Total Membrane Fraction Research Articles

Identification of GTP‐ binding proteins in myelin and oligodendrocyte membranes

Myelin membranes purified from mouse and rat brain are associated with alpha subunits of four signal transducing guanosine triphosphate (GTP)-binding proteins: Go, Gi, Gs, and ras. Four low-molecular-weight (Mr) GTP-binding proteins are also present, as demonstrated by the binding of GTP to proteins immobilized in nitrocellulose. This latter group is more prominent at early stages of myelination and remains associated with isolated myelin membranes despite repetitive cycles of purification. At least one nonmyelin subcellular membrane fraction possesses the same proteins. The total membrane fraction of cultured oligodendrocytes is associated with both groups of GTP-binding proteins. None of the well-known myelin proteins bound GTP by the procedure described.

Sterol effects on phospholipid biosynthesis in the yeast strain GL7.

Cells of the yeast sterol auxotroph GL7 were grown on either ergosterol or cholesterol to mid-logarithmic phase and total membrane fractions prepared. Activities of phospholipid biosynthetic enzymes in the two cell types were determined. The rates of phosphatidyl-ethanolamine-phosphatidyl-choline-N-methyl transferase and acyl-CoA-alpha-glycerol-3-phosphate transcylase were significantly greater in ergosterol-grown than in cholesterol-grown cells. These reactions were also inhibited by the polyene antibiotic filipin. By contrast the activities of long-chain fatty acyl-CoA synthetase, CTP-phosphatidate-cytidyl transferase, phosphatidylserine decarboxylase and of phosphatidylinositol synthetase were identical in the two (ergosterol and cholesterol) cultures and unaffected by filipin. The ergosterol effect on phosphatidyl-ethanolamine N-methyl transferase was greatest in cells harvested in early log phase, intermediate in the mid-log phase cells, and not significant in stationary phase cells.

Dexamethasone regulates the glucose transport system in primary cultured adipocytes: different mechanisms of insulin resistance after acute and chronic exposure.

We have studied the ability of dexamethasone to regulate the glucose transport system in primary cultured adipocytes and delineated the mechanisms of insulin resistance after both acute and chronic treatment. Acutely, 20 nM dexamethasone led to a 65% decrease in basal and a 31% decrement in maximally insulin-stimulated glucose transport (ED50 = 3-4 nM; t1/2 = 50 min). These effects were maximal by 90-120 min, and a plateau was maintained over an additional 1-1.5 h. Chronic dexamethasone exposure (24 h) led to a more profound decrease in basal (77%; ED50 = 0.4 nM) and maximally stimulated (55%; ED50 = 1.0 nM) rates of glucose transport and shifted the transport: insulin dose-response curve to the right by increasing the half-maximally effective insulin concentration from 0.2 to 0.4 ng/ml. Dexamethasone did not affect cell surface insulin binding over 24 h. Both the short and long term effects of dexamethasone were partially blocked by the combined presence of insulin during preincubation and were not modulated by glucose. We also assessed effects on the number and cellular distribution of glucose transporter proteins using the cytochalasin-B binding assay. After 2 h, dexamethasone (30 nM) decreased the number of glucose transporters in plasma membranes by 30% in basal cells and by 41% in maximally insulin-stimulated cells, while increasing the number of low density microsomal transporters by 22-23% (P = NS). Transporter number in a total cellular membrane fraction was unaltered by short term dexamethasone. Chronic dexamethasone exposure (24 h) decreased plasma membrane and low density microsomal transporters by 30-50% in both basal and insulin-stimulated cells and depleted transporters by 43% in a total cellular membrane fraction. In conclusion, 1) dexamethasone induces progressive insulin resistance by sequentially regulating multiple aspects of the insulin-responsive glucose transport system. At early times (2 h) dexamethasone impairs insulin's ability to translocate intracellular glucose transporters to the cell surface and with more chronic exposure (24 h), depletes the total number of cellular transporters. 2) Glucose modulates desensitization of the glucose transport system by insulin, but not by dexamethasone, and thus, there are both glucose-dependent and -independent mechanisms of insulin resistance. 3) Insulin can heterologously inhibit dexamethasone's effects on glucose transport at both early and late phases of desensitization. These studies highlight the complex hormonal regulation at the glucose transport system.

