Glycosaminoglycans of the plasma membranes of renal tubule and liver cells

Glycosaminoglycans were isolated from plasma membranes of hepatic and renal tubule cells of guinea pig. Plasmalemma of renal tubule cells contained more total glycosaminoglycans, hyaluronic acid, chondroitin-4 sulfates and chondroitin-6 sulfates, and less dermatan sulfates and heparin sulfates than liver plasma membranes. These glycocalyx components, owing to their polyanionic properties, may have a role in the transport of water, ions, and macromolecules across the cell membrane.     

Separate and shared lysosomal transport of branched and aromatic dipolar amino acids

Transport systems analogous to the T and L carriers for aromatic and bulky dipolar amino acids in plasma membranes have been characterized in the membranes of intact lysosomes isolated from human fetal skin fibroblasts. While system L appears ubiquitous in plasma membranes, system T has previously been discriminated only in the plasmalemma of human red blood cells and freshly isolated rat hepatocytes. Our findings with the lysosomal systems, provisionally designated t and l, reveal both shared and dissimilar properties with the plasma membrane systems. These properties include a lack of dependency on extralysosomal Na+, differential sensitivities to the classical system L analog, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), and the system T analog, D-tryptophan, as well as susceptibility to thiol modification at the membrane by reactivity with N-ethylmaleimide. A transport system in lysosomes from the FRTL-5 rat thyroid cell line has been described by Bernar et al. ((1986) J. Biol. Chem. 261, 17107-17112) resembles a composite of both carrier systems reported in this work.     

Evidence for coenzyme Q function in transplasma membrane electron transport

Transplasma membrane electron transport activity has been associated with stimulation of cell growth. Coenzyme Q is present in plasma membranes and because of its lipid solubility would be a logical carrier to transport electrons across the plasma membrane. Extraction of coenzyme Q from isolated rat liver plasma membranes decreases the NADH ferricyanide reductase and added coenzyme Q10 restores the activity. Piericidin and other analogs of coenzyme Q inhibit transplasma membrane electron transport as measured by ferricyanide reduction by intact cells and NADH ferricyanide reduction by isolated plasma membranes. The inhibition by the analogs is reversed by added coenzyme Q10. Thus, coenzyme Q in plasma membrane may act as a transmembrane electron carrier for the redox system which has been shown to control cell growth.     

Separate and shared lysosomal transport of branched and aromatic dipolar amino acids

Transport systems analogous to the T and L carriers for aromatic and bulky dipolar amino acids in plasma membranes have been characterized in the membranes of intact lysosomes isolated from human fetal skin fibroblasts. While system L appears ubiquitous in plasma membranes, system T has previously been discriminated only in the plasmalemma of human red blood cells and freshly isolated rat hepatocytes. Our findings with the lysosomal systems, provisionally designated tandl, reveal both shared and dissimilar properties with the plasma membrane systems. These properties include a lack of dependency on extralysosomal Na⁺, differential sensitivities to the classical system L analog, 2-aminobicyclo[2.2.1]heptane-2-car☐ylic acid (BCH), and the system T analog, d-tryptophan, as well as susceptibility to thiol modification at the membrane by reactivity with N-ethylmaleimide. A transport system in lysosomes from the FRTL-5 rat thyroid cell line has been described by Bernar et al. ((1986) J. Biol. Chem. 261, 17107–17112) resembles a composite of both carrier systems reported in this work.     

Regulation of glucose transport by insulin and non-hormonal factors

Glucose uptake by nucleated cells is mediated by facilitated diffusion. In adipocytes, fibroblasts and muscle fibers uptake is regulated by a variety of hormones, environmental factors, and metabolic conditions. Glucose uptake by mammalian red cells also occurs by facilitated diffusion, but is not regulated by the same factors and conditions as in nucleated cells; yet the pharmacological and selectivity properties of this transport system resemble those of glucose uptake in regulated cells. The glucose transporter in the human red cell is a 55, 000 dalton protein, which has been purified to homogeneity and functionally reconstituted in artificial systems. Little is known about the molecular identity of the sugar carrier in other cell types. Glucose uptake is stimulated by insulin in muscle, fat and skin cells but not in bone, brain, placenta, erythrocytes nor probably lymphocytes. In responsive cells, stimulation occurs within seconds of exposure to the hormone; it requires cellular integrity but once elicited, it persists in isolated membranes; protein synthesis is not required for either the onset of the response or the return to basal conditions after hormone removal; on the other hand, intracellular energy is required for both steps; the cytoskeleton does not seem to be involved in the regulation of glucose uptake by insulin. In general, insulin increases V_t while K_t is unaffected. The hormone could affect the rate of turnover of the transporter in the membrane, and/or the number of transporters active at any time. An increase in the number of transport sites in the plasma membrane, due to incorporation of additional sites originating from intracellular membranes, has recently been proposed on the basis of both ³H-cytochalasin B binding and glucose transport determinations in isolated plasma and intracellular membranes. The feasibility and implications of a rapid and reversible translocation of glucose transport sites from specific intracellular pools to the plasma membrane are discussed.     

