Isolation Of Plasma Membrane Vesicles Of Human Platelet By Affinity Chromatography

1981 ◽  
Author(s):  
T Kobayashi ◽  
M Sakon ◽  
H Ohno ◽  
J Kambayshi ◽  
G Kösaki
Keyword(s):  
Plasma Membrane ◽  
Affinity Chromatography ◽  
Morphological Changes ◽  
Membrane Vesicles ◽  
Dry Weight ◽  
Appreciable Amount ◽  
Marker Enzyme ◽  
Plasma Membrane Vesicles ◽  
Platelet Suspension ◽  
Centrifugal Method

Platelets undergo a unique morphological changes leading to the formation of hemostatic plug. In recent years, its intermediaty metabolism has been extensively studied and the important function of plasma membrane in the platelet reaction has been recognized. The method of Barber and Jamieson has been employed in order to prepare plasma membrane vesicles of platelet of excellent quality but it is rather time consuming and the yield is relatively low. In this study, an attempt was made to isolate plasma membrane vesicles of human platelets by wheat germ agglutinin affinity chromatography.Freshly collected human citrated blood was subjected to glycerol loading and hypotonic lysis to obtain lysed platelet suspension. Then, it was applied to the affinity chromatography and the fraction of plasma membrane vesicles was eluted by 0.2 M N-acetyl glucosamine. Electron micrograph of the fraction showed round membrane vesicles with some scattered intracellular organelles. Several marker enzymes were assayed in the fraction. No appreciable amount of β-glucuronidase or cytochrome c oxidase was detected in the fraction, indicating no contamination of mitochondria or α-granules. Relatively high activity of G-6-Pase was detected, suggesting possible contamination of endoplasmic reticulum. The yield was 11.6% in dry weight and 7.9% in protein.By this method, the isolation was much faster than the centrifugal method and as low as 20 ml of human citrated whole blood may be used as starting material. Upon characterization of the plasma membrane fraction by electron microscopy and marker enzyme assays, the quality of the fraction was found comparable with the centrifugal method. The yield by this method was approximately two times higher than by the conventional method.

Taurocholate transport by basolateral plasma membrane vesicles isolated from developing rat liver

1985 ◽  
Vol 248 (6) ◽  
pp. G648-G654
Author(s):  
F. J. Suchy ◽  
S. M. Courchene ◽  
B. L. Blitzer
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Plasma Membrane Vesicles ◽  
Adult Rat ◽  
Basolateral Plasma Membrane ◽  
Taurocholate Transport

Taurocholate transport was characterized in basolateral plasma membrane vesicles prepared from the livers of 14-day-old Sprague-Dawley rats using a self-generating Percoll gradient method. Liver plasma membrane protein yield, intravesicular volume, and enrichments of various marker enzymes were similar to those obtained for vesicles from adult rat liver. The basolateral marker enzyme Na+-K+-ATPase was enriched 26-fold in the suckling rat basolateral membrane fraction while the bile canalicular marker enzymes alkaline phosphatase and Mg2+-ATPase were enriched only 3- and 5-fold, respectively. The activities of marker enzymes for endoplasmic reticulum, mitochondria, or lysosomes were not enriched compared with homogenate. In the presence of an inwardly directed 100 mM Na+ gradient, vesicle accumulation of taurocholate transiently reached a concentration 1.5- to 2-fold higher than that at equilibrium ("overshoot") in suckling and adult membrane vesicles, but the initial rate of taurocholate entry and peak intravesicular accumulation were markedly decreased in suckling compared with adult membrane vesicles. In the presence of an inwardly directed 100 mM K+ gradient, the rate of uptake was slower, and no overshoot occurred in either suckling or adult rat vesicles. The decreased rate of Na+-coupled taurocholate uptake by membrane vesicles from suckling rat liver could not be explained on the basis of more rapid dissipation of the transmembrane Na+ gradient. Kinetic studies demonstrated saturable, Na+-dependent taurocholate uptake for both suckling and adult vesicles. However, the Vmax for taurocholate uptake in suckling rat vesicles was less than half of the adult rate (2.46 +/- 0.13 vs. 5.25 +/- 0.22 nmol X mg prot-1 X min-1, respectively, P less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The lipoprotein lipase of white adipose tissue. Studies on the intracellular distribution of the adipocyte-associated enzyme

