Isolation of plasma membrane vesicles, derived from transverse tubules, by selective homogenization of subcellular fractions of frog skeletal muscle in isotonic media.

A new technique for isolating fragmented plasma membranes from skeletal muscle has been developed that is based on gentle mechanical disruption of selected homogenate fractions. (Na+ + K+)-stimulated, Mg2+-dependent ATPase was used as an enzymatic marker for the plasma membrane, Ca2+-stimulated, Mg2+-dependent ATPase as a marker for sarcoplasmic reticulum, and succinate dehydrogenase for mitochondria. Cell segments in an amber low-speed (800 x g) pellet of a frog muscle homogenate were disrupted by repeated gentle shearing with a Polytron homogenizer. Sarcoplasmic reticulum was released into the low-speed supernatant, whereas most of the plasma membrane marker remained in a white, fluffy layer of the sediment, which contained sarcolemma and myofibrils. Additional gentle shearing of the white low-speed sediment extracted plasma membranes in a form that required centrifugation at 100,000 x g for pelleting. This pellet, the fragmented plasma membrane fraction, had a relatively high specific activity of (Na+ + K+)-stimulated ATPase compared with the other fractions, but it had essentially no Ca2+-stimulated ATPase activity and only a small percentage of the succinate dehydrogenase activity of the homogenate. Experimental evidence suggests that the fragmented plasma membrane fraction is derived from delicate transverse tubules rather than from the thicker, basement membrane-coated sarcolemmal sheath of muscle cells. Electron microscopy showed small vesicles lined bu a single thin membrane. Hydroxyproline, a characteristic constituent of collagen and basememt membrane, could not be detected in this fraction.     

Studies on the fractionation of mucosal homogenates from the small intestine

1. Homogenates of the mucosa of the small intestine of the guinea pig were separated by fractional sedimentation into seven different fractions. The enzymic properties of some of these subcellular fractions were compared with those obtained from the mucosa of the small intestine of the rabbit and cat. 2. The enzymic properties of the low-speed sediment (15000g-min.) were investigated and it was shown that invertase and alkaline ribonuclease were predominantly located in this subcellular fraction, whereas alkaline phosphatase, aryl-amidase, acid phosphatase, acid ribonuclease and phosphoprotein phosphatase, though true constituents of this fraction, occurred to varying degrees in other subcellular structures also. 3. It was shown that the most probable source of the enzymic activities observed in the low-speed sediment was the brush border. Electron micrographs of the purified brush-border fraction indicated vesicles derived from the brush-border membrane. 4. A method is described for the fractionation of mucosal homogenates into a brush border-plus-nuclei fraction, a mitochondrial fraction, a microsomal fraction and a particle-free supernatant. The fractions were shown to be relatively pure, as indicated by the distribution of invertase, DNA, succinate dehydrogenase, glucose 6-phosphatase and 6-phosphogluconate dehydrogenase. 5. Most of the activity of four lysosomal enzymes present in the nuclei-free homogenate was sedimented at 375000g-min., suggesting the occurrence of lysosomal particles in mucosal homogenates. 6. Further fractionation of the microsomal membranes into three fractions is described. The enzymic composition of the membrane fractions is given and discussed in relation to their structure as seen in electron micrographs.     

Isolation of plasma membranes and Golgi apparatus from a single chicken liver homogenate

Chicken liver plasma membranes, minimally contaminated with Golgi apparatus-derived vesicles, were prepared from a low-speed (400 g) pellet by means of flotation in isotonic Percoll solution, followed by a hypotonic wash and flotation in a discontinuous sucrose gradient. Based on the analysis of suitable marker enzymes, alkaline phosphatase and alkaline phosphodiesterase, two plasma membrane fractions were isolated with enrichments, depending on the equilibrium density and marker of 28-97 and with a total yield of 4-5%. Golgi apparatus fractions were prepared by flotation of microsomes, obtained from the same homogenate as the low-speed pellet, in a discontinuous sucrose gradient. The trans-Golgi marker galactosyltransferase was 27-fold enriched in a fraction of intermediate density (d=1.077-1.116 g/ml). Approximately 12% of galactosyltransferase was recovered in the membranes equilibrating d=1.031-1.148 g/ml. Contamination with plasma membrane fragments was low in the light (d=1.031-1.077 g/ml) and intermediate density Golgi vesicles. The isolation of purified plasma membranes and Golgi vesicles from one liver homogenate will enable future studies on receptor cycling between these cell organelles.     