Characterization of phospholipase C-mediated polyphosphoinositide hydrolysis in rat heart ventricles

Phospholipase C (PLC)-mediated degradation of polyphosphoinositides (phosphatidylinositol 4,5-bisphosphate (PIP 2) and phosphatidylinositol 4-phosphate (PIP)) was found to be present in rat heart ventricular soluble and total membrane fractions (100,000 g supernatant and pellet). Distribution of polyphosphoinositide-specific phospholipase C activity between the membrane and soluble fraction was approximately 63 and 33% of total activity, respectively, whereas, phosphatidylinositol (PI) degradation could be detected only in the soluble fraction. Optimal PIP 2-PLC activity occurred at a pCa 2+ of 4.5. A similar peak in PIP-PLC activity could be demonstrated in soluble and membrane preparations; however, the rate of PIP degradation in the soluble fraction continued to increase at the highest calcium level tested ( pCa 2+ 3). With the exception of Sr 2+, other noncalcium polycations did not support homogenate PIP 2-PLC activity. In the presence of Ca 2+, addition of Mg 2+, La 3+, or Sr 2+ (10 −3 m) inhibited PIP 2-PLC while Mn 2+ and Gd 3+ stimulated activity. In both the total membrane and soluble fractions, maximal polyphosphoinositide degradation occurs at pH 5.5 and 6.8. The detergents deoxycholate, cholate, and saponin exert a biphasic effect on PIP 2-PLC activity (stimulating at lower concentrations and inhibiting at higher concentrations). The deoxycholate effect is observed in both the cytosolic and membrane fractions. Neutral and cationic detergents inhibit PIP 2-PLC activity in a concentration-dependent manner. Similar to cytosolic PI-PLC activity, PIP 2-PLC appears to depend on intact sulfhydryl groups. In the presence of a mixture of all three inositol phospholipids or the three phosphoinositides plus noninositol phospholipids, polyphosphoinositides are preferentially degraded.

In Vitro Effects of Sulfonylurea on Glucose Transport and Translocation of Glucose Transporters in Adipocytes From Streptozocin-Induced Diabetic Rats

The in vitro effects of the sulfonylurea glyburide on insulin binding and action were compared in adipocytes from control and nonketotic streptozocin-induced diabetic rats. Adipose tissue from control and diabetic animals was maintained in the absence or presence of 2 micrograms/ml glyburide for 20 h. Insulin binding and insulin-stimulated glucose transport were examined in adipocytes prepared from this tissue. As expected, insulin binding was increased in adipocytes from diabetic animals. Exposure of tissue to glyburide did not influence insulin binding in either control or diabetic cells. Glucose transport activity of diabetic cells, assessed with 2-deoxyglucose, was decreased 30-40% in both the absence (basal) and presence of insulin compared with controls. Glyburide potentiated insulin's effects in both control (15-20%) and diabetic (30-40%) adipocytes. As a result, glucose transport activity in glyburide-treated diabetic cells was restored to a level similar to that of control cells not exposed to the drug. The mechanism by which glyburide potentiated glucose transport activity was examined with the D-glucose-displaceable cytochalasin B-binding technique to measure glucose-transporter concentration in membranes prepared from control and diabetic adipocytes exposed to the drug. Adipocytes from this model of diabetes are known to have a decreased cellular content of glucose transporters. The concentration of glucose transporters was decreased by 31% in plasma membranes from insulin-treated diabetic cells. There were corresponding decreases in diabetic microsomal and total membrane fractions. There was also a 40% decrease in the translocation of transporters from the microsomes to the plasma membrane in response to insulin in diabetic cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity and regulation of calcium-, phospholipid-dependent protein kinase in differentiating chick myogenic cells.

The activity of calcium-, phospholipid-dependent protein kinase (PKc) was measured in (a) total extracts, (b) crude membrane, and (c) cytosolic fractions of chick embryo myogenic cells differentiating in culture. Total PKc activity slowly declines during the course of terminal myogenesis in contrast to the activity of cAMP-dependent protein kinase, which was also measured in the same cells. Myogenic cells at day 1 of culture possess high particulate and low soluble PKc activity. A dramatic decline of particulate PKc activity occurs during myogenic cell differentiation and is accompanied, through day 4, by a striking rise of the soluble activity. The difference in the subcellular distribution of PKc between replicating myoblasts and myotubes is confirmed by phosphorylation studies conducted in intact cells. These studies demonstrate that four polypeptides whose phosphorylation is stimulated by the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate in myotubes, are spontaneously phosphorylated in control myoblasts. Phosphoinositide turnover under basal conditions in [3H]inositol-labeled cells is faster in myoblasts than in myotubes, a finding that may in part explain the different distribution of PKc observed during the course of myogenic differentiation.

