scholarly journals Further characterization of the plasma membrane- and intracellular membrane-associated platelet Ca2+ transport systems

1989 ◽  
Vol 263 (2) ◽  
pp. 547-552 ◽  
Author(s):  
J Enouf ◽  
R Bredoux ◽  
N Bourdeau ◽  
B Sarkadi ◽  
S Levy-Toledano
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Ph Dependence ◽  
Transport Systems ◽  
Intracellular Membrane ◽  
Activity Maximum ◽  
Membrane Enzyme ◽  
Molecular Masses

Biochemical characterization of the Ca2+-ATPases isolated from human platelet intracellular and plasma membranes is reported. A comparative study of the previously partly described plasma membrane Ca2+-ATPase [Enouf, Bredoux, Bourdeau & Levy-Toledano (1987) J. Biol. Chem. 261, 9293-9297] and the intracellular membrane Ca2+-ATPase obtained simultaneously shows differences in the following parameters: (1) different kinetics of the two enzymes; (2) similar apparent affinity towards Ca2+ (10(-7) M), though the intracellular membrane enzyme was inhibited at Ca2+ concentrations above 10(-6) M; (3) different pH dependence with an activity maximum at pH 7 for the intracellular membrane Ca2+-ATPase and no detectable pH maximum for the plasma membrane Ca2+-ATPase; (4) a 10-fold difference in the ATP requirement of the two Ca2+-ATPases; (5) different patterns of inhibition by vanadate. Finally, the possible regulation of the Ca2+-ATPases was examined by studying the effect of chlorpromazine on the two Ca2+-ATPase activities, with only the plasma membrane enzyme being inhibited. It is concluded that the two platelet Ca2+ transport systems show biochemical differences in spite of the previously shown similarity in the molecular masses of their Ca2+-ATPases, thus conferring a definite specificity to the platelet system.

EVIDENCE FOR TWO DIFFERENT Ca2+TRANSPORT SYSTEMS ASSOCIATED WITH PLASMA AND INTRACELLULAR HUMAN PLATELET MEMBRANES

1987 ◽  
Author(s):  
J Enouf ◽  
R Breadux ◽  
N Bourdeau ◽  
S Levy-Toledano
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Plasma Membranes ◽  
Human Platelets ◽  
Transport Systems ◽  
Maximal Response ◽  
Potassium Oxalate ◽  
Intracellular Enzyme ◽  
Intracellular Membranes ◽  
Membrane Enzyme

The regulation of Ca2+ concentration in different cells involves two Ca2+ pumps. The presence of such mechanisms in human platelets is still controverted. We then investigated this question by using plasma and intracellular membranes obtained after simultaneous isolation by centrifugation ca 40% sucrose from a mixed 100,000 g membrane fraction.The Ca2+ uptake by the different membrane vesicles has been studied. Both membrane fractions took up Ca2+ and the Ca2+ transport systems exhibited a high affinity towards Ca 2+.However, the two associated Ca2+ transport systems showed a different time course and exhibited different oxalate sensitivity. The plasma membranes are not permeable to potassium oxalate, while the Ca2+ uptake was stimulated by potassium oxalate in intracellular membranes.Two Ca2+ activated ATPase activities are associated with the isolated membrane fractions and appeared different for the following parameters : 1) a different time course of the two enzyme activities; 2)a similar apparent affinity towards Ca2+ (10−7 M), though inhibition of the Ca2+ ATPase activity was only observed in intracellular membranes at 10−6 M Ca2+ ; 3)a different pH dependence with a maximum at pH 7 for the enzyme of intracellular membranes and pH 8 for the enzyme of plasma membranes; 4)a 10 fold difference in the ATP requirement of the enzymes, thus the maximal response was obtained with 20 uM for the intracellular membrane enzyme and with 200 uM for the plasma membrane enzyme ; 5) a different affinity for various nucleotides as energy donors with a higher specificity of the plasma membrane enzyme towards ATP ; 6) a different vanadate inhibition-dose reponse which did not exceed 60% for the plasma enzyme while it reached 100% for the intracellular enzyme.Taken together, these studies agree with the possible role of both a plasma membrane and a dense tubular system Ca2+ -ATPases in the regulation of Ca2+ concentration in human platelets.