Localization of the membrane-associated thiol oxidase of rat kidney to the basal-lateral plasma membrane.

The localization of the membrane-associated thiol oxidase in rat kidney was investigated. Fractionation of the kidney cortex by differential centrifugation demonstrated that the enzyme is found in the plasma membrane. The crude plasma membrane was fractionated by density-gradient centrifugation on Percoll to obtain purified brush-border and basal-lateral membranes. Gamma-Glutamyltransferase, alkaline phosphatase and aminopeptidase M were assayed as brush-border marker enzymes, and (Na+ + K+)-stimulated ATPase was assayed as a basal-lateral-membrane marker enzyme. Thiol oxidase activity and distribution were determined and compared with those of the marker enzymes. Its specific activity was enriched 18-fold in the basal-lateral membrane fraction relative to its activity in the cortical homogenate, and its distribution paralleled that of (Na+ + K+)-stimulated ATPase. This association indicates that thiol oxidase is localized in the same fraction as (Na+ + K+)-stimulated ATPase, i.e. the basal-lateral region of the plasma membrane of the kidney tubular epithelium.     

Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system.

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.     

Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta.

A method for the simultaneous preparation of microvillous and basal membrane vesicles from human placental syncytiotrophoblast is described. Mg2(+)-aggregated basal membranes are separated from microvillous membranes by low-speed centrifugation after initial homogenization and centrifugation steps. Microvillous membranes (MVM) are obtained from the low speed supernatant while basal membranes (BM) contained in the Mg2(+)-aggregated material are resuspended and further purified on a sucrose step gradient. MVM and BM prepared by this method were enriched 20-fold and 11-fold as determined by the membrane marker enzymes, alkaline phosphatase (MVM) and adenylate cyclase (BM). There was minimal cross-contamination of the two isolated plasma membrane fractions and the yields obtained were 26% (MVM) and 21% (BM) compared to the initial homogenate. The MVM and BM fractions were free from contamination by mitochondrial or lysosomal membranes and showed only minor contamination by microsomal membranes. The two membrane fractions were also tested for the presence of non-syncytial plasma membranes by electrophoretic immunoblotting. Contamination of both MVM and BM by fibroblast, endothelial, macrophage and cytotrophoblast plasma membranes amounted to less than 15% of the total membrane protein as determined by immunoblotting. Vesicle orientation, determined from the latency of specific concanavalin A binding, was 88 +/- 4% right-side out for MVM and 73 +/- 12% right-side out for BM. This simple preparative procedure produces a high yield of both MVM and BM from human placenta. The analytical data demonstrates that 'paired' MVM and BM fractions derived from the same placental tissue have a high purity in terms not only of contamination by intracellular membranes, but also in terms of contamination by non-syncytial plasma membranes.     

Preparation of rat liver plasma membranes in a high yield

Existing procedures for the preparation of rat liver plasma membranes are time consuming and generally produce low yields. A method is described in which a rat liver homogenate low-speed pellet is fractionated on a self-forming Percoll gradient. Plasma membranes can be removed from the gradient in a high yield along with much of the DNA in the liver homogenate. A second Percoll step performed in the presence of a low concentration of calcium ions separates the DNA from the plasma membranes. The final membrane fraction has high specific activities of marker enzymes with little contamination with microsomal, mitochondrial, Golgi, or lysosomal markers.     

Extraction of plasma membranes from smooth muscle cells of the rabbit small intestine

A method is offered for isolation of subcellular fractions from small intestinal smooth muscle cells enriched by plasma membranes (PM). The method is based on differential centrifugation over sucrose density gradient. According to the localization of marker enzymes, the membrane fraction obtained with the use of 30% sucrose is considered to be optimal. The PM fraction is superior to the homogenate 10-fold on the average in the magnitude of Na, K-ATPase, 17-fold in Mg2+-ATPase, and 15-fold in that of 5'-nucleotidase activity. ATPase of PM is activated by Ca2+ in micro- and millimolar concentrations. It is suggested that Mg2+-dependent Ca-activated ATPase of PM is related to the Ca2+ content control in the cell.     