Cyclic 3',5'-adenosine monophosphate-dependent kinase and phosphoproteins in human amnion.

3',5'-Cyclic adenosine monophosphate (cAMP) modulates prostaglandin production in human amnion membranes. The major effects of cAMP are presumably mediated through the phosphorylation of specific regulatory phosphoproteins following cAMP activation of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase and phosphoproteins have not previously been characterized in human amnion. Total homogenates, cytosol, and membrane fractions from human amnion were examined for [3H]cAMP binding activity and cAMP-dependent kinase activity. cAMP-dependent kinase activity was barely detectable in crude amnion fractions. Cytosol was therefore partially purified by DEAE column chromatography for further examination. Two peaks of coincident [3H]cAMP binding and cAMP-dependent kinase activity were demonstrated at 70 and 140 mM NaCl, characteristic of the Type I and Type II cAMP-dependent protein kinase isozymes. [3H]cAMP binding to the material from both peak fractions was saturable and reversible. Scatchard analysis of [3H]cAMP binding to the peak fractions was linear for peak I and curvilinear for peak II. Assuming a one-site model, [3H]cAMP binding to the Type I isozyme showed a KD = 4.17 x 10(-8) M and Bmax = 73 pmole/mg protein; using a two-site model, [3H]cAMP binding to the high-affinity site for the Type II isozyme had a KD = 3.94 x 10(-8) M and Bmax = 6.3 pmole/mg protein. Other cyclic nucleotides competed for these [3H]cAMP binding sites with a potency order of cAMP much greater than cGMP greater than (BU)2cAMP.cAMP caused a dose-dependent increase in cAMP-dependent kinase activity in the peak fractions; half-maximal activation was observed with 5.0 x 10(-8) M cAMP. The ability of cAMP to increase phosphorylation of endogenous proteins in both crude amnion cytosol and cytosol from cultures of amnion epithelial cells was assessed using [32P]ATP, SDS-polyacrylamide gel electrophoresis and autoradiography. cAMP stimulated 32P incorporation into three proteins having Mr = 80,000, 54,000, and 43,000 (P less than .01). Half-maximal 32P incorporation into these proteins occurred at 1.0 x 10(-7) M cAMP. cAMP-dependent kinase is present in human amnion; specific cAMP-enhanced phosphoproteins are also present. Hormones elevating cAMP levels in amnion may exert their effects by activating cAMP-dependent kinase and phosphorylating these phosphoproteins.

Pertussis toxin stimulates delayed-onset, Ca 2+-dependent catecholamine release and the ADP-ribosylation of a 40 kDa protein in bovine adrenal chromaffin cells

Pertussis toxin was found to stimulate catecholamine release from bovine adrenal chromaffin cells in a Ca 2+ -dependent manner and in the absence of any stimulatory or inhibitory agonists for this cell. The release of catecholamine was associated with the ADP-ribosylation of an approx. 40 kDa protein present in the total membrane fraction. These results are consistent with the existence of an exocytosis-linked G-protein.

Effects of radioligand oxidation and ascorbate-induced lipid peroxidation on serotonin-1 receptor assay: use of ascorbate and ethylenediamine tetraacetic acid buffers to prevent (3H)-5-HT binding artifacts.

The effects of ascorbic acid and ascorbate-induced lipid peroxidation upon (3H)-5-HT binding were examined in total membrane fractions prepared from mouse cortex and hippocampus. Low concentrations of ascorbic acid promoted lipid peroxidation of membranes as assessed by malondialdehyde production relative to intermediate concentrations of ascorbate. The actual concentration of ascorbate required for expression of pro-oxidative and antioxidative properties was dependent upon assay conditions such as temperature and ionic constituents. Ascorbate-induced lipid peroxidation was completely prevented by addition of 1 mM ethylenediamine tetraacetic acid (EDTA) and enhanced by GTP at concentrations typically used in binding assays. Marked lipid peroxidation was associated with loss of (3H)-5-HT binding sites with little effect upon affinity of the receptor for the ligand. In contrast, mild lipid peroxidation occurring during the binding assay with the ligand present was associated with a decreased affinity for (3H)-5-HT and the appearance of a curvilinear Scatchard plot. Under assay conditions that prevented ascorbate-induced lipid peroxidation, Scatchard analysis indicated only a single high-affinity binding site for (3H)-5-HT even when assayed with an expanded range of ligand concentrations. Preparation of ligand and assay of (3H)-5-HT binding in the absence of ascorbate resulted in shallow, markedly curvilinear Scatchard plots. These data support the continued use of ascorbate in (3H)-5-HT binding assays to prevent ligand degradation and the addition of 1 mM EDTA to prevent lipid peroxidation from occurring during the binding assay.