In the light of stomatal opening: new insights into 'the Watergate'

Stomata can be regarded as hydraulically driven valves in the leaf surface, which open to allow CO2 uptake and close to prevent excessive loss of water. Movement of these 'Watergates' is regulated by environmental conditions, such as light, CO2 and humidity. Guard cells can sense environmental conditions and function as motor cells within the stomatal complex. Stomatal movement results from the transport of K+ salts across the guard cell membranes. In this review, we discuss the biophysical principles and mechanisms of stomatal movement and relate these to ion transport at the plasma membrane and vacuolar membrane. Studies with isolated guard cells, combined with recordings on single guard cells in intact plants, revealed that light stimulates stomatal opening via blue light-specific and photosynthetic-active radiation-dependent pathways. In addition, guard cells sense changes in air humidity and the water status of distant tissues via the stress hormone abscisic acid (ABA). Guard cells thus provide an excellent system to study cross-talk, as multiple signaling pathways induce both short- and long-term responses in these sensory cells.     

Energy transduction in Escherichia coli. Genetic alteration of a membrane polypeptide of the (Ca2+,Mg2+)-ATPase.

Recent genetic analyses of the membrane components involved in energy transduction in Escherichia coli have concentrated on the (Ca2+, Mg2+)-ATPase complex (EC 3.6.1.3). Many mutants have been described with altered biochemical properties and defects in energy-requiring processes such as oxidative phosphorylation, transhydrogenase activity, and active transport of several solutes. This report describes the isolation of a mutant strain of E. coli that is defective in several energy-requiring processes. The strain BG-31 was obtained by "localized mutagenesis" using phage P1c1. The mutation maps at approximately 73.5 min on the E. coli chromosome. Reversion and suppression analyses indicate that the defect is the result of a single amber mutation. This strain is unable to utilize succinate, D-lactate, or malate for growth. Mutant cells are unable to couple the energy derived from the hydrolysis of ATP to the active transport of proline, although coupling of energy derived from electron transport to solute transport appears normal when examined in both cells and isolated membrane vesicles. Isolated membranes of the mutant are unable to couple the energy derived from the hydrolysis of ATP to transhydrogenase activity while they can utilize the energy generated from electron transport to drive transhydrogenase activity. Extracts of strain BG-31 have normal levels of (Ca2+, Mg2+)-ATPase activity. The ATPase portion of the complex, bacterial F1 (BF1), is poorly attached to the membrane portion of the complex. In vitro reconstitution of transhydrogenase activity with stripped membrane fractions and crude preparations of BF1 localize the defect in strain BG-31 to the membrane portion of the complex. Analysis of membranes of the strain BG-31 by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrate the absence of a single polypeptide of molecular weight about 54,000 and the appearance of a new polypeptide of lower molecular weight, about 25,000. Analysis of a spontaneous revertant of BG-31 shows complete restoration of the parental phenotype including the gel patterns. The characterization of this mutant provides the first demonstration of the consequences of a structural gene mutation on a polypeptide in the membrane portion of the complex and represents the initial stages in what we hope will be the biochemical definition and functional characterization of this important energy-transducing system.     

