Plasma Membrane Marker Enzyme Activities Research Articles

Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

Characterization of plasma membrane fraction from filamentous fungus Rhizopus nigricans.

In the filamentous fungus Rhizopus nigricans a steroid hydroxylating multienzyme system is inducible by progesterone and by several other steroids. The biological signal carried by progesterone might be mediated by receptors, located either in the plasma membrane or inside the cell. To elucidate the first possibility, plasma membrane fraction was examined for the presence of progesterone receptors. The isolation of plasma membrane from fungal homogenate containing different other membranes is difficult because of the rigid cell wall. Three different membrane fractions were prepared by differential centrifugation of the fungal homogenate and characterized by plasma membrane and mitochondrial membrane marker enzymes, H+-ATPase and mit-ATPase, respectively. The same fractions were examined for the presence of specific progesterone-binding molecules. Two of these fractions comprising the highest level of plasma membrane enzyme activity contained also the highest level of specific progesterone-binding compounds: 27,6 fmol/mg protein and 18,8 fmol/mg protein. The correlation between plasma membrane marker enzyme activity and the amount of progesterone-binding proteins in plasma membrane fraction of Rhizopus nigricans might indicate the involvement of these molecules in the induction process.

LLC-PK1 epithelia as a model for in vitro assessment of proximal tubular nephrotoxicity.

LLC-PK1 cells, an established epithelial cell line derived from pig kidney, were used as a model system for assessment of nephrotoxic side effects of three cephalosporin antibiotics: cephaloridine, ceftazidime, and cefotaxime. Toxic effects of these xenobiotics were monitored on confluent monolayers by light and electron microscopy and by the release of cellular marker enzyme activities into the culture medium. In addition, LLC-PK1 cells were grown on microporous supports, and cephalosporin-induced alteration of epithelial functional integrity was monitored by a novel electrophysiologic approach. For this purpose, an Ussing chamberlike experimental setup was used. The dose-dependent effects on transepithelial ionic permselectivity were monitored under conditions in which defined fractions of the apical culture medium NaCl contents were replaced iso-osmotically by mannitol. This method of determining the functional intactness of the epithelial barrier by measuring dilution potentials was found to be far more sensitive than monitoring cell injury by means of morphology or measurement of enzyme release. As expected from animal experimental data, a dose-dependent disruption of monolayer integrity was detected with all three methodologies applied. Cephaloridine was found the most toxic compound followed by ceftazidime, where a 3-fold, and cefotaxime, where a 10-fold dose of that of cephaloridine was needed to produce cell injury. Measurement of transepithelial dilution potentials was more sensitive as compared to the release of the apical plasma membrane marker enzyme activities alkaline phosphatase and gamma-glutamyltranspeptidase, the cytosolic lactate dehydrogenase, or the mitochondrial glutamate dehydrogenase. The data were compared to the effects of the aminoglycoside antibiotic gentamicin, which at least with respect to its effects on LLC-PK1 morphology and enzyme release, but not transepithelial electrical properties, was already investigated.

Protein Kinase C Located in Rat Liver Nuclei: Partial purification and biochemical and immunochemical characterization

In the rat liver homogenate, maximal protein kinase C activity was found at two calcium concentrations (1.75 and 3.5 mM). Subcellular fractionation of the liver homogenate revealed that the protein kinase C activity requiring 1.75 mM calcium was present only in the cytosolic and particulate subcellular fractions. The protein kinase C activity requiring 3.5 mM calcium concentration was mainly located in the rat liver nuclei preparation. About 19% of the liver homogenate protein kinase C activity requiring 3.5 mM calcium was present in the nuclei. Goat anti-rat brain protein kinase C antibodies revealed a single immunoreactive band at 80-82 kDa in the rat liver nuclear, particulate, or cytosolic fractions. Based on the ratio of plasma membrane marker enzyme activity determined in the nuclear preparation, the purity of the isolated nuclei was ascertained. Rat liver nuclear protein kinase C activity has been partially purified. The purification steps sequentially employed were Triton X-100 extraction of isolated nuclei, DEAE-cellulose chromatography, Phenyl-Superose, and Mono Q (fast protein liquid) chromatography. The final purification step revealed, by silver nitrate staining on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two protein bands at 80 and 66 kDa, respectively. These findings provide definitive data regarding the nuclear location of protein kinase C. The nuclear location of protein kinase C may lead to an understanding of the molecular pathway involved in signal transduction from the plasma membrane to the nucleus.