Brain Ca2+/Mg(2+)-ATPase activity and seasonal adaptation of the Djungarian dwarf hamster Phodopus sungorus.

1. Of three sets of Djungarian dwarf hamster, two groups were raised during winter under greatly differing circumstances. One winter group was raised within a climate controlled cage in which the ambient temperature was maintained at 22 degrees C and whereby conditions of light vs darkness were maintained in a constant 12 hr cycle. The second winter group was raised out of doors whereby the hamsters were subjected to prevailing seasonal environmental conditions. A third group was studied under summer conditions, as well. Ca(2+)-, Mg(2+)- and (Ca2+/Mg2+)-ATPase activity was analysed in cellular (= total homogenate) and subcellular fractions (P1-, synaptosomal fraction, synaptic membranes) from cortex, cerebellum and basal brain. 2. The data obtained indicate similar ATPase activity in the cortical homogenates of the winter indoor and summer hamsters. 3. Winter outdoor animals experiencing normal torpidity, however, exhibited reduced ATPase activity by about 50%. 4. Cortical subcellular fractions yielded different results: both the winter and the summer groups showed high ATPase activity in the synaptosomal and synaptic membrane fractions. 5. In the total cerebellar homogenate, the hamsters raised under summer and winter conditions showed the greatest enzyme activity, although less activity was seen in the subcellular fractions. 6. The ATPase activity in the basal brain was found to be nearly identical in all three hamster groups.     

Polarized subcellular distribution of the 1-, 4- and 5-phosphatase activities that metabolize inositol 1,4,5-trisphosphate in intestinal epithelial cells.

In intestinal epithelial cells, Ins(1,4,5)P3 is metabolized both by an intracellular 5-phosphatase and by less specific extracellular phosphatases [Rubiera, Velasco, Michell, Lazo & Shears (1988) Biochem. J. 255, 131-137]. A total of 91% of intracellular Ins(1,4,5)P3 5-phosphatase was particulate, and was preferentially associated with plasma membranes rather than with other subcellular organelles. A soluble Ins(1,4,5)P3 3-kinase activity was also characterized, further supporting the idea that inositol phosphates are important in enterocyte function. We have studied the distribution of Ins(1,4,5)P3 phosphatase activities in basolateral and brush-border domains of the plasma membrane. Compared with homogenates, the extracellular phosphatases were 13-17-fold enriched in brush-border membranes, but only 2-fold enriched in basolateral membranes. The 1- and 4-phosphates of Ins(1,4,5)P3 were hydrolysed at equal rates by the extracellular phosphatases; these enzymes are proposed to have digestive functions. The intracellular particulate 5-phosphatase was 2-fold enriched in brush-border membranes and 13-fold enriched in basolateral membranes, at the same pole of the cell where Ins(1,4,5)P3 is believed to be generated. This is opposite to the polarized distribution of particulate 5-phosphatase in hepatocytes [Shears, Evans, Kirk & Michell (1988) Biochem. J. 256, 363-369]; these differences in subcellular distribution may be important in determining cell-specific metabolism of Ins(1,4,5)P3.     

Presence of Base-Exchange Activity in Rat Brain Nerve Endings: Dependence on Soluble Substrate Concentrations and Effect of Cations