B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes

Fractions of plasma membranes, Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope were isolated from rat liver and were characterized by electron microsocpe and biochemical methods. The purity of the fractions was controlled by morphometry and by marker enzyme activities. Amounts of cytochromes b5, P-450, and P-420 were measured, as well as the NADPH- and NADPH-cytochrome c reductase activities. The pigments of the microsomal electron transport system were found in all membrane fractions in relatively high amounts, thus excluding an origin by microsomal contamination. Purified preparations of plasma membrane and Golgi apparatus contained approximately 30% of the cytochrome b5 and cytochrome P-450 + P-420 found in ER membranes. Plasma membranes were also characterized by a high ratio of P-420/450. Degradation of cytochromes P-450 and P-420 was relatively rapid in all fractions, except in the ER. Cytochrome b5 extracted from plasma membranes was spectrophotometrically and enzymatically indistinguishable from ER cytochrome b5. However, immunnlogical characterization with rabbit antibodies against the trypsin-resistant core of microsomal cytochrome b5 showed the presence of at least two types of cytochrome b5 in ER membranes, in contrast to the plasma membranes in which only one of these components was detected. This immunological differentiation also demonstrates that the plasma membrane-bound cytochrome b5 is endogenous to this membrane and does not reflect contamination by ER elements. We conclude that cytochromes b5, P-450, and P-420 are not confined only to ER and nuclear membranes but also occur in signficant amounts in Golgi apparatus and plasma membranes. The findings are discussed in relation to observations of similar redox components in Golgi apparatus, secretory vesicles, and plasma membranes of other cells.     

Analysis of hexose transport in untransformed and sarcoma virus-transformed mouse 3T3 cells by photoaffinity binding of cytochalasin B

The effect of simian virus 40 transformation on the hexose transport system in mouse embryo fibroblast Swiss 3T3 cells was examined. The concentration of hexose transporters was estimated by measuring D-glucose-inhibitable cytochalasin B binding. The binding of cytochalasin B to the plasma membranes of simian virus 40-transformed mouse 3T3 cells (SV3T3 cells) was significantly greater than that of 3T3 cells. On the other hand, cytochalasin B binding to the microsomal membranes of SV3T3 cells was decreased, and the total amount of binding to plasma and microsomal membranes was not significantly changed in both cell lines. The electrophoretic analysis demonstrated that both hexose-transporter components of Mr 46 000 and Mr 58 000 affinity labeled were responsible for an increase in the hexose transport by viral transformation. These results suggested that the higher hexose-transport activity of transformed cells is caused by a redistribution of transporter from intracellular membranes to plasma membranes.     

Bile acid binding proteins in hepatocellular membranes of newborn and adult rats. Identification of transport proteins with azidobenzamidotauro[14C]cholate ([14C]ABATC)

Neonatal hepatocytes are less active in uptake of bile acids than are mature hepatocytes. This phenomenon has been further investigated by transport studies with azidobenzamidotaurocholate (ABATC). Taurocholate, cholate and the photolabile ABATC were taken up by liver cells of adult rats by a sodium-dependent and by an additional sodium-independent mechanism. In the dark, ABATC inhibited the uptake of taurocholate and cholate. Taurocholate decreased the transport of ABATC in a competitive manner, both in the presence and absence of sodium. In neonatal hepatocytes the Vmax for taurocholate and for ABATC was similar but was lower than in mature liver cells. In contrast, the Km was similar for neonatal and mature hepatocytes. For identification of binding proteins in both kinds of cells ABATC was photolysed after preincubation with isolated hepatocytes. Under our experimental conditions (single ultraviolet flash) about 80% of the azido groups was converted to nitrene. The covalently binding nitrene derivative inhibited bile salt transport irreversibly. Photolabeling of intact hepatocytes or of isolated plasma membranes with ABATC resulted in radioindication of membrane proteins with 67, 60, 54, 50 and 43 kDa in mature plasma membranes but of proteins with masses of 67, 54, 43 and 37 kDa in neonatal basolateral membranes. The 50 kDa protein in largely lacking in membranes of 9-day-old rats. The process of photolabeling itself was sodium-independent when isolated cells were treated with ABATC. In contrast, the degree of labeling of intact hepatocytes was markedly reduced in the absence of sodium and chloride. 100-fold molar excess of taurocholate, benzamidotaurocholate (BATC), phalloidin or cyclosomatostatin protected isolated plasma membranes against coupling of ABATC. Photolabeling of hepatoma cells known to be deficient in bile salt transport did not result in radiomodification of membrane proteins.     