Differential expression of phenotype by resting zone and growth region costochondral chondrocytes in vitro

This study establishes an in vitro model for examining endochondral cartilage cell metabolism. Chondrocytes derived from the resting cell zone and adjacent growth zone of rat costochondral cartilage were compared for retention of phenotype in culture. At third passage confluence, two cell populations differ morphologically and biochemically. Resting zone cells are fibroblast-like, with smooth cell membranes and little rough endoplasmic reticulum. Growth zone cells are more polygonal, smaller in diameter, with numerous cytoplasmic extentions of the plasma membranes and abundant rough endoplasmic reticulum. Both cell populations produce matrix vesicles that are comparable morphologically to matrix vesicles isolated enzymatically from epiphyseal cartilage. While membrane vesicles are released into the media by cells derived from the resting zone as well as from the growth cartilage, alkaline phosphatase activity is enriched in media vesicles produced by growth cartilage cells. Alkaline phosphatase enriched vesicles appear to be preferentially incorporated into the extracellular matrix. Both the plasma membrane marker enzyme activity and the membrane phospholipid composition are differentially expressed in matrix vesicles and plasma membranes and are cell specific. Matrix vesicles produced by resting zone cells are enriched in alka-line phosphatase, 5′-nucleotidase, ouabain sensitive Na +/K + ATPase and cardiolipin when compared to the cell membrane. In addition, the plasma membranes of these cells contain more phosphatidylcholine plus sphingomyelin than do growth cartilage plasma membranes. Resting zone cell matrix vesicles have less phosphatidylethanolamine than do vesicles from growth cartilage cultures. Matrix vesicles produced by growth cartilage cells contain one proteolipid at 43,000 M r which comigrates with plasma membrane proteolipid and an additional proteolipid at approximately 3,000 M r. These data indicate that both cells retain differential expression of phenotype in culture and that one expression of this phenotype is production of specific extracellular matrix vesicles.

Subcellular localization of a membrane-associated transglutaminase activity in rat liver

Fractionation of rat liver by homogenization and differential centrifugation revealed that only about 83% of the transglutaminase activity in the tissue is in a soluble form, and that the remainder is associated with the particulate fraction. This latter activity remained with the membranes even after they were extensively washed to remove 99% of such soluble enzymes as lactate dehydrogenase and aldolase. Subsequent fractionation of the membranes by isopycnic density gradient centrifugation in sucrose resulted in a single band of transglutaminase activity at a density of 1.194 g/cm 3. This activity was coincident with the major band of plasma membranes, which was identified by its content of 5′-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and leucine aminopeptidase activities. After treatment with digitonin and fractionation on sucrose gradients, the transglutaminase activity and the plasma membrane marker enzyme activities were found at a new density of 1.210 g/cm 3, while the enzyme markers for the other membrane fractions remained unchanged. From these data, we conclude that approximately 17% of the transglutaminase activity in rat liver is specifically associated with the plasma membranes.

A Mg 2+-independent high-affinity Ca 2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg 2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca 2+-stimulated ATPase activity in the absence of Mg 2+. The Ca 2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity ( K m = 0.4 μM ) and a low affinity ( K m = 0.6 mM ) for Ca 2+. Distribution of these high-affinity and low-affinity Ca 2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg 2+ also stimulates the ATPase in the absence of Ca 2+. Unlike the Mg 2+-ATPase and low-affinity Ca 2+-ATPase, the plasmalemmal high-affinity Ca 2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca 2+-ATPase is noncompetitively inhibited by Mg 2+ with respect to Ca 2+ stimulation. Such an inhibitory effect of Mg 2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca 2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg 2+-independent, high-affinity Ca 2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg 2+-dependent, high-affinity Ca 2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca 2+-pump.