The calcium-dependent, energy-independent incorporations of 14C-labeled bases, choline, ethanolamine, and serine, into their corresponding membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, were compared in microsomes and in subcellular fractions prepared from a lysed crude mitochondrial (P2) pellet of whole rat brain. When activities were measured in the presence of an extracellular (1.25 mM) concentration of Ca2+, recovered activities were highest in the microsomal fraction, although substantial activity remained associated with the P2 homogenate even after repeated washing of the pellet. When this washed P2 homogenate was subfractionated, enrichment of all three exchange activities was obtained only in a fraction that was fivefold enriched over the homogenate and sevenfold enriched over the microsomal fraction in Na+, K+-ATPase, a plasma membrane marker. This strongly suggests that the base-exchange enzymes are normal constituents of synaptosomal plasma membranes. The three exchange activities were measured in synaptosomes prepared from whole rat brain in the presence of various substrate (base) concentrations, and kinetic constants were calculated. The Vmax values for choline, ethanolamine, and serine exchange were, respectively, 1.27 +/- 0.09, 1.60 +/- 0.17, and 0.56 +/- 0.06 nmol/mg of protein/h; the respective Km (apparent) values were 241 +/- 29, 65 +/- 18, and 77 +/- 22 microM. Endogenous levels of the three bases, choline, ethanolamine, and serine, in whole (microwaved) rat brains were 20 +/- 8, 78 +/- 28, and 639 +/- 106 nmol, respectively. That ethanolamine and serine incorporations had lower Km values than choline incorporation suggests that these bases are preferentially incorporated into their respective phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved yield of plasma membrane from mammalian cells through modifications of the two-phase polymer isolation procedure

Modifications to the two-phase polymer gradient procedure for isolating plasma membrane from mammalian cells have resulted in greatly increased yields of purified plasma membrane. First, the cells were not treated with a membrane stabilizer (ZnCl₂) prior to homogenization. This reduced the severity of homogenization required for disruption and allowed a greater proportion of the surface membrane to form large, flattened sheets that are more easily purified than the smaller fragments formed during more severe homogenization. Second, three crude fractions obtained from the homogenate (600g, 2000g, and 12,000g pellets), rather than a single, low-speed pellet (600g) containing only large sheets of membrane, were subjected to gradient centrifugation to obtain plasma membrane. This modification allowed purification of small as well as large fragments of plasmalemma and greatly increased the yield of purified membrane. Mg⁺²-dependent, Na⁺-K⁺-stimulated ATPase, a marker enzyme for plasma membrane, was enriched in the purified fraction by ≈17-fold relative to homogenate on a specific activity basis, and the yield of isolated plasma membrane averaged 70%, and was occasionally as high as 90%.     

A comparison of brush-border membranes prepared from rabbit small intestine by procedures involving Ca2+ and Mg2+ precipitation

Brush-border membranes were isolated from rabbit small intestine by procedures involving precipitation of undesired membranes with either 10 mM MgCl2 or 10 mM CaCl2. The membranes were compared on the basis of marker enzyme content and lipid composition. Ca2+-prepared membranes displayed a greater enrichment of alkaline phosphatase and sucrase activity compared to homogenate than did the Mg2+-prepared membranes. The former also displayed an impoverishment of (Na+ + K+)-ATPase activity, the specific activity of which increased several-fold in Mg2+-prepared membranes. Membranes prepared with Ca2+ were characterized by a lower phosphoacylglycerol-protein ratio and a higher phosphatidylethanolamine-phosphatidylcholine ratio. Although lysophosphoacylglycerols accounted for about 6% of the total phospholipids in these membranes compared to 2% in Mg2+-prepared membranes, the free fatty acid content was similar in both types of membranes. It was concluded that Ca2+ prepared membranes were less contaminated by basolateral membranes than were Mg2+-prepared membranes and the use of Ca2+ did not notably enhance degradation of endogenous lipids by brush-border membrane phospholipase A.     

Rapid Method for the Isolation of Plasma Membranes from a Murine Fibrosarcoma Using an Aqueous Two-Phase Polymer System

An aqueous two-phase polymer system was used to isolate plasma membranes from a palpable mouse fibrosarcoma. The excised tumor tissue was washed with sterile saline and pushed through nylon screens of decreasing mesh size. This cell suspension was placed in Tris-buffered, isotonic sucrose plus MgSo₄ and homogenized by nitrogen cavitation. A pellet was collected from the homogenate by low-speed centrifugation and was added to the aqueous two-phase polymer system. After several brief, low-speed centrifugations, the interfacial material between the polymer phases was collected. Data from enzyme and biochemical assays demonstrated that this fraction was plasma membrane. This method provided a high yield of the surface membrane in less than three hours.     