Insulin-like growth factor I (IGF-I) receptors on Sertoli cells from immature rats and age-dependent testicular binding of IGF-I and insulin

Insulin-like growth factor I (IGF-I) binding to cultured Sertoli cells from immature rats was quantitatively evaluated. The binding of 125I-IGF-I to the Sertoli cells was specific, time- and pH-dependent and reversible. Scatchard analysis yielded a K d of 3.5 × 10 −9 M and a binding capacity of 2080 fmol/mg protein. Competition with IGF-I resulted in a half-maximal displacement by 2 nM IGF-I, whereas insulin up to a concentration of 100 nM gave virtually no displacement of IGF-I binding. Similarly, the gonadotropic hormones follitropin and lutropin did not compete with 125I-IGF-I binding. In previous studies, it was shown that cultured Sertoli cells from immature rats bind insulin with a K d of 1.8 × 10 −9 M and a binding capacity of 8.5 fmol/mg protein. The binding of IGF-I and insulin to a total testis membrane fraction was studied using testes from immature and adult rats. In testis from 21-day-old rats, the maximal specific binding was relatively high for IGF-I (871 ± 50 fmol/g wet weight) and relatively low for insulin (118 ± 11 fmol/g wet weight). In adult testis, the maximal specific binding of IGF-I was 324 ± 40 fmol/g wet weight and that of insulin was 330 ± 17 fmol/g wet weight. The binding of IGF-I and insulin expressed as fmol bound per testis was increased 6-fold and 45-fold, respectively, between the age of 21 days and adult age. It is discussed that the numbers of receptors for IGF-I and insulin in testis may be developmentally regulated, and that IGF-I may be more important than insulin with respect to testis development and Sertoli cell maturation in the immature rat.

Heat shock response ofChlorella protothecoides during greening

Heterotrophically grown cells ofChlorella protothecoides were transferred to autotrophic medium and allowed to green at 25°C. The protein synthetic activity of the greening cells measured in terms of incorporation of [35S]-methionine showed a maximum around 20 h of greening and thereafter started declining. Similarly, an analysis of densitometric tracings of the fluorographic profile of the polypeptides associated with both total cellular fraction and membrane fractions during different hours of greening revealed that maximum number of polypeptides were getting labelled around 20 h of greening. At 20 h of greening, the cells were shifted to 40°C and the effect of heat shock on protein synthesis was studied. The heat shock treatment caused a definite decrease in the incorporation of [35S]-methionine into proteins. Due to heat shock, the synthesis of total soluble proteins was affected much more than that of the thylakoid membrane bound proteins. When the cells were transferred back to 25°C after a brief period of heat shock at 40°C, there was a considerable recovery in the protein synthesis and this recovery was found to be significant in the case of soluble proteins, while there was no such definite recovery in the synthesis of thylakoid membrane bound proteins.

Isolation and Characterization of the Outer Membrane Proteins of Azospirillum brasilense

SUMMARY: The outer membrane of Azospirillum brasilense was isolated from the total membrane fraction by sucrose density gradient centrifugation and by Sarkosyl extraction; both preparations showed an identical outer membrane protein profile in slab gels after electrophoresis under denaturing conditions. The profile showed a major 42 kDa protein constituting about 60% of the total outer membrane proteins. This major protein and a minor 40 kDa protein were tightly but non-covalently associated with peptidoglycan. In addition, a 23 kDa heat-modifiable protein, resistant to trypsin digestion, was detected in the outer membrane. Growth of A. brasilense under iron deficiency induced four additional high molecular mass proteins (87, 83, 78 and 72 kDa) in the outer membrane. A comparison of the outer membrane protein profiles of the three different Azospirillum species suggests that such profiles might be useful in taxonomic classification.

Measurement of proton pump activity of the thermophilic bacterium PS3 and Nitrobacter agilis at the cytochrome oxidase level using total membrane and heptyl thioglucoside.

