Crude Plasma Membrane Research Articles

Modulation of Morphology and Plasma-Membrane Enzyme Activities of Rat Thymocytes Induced by Specific Antisera

Lewis rat thymocytes were incubated with different ligands: specific rat alloantisera, rabbit xenoantisera against whole-rat thymocytes or against thymocyte plasma-membrane vesicles and the two mitogens: concanavalin A and the Ca ++ ionophore A 23187. After treatment, a crude plasma-membrane fraction was prepared, and the activities of two plasma-membrane marker enzymes, alkaline phosphatase and y-glutamyl transferase, and a general membrane marker enzyme, lysolecithin acyltransferase, were determined. An increase of all marker enzyme activities was observed only when thymocytes had been incubated with alloantiserum directed against the gene products of their major histocompatibility complex (MHC) or with rabbit antiserum against syngeneic thymocytes. Anti-MHC alloantiserum against a nonrelevant haplotype increased moderately the y-glutamyltransferase activity. Alloantiserum directed against the weak histocompatibility antigens had no significant effect as had rabbit antiserum raised against thymocyte plasma-membrane vesicles. The mitogens concanavalin A and A 23187 both increased the activity of the alkaline phosphatase and lysolecithin acyltransferase. Scanning electron microscopy showed that treatment with alloantisera did not alter the cell shape drastically. In contrast, incubation with rabbit xenoantiserum against thymocytes resulted in cell rounding and deformation. Rabbit xenoantiserum against the plasma-membrane vesicles of thymocytes resulted in markedly disturbed or damaged plasma membranes.

Temperature dependence of [ 3H]dihydroalprenolol binding in rat myocardium

The β-adrenergic antagonist, [ 3H]dihydroalprenolol, was used to label binding sites in crude rat myocardial plasma membranes. The specificity of binding was dependent on the temperature of the assay. Specific binding at 22 and 37°C and at concentrations of radioligand less than 5 nM was consistent with binding to the myocardial β-receptor. Binding sites labeled at 4°C possessed quite different properties. Binding was non-stereoselective and of lower affinity. Agonist compounds were much less effective at competing for the labeled myocardial sites at 4°C than at 22°C. Those β-antagonists which additionally possess pharmacological ‘quinidine-like’ activity (e.g. propranolol, alprenolol) were potent competitors at 4°C, but competition was non-stereoselective. In contrast atenolol, a β-antagonist devoid of ‘quinidine-like’ activity was ineffective at 4°C. Furthermore, procaine, and quinidine itself were potent competitors of [ 3H]dihydroalprenolol binding at 4°C. Thus the specificity of [ 3H]dihydroalprenolol binding to rat heart membranes at 4°C appears to be directed non-stereoselectively at that portion of the competing molecule which recognized ‘quinidine-like’ as opposed to adrenergic activity.

A new in vitro assay for human thyroid stimulator using cultured thyroid cells: effect of sodium chloride on adenosine 3',5'-monophosphate increase.

A new sensitive in vitro assay for human thyroid stimulator (HTS) was developed using human thyroid adenoma cells in monolayer culture. After being cultured for 2 days, the cells were incubated in 0.3 ml Hank's solution without 0.8% NaCl (medium 1) and with thyroid stimulator (bovine TSH or 3 mg patient serum immunoglobulin G) at 37 C for 2 h. The cAMP generated in the cells and the medium during the incubation was measured by RIA. The assay was sensitive enough to elicit a 1.7- to 7.9-fold increase in cAMP at a TSH concentration of 10 microU/ml. HTS was detected in 33 (82.5%) of the 40 patients with untreated graves' disease using this assay system. In Hank's solution (medium 2), however, HTS was detected in only 5 (23.8%) of the 21 patients with untreated GRaves' disease. cAMP increment upon stimulation by either TSH or HTS in medium 1 was larger than that in medium 2, and the difference in the response to HTS using the two media was much greater than that in the response to TSH. Therefore, all HTS-immunoglobulin G studies showed higher activity using medium 1 than using medium 2 when expressed as bovine TSH equivalent. Analysis by the Lineweaver-Burk plot of dose-response curves of the effect of TSH and HTS stimulation on cAMP increment showed an increase in the Km upon the addition of NaCl to the medium. A similar inhibitory effect of NaCl (150 mM) was also observed in the assay system of human thyroid adenylate cyclase stimulator using crude plasma membrane fractions. In summary: 1) an assay for HTS measuring cAMP production in cultured thyroid adenoma cells was developed and the assay using low NaCL medium was found to be the most sensitive, and 2) the inhibitory effect of NaCl on the response to HTS was much greater than that on the response to TSH. These data suggest different behaviors of these two stimulators at their receptor sites.