Boophilus microplus: characterization of larval phosphomonoesterases and isolation of subcellular fractions with high phosphatase activity.

Phosphomonoesterase activity was determined for a 115,000g pellet and soluble fractions resulting from a subcellular fractioning of a homogenate of larval Boophilus microplus. Both fractions showed maximum phosphatase activity at pH 5.5 and 10. Acid phosphatase (EC 3.1.3.2) activity was found to be greatest in the soluble fraction. When the reaction rate was plotted against homogenate concentration, the soluble acid phosphatase deviated from the linear relationship. For both fractions different thermostability patterns were obtained, inactlvation beginning for the alkaline phosphatase (EC 3.1.3.1) at 45–55 C. When the effect of substrate concentration on activity was studied, deviations from the typical hyperbolic behavior were observed. Homogenization of larvae with 5 mm EDTA buffer failed to yield a low-speed pellet with high alkaline phosphatase activity, as it is expected if absorptive structures sediment. Moreover, total alkaline phosphatase activity recovered by this method is significantly lower than activity recovered when homogenization is carried out without EDTA. Alternately, homogenization with 10 mM Tris buffer and 0.25 M sucrose gave 27,000g and 115,000g fractions with high phosphatase activity when fractioned by centrifugation. Alkaline treatment of the 115,000g fraction with 10 mM Tris buffer, pH 7.8, failed to separate endoplasmic reticulum contaminants without loss of phosphatase activity. When the 115,000g fraction was centrifuged in a sucrose density gradient, two activity peaks, coincident for both acid and alkaline phosphatases, were obtained. Antigenic analysis showed the existence of similar antigenic determinants in both peaks “immunologically” presented in different ways.     

Partial purification and characterization of the (Ca2+ + Mg2+)-ATPase from squid optic nerve plasma membrane

A membrane fraction enriched in axolemma was obtained from optic nerves of the squid (Sepiotheutis sepioidea) by differential centrifugation and density gradient fractionation. The preparation showed an oligomycin- and NaN3-insensitive (Ca2+ + Mg2+)-ATPase activity. The dependence of the ATPase activity on calcium concentration revealed the presence of two saturable components. One had a high affinity for calcium (K1 1/2 = 0.12 microM) and the second had a comparatively low affinity (K2 1/2 = 49.5 microM). Only the high-affinity component was specifically inhibited by vanadate (K1 = 35 microM). Calmodulin (12.5 micrograms/ml) stimulated the (Ca2+ + Mg2+)-ATPase by approx. 50%, and this stimulation was abolished by trifluoperazine (10 microM). Further treatment of the membrane fraction with 1% Nonidet P-40 resulted in a partial purification of the ATPase about 15-fold compared to the initial homogenate. This (Ca2+ + Mg2+)-ATPase from squid optic nerve displays some properties similar to those of the uncoupled Ca2+-pump described in internally dialyzed squid axons, suggesting that it could be its enzymatic basis.     

Proteins of the kidney microvillar membrane. Biosynthesis of endopeptidase-24.11, dipeptidylpeptidase IV and aminopeptidases N and A in pig kidney slices.

An organ culture employing slices of renal-cortex tissue from piglets of the Yucatan strain was used to study the biogenesis of four microvillar peptidases: endopeptidase-24.11 (EC 3.4.24.11), dipeptidyl peptidase IV (EC 3.4.14.5), aminopeptidase N (EC 3.4.11.2) and aminopeptidase A (EC 3.4.11.7). The viability of the culture system was confirmed by the preservation of ultrastructural integrity and by an unchanged uptake of [3H]alanine into cells during the period of the experiments. After labelling with [35S]methionine, treatment with Mg2+ yielded two fractions, one containing microvilli and another, the Mg2+ pellet, containing intracellular and basolateral membranes. The labelled forms of the peptidases, isolated by immunoprecipitation, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The Mg2+ pellet contained the earliest detectable forms of the enzymes. In each case, a polypeptide of lower Mr than the mature form and sensitive to treatment with endo-beta-N-acetylglucosaminidase H was the first form to be detected. These high-mannose forms were followed, about 30 min after the pulse, by a complex glycosylated form of higher Mr. Only the latter form was observed in microvilli and then only after 90 min of the chase period. A quantitative study of dipeptidyl peptidase IV showed that the forms observed in the Mg2+ pellet were precursors of those in the microvillar fraction. No labelled forms were observed in the cytosol. All four peptidases were thus synthesized within membrane compartments and glycosylated in two steps before assembly in microvilli.     