scholarly journals Isolation and biochemical characterization of the α- and β-subunits of glycoprotein IIb of human platelet plasma membrane

1986 ◽  
Vol 240 (1) ◽  
pp. 155-161 ◽  
Author(s):  
J J Calvete ◽  
J González-Rodríguez
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Human Platelet ◽  
Size Exclusion ◽  
Beta Subunits ◽  
Exclusion Chromatography ◽  
Stepwise Reduction ◽  
Acidic Nature

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.

scholarly journals Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum α-l-fucosidase from rat testis and epididymal spermatozoa

1996 ◽  
Vol 318 (3) ◽  
pp. 821-831 ◽  
Author(s):  
Manuel AVILÉS ◽  
Irene ABASCAL ◽  
José Angel MARTÍNEZ-MENÁRGUEZ ◽  
María Teresa CASTELLS ◽  
Sheri R. SKALABAN ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Gradient Centrifugation ◽  
Light And Electron Microscopy ◽  
Enzymic Activity ◽  
Epididymal Sperm ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Neutral Ph

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum α-l-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for α-l-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the α-l-fucosidase activity was associated with the 48000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of α-l-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH–activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm–egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm–egg interactions.

What Can Epitope Specific Antibodies Tell us About the Organization of Caveolin in Cells?

2001 ◽  
Vol 7 (S2) ◽  
pp. 1030-1031
Author(s):  
J.M. Robinson
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Biochemical Characterization ◽  
Cell Types ◽  
Membrane Microdomains ◽  
Truncated Form ◽  
Coated Pits ◽  
N Terminus ◽  
Plasma Membrane Microdomains

There are three members of the caveolin (CAV) gene family that give rise to four polypeptides. These polypeptides are CAV-1α, CAV-1β, CAV-2, and CAV-3. The CAV-1β isoform is a truncated form of CAV-1α that lacks 31 amino acids at the N-terminus of the molecule. The CAV- 1β molecule arises through an alternative splicing mechanism.Caveolae are specialized plasma membrane microdomains that are expressed at high levels in some cell types (e.g., endothelium, adipocytes, fibroblasts). These specialized regions of the plasma membrane have a characteristic omega-shaped appearance with diameters ranging from 40-90 run. They are distinct from clathrin-coated pits since they lack the characteristic coated appearance in electron microscopy. Caveolae were among the first structures to be discovered by biological electron microscopy. However, biochemical characterization of these structures did not begin in earnest until a marker protein was identified. The initial marker was the 22-kDa protein known as caveolin.

scholarly journals Secretory vesicles externalize the major plasma membrane ATPase in yeast.

1988 ◽  
Vol 106 (3) ◽  
pp. 641-648 ◽  
Author(s):  
C L Holcomb ◽  
W J Hansen ◽  
T Etcheverry ◽  
R Schekman
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Biochemical Characterization ◽  
Plasma Membrane Atpase ◽  
Membrane Assembly ◽  
Yeast Cell Surface ◽  
Membrane Atpase ◽  
Constitutive Secretion ◽  
Secretory Enzyme

Yeast cell surface growth is accomplished by constitutive secretion and plasma membrane assembly, culminating in the fusion of vesicles with the bud membrane. Coordination of secretion and membrane assembly has been investigated by examining the biogenesis of plasma membrane ATPase (PM ATPase) in secretion-defective (sec) strains of Saccharomyces cerevisiae. PM ATPase is synthesized as a approximately 106-kD polypeptide that is not detectably modified by asparagine-linked glycosylation or proteolysis during transit to the plasma membrane. Export of the PM ATPase requires the secretory pathway. In sec1, a mutant defective in the last step of secretion, large amounts of Golgi-derived vesicles are accumulated. Biochemical characterization of this organelle has demonstrated that PM ATPase and the secretory enzyme, acid phosphatase, are transported in a single vesicle species.