NADH diferric transferrin reductase in liver plasma membrane

Evidence is presented that rat liver plasma membranes contain a distinct NADH diferric transferrin reductase. Three different assay procedures for demonstration of the activity are described. The enzyme activity is highest in isolated plasma membrane, and activity in other internal membranes is one-eighth or less than in plasma membrane. The activity is inhibited by apotransferrin and antitransferrin antibodies. Trypsin treatment of the membranes leads to rapid loss of the transferrin reductase activity as compared with NADH ferricyanide reductase activity. Erythrocyte plasma membranes, which lack transferrin receptors, show no diferric transferrin reductase activity, although NADH ferricyanide reductase is present. The transferrin reductase is inhibited by agents that inhibit diferric transferrin reduction by intact cells and is activated by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate) detergent. Inhibitors of mitochondrial electron transport have no effect on the activity. We propose that the NADH diferric transferrin reductase in plasma membranes measures the activity of the enzyme that causes the reduction of diferric transferrin by intact cells. This transmembrane electron transport system requires the transferrin receptor for diferric transferrin reduction. Because the transmembrane electron transport has been shown to stimulate cell growth, the reduction of diferric transferrin at the cell surface may be an important function for diferric transferrin in stimulation of cell growth, in addition to its role in iron transport.     

Retinoic acid inhibition of transplasmalemma diferric transferrin reductase

All trans retinoic acid inhibited diferric transferrin reduction by HeLa cells. The NADH diferric transferrin reductase activity of isolated liver plasma membranes was also inhibited by retinoic acid. Retinol and retinyl acetate had very little effect. Transplasma membrane ferricyanide reduction by HeLa cells and NADH ferricyanide reductase of liver plasma membrane was also inhibited by retinoic acid, therefore the inhibition was in the electron transport system and not at the transferrin receptor. Since the transmembrane electron transport has been shown to stimulate cell growth, the growth inhibition by retinoic acid thus may be based on inhibition of the NADH diferric transferrin reductase.     

Insulin-mediated translocation of glucose transporters from intracellular membranes to plasma membranes: sole mechanism of stimulation of glucose transport in L6 muscle cells

Plasma membranes and light microsomes were isolated from fused L6 muscle cells. Pre-treatment of cells with insulin did not affect marker enzyme or protein distribution in isolated membranes. The number of glucose transporters in the isolated membranes was calculated from the D-glucose-protectable binding of [3H]cytochalasin B. Glucose transporter number was higher in plasma membranes and lower in intracellular membranes derived from insulin-treated cells than in the corresponding fractions from untreated cells. The net increase in glucose transporters in plasma membranes was identical to the net decrease in glucose transporters in light microsomes (2 pmol/1.23 x 10(8) cells). The fold increase in glucose transporter number/mg protein in plasma membranes (2-fold) was similar to the fold increase in glucose transport caused by insulin. This suggests that recruitment of glucose transporters from intracellular membranes to the plasma membrane is the major mechanism of stimulation of hexose transport in L6 muscle cells. This is the first report of isolation of the two insulin-sensitive membrane elements from a cell line, and the results indicate that, in contrast to rat adipocytes, there is not change in the intrinsic activity of the transporters in response to insulin.     

Properties of a polarized primary culture from rat renal inner medullary collecting duct (IMCD) cells

Summary A primary culture from rat renal IMCD cells was established to investigate the permeability characteristics of the luminal and contraluminal plasma membranes of the papillary collecting duct in vitro. Freshly isolated IMCD cells were grown on filters in a special “epithelial cell” medium. Confluency was proved with an epithelial volt/ohm meter. After 7 d of culture the transepithelial resistance reached more than 1000 Ω×cm². A polarization of the cells with regard to a basolateral localization of a lactate efflux system, and an l-alanine transport system was achieved. The hypotonicity-activated release systems for the organic osmolytes sorbitol and betaine were also located basolaterally, whereas taurine, glycerophosphorylcholine, and myo-inositol left the cells at both cell poles but with different capacity. Morphological observations revealed also that the monolayer was well differentiated. Thus, a model of a renal collecting duct epithelium was established which can be used to analyze polarized and differentiated transport processes across the epithelial cells and their plasma membranes.     