Phosphatidylinositol kinase and diphosphoinositide kinase of rat kidney cortex: properties and subcellular localization.

The properties of phosphatidylinositol kinase and diphosphoinositide kinase from rat kidney cortex were studied. The enzymes were completely Mg2+-dependent. Cutscum detergent activated phosphatidylinositol kinase, but diphosphoinositide kinase was inhibited by all detergents tested. The pH optima were 7.7 for phosphatidylinositol kinase and 6.5 for diphosphoinositide kinase. On subcellular fractionation of kidney-cortex homogenates by differential centriflgation, the distribution of phosphatidylinositol kinase resembled that of the marker enzymes for brush-border, endoplasmic-reticulum and Golgi membranes. Diphosphoinositide kinase distribution resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), diphosphoinositide phosphatase and triphosphoinositide phosphatase. Activities of both kinases were low in purified brush-border fragments. Diphosphoinositide kinase is probably localized in the Golgi complex.     

Isolation of brush-border membranes from rat and rabbit colonocytes: Is alkaline phosphatase a marker enzyme?

Summary A method for the isolation of brush-border membranes of large intestinal epithelial cells was developed, which is based on the purification of intact brush-border caps by Percoll^® density-gradient centrifugation followed by separation of the vesiculated brush-border membranes on sucrose gradients. The procedure has two major advantages in comparison to known methods: 1) its first step does not depend on the determination of marker enzymes and 2) the method is applicable to rats as well as rabbits without major modifications. Due to the lack of an accepted marker for the colonic brush-border membrane the validity of the isolation procedure was tested by its application to the small intestine. Rat small intestinal brush-border membranes were enriched 21-fold when compared to the homogenate. The method was used to evaluate alkaline phosphatase as a marker enzyme for the colonic brush-border membrane. The results suggest that alkaline phosphatase is not exclusively localized in the brush-border membrane since this enzyme was also associated with membranes having different physical properties.     

Hepatic alpha-adrenergic receptors. Identification and subcellular localization using [3H]dihydroergocryptine.

Recently, several workers have shown that adrenergic control of hepatic carbohydrate metabolism has the characteristics of an alpha-receptor-mediated process. Using the rat liver membrane preparation of Neville (Neville, D. (1968) Biochim. Biophys. Acta 154, 540-552), alpha-adrenergic receptors have been identified using the ligand [3H]dihydroergocryptine. The receptors are saturable and of high affinity. Scatchard analysis yields a KD of 1.8 nM with 1.7 +/- 0.55 pmol of sites/mg of protein. Competition of dihydroergocryptine binding with various pharmacologic agents yields the typical (alpha-adrenergic potency series: (-)-epinephrine greater than (-)-norepinephrine greater than (-)-isoproterenol. (-)-Isomers are more potent than (+)-isomers. The alpha-blocker phentolamine is 3.4 orders of magnitude more potent than the beta-blocker propranolol. To determine subcellular localization of alpha-adrenergic receptors, livers were fractionated into a crude homogenate, a 1500 X g pellet, and the purified membrane preparation used previously for binding. Specific dihydroergocryptine binding, ouabain-inhibitable (Na,K)-ATPase, and F--stimulated adenylate cyclase activities, were followed in these fractions. Specific binding was enriched, relative to that in the crude homogenate, 2.88-fold in the pellet and 6.28-fold in the membranes. Similarly, (Na,K)-ATPase acticity was enriched 2.6-fold in the pellet and 7.1-fold in the membranes while adenylate cyclase activity was enriched 2.9-fold in the pellet and 3.5-fold in the membranes. It is concluded that hepatic alpha-adrenergic receptors are likely concentrated in the plasma membranes.