capacitation and AR. In addition, our findings also reveal one of the mechanisms by which calpain and calcium exert its function in sperm.

scholarly journals Isolation and characterization of the urothelial lumenal plasma membrane.

1977 ◽  
Vol 73 (2) ◽  
pp. 382-399 ◽  
Author(s):  
J S Caruthers ◽  
M A Bonneville
Keyword(s):  
Plasma Membrane ◽  
Sodium Dodecyl ◽  
Modified Technique ◽  
Sialic Acid Content ◽  
Polarity Index ◽  
Total Sialic Acid ◽  
Isolation And Characterization ◽  
A Value

The lumenal plasma membrane has been isolated from transitional epithelial cells (urothelium) lining the urinary bladder in sheep by a modified technique involving treatment with hypotonic thioglycolate. The isolated membranes, like those in situ, are distinguished morphologically by arrays of hexagonal particles (in plague regions) separated by smooth interplaque regions. These plaque regions, specifically, can be isolated from the lumenal plasma membrane. Of the proteins constituting the lumenal plasma membrane, five were found to characterize the plaque regions and, in particular, the 33,000-dalton species appears to be most heavily concentrated in the sodium dodecyl sulfate-polyacrylamide gel pattern of the isolated plaque regions. Lipid analyses showed that there are approximately 0.93 mg of phospholipid and 0.27 mg of cholesterol for each milligram of protein, giving a value of 55% lipids and 45% proteins for the composition of the lumenal plasma membrane. The total sialic acid content was measured to be approximately 0.038 micronmol/mg protein for the plasma membrane. Several plasma membrane marker enzymes were found to be associated with the lumenal plasma membrane fraction, but only the 5'-nucleotidase activity was found to be further enriched in the plaque region fraction. Amino acid analysis of the intrinsic proteins of the plaques indicated a polarity index of 45%.

scholarly journals Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

1977 ◽  
Vol 74 (2) ◽  
pp. 561-577 ◽  
Author(s):  
DS Friend ◽  
L Orci ◽  
A Perrelet ◽  
R Yanagimachi
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Acrosome Reaction ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Phosphatidylcholine Liposomes ◽  
Acrosomal Membrane ◽  
Large Particles ◽  
Fracture Appearance ◽  
Preparatory Stage

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.

scholarly journals THE POLYMERIZATION OF ACTIN: ITS ROLE IN THE GENERATION OF THE ACROSOMAL PROCESS OF CERTAIN ECHINODERM SPERM

1973 ◽  
Vol 59 (1) ◽  
pp. 109-126 ◽  
Author(s):  
Lewis G. Tilney ◽  
Sadashi Hatano ◽  
Harunori Ishikawa ◽  
Mark S. Mooseker
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acetone Powder ◽  
Heavy Meromyosin ◽  
Skeletal Muscle Myosin ◽  
Sperm Extract ◽  
Triton X ◽  
Muscle Myosin

When Asterias or Thyone sperm come in contact with egg jelly, a long process which in Thyone measures up to 90 µm in length is formed from the acrosomal region. This process can be generated in less than 30 s. Within this process is a bundle of microfilaments. Water extracts prepared from acetone powders of Asterias sperm contain a protein which binds rabbit skeletal muscle myosin forming a complex whose viscosity is reduced by ATP. Within this extract is a protein with the same molecular weight as muscle actin. It can be purified either by collecting the pellet produced after the addition of Mg++ or by reextracting an acetone powder of actomyosin prepared by the addition of highly purified muscle myosin to the extract. The sperm actin can be polymerized and by electron microscopy the polymer is indistinguishable from muscle F-actin. The sperm actin was shown to be localized in the microfilaments in the acrosomal processes by: (a) heavy meromyosin binding in situ, (b) sodium dodecyl sulfate (SDS) gel electrophoresis of the isolated acrosomal processes and a comparison to gels of flagella which contain no band corresponding to the molecular weight of actin, and (c) SDS gel electrophoresis of the extract from isolated acrosomal caps. Since the precursor for the microfilaments in the unreacted sperm appears amorphous, we suspected that the force for the generation of the acrosomal process is brought about by the polymerization of the sperm actin. This supposition was confirmed, for when unreacted sperm were lysed with the detergent Triton X-100 and the state of the actin in the sperm extract was analyzed by centrifugation, we determined that at least 80% of the actin in the unreacted sperm was in the monomeric state.