Membrane proteins and phospholipids as effectors of reverse cholesterol transport

The review highlights the membrane aspect of cholesterol efflux from cell membranes to high density lipoproteins (HDL), an initial stage of reverse cholesterol transport to liver. In addition to traditional viewpoints considering cholesterol transport as the step of sequential lipoprotein transformation, which involves blood plasma apoproteins and proteins transporters, employment of proteomic approaches has shown the active role of cell plasma membranes as cholesterol donors and plasma membrane bound proteins in cholesterol transport. These include ATP-binding ABC-A1 transporter and membrane receptor SR-B1. There is experimental and clinical evidence that impairment of genes encoding these proteins cause impairments of reverse cholesterol transport (e.g. Tangier disease and genetic manipulations with experimental animals.) Although precise mechanism involving these membrane proteins remains unknown it is suggested that ABC-AI with free plasma apoA1 facilitates the efflux of membrane phospholipids and formation of their complex with apoAI. This complex accepts membrane cholesterol, with simultaneous formation of a full HDL particle. In certain cells there is correlation between cholesterol efflux into HDL and expression of SR-BI, which reversibly binds to HDL. This receptor protein may influence molecular organization of membrane phospholipids and cholesterol, facilitating cholesterol efflux. The review also deals with properties of ABC-A1 and SR-B1, putative mechanisms of their effects, the role of these proteins in reverse cholesterol transport and their functional coupling to the phospholipid matrix of biomembranes.     

Cell culturing in a three-dimensional matrix affects the localization and properties of plasma membrane cholesterol

Most in vitro studies use 2-dimensional (2D) monolayer cultures, where cells are forced to adjust to unnatural substrates that differ significantly from the natural 3-dimensional (3D) extracellular matrix that surrounds cells in living organisms. Our analysis demonstrates significant differences in the cholesterol and sphingomyelin content, structural organization and cholesterol susceptibility to oxidation of plasma membranes isolated from cells cultured in 3D cultures compared with conventional 2D cultures. Differences occurred in the asymmetry of cholesterol molecules and the physico-chemical properties of the 2 separate leaflets of plasma membranes in 2D and 3D cultured fibroblasts. Transmembrane distribution of other membrane phospholipids was not different, implying that the cholesterol asymmetry could not be attributed to alterations in the scramblase transport system. Differences were also established in the chemical activity of cholesterol, assessed by its susceptibility to cholesterol oxidase in conventional and “matrix” cell cultures. The influence of plasma membrane sphingomyelin and phospholipid content on cholesterol susceptibility to oxidation in 2D and 3D cells was investigated with exogenous sphingomyelinase (SMase) and phospholipase C (PLC) treatment. Sphingomyelin was more effective than membrane phospholipids in protecting cholesterol from oxidation. We presume that the higher cholesterol/sphingomyelin molar ratio is the reason for the higher rate of cholesterol oxidation in plasma membranes of 3D cells.     

Plasma membrane of a murine T cell lymphoma: surface labelling, membrane isolation, separation of membrane proteins and distribution of surface label amongst these proteins.

Two established techniques for analysis of plasma membranes, namely, lactoperoxidase catalyzed surface radioiodination of intact cells and bulk membrane isolation following disruption of cells by shear forces, were applied in studies of membrane proteins of continuously cultured cells of the monoclonal T lymphoma line WEHI-22. It was found that macromolecular 125I-iodide incorporated into plasma membrane proteins of intact cells was at least as good a marker for the plasma as was the commonly used enzyme 5'-nucleotidase. T lymphoma plasma membrane proteins were complex when analysed by polyacrylamide gel electrophoresis in sodium dodecylsulphate-containing buffers and more than thirty distinct components were resolved. More than fifteen of the components observed on a mass basis were also labelled with 125I-iodide. Certain bands, however, exhibited a degree of label disproportionate to their staining properties with Coomassie Blue. This was interpreted in terms of their accessibility to the solvent in the intact cells.     

Isolation and characterization of plasma membranes from strains of Neurospora crassa with wild type morphology