It is possible to prepare liposomal vesicles by solubilization of total bacterial membranes with n-heptyl beta-D-thioglucoside followed by reconstitution into proteoliposomes by a freeze-thaw-sonication procedure with soybean phospholipids. The resulting proteoliposomes from total membrane fraction of sufficiently aerated cells of the thermophilic bacterium PS3 containing cytochrome aa3 showed a reasonable H+ pumping activity upon addition of reduced cytochrome c. On the other hand, the proteoliposomes reconstituted from air-limited PS3 cells containing cytochrome o and those from Nitrobacter agilis cells containing cytochrome aa3 did not show H+ pumping upon addition of reduced cytochrome c, although the vesicles showed "respiratory control"; 3-4-fold stimulation of oxygen consumption took place upon addition of an uncoupler. In proteoliposomes prepared from PS3 membranes by this method, H+-translocating ATPase (F0 X F1) was successfully reconstituted as well, suggesting that this method has wide applicability for investigation of enzymes catalyzing transmembrane processes.

Crystalline arrays of the Escherichia coli sn-glycerol-3-phosphate acyltransferase, an integral membrane protein.

The gene encoding the Escherichia coli sn-glycerol-3-phosphate acyltransferase, plsB, was inserted into hybrid plasmids under transcriptional control of the lambda PL and tac promoters. Enzymatic activities 35-50-fold above wild type and a large increase in glycerol-P acyltransferase polypeptide were obtained. Thin section electron microscopy of the cells overproducing the glycerol-P acyltransferase revealed 235-245-A diameter tubular structures associated with the cytoplasmic membrane. These structures were released from the cell by osmotic lysis and purified on Matrex Gel Green A. Subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the dominant protein constituent of the tubules was the glycerol-P acyltransferase. Analysis of tubule-enriched fractions isolated by differential centrifugation revealed a decreased phospholipid to protein ratio as compared to total and cytoplasmic membrane fractions. At high magnification, negative stained tubules displayed ordered arrays of stain-excluding components projecting 50-60 A from the cytoplasmic surface. Optical diffraction patterns from the micrographs contained intense layer lines at (1/78 A) and (1/39 A) along the tubule axis and a prominent spot at (1/62 A) near the equator. From compositional and structural data, 18-37% of the polypeptide volume is estimated to lie within the hydrophobic domain of the tubule membrane.

Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa.

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.

The transferrin receptor in hepatocyte nodules: binding properties, subcellular distribution and endocytosis.

Transferrin binding was found to be around 60-fold higher in hepatocyte nodules compared to normal liver, with no apparent differences in binding affinity or molecular weight of binding proteins, indicating that the increase in [125I]transferrin binding was the result of an increased number of binding sites with similar properties as in normal liver. The relative 'induction' of transferrin receptors was most marked in the total membrane fraction followed by membrane subfractions comprising endoplasmic reticulum, the Golgi complex and endocytic vesicles. Despite the increased number of transferrin receptors, the in vivo endocytosis of transferrin, measured as uptake of [125I]ferrotransferrin, was quantitatively similar in nodular cells compared to normal liver. The number of transferrin receptors in regenerating liver, 48 h after partial hepatectomy, was increased 20-fold over normal levels, but binding affinity, receptor structure and kinetics of transferrin uptake were normal. A slower than normal rate of 59Fe accumulation in hepatocyte nodules may suggest an alteration in the dissociation of iron from ferrotransferrin, thereby suggesting one mechanism relevant to the iron storage deficiency in nodular cells.

Effects of Peptidase Inhibitors on the (Met5)-Enkephalin Hydrolysis in Ileal and Striatal Preparations of Guinea-Pig: Almost Complete Protection of Degradation by the Combination of Amastatin, Captopril and Thiorphan

AbstractThe contents of [Met5]-enkephalin and its four hydrolysis products. Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Gly-Gly-Phe, in the sample obtained after enkephalin was incubated with tissues in either the absence or the presence of the peptidase inhibitor were estimated by high performance liquid chromatography combined with electrochemical detection in order to elucidate the effect of the peptidase inhibitor on enkephalin hydrolysis. Free Tyr was the major degradative product in the absence of the peptidase inhibitor, while the major hydrolysis product was the Tyr-Gly-Gly fragment in the presence of amastatin in both total homogenates and membrane fractions prepared from either the myenteric plexus-longitudinal muscle strip of guinea-pig ileum or the striatum of guinea-pig brain. Additionally. Captopril significantly decreased the generation of Tyr-Gly-Gly in the presence of amastatin in both ileal and striatal membrane fractions. Moreover, thiorphan significantly prevented the formation of Tyr-Gly-Gly in the presence of both amastatin and Captopril in both membrane preparations. Finally, when [Met6]-enkephalin was incubated with either an ileal or a striatal membrane fraction for 60 min at 37°C in the presence of three peptidase inhibitors, amastatin. Captopril, and thiorphan. approximately 98 or 94%, respectively, of enkephalin remained intact. The results indicated that [Met6]-enkephalin was almost exclusively hydrolyzed by three distinct enzymes, amastatin-sensitive ammopeplidase. Captopril-sensitive peptidyl dipeptidase A. and thiorphan-sensitive endopeptidase-24.11 in both ileal and striatal membrane fractions.