Membrane fractionation of canine aortic smooth muscle: Subcellular distribution of calcium transport activity

Various subcellular membrane fractions were isolated from dog aortic smooth muscle by conventional differential centrifugation followed by isopycnic centrifugation on a sucrose density gradient. These subcellular fractions were characterized by membrane marker enzyme activities, morphological features and the electrophoretic patterns on a sodium dodecyl sulfate polyacrylamide gel. Our results showed that the microsomal membrane fraction isolated by differential centrifugation was very heterogeneous and contained substantial amount of plasma membranes which could be further enriched as a light density fraction on the sucrose density gradient. The subcellular distribution of Ca2+ binding in the absence of ATP and Ca2+ transport in the presence of ATP closely paralleled the distribution of plasma membrane markers. The ATP-supported Ca2+ transport was inhibited by several Ca2+ ionophores, enhanced by inorganic phosphate and oxalate ions and cosedimented toward higher density in a continuous source density gradient with plasma membrane marker enzyme activity in the presence of digitonin. Our present work strongly suggests that plasma membrane is the predominant component of microsomal fraction and responsible for most, if not all, of the azide-insensitive ATP-supported Ca2+ accumulation.

An analytical approach to the preparation and characterization of subcellular membranes from canine mesenteric arteries.

Subcellular membrane fractions were isolated from dog mesenteric arteries by differential and isopynic sucrose density gradient centrifugations. Isolated membrane fractions were characterized by marker enzyme activities, morphological features and sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns. Our results show that the microsomal fraction isolated by conventional differential centrifugation was highly heterogenous and contained substantial amount of plasma membranes which could be further enriched as a light density membrane fraction on a discontinuous sucrose density gradient. The microsomal fraction and its subfractions were vesicular in appearance under electron microscope and were capable of binding and actively transporting Ca2+. The binding of Ca2+ and ATP-supported Ca2+-transport in the presence or absence of oxalate paralleled the distribution of plasma membrane marker enzyme activities suggesting that plasma membranes in vascular smooth muscle may play a major role in handling Ca2+ and thus the control of contractile function.

Chenodeoxycholate feeding increases the output of plasma-membrane marker enzymes into rat bile

The naturally occurring bile acid chenodeoxycholate is used clinically at doses of 14-15 mg/kg per day for the dissolution in vivo of small radiolucent gallstones (Bouchier, 1980; Hofmann, 1980; Iser & Sali, 1981). A consequence of chenodeoxycholate administration is that the amount of chenodeoxycholate in human bile increases to 75-90% of biliary bile acids; this occurs at the expense of the other major human bile acids, deoxycholate and cholate (Bouchier, 1980; Iser & Sali, 1981). Several plasma-membrane marker enzymes have been identified in the biles of many mammalian species (Holdsworth & Coleman, 1975; Evans et at., 1976; Coleman et a!., 1979). These enzymes have been shown to be released from the plasma membranes of isolated hepatocytes before the onset of lysis by treatment with bile salts (Billington et al., 1980). Dihydroxy bile salts were more membrane-damaging than trihydroxy bile salts in that they released significant amounts of these enzymes at approx. 10-fold lower concentrations. These results, together with the observation of Mullock et al. (1977) that rat bile 5'-nucleotidase is immunologically identical with the liver enzyme, have led to the suggestion that plasma-membrane enzymes occur in bile as a result of bile salt attack on the plasma membrane in the region of the bile canaliculus (Coleman et al., 1977). Chenodeoxycholate was administered to female Wistar rats (approx. 220g) by gastric intubation for 14 days at two dosages: 90 mg/kg per day [roughtly equivalent to the therapeutic dose in man on the basis of body surface area (Freireich et al., 1966)l and 300mg/kg per day. The bile acid was dissolved in a final volume of 1 ml and control animals were similarly intubated with 1 ml of saline (0.9% NaCl). Bile-duct cannulation was performed while the animals were under pentobarbitone anaesthesia. Bile was collected for 2h on ice and then stored at -20°C until analysed; a 0-2 h collection period was used, since the changes in the composition of rat bile owing to interruption of the enterohepatic circulation are minimal over this time period (Kakis & Yousef, 1978; Godfrey et al., 1981). Alkaline phosphatase (EC 3.1.3.1), alkaline phosphodiesterase I (EC 3.1.4. I), 5'-nucleotidase (EC 3.1.3.5), L-leucine p-naphthylamidase (EC 3.4.1 1.1) and lactate dehydrogenase (EC 1.1.1.27) were assayed in bile samples as described previously (Coleman et al., 1979). Chenodeoxycholate feeding did not significantly alter the bile-flow rate or the total protein concentration of bile, showing that total biliary protein output was unaffected. However, the specific activities of some plasma-membrane marker enzymes in bile from chenodeoxycholate-fed rats was markedly increased (Table 1). Alkaline phosphatase and 5'-nucleotidase were increased 3-fold in rats given 300mg/kg per day. Alkaline phosphodiesterase I activity in bile was unaffected whilst a small but insignificant increase was seen with L-leucine p-naphthylamidase. Lactate dehydrogenase was absent from the biles of both control and chenodeoxycholate-fed rats, showing that these plasma-membrane enzymes were released into bile in the absence of gross cellular damage. Sodium dodecyl sulphate/ Table 1. Spec@ activities of plasma-membrane marker enzymes in bile from control and chenodeoqrholatefed rats