Plasma membrane alterations of maturing goat (Capra indicus) spermatozoa: Lectin-binding and freeze-fracture study

1993 ◽  
Vol 271 (1) ◽  
pp. 159-168 ◽  
Author(s):  
H. K. Bains ◽  
S. R. Bawa ◽  
M. A. Pabst ◽  
S. Sehgal
Keyword(s):  
Plasma Membrane ◽  
Lectin Binding ◽  
Freeze Fracture ◽  
Fracture Study

Effect of anionic and nonionic surfactants on sorption and micellar solubilization of monocyclic aromatic compounds

1996 ◽  
Vol 34 (7-8) ◽  
pp. 327-334 ◽  
Author(s):  
Ruey-an Doong ◽  
Wen-gang Lei ◽  
Tsu-feng Chen ◽  
Chen-yeu Lee ◽  
Jee-hua Chen ◽  
...  
Keyword(s):  
Adsorption Capacity ◽  
Aromatic Compounds ◽  
Nonionic Surfactants ◽  
Sodium Dodecyl ◽  
Maximum Adsorption Capacity ◽  
Micellar Solubilization ◽  
Maximum Sorption Capacity ◽  
Maximum Sorption ◽  
Triton X

The effect of anionic and nonionic surfactants on the sorption and micellar solubilization of monocyclic aromatic compounds in soil-free and soil-water systems was investigated at 25 °C to examine the feasibility of in situ remediation. Benzene, chlorobenzene and styrene (BCS) were selected as the target compounds due to their suspected carcinogenic and mutagenic properties. Sodium dodecyl sulfate (SDS) and Triton X-100 were used to represent the anionic and nonionic surfactants, respectively. The addition of Triton X-100 had little effect on the micellar solubilization of BCS. However, the solubilization of aromatic compounds increased significantly with the increase of SDS concentration. A 20% to 43% enhancement of the solubilization in SDS-amended systems was demonstrated. The adsorption isotherms of BCS with Triton X-100 can conveniently be fitted by Langmuirian expression. However, multilayer adsorption of chlorobenzene and styrene was observed in SDS-amended systems. The values of maximum adsorption capacity ranged from 323 to 736 μg/g. Also, the effect of Triton X-100 on maximum adsorption capacity was greater than that of SDS. Moreover, a correlation between the maximum sorption capacity and partition coefficient was established. The results of this study demonstrate that surfactants can be effectively used as chemical amendments to minimize the volatilities of monocyclic aromatic compounds and enhance sorption and solubilization in soil environments contaminated by proper selection of surfactant type and concentration.

Structural studies of purified pig lymph node plasma membrane: association of cytoskeletal components with the plasma membrane and the effect of detergent solubilization

1982 ◽  
Vol 60 (1) ◽  
pp. 57-70 ◽  
Author(s):  
Robert J. Allore ◽  
Brian H. Barber
Keyword(s):  
Plasma Membrane ◽  
Lymph Node ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Sodium Deoxycholate ◽  
Insoluble Fraction ◽  
Plasma Membrane Vesicles ◽  
Detergent Solubilization ◽  
Lentil Lectin

The reproducibility of preparation, stability at 4 °C, and detergent solubilization characteristics of plasma membrane vesicles purified from domestic pig mesenteric lymph node tissue have been examined. It was found mat 2% (w/v) Nonidet P-40 solubilized 50–60% and 2% (w/v) sodium deoxycholate solubilized 60–70% of the total membrane protein. As judged by 125I-labelled lentil lectin staining of the sodium dodecyl sulfate – polyacrylamide gel electrophoresis patterns, 2% (w/v) Nonidet P-40 solubilized approximately 73%, and 2% (w/v) sodium deoxycholate approximately 82% of the total glycoprotein. Actin and a myosin-like component were identified as major constituents of both the Nonidet P-40 and the sodium deoxycholate insoluble fractions, suggesting the possibility that the detergent-insoluble fraction may represent a membrane-associated cytoskeletal network analogous to that which has been demonstrated for the erythrocyte membrane. Consistent with such an intimate association between actin and the plasma membrane, it was found mat very little actin could be eluted from the intact membrane vesicles by dialysis against low ionic strength ATP solutions, 0.6 M KCl, or by incubation with DNase I.