Affinity labeling the bovine gallbladder cholecystokinin receptor using a battery of probes

1988 ◽  
Vol 255 (5) ◽  
pp. G579-G586
Author(s):  
B. Schjoldager ◽  
S. P. Powers ◽  
L. J. Miller
Keyword(s):  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Peptide Hormone ◽  
Molecular Probes ◽  
Affinity Labeling ◽  
Molecular Heterogeneity ◽  
Cck Receptors ◽  
Amino Terminal ◽  
Binding Subunit

Although the gallbladder was the first recognized target of the peptide hormone cholecystokinin (CCK) and is a physiologically important target, only one preliminary report of the biochemical characterization of this receptor exists. Recently, a series of molecular probes for the affinity labeling of different domains of the pancreatic CCK receptor have been developed. In this work we report the application of several of those probes toward the biochemical characterization of the bovine gallbladder muscularis receptor. These include "long" (125I-Bolton-Hunter-CCK-33) and "short" (125I-D-Tyr-Gly-[Nle28,31)CCK-(26-33)]) probes chemically cross-linkable through their amino-terminal amino groups and monofunctional probes with their photolabile moieties at their amino terminus (2-diazo-3,3,3-trifluoropropionyl-125I-D-Tyr-Gly-[(Nle28,31) CCK-(26-33)]) and carboxyl terminus (125I-D-Tyr-Gly-[(Nle28,31,pNO2-Phe33)CCK-(26-33)]), that span the receptor-binding region. Each of these bound specifically and saturably to a preparation enriched in plasma membranes from bovine gallbladder muscularis (mean inhibitor constants: 5.2, 1.1, 0.8, and 1.8 nM, respectively). A major relative molecular weight (Mr) 70,000-85,000 band was specifically and reproducibly labeled with the appropriate apparent affinity by each of the probes, whereas labeling of minor bands of Mr 40,000-50,000, Mr 92,000, Mr 120,000, and Mr 200,000 was dependent on cross-linker type or concentration. These observations support the identification of the Mr 70,000-85,000 protein as the bovine gallbladder CCK-binding subunit and, since this is a different size from the pancreatic CCK-binding subunit, provide biochemical evidence for molecular heterogeneity of peripheral CCK receptors.

scholarly journals Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis

1997 ◽  
Vol 321 (2) ◽  
pp. 487-495 ◽  
Author(s):  
Peter J. A. van den BROEK ◽  
Angeline E. van GOMPEL ◽  
Marijke A. H. LUTTIK ◽  
Jack T. PRONK ◽  
Carla C. M. van LEEUWEN
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Proton Motive Force ◽  
Motive Force ◽  
Transport Systems ◽  
Sugar Accumulation ◽  
Plasma Membrane Vesicles ◽  
Maltose Transport

Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome coxidase as a proton-motive-force-generating system. Addition of reduced cytochrome cgenerated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40Ő50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+Őglucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+Őmaltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilisthe transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different.

Proton conductance of the plasma membrane: properties, regulation, and functional role

1993 ◽  
Vol 265 (1) ◽  
pp. C3-C14 ◽  
Author(s):  
G. L. Lukacs ◽  
A. Kapus ◽  
A. Nanda ◽  
R. Romanek ◽  
S. Grinstein
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Second Messengers ◽  
Cell Types ◽  
Membrane Properties ◽  
Transport Systems ◽  
Extracellular Acidification ◽  
Phagocyte Nadph Oxidase ◽  
Selective Permeability ◽  
Conductive Pathway

H+ conductive pathways have been detected in the plasma membranes of a variety of cell types. The large exquisitely H(+)-selective permeability of the conductive pathway can support sizable net H+ fluxes. Although subtle differences exist among tissues and species, certain common features suggest that related transport systems are involved in all cases. The H+ conductance is gated by depolarizing voltages and is promoted by intracellular acidification. Conversely, extracellular acidification inhibits the conductance. These features facilitate net H+ efflux, while precluding potentially deleterious H+ uptake. In some cell types, activation of the conductance is additionally controlled by physiological ligands and by second messengers. The conductance most likely functions in the regulation of intracellular pH, contributing to the extrusion of H+ during repetitive depolarization of the plasma membrane, as occurs in neurons and muscle cells. This pathway may be particularly relevant in the case of phagocytes. When stimulated, these cells undergo a sustained depolarization, while generating large amounts of metabolic acid. In addition, conductive H+ fluxes may also provide counterions to neutralize the activity of electrogenic enzymes, as suggested for the phagocyte NADPH oxidase.

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