Immunologic inhibition of estrogen binding and action in preputial-gland cells and their subcellular fractions

To assess the specific binding and actions of estradiol-17β (E 2β) in preputial gland of ovariectomized rats, isolated cells, consisting of parenchymal- and ductal-cell types were prepared by a collagenase procedure. Such cells exhibited substantial oxidation to 14CO 2 of [ 14C]-cholesterol. The latter function was stimulated 15-fold by treatment for 3 h in vitro with 1 × 10 −9M E 2β but not E 2α. Marked inhibition of such estrogen action was elicited by prior exposure of cells to antibodies directed against preputial-gland lysosomal lipoproteins with high-affinity binding activity for E 2β (anti-PHDLP). Retention of E 2β by preputial-gland cells was saturable, with an association constant of 6.4 × 10 8 M −1. At saturation, E 2β receptors corresponded to ~ 10,900 sites per cell. Treatment of 2 × 10 7 cells with anti-PHDLP, but not preimmune, IgG at 20 μg/ml of protein reduced the specific binding of E 2β by effecting a 4- to 5-fold decrease in the magnitude of the binding constant. The subcellular distribution and immunochemical reactivity of E 2β-binding components in their native state was investigated by disruption of preputial-gland cells in the absence of hormone. Cells were homogenized under carefully monitored conditions in buffered isotonic sucrose with Cacl 2; and proteinase inhibitor and fractionated using isotonic media. Fractions were characterized by determinations of selected enzyme activities and DNA. Activities of plasma membrane marker-enzymes as well as specific binding-sites for E 2β were present principally in paniculate fractions. Plasma membranes of low density ( ρ = 1.13–1.16) were purified from crude nuclear fractions. Activities of appropriate marker-enzymes were enriched in plasma membranes to about 10 times that of the homogenate, while binding-sites for E 2β were concentrated to about 11 times the homogenate. Specific binding of E 2β in membranes was saturable, with an association constant of 2.6 × 10 9 M −1. At saturation, E 2β receptors in plasma membrane corresponded to 0.6 pmol/mg of protein. Specific binding of [ 3H]-E 2β in plasma membrane and other major subcellular fractions was reduced by a 200-fold molar excess of unlabeled E 2β or DES, but not by E 2α, testosterone, or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 5 min heating to 65°C, but was diminished by no more than 9.8% by extraction of membranes with either hypotonic or high-salt buffers. Significant inhibition of E 2β binding in plasma membrane, microsomerich, mitochondria-lysosome and cytosol fractions was elicited by treatment of each fraction with anti-PHDLP, but not preimmune, IgG. These findings provide evidence of an immunochemical similarity of specific E 2β binding-sites which occur in multiple cellular loci before hormonal stimulation.

Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes.

To assess the subcellular distribution of oestrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [(3)H]oestradiol-17beta. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl(2) and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2nm-[(3)H]oestradiol-17beta to intact cells and their fractions was detemined after equilibration for 1.5h at 4 degrees C. More than 92% of the radioactivity from representative preparations was verified as authentic oestradiol by thin-layer chromatography. Activities of plasma-membrane marker enzymes as well as binding sites for oestrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105000g-supernatant fractions. However, by using alternative homogenization procedures (i.e. hypotonic media), known to fragment and strip structural components, oestradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (rho=1.13-1.16) and high (rho=1.16-1.18) density were purified from crude nuclear fractions. A second low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to about 28 and four times that of the homogenate in plasma membranes of low and high density respectively. Binding sites for wheat germ agglutinin and oestradiol were concentrated in low-density plasma membranes to 46-63 times that of the homogenate. Specific binding of oestrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5nm. At saturation, such oestradiol receptors corresponded to 526fmol/mg of membrane protein. A Hill plot showed a moderate degree of positive co-operativity in the interaction of hormone with plasma membranes. Specific binding of [(3)H]oestradiol-17beta was reduced by a 200-fold molar excess of unlabelled oestradiol-17beta, oestriol or diethylstilbestrol, but not by oestradiol-17alpha, cortisol, testosterone or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 60 degrees C, but remained essentially undiminished by extraction of membranes with either hypotonic or high-salt buffers. Extraction with 0.1% (v/v) Triton X-100 partially solubilized the oestrogen-binding component(s) of plasma membranes. Particle-free extracts were resolved on 5-20% (w/v) sucrose density gradients with either 0.01m- or 0.4m-KCl, and the fractions were analysed by adsorption to hydroxyapatite. In low-salt gradients macromolecule-bound oestrogen sedimented at predominantly 7.4S and binding was 1560 times that of the homogenate. Under high-salt conditions oestradiol-binding activity occurred at both 3.6S and 4.9S.

Plasma membrane isolation on DEAE-Sephadex beads

A much-simplified method for the purification of plasma membranes of cultured cells is presented, based upon the attachment of viable cells to nitrocellulose-treated DEAE-Sephadex beads, and their subsequent shearing by hypotonic lysis, agitation on a vortex mixer and sonication. The method is suggested by an older procedure involving attachment to poly-( L-lysine)-coated glass or polyacrylamide beads; the preparation involved in the present method, however, is considerably easier, more rapid and less expensive. Recovery of L-cell plasma membrane marker enzyme activities is approx. 25%, while contamination by internal membrane markers is much less than 1%.

An improved procedure for the isolation of plasma membranes from rat mesenteric arteries

A plasma membrane enriched fraction has been prepared from rat mesenteric arteries by an improved procedure which eliminated the contamination by cells other than smooth muscle; i.e. those derived from mesenteries and fat tissues. Morphological and enzymatic characterization of the plasma fraction revealed only minimal contamination by other intracellular membrane fragments in the smooth muscle cell. The plasma membrane was concentrated approximately 8-fold from the post nuclear supernatant fraction as suggested by the enrichment of 5′-nucleotidase, alkaline phosphatase and ATP-dependent calcium accumulation activities. Calcium accumulation by various membrane fractions in the presence of ATP was closely associated with plasma membrane marker enzyme activities, and was not affected by azide and oxalate. The ATP-dependent calcium accumulation by plasma membrane enriched fraction was not affected by the sodium ionophore, monensin, but was substantially inhibited by calcium ionophores X537A and A23187. This suggests that the functional properties of the plasma membrane of vascular smooth muscle include calcium extrusion across the cellular membrane via an energy-dependent transport process and that the plasma membrane plays the major role in regulating the intracellular calcium in vascular smooth muscle.

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient.

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.

Preparation of plasma-membrane subfractions from isolated rat hepatocytes.

1. Rat livers were dissociated into their constituent cells by perfusion through the portal vein with a medium containing collagenase, and hepatocytes separated from non-parenchymal cells. 2. It is shown that the procedure described by Wisher & Evans [(1975) Biochem. J. 146, 375-388] for preparation of plasma membranes from liver tissue when applied to isolated hepatocytes also yielded subfractions of similar morphology and marker-enzyme distribution. 3. Thus the distribution of alkaline phosphodiesterase, 5'-nucleotidase and the basal and glucagon-stimulated adenylate cyclase among two 'light' vesicular and one 'heavy' junction-containing plasma-membrane subfractions paralleled that reported for tissue-derived plasma-membrane subfractions. 4. Increased recoveries and specific activities of plasma-membrane marker enzymes were obtained when soya-bean trypsin inhibitor was included in the collagenase-containing perfusion media used to dissociate the liver. 5. Polyacrylamide-gel-electrophoretic analysis of the corresponding plasma-membrane subfractions prepared from liver tissue and isolated hepatocytes were generally similar. 6. The results indicate that the functional polarity of the hepatocyte's plasma membrane is retained after tissue dissociation. The damage occurring to plasma-membrane ectoenzymes by the collagenase-perfusion procedure is discussed.