scholarly journals ACROSOMAL DISRUPTION IN SPERM

1974 ◽  
Vol 63 (2) ◽  
pp. 466-479 ◽  
Author(s):  
Daniel S. Friend ◽  
Irene Rudolf
Keyword(s):  
Plasma Membrane ◽  
Guinea Pig ◽  
Plasma Membranes ◽  
Freeze Fracture ◽  
Direct Consequence ◽  
Thin Sections ◽  
Geometric Patterns ◽  
Acrosomal Membrane ◽  
Principal Piece

"Capacitation" is a physiological event which alters sperm to permit rapid penetration through oocyte investments and fusion between gametes. Acrosomal "reaction," the physiological release of acrosomal contents, occurs after this facilitating process. In this study, acrosomal "disruption" of guinea pig and rat sperm was achieved in vitro by incubating sperm together with the follicular contents of superovulated mice. The samples contained both "reacted" and "disrupted" sperm. Thin sections of affected sperm revealed rupture and vesiculation of the plasma membrane overlying the acrosome, as well as loss of both the outer acrosomal membrane and the acrosomal content. Freeze-fracture revealed disintegration of the characteristic geometric patterns in regions of the acrosomal and plasma membranes thus disrupted and major modifications in particle distribution in the sperm tail. In the guinea pig, strands of 6–8-nm particles, usually confined to the plasma membrane of the midpiece, which overlies mitochondria, also appeared in the principal piece. Likewise, in rat sperm, bands of similarly small particles formed acute angles throughout the membrane of the principal piece. Compared with the membranes of control preparations, these membrane alterations are apparently a direct consequence of incubation with ovarian follicular contents.

scholarly journals Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 537-545 ◽  
Author(s):  
JE Fox ◽  
CC Reynolds ◽  
JS Morrow ◽  
DR Phillips
Keyword(s):  
Plasma Membrane ◽  
Platelet Activation ◽  
Actin Filaments ◽  
Sodium Dodecyl ◽  
Actin Binding ◽  
Ionophore A23187 ◽  
Unidentified Component ◽  
Membrane Bound ◽  
A Minor ◽  
Triton X

We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)- polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X- 100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta- aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), or N- ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.

scholarly journals Spectrin is associated with membrane-bound actin filaments in platelets and is hydrolyzed by the Ca2+-dependent protease during platelet activation

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 537-545 ◽  
Author(s):  
JE Fox ◽  
CC Reynolds ◽  
JS Morrow ◽  
DR Phillips
Keyword(s):  
Plasma Membrane ◽  
Platelet Activation ◽  
Actin Filaments ◽  
Sodium Dodecyl ◽  
Actin Binding ◽  
Ionophore A23187 ◽  
Unidentified Component ◽  
Membrane Bound ◽  
A Minor ◽  
Triton X

Abstract We recently showed that platelets contain submembranous actin filaments that are linked to glycoprotein (GP) Ib on the plasma membrane. In the present study, experiments were performed to determine whether spectrin was associated with these filaments. The membrane-bound filaments were isolated from Triton X-100 (Sigma, St Louis) lysates of unstimulated platelets by differential centrifugation. Platelet spectrin was detected immunologically by using antibodies against human brain and RBC spectrin. Immunoblots showed that platelet spectrin consisted of two polypeptides (mol wt 240,000 and 235,000) that were similar in apparent mol wt to those of the alpha and beta chains of brain spectrin but differed slightly from those of RBC spectrin (mol wt 240,000 and 220,000). Immunoprecipitation experiments identified platelet spectrin as two minor polypeptides migrating on sodium dodecyl sulfate (SDS)- polyacrylamide gels between actin-binding protein (mol wt 250,000) and the platelet polypeptide P235 (mol wt 235,000). Immunoblots of fractions isolated from Triton X-100-lysed platelets revealed that the alpha and beta chains of platelet spectrin were associated almost entirely with the actin filaments that were linked to the plasma membrane. Little spectrin was recovered in the Triton X-100-soluble fraction or with the actin filaments that were not membrane bound. During activation of platelets with thrombin or ionophore A23187, the alpha and beta chains of spectrin were hydrolyzed, generating a major degradation product of mol wt 160,000 and a minor one of mol wt 170,000. These two hydrolytic products were also generated in Triton X- 100 lysates incubated in the presence of Ca2+ but were not produced when lysates were treated with leupeptin, ethylene glycol bis(beta- aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), or N- ethylmaleimide, known inhibitors of the Ca2+-dependent protease. These experiments show that spectrin is a previously unidentified component of the membrane-bound actin filament network and that hydrolysis of spectrin by the Ca2+-dependent protease may regulate the interactions of the filaments during platelet activation.

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