A variety of commercially available cell wall hydrolytic enzyme preparations were screened alone and in various combinations for their ability to degrade the cell wall of Neurospora crassa wild type strain 1A. A combination was found which causes complete conversion of the normally filamentous germinated conidia to spherical structures in about 1.5 h. Examination of these spheroplasts by scanning electron microscopy indicated that, although they are spherical, they retain a smooth coat that can only be removed upon prolonged incubation in the enzyme mixture (about 10 h). The 10-h incubation in the enzyme mixture appears to have no obvious detrimental effects on the integrity of the plasma membrane since the activity and regulatory properties of the glucose active transport system in 10-h spheroplasts are essentially unimpaired. Importantly, plasma membranes can be isolated from the 10-h spheroplasts by an adaptation of the concanavalin A method developed previously in this laboratory for cells of the cell wall-less sl strain, which is not the case for the 1.5-h spheroplasts. The yield of plasma membrane vesicles isolated by this procedure is 18-36% as indicated by surface labeling with diazotized [125I]iodosulfanilic acid, and the preparation is less than 1% contaminated with mitochondrial protein. The chemical composition of the wild type plasma membranes is similar to that previously reported for membranes of the sl strain of Neurospora. The isolated wild type plasma membrane vesicles also exhibit all of the functional properties that have previously been demonstrated for the sl plasma membrane vesicles. The wild type vesicles catalyze MgATP-dependent electrogenic proton translocation as indicated by the concentrative uptake of [14C]SCN- and [14C]imidazole under the appropriate conditions, which indicates that they contain the plasma membrane H+-ATPase previously shown to exist in the sl plasma membranes and that they possess permeability barrier function as well. The vesicles also contain a Ca2+/H+ antiporter as evidenced by their ability to catalyze protonophore-inhibited MgATP-dependent 45Ca2+ accumulation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of the isolated vesicles indicate that the protein composition of the wild type vesicles is roughly similar to that of the sl plasma membranes with the H+-ATPase present as a major band of Mr approximately 105,000. The wild type plasma membrane ATPase forms a phosphorylated intermediate similar to that of the sl ATPase, and the specific activity of the H+-ATPase in both wild type and sl membranes is approximately 3 mumol of Pi released/mg of protein/min.(ABSTRACT TRUNCATED AT 400 WORDS)     

An Analysis of γ-Aminobutyrate Receptors and Uptake by Isolated Purkinje Cells

Abstract: An isolated fraction of Purkinje perikarya was prepared. This fraction had [³H]GABA receptor binding and [³H]muscimol binding analogous to that reported for heterogeneous cerebellar membranes. The finding of two binding components with each ligand suggests that these components do not represent two binding sites, one presynaptic and the other postsynaptic, since both are clearly seen on purified Purkinje cell bodies. Subcellular fractionation indicates that synaptic endings and plasma membranes are enriched in GABA receptors compared with intracellular organelles. Purkinje cell bodies were also found to possess a high-affinity transport system for GABA, which was sensitive to inhibition by diaminobutyric acid (DABA) but not by β-alanine. They showed no evidence of homoexchange (the movement of label without net transport). This supports our suggestion that homoexchange is an artifact of synaptic particle formation.     

Insulin-stimulated glucose transport in rat adipose cells. Modulation of transporter intrinsic activity by isoproterenol and adenosine

The mechanism of modulation of insulin-stimulated glucose transport activity in isolated rat adipose cells by lipolytic and antilipolytic agents has been examined. We have measured glucose transport activity in intact cells with 3-O-methylglucose and in plasma membranes with D-glucose, and the concentration of glucose transporters in plasma membranes using a cytochalasin B binding assay. In intact cells, isoproterenol reduced insulin-stimulated transport activity by 60%. This effect was lost after cooling and washing the cells with homogenization buffer, and neither the concentration of glucose transporters nor transport activity in the plasma membranes differed from control. However, treatment of cells with KCN prior to homogenization preserved the isoproterenol effect through the fractionation procedure. Plasma membranes from these cells contained an unchanged number of transporters (31 +/- 7, mean +/- S.E., versus 31 +/- 4 pmol/mg of protein in controls) but transported glucose at a reduced rate (19 +/- 6 versus 48 +/- 9 pmol/mg of protein/s). Conversely, incubation of intact cells in the presence of adenosine stimulated plasma membrane glucose transport activity compared to that in the absence of adenosine (44 +/- 6 versus 36 +/- 6 pmol/mg of protein/s). Kinetic studies of isoproterenol-inhibited glucose transport in plasma membranes revealed a 60% decrease in Vmax (2900 +/- 350 versus 7200 +/- 1000 pmol/mg of protein/s) and a small increase in Km (15.1 +/- 1 versus 13.0 +/- 0.6 mM). These data indicate that modifications of glucose transport activity produced by lipolytic and antilipolytic agents in intact adipose cells can be fully retained in plasma membranes isolated under appropriate conditions. Furthermore, the effects of these agents occur through a modification of the glucose transporter intrinsic activity.