Association of endogenous substrate with solubilized bovine brain sialidase.

Nonidet P40 solubilized up to 90% of the sialidase, active towards added ganglioside substrate, that was associated with the total membrane fraction prepared from gray matter of bovine brains. Solubilized sialidase acted upon endogenous substrate (sialic acid containing compounds solubilized with the enzyme), hydrolyzing approximately 50% of the readily available sialosyl residues within 20 min. During a 2-hr reaction time 80% of the polysialylated gangliosides solubilized with the enzyme were acted upon. A 20-min lag was observed before sialidase acted upon added ganglioside substrate. The lag could be reduced to less than 2 min when the enzyme was allowed to act on endogenous substrate prior to exposure to exogenous substrate, suggesting that the solubilized enzyme acted preferentially on endogenous substrate. A protease inhibitor prevented much of the 86% loss of activity towards added substrate that was seen when the enzyme was stored at 4 degrees C for 6 days; activity towards endogenous substrate decreased only 34%.

Effect of glucocorticoids on hexose transport in rat adipocytes. Evidence for decreased transporters in the plasma membrane.

Glucocorticoids are known to rapidly inhibit glucose transport when added to isolated rat adipocytes. To determine whether this inhibition of transport persists following isolation of the plasma membranes, adipocytes were incubated in the absence or presence of a maximally inhibitory concentration of dexamethasone, a synthetic glucocorticoid, and plasma membrane vesicles were prepared. D-Glucose uptake into vesicles from steroid-treated cells was inhibited by an average of 40%. The ability of dexamethasone to inhibit transport depended upon pretreatment of cells with hormone prior to membrane isolation. Furthermore, the decreased rate of transport was prevented by the simultaneous addition to the cell of actinomycin D or cycloheximide with dexamethasone, indicating a requirement for RNA and protein synthesis. The effect of dexamethasone on glucose transport was further investigated using our recently developed cytochalasin B affinity-labeling protocol to identify the transporter on sodium dodecyl sulfate-polyacrylamide gels. A peak of radioactivity having Mr = 54,000 was identified which exhibited the properties expected for the glucose transporter, in that label incorporation was prevented by D-glucose and unlabeled cytochalasin B, but not by D-sorbitol or unlabeled cytochalasins A, D, or E. Dexamethasone was found to cause a significant (average 33%) decrease in the amount of labeled transporter in the plasma membrane which was prevented by the simultaneous addition of actinomycin D with dexamethasone to the cells. A similar percentage decrease was not found in a microsomal membrane fraction nor in a total cellular membrane fraction. These results suggest that glucocorticoids may decrease glucose transport in rat adipocytes by selectively decreasing the number of transporters in the plasma membrane.

Human lactase and the molecular basis of lactase persistence

Human lactase purified from detergent extracts of the total membrane fraction of postmortem jejunum by means of monoclonal immunoadsorbent chromatography appears to be a dimer of subunits identical in Mr (160K). Trypsin or papain removes a small hydrophobic anchoring peptide from each subunit to give a hydrophilic enzyme which no longer interacts with detergent micelles. Lactase hydrolyzes, besides lactose, cellobiose and the synthetic substrates, 4-methylumbelliferyl-beta-galactoside and beta-glucoside, as well as phlorizin; but it does not hydrolyze glucocerebroside. Phlorizin hydrolase is associated with lactase under all conditions investigated; coincident staining on immunodiffusion and immunoelectrophoresis, coincident elution on immunoadsorbent chromatography and on gel filtration in a dissociating buffer, and correlated reduction in activity in lactase-nonpersistent individuals. Adult and infant lactases are indistinguishable by titration or immunodiffusion against polyclonal rabbit antibodies. Adult individuals low in lactase activity also show a corresponding reduction in cross-reacting material. These observations suggest that lactase persistence is due to the continued synthesis of the infant enzyme.