1986 ◽  
Vol 236 (3) ◽  
pp. 749-756 ◽  
Author(s):  
A A Al-Jafari ◽  
A Cryer
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Guinea Pig ◽  
Lipoprotein Lipase ◽  
Membrane Vesicles ◽  
Capillary Endothelium ◽  
Enzyme Analysis ◽  
Marker Enzyme ◽  
Igg Antibodies ◽  
Plasma Membrane Vesicles

The separation of rat epididymal adipocytes into plasma-membrane, mitochondrial, microsomal and cytosol fractions is described. The fractions, which were characterized by marker-enzyme analysis and electron-micrographic observation, from the cells of fed and 24 h-starved animals were used to prepare acetone/diethyl ether-dried powders for the measurement of lipoprotein lipase activities. The highest specific activities and proportion of recovered lipoprotein lipase activity were found in the plasma-membrane and microsomal fractions. The two fractions from the cells of fed rats showed similar activities and enrichments of the enzyme, these activities being higher than the plasma-membrane and lower than the microsomal activities recovered from the cells of starved animals. Chicken and guinea-pig anti-(rat lipoprotein lipase) sera were prepared, and an indirect labelled-second-antibody cellular immunoassay, using 125I-labelled rabbit anti-(chicken IgG) or 125I-labelled sheep anti-(guinea-pig IgG) antibodies respectively, for the detection of cell-surface enzyme was devised and optimized. The amount of immunodetectable cell-surface lipoprotein lipase was higher for cells isolated from fed animals than for cells from 24 h-starved animals, when either anti-(lipoprotein lipase) serum was used in the assay. The amount of immunodetectable cell-surface lipoprotein lipase fell further when starvation was extended to 48 h. The lipoprotein lipase of plasma-membrane vesicles was shown to be a patent activity and to be immunodetectable in a modification of the cellular immunoassay. Although the functional significance of the adipocyte surface lipoprotein lipase is not known, the possibility of it forming a pool of enzyme en route to the capillary endothelium is advanced.

Isolation of plasma membrane vesicles of human platelets by affinity chromatography

1981 ◽  
Vol 21 (1-2) ◽  
pp. 129-135 ◽  
Author(s):  
J. Kambayashi ◽  
M. Sakon ◽  
H. Ohno ◽  
G. Kòsaki
Keyword(s):  
Plasma Membrane ◽  
Affinity Chromatography ◽  
Membrane Vesicles ◽  
Human Platelets ◽  
Plasma Membrane Vesicles

scholarly journals Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver.

1984 ◽  
Vol 98 (3) ◽  
pp. 991-1000 ◽  
Author(s):  
P J Meier ◽  
E S Sztul ◽  
A Reuben ◽  
J L Boyer
Keyword(s):  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Rat Liver ◽  
High Speed ◽  
Membrane Vesicles ◽  
Fold Increase ◽  
High Yield ◽  
Marker Enzyme ◽  
Gamma Glutamyl Transpeptidase ◽  
Plasma Membrane Vesicles

A method has been developed for routine high yield separation of canalicular (cLPM) from basolateral (blLPM) liver plasma membrane vesicles of rat liver. Using a combination of rate zonal floatation (TZ-28 zonal rotor, Sorvall) and high speed centrifugation through discontinuous sucrose gradients, 9-16 mg of cLPM and 15-28 mg of blLPM protein can be isolated in 1 d. cLPM are free of the basolateral markers Na+/K+-ATPase and glucagon-stimulatable adenylate cyclase activities, but are highly enriched with respect to homogenate in the "canalicular marker" enzyme activities leucylnaphthylamidase (48-fold), gamma-glutamyl-transpeptidase (60-fold), 5'-nucleotidase (64-fold), alkaline phosphatase (71-fold), Mg++-ATPase (83-fold), and alkaline phosphodiesterase I (116-fold). In contrast, blLPM are 34-fold enriched in Na+/K+-ATPase activity, exhibit considerable glucagon-stimulatable adenylate cyclase activity, and demonstrate a 4- to 15-fold increase over homogenate in the various "canalicular markers." cLPM have a twofold higher content of sialic acids, cholesterol; and sphingomyelin compared with blLPM. At least three canalicular-(130,000, 100,000, and 58,000 mol wt) and several basolateral-specific protein bands have been detected after SDS PAGE of the two LPM subfractions. Specifically, the immunoglobin A-binding secretory component is restricted to blLPM as demonstrated by immunochemical techniques. These data indicate virtually complete separation of basolateral from canalicular LPM and demonstrate multiple functional and compositional polarity between the two surface domains of hepatocytes.

Plasma membrane vesiculation in 3T3 and SV3T3 cells. I. Morphological and biochemical characterization

1979 ◽  
Vol 35 (1) ◽  
pp. 229-243
Author(s):  
R.E. Scott ◽  
R.G. Perkins ◽  
M.A. Zschunke ◽  
B.J. Hoerl ◽  
P.B. Maercklein
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Biochemical Characterization ◽  
Succinic Dehydrogenase ◽  
Membrane Vesicles ◽  
Marker Enzyme ◽  
Plasma Membrane Vesicles ◽  
Vesicle Membrane ◽  
Morphological Studies ◽  
Embryo Cells

3T3 and SV3T3 mouse embryo cells and a variety of other monolayer cell lines can be induced to form and shed plasma membrane vesicles by exposure to sulphydryl blocking agents including formaldehyde and N-ethyl malemide. Morphological studies show that multiple vesicles are formed and released from individual cells and that the vesicle membrane is continuous with the plasma membrane of the cell. Vesicles measure from o.1 to 15 micrometer in diameter and are free of detectable contamination with cytoplasmic membranes and organelles. Vesicles also show a 10-fold enrichment in the plasma membrane marker enzyme 5′-nucleotidase and are devoid of detectable NADH-cytochrome C reductase and succinic dehydrogenase activity which are marker enzymes for endoplasmic reticulum and mitochondria, respectively. Vesicles have a high cholesterol: phospholipid ratio and show enrichment in sphingomyelin content. They contain receptors for Con A and WGA, approximately 20 size class polypeptides and intramembranous particles. These results suggest that vesicles are derived from and have the general characteristics of plasma membranes.

Taurocholate transport and Na+-K+-ATPase activity in fetal and neonatal rat liver plasma membrane vesicles

1986 ◽  
Vol 251 (5) ◽  
pp. G665-G673 ◽  
Author(s):  
F. J. Suchy ◽  
J. C. Bucuvalas ◽  
A. L. Goodrich ◽  
M. S. Moyer ◽  
B. L. Blitzer
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Rat Liver ◽  
Maximum Velocity ◽  
Membrane Vesicles ◽  
Neonatal Rat ◽  
Marker Enzyme ◽  
Marker Enzymes ◽  
Plasma Membrane Vesicles ◽  
Early Maturation

The ontogenesis of Na+-K+-ATPase activity and Na+-taurocholate cotransport was studied in basolateral plasma membrane vesicles from fetal and neonatal rat liver. Membrane vesicles from each age group were 30-fold enriched in the basolateral marker enzyme Na+-K+-ATPase, 4- to 7-fold enriched in the bile canalicular membrane marker enzymes alkaline phosphatase and Mg2+ ATPase, and not significantly enriched in activities of marker enzymes for intracellular organelles. Na+-K+-ATPase activity was significantly lower in basolateral membranes from late fetal (day 21–22) and neonatal (day 1) rat liver. Kinetic analysis of Na+-K+-ATPase activity at various concentrations of ATP revealed that the maximum velocity of enzyme reaction (Vmax) for Na+-K+-ATPase was 70 and 90% of adult activity in the fetus and the neonate, respectively. The ATP Km was significantly lower in the neonate than the adult, suggesting a higher affinity of the neonatal enzyme for ATP. In contrast to the early maturation of Na+-K+-ATPase, transport of taurocholate was markedly lower in both fetal and neonatal vesicles compared with the adult. Taurocholate uptake on day 19 of gestation did not differ in the presence of a Na+ or K+ gradient, and uphill transport, as indicated by an overshoot, did not occur. On day 20 taurocholate uptake was stimulated by a Na+ compared with a K+ gradient, and accumulation of isotope above equilibrium was demonstrated. Na+-dependent transport of taurocholate by late fetal (day 22) and neonatal vesicles was saturable but the Vmax at each age was significantly lower and the apparent Km higher in developing compared with adult membrane vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

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