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.

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)

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)

scholarly journals Albumin binding of unconjugated [3H]bilirubin and its uptake by rat liver basolateral plasma membrane vesicles

1996 ◽  
Vol 316 (3) ◽  
pp. 999-1004 ◽  
Author(s):  
Lorella PASCOLO ◽  
Savino DEL VECCHIO ◽  
Ronald K. KOEHLER ◽  
J. Enrique BAYON ◽  
Cecile C. WEBSTER ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Experimental Conditions ◽  
Saturation Kinetics ◽  
Basolateral Plasma Membrane ◽  
Component C ◽  
Uptake Studies

Using highly purified unconjugated [3H]bilirubin (UCB), we measured UCB binding to delipidated human serum albumin (HSA) and its uptake by basolateral rat liver plasma membrane vesicles, in both the absence and presence of an inside-positive membrane potential. Free UCB concentrations ([Bf]) were calculated from UCB–HSA affinity constants (K´f), determined by five cycles of ultrafiltration through a Centricon-10 device (Amicon) of the same solutions used in the uptake studies. At HSA concentrations from 12 to 380 μM, K´f (litre/mol) was inversely related to [HSA], irrespective of the [Bt]/[HSA] ratio. K´f was 2.066×106+(3.258×108/[HSA]). When 50 mM KCl was iso-osmotically substituted for sucrose, the K´f value was significantly lower {2.077×106+(1.099×108/[HSA])}. The transport occurred into an osmotic-sensitive space. Below saturation ([Bf] ⩽ 65 nM), both electroneutral and electrogenic components followed saturation kinetics with respect to [Bf], with Km values of 28±7 and 57±8 nM respectively (mean±S.D., n = 3, P < 0.001). The Vmax was greater for the electrogenic than for the electroneutral component (112±12 versus 45±4 pmol of UCB·mg-1 of protein·15 s-1, P < 0.001). Sulphobromophthalein trans-stimulated both electrogenic (61%) and electroneutral (72%) UCB uptake. These data indicate that: (a) as [HSA] increases, K´f decreases, thus increasing the concentration of free UCB. This may account for much of the enhanced hepatocytic uptake of organic anions observed with increasing [HSA]. (b) UCB is taken up at the basolateral membrane of the hepatocyte by two systems with Km values within the range of physiological free UCB levels in plasma. The electrogenic component shows a lower affinity and a higher capacity than the electroneutral component. (c) It is important to calculate the actual [Bf] using a K´f value determined under the same experimental conditions (medium and [HSA]) used for the uptake studies.

scholarly journals Carrier-mediated uptake of L-(+)-lactate in plasma membrane vesicles from rat liver

FEBS Letters ◽  
1988 ◽  
Vol 235 (1-2) ◽  
pp. 224-228 ◽  
Author(s):  
Irene Quintana ◽  
Antonio Felipe ◽  
Xavier Remesar ◽  
Marçal Pastor-Anglada
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles

Characterization of two Mg2+transporters in sealed plasma membrane vesicles from rat liver

1998 ◽  
Vol 275 (4) ◽  
pp. C995-C1008 ◽  
Author(s):  
Christie Cefaratti ◽  
Andrea Romani ◽  
Antonio Scarpa
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Intracellular Signaling ◽  
Mammalian Cells ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Channel Blockers ◽  
Transport Mechanisms ◽  
Plasma Membrane Vesicles ◽  
New Information

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ ⋅ μm−2 ⋅ min−1after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.

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.

Thiamine transport by basolateral rat liver plasma membrane vesicles

1992 ◽  
Vol 103 (3) ◽  
pp. 1056-1065 ◽  
Author(s):  
Richard H. Moseley ◽  
Pankaj G. Vashi ◽  
Suzanne M. Jarose ◽  
Chris J. Dickinson ◽  
Patricia A. Permoad
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles ◽  
Liver Plasma Membrane ◽  
Thiamine Transport
Sign in / Sign up

Export Citation Format

Share Document