Glucocorticoid-induced proteins in rat thymus cells.

The possibility that hormone-induced changes in the synthesis of individual proteins may serve as initiating events for the rapidly evolving glucocorticoid-induced metabolic suppressions in rat thymus cells was examined. Synthetic rates of about 2500 individual proteins were screened using giant two-dimensional gel electrophoresis. Dexamethasone induces large, rapid increases in the rates of synthesis of at least three and more subtle increases in another three proteins. Increases in the rates of synthesis of four of these six proteins occur within 15 to 45 min. The time course suggests that changes in the synthesis of some or all of these four proteins may serve to generate the rapidly evolving hormone-induced inhibition of glucose transport. More slowly emerging changes in two other proteins parallel the latter metabolic effects of decreased mitochondrial ATP production and increased nuclear fragility. Cordycepin, an inhibitor of new mRNA processing, prevents the dexamethasone-induced increases in protein labeling. Also, when used alone, cordycepin selectively slows the synthesis of those early proteins that can be induced by dexamethasone, suggesting that the mRNAs coding for the early induced proteins have unusually short half-lives. Short-lived mRNAs for such putative regulatory proteins may allow the cell to respond rapidly to changing needs. Preliminary experiments using giant gel separations of subcellular fractions indicate that two of the induced proteins co-purify with crude plasma and nuclear membrane fractions. None of the induced proteins was detected in gel separations of mitochondria.

Immunological and biochemical properties of transverse tubule membranes isolated from rabbit skeletal muscle.

A new method for isolating transverse tubule membranes from rabbit skeletal muscle has been developed. This procedure has the advantage of being mild, fast, and producing with good yields a purified membrane fraction. The transverse tubule membranes are purified by a discontinuous sucrose density centrifugation after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. Immunofluorescence staining of cryostat sections of rabbit psoas muscle with purified goat antibodies directed against the purified membranes shows that the reacting antigens are distributed at the boundary of the A and I bands of the myofibrils where transverse tubules are localized in mammalian muscle. The purified antibodies showed no cross-reactivity with sarcoplasmic reticulum, nor did they show any fluorescence staining of the muscle plasma membrane, indicating that the isolated membranes indeed originate from the transverse tubules. The transverse tubule fraction has a characteristic protein composition distinguishable from that of sarcoplasmic reticulum, a much higher cholesterol content than that of the crude microsomes, plasma membrane, and sarcoplasmic reticulum, and a phospholipid content about twice as high as that of sarcoplasmic reticulum and plasma membrane. The purified transverse tubule membrane has a distinct phospholipid composition with high contents of sphingomyelin and phosphatidylserine. A Mg2+-activated ATPase characteristic of the transverse tubule fraction undergoes a 20-30-fold increase in specific activity during purification. The levels of Ca2+-ATPase activity present in the purified transverse tubule fraction remain comparable to those of sarcoplasmic reticulum even after extensive removal of the latter.

Plasma membrane insulin receptors in fetal rabbit lung.

Summary: Previous studies have suggested that fetal hyperinsulinemia which occurs in offspring of diabetic mothers is responsible for diminished surfactant production and respiratory distress syndrome. Recognition of specific insulin effects on fetal lung tissue prompted us to characterize insulin receptors on plasma membranes of fetal rabbit lung tissue and to investigate the effects of maternal diabetes on such receptors. Six pairs of pregnant New Zealand rabbits were studied. One of each pair received alloxan (60 mg/kg) intravenously on day 14 of pregnancy, whereas the controls received saline. Animals were sacrificed on day 28 of gestation, and for each experiment, crude plasma membranes were prepared from maternal and pooled fetal lung tissue for 125I-insulin binding studies. Plasma glucose values were elevated for both maternal diabetic (246 ± 81 versus 98.5 ± 7.1 mg/dl; P < 0.001) and fetal diabetic offspring (160 ± 69 versus 55 ± 12 mg/dl; P < 0.02) in comparison to controls. Fetal diabetic offspring had plasma insulin values significantly higher than control fetuses (84.8 ± 25 versus 23.2 ± 3.7 μU/ml), (mean ± S.D.); P < 0.05). Insulin was undetectable in diabetic mothers.Lung membranes from fetuses of diabetic animals bound significantly more insulin than did those of control fetuses. Scatchard analysis yielded curvilinear plots of bound fractions versus total amount of insulin bound suggesting the presence of more than one class of receptors or negative cooperativity. Assuming two classes of receptors, one of high affinity and low capacity and another of low affinity and high capacity, we found that fetal membranes had a five-fold increase in binding capacity of high-affinity receptors as compared to adult membranes. In spite of marked hyperinsulinemia in the offspring of the diabetic animal, the fetal lung, far from experiencing a down-regulation of insulin receptor binding, showed increased insulin binding. This finding is consistent with observations made previously on circulating monocytes of infants of diabetic mothers.Speculation: Hyperinsulinemia occurring postnatally appears to lead to down-regulation of insulin binding. Although the mechanisms of down-regulation are not clearly understood, from a teleological standpoint the phenomenon may be viewed as partially protecting the organism from the effects of hyperinsdinism because less binding sites are available for insulin to exert its effects on target tissues. In the fetus, this protective mechanism does not appear to exist in at least two tissues examined thus far, circulating monocytes of the human and lung tissue of the rabbit. The fetus with hyperinsulinemia may therefore be subject to the double jeopardy of higher concentrations of insulin and higher binding of insulin to its target tissues.

Characterization of a membrane-associated receptor from bovine liver that binds phosphomannosyl residues of bovine testicular beta-galactosidase.

A receptor that binds the phosphomannosyl recognition marker of bovine testicular beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was isolated from bovine liver membranes. The receptor was extracted from crude plasma membrane preparations with Triton X-100 and immunoprecipitated as a receptor--beta-galactosidase complex with anti-beta-galactosidase. The receptor was dissociated from the precipitate with mannose 6-phosphate, labeled with 125I, and purified on a beta-galactosidase-Sepharose 4B affinity matrix. A quantitative binding assay employing anti-beta-galactosidase and IgGsorb (formalin-fixed Staphylococcus aureus) was devised to study the binding of 125I-labeled receptor to beta-galactosidase. Maximal binding of receptor to enzyme occurred at pH values between 5.7 and 6.5. Divalent cations were not required for binding. The values of the dissociation constant obtained for beta-galactosidase varied between 200 nM observed with "lower uptake" forms and 20 nM for "higher uptake" forms of the enzyme. A number of phosphorylated monosaccharides were tested as inhibitors of binding of enzyme to receptor; mannose 6-phosphate and fructose 1-phosphate served as inhibitors and exhibited Ki values of 0.064 mM and 0.24 mM, respectively. The receptor has a subunit molecular weight of 215,000. Similar receptors were also demonstrated in Triton X-100 extracts of human skin fibroblasts, Chinese hamster ovary cells, and rat hepatocytes. These cell types are known to assimilate lysosomal enzymes containing covalently bound mannose 6-phosphate residues.

Benzamil binding to kidney cell membranes

Tile binding of [ 3H]benzamil ( N-benzylamidino-3,5-diamino-6-chloropyrazine carboxamide), a blocker of passive sodium channels, was investigated in tissues from several species (rat, guinea-pig, rabbit, chicken, frog and two species of fish). Tissues used were kidney, colon epithelium, cardiac and skeletal muscle, brain, liver, epididymis and red cell membranes. Displaceable binding was detected principally in target tissues, and particularly in the kidney. In rat and rabbit kidney considerably more binding activity was present in the cortex than in the medulla. In rat kidney cortex, a saturable binding component (2 nmoles g −1 protein) with high affinity (approx. 5 × 10 7 M −1) was detected. Other agents which are known to interact with sodium channels competed with [ 3H]benzamil for the binding site. The affinity of amiloride detected in this way was 10 5 M −1. Neither adrenalectomy nor pretreatment with aldosterone affected the amount of benzamil binding activity. Sodium had no effect on either the affinity or the capacity of binding. The binding activity appeared not to be associated with the base carrier known to be present in the proximal convoluted tubule. Binding activity was increased in both crude plasma membrane fractions and in fractions enriched in brush border membranes. The possible physiological role of the diuretic receptor is discussed.

Interaction between thyrotropin (TSH) binding inhibitor immunoglobulins (TBII) and soluble TSH receptors in fat cells.

To clarify whether TSH binding inhibitor immunoglobulins (TBII) are antibodies to the membrane site associated with the TSH receptor itself or its neighboring sites, the interactions of TSH and TBII with soluble TSH receptor were investigated with a TSH radioreceptor assay using labeled highly purified bovine TSH (bTSH) and Triton extracts from guinea pig crude fat cell membranes (800-10,000 x g fraction). Treatment of the crude fat cell membranes with 0.5% (vol/vol) Triton X-100 resulted in solubilization of membrane proteins with recoveries of 25-30%, whereas increasing the concentration of Triton X-100 in the assay medium caused a decrease of [125I]bTSH binding to the solubilized membranes. Thus, the solubilized membranes were found to retain the capacity to bind [125I]bTSH below the Triton X-100 concentration of 0.4% in the assay medium. Incubation of the solubilized membranes with [125I]bTSH for 24 h at 4 C led to a steady state of specific binding, while incubation at 37 C resulted in more rapid but less specific binding, with a shorter duration of the steady state. Maximum binding occurred within the physiological pH range. Both TSH and Graves' immunoglobulins (Igs) specifically inhibited [125I]bTSH binding to the solubilized membranes, as demonstrated by polyethylene glycol separation of the [125I]bTSH-solubilized membrane complex from unbound [125I]-bTSH. Scatchard analysis of [125I]bTSh displacement curves for both TSH and Graves' Igs indicated a single class of binding site for each, with an affinity constant of 1.8 x 10(9) M-1. In addition, Igs capable of inhibiting [125I]bTSH binding to the solubilized membranes were detected predominantly in the serum of patients with Graves' disease. These results strongly suggest that TBII are antibodies directed to the TSh receptor itself without strict organ and species specificity.

Plasma membrane enzymes in BALB/c lymphomas with either T or B cell properties, I. 5'-Nucleotidase.

Comparison of membrane bound 5'-nucleotidase activity has been made in crude extracts and plasma membrane fractions from Abelson virus transformed lymphomas, IgM, IgG and IgA producing plasmacytomas and from thymomas with different surface antigen markers. 5'Nucleotidase activity was characterised by the following criteria : 1) optimal pH for enzyme activity, 2) specificity of 5'AMP as a substrate at the optimal pH, 3) specific inhibition of the enzyme by alpha beta methylene ADP, 4) inhibition by EDTA. 5'-Nucleotidase was found to be present on the following B cells : Abelson virus transformed lymphomas, plasmacytomas and LPS-stimulated blasts from nu/nu spleens. The enzyme was also found in one thymoma Lut 13, but it was absent from all the other thymomas studied. A possible relationship between 5'-Nucleotidase and purine metabolism in lymphocyte subpopulations is suggested by the results.

Phosphorylation of the beta subunit of Na+K+-ATPase in Ehrlich ascites tumor by a membrane-bound protein kinase.

We have shown previously that proteoliposomes reconstituted with purified Na+K+-ATPase from Ehrlich ascites tumor cells, transport Na+ with low efficiency (Spector, M., O'Neal, S. and Racker, E. (1980) J. Biol. Chem., 255, 5504-5507). We now present evidence that this low efficiency (expressed in the ratio of Na+-transported/ATP-hydrolyzed) is caused by the phosphorylation of the beta subunit of the Na+K+-ATPase by an endogenous protein kinase. On addition of [gamma-32P]ATP, crude tumor plasma membrane preparations phosphorylated the beta subunit of the ATPase, whereas crude mouse brain plasma membranes did not. However, solubilized Na+K+-ATPase from either tumor or brain wre phosphorylated by purified protein kinase from the tumor plasma membrane and dephosphorylated by a phosphatase. In both cases, the phosphorylated enzyme was inefficient; the dephosphorylated enzyme was efficient after reconstitution into liposomes. During isolation of the Na+K+-ATPase from Ehrlich ascites tumor or mouse brain, an endogenous protease partially cleaved from the beta subunit a polypeptide of 29,000 daltons that contained the phosphorylation site. The proteolytic cleavage of the beta subunit was partially inhibited by phenylmethylsulfonyl fluoride and the major site of phosphorylation was then seen in the 53,000-dalton beta subunit of the enzyme. The isolated 29,000-dalton polypeptide from mouse brain ATPase was phosphorylated by tumor protein kinase with a stoichiometry of 1 mol of phosphate/mol of protein. When this 29,000-dalton polypeptide from mouse brain was incorporated into the tumor Na+K+-ATPase after mild proteolytic digestion, a marked increase in efficiency was observed after reconstitution of the Na+ pump.

Adenylate cyclase activity in thyroid tissue from patients with untreated Graves' disease.

Both basal and TSH-stimulated (10 mU/ml) adenylate cyclase (AC) activities were measured in a crude plasma membrane function (800–10,000 × g) prepared from 3–10 mg (wet wt) thyroid tissue, which was obtained from patients with Graves disease and thyroid adenoma and from normal subjects. Twelve thyroid tissues of patients with untreated Graves disease and 4 adenoma tissues were obtained by needle biopsy, while 15 normal thyroid tissues, 20 tissues of patients with Graves disease under treatment, and 8 adenoma tissues were obtained at the time of operation. AC activities measured in 4 thyroid adenoma tissues obtained by needle biopsy did not differ significantly from those in 8 adenoma tissues obtained at the time of operation. In normal thyroid tissue the basal AC activity and the percent stimulation by TSH were 1.25 ± 0.46 (mean ± SD) pmol cAMP generated/mg wet wt-10 min and 134.0 ± 23.8%, respectively. In untreated Graves thyroid tissue, the mean basal AC activity (2.76 ± 0.57) was significantly high...

Binding of adenosine to the crude plasma membrane fraction isolated from dog coronary and carotid arteries.

Binding of adenosine to the crude plasma membrane fraction isolated from dog coronary and carotid arteries.

Interactions of glucagon with isolated rat-liver cells fate and subcellular localization of cell-associated hormone

The interaction of glucagon with isolated rat-liver cells was studied, and some results were compared with those obtained in parallel experiments with insulin. Attempts were made, in particular, to examine the functional relationships between receptor-binding and degradation, to assess the subcellular localization of cell-associated glucagon, and to compare the glucagonbinding properties of intact liver cells and whole-liver tissue. Binding determinations at the steady state are compatible with the existence of about 1.3 × 10 −5 sites per cell with a dissociation constant of 1.5 nM. Unlike that of liver membranes, the dissociation of bound glucagon is little affected by guanyl nucleotides. Glucagon in the incubation medium undergoes rapid inactivation and physical changes that suggest cleavage into iodotyrosyl peptides and free iodotyrosines. Most of the degrading activity is unrelated to the receptors as indicated by its low affinity (apparent K m 4 μM), its inhibition by polypeptides chemically unrelated to glucagon, and its partial association (about 50% of the total) with degradative components released extracellularly. In the absence of inhibitors, cell-associated glucagon also undergoes a progressive degradation, with a concomitant release of degraded products in the medium; this process may be, at least in part, receptor-mediated. However, in contrast with insulin, it represents only a minor fraction of the total degrading activity. About 80% of the glucagon, and 90% of the insulin associated to liver cells is recovered in the particulate fraction. On differential centrifugation, this material is almost exclusively recovered in the microsomal fraction; on sucrose density-gradient centrifugation, it is present to the extent of 70% in unidentified membranes of density 1.11–1.17. This pattern of distribution is unaffected by the time of incubation, indicating that if labeled hormone undergoes translocation, the latter is not detectable under the conditions used here. The subcellular distribution of the glucagon and insulinbinding activities is nearly the same whether the binding reaction is performed on intact cells or on isolated fractions, and, at saturation, intact cells bind about the same amount of hormone as does the particulate fraction derived from the cells. Thus, cell disruption does not affect the apparent accessibility of the binding sites. Crude particles and plasma membranes prepared from liver cells show a decrease in their ability to bind insulin and glucagon as compared with the same subcellular fractions obtained from whole liver; this is most pronounced with plasma membranes. Evidence that the collagenase preparation may be involved in these changes is presented.

Cellular fractionation and isolation of the plasma membrane of Burkitt's lymphoma cells

A procedure for cellular fractionation and preparation of plasma membrane from a Burkitt's lymphoma cell line is described. This procedure involves homogenization with a Polytron in buffered isotonic sucrose, and separation of cellular fractions by differential and isopycnic centrifugation in sucrose. The isolated plasma membrane fraction contains 44% of the cellular cholesterol, 50% of the ouabain-sensitive (Na + + K +)-ATPase activity, 43% of the γ-glutamyltranspeptidase activities and 16% of the phospholipid. This fraction contains only 3% of cellular protein and is contaminated with less than 4% of the total cellular activities of microsomal, lysosomal, mitochondrial, Golgi and soluble marker enzymes. The cholesterol : phospholipid molar ratio of the crude plasma membrane is 0.56. The membranes in this fraction are in the form of vesicles. Further purification of plasma membrane is achieved by sucrose density gradient centrifugation and results in a 25- to 30-fold enrichment of plasma membrane markers. Plasma membrane markers band in these gradients between 1.10 and 1.15 g/cm 3. The distribution patterns in the cell fractions of 18 cellular constituents are quantitatively determined. Most constituents are found to distribute in a fashion consistent with the results obtained in other systems. Thymidine-5′-phosphodiesterase (phosphodiesterase I), esterase, nucleoside diphosphatase and glucose-6-phosphatase, however, are shown to be poor markers of membrane fractions in this system. Lactoperoxidase-catalyzed iodination was used to identify several plasma membrane proteins which are exposed at the surface. After separation of labeled polypeptides by sodium dodecyl sulfate gel electrophoresis, the predominant labeled protein was identified as the heavy chain of IgM. Several lesser labeled proteins were observed.

The connexon order in isolated lens gap junctions

We have purified plasma membranes from calf eye lens fiber cells and have examined the arrangement of the connexons in the gap junctions by the freeze-fracture method. Lens gap junctions with almost entirely random connexon arrangement were obtained when 10 mM EDTA was present throughout the isolation protocol. However we failed to isolate lens gap junctions in which the connexon order was entirely crystalline: a maximum yield of approximately 50% crystalline gap junctions was achieved by solubilization of lens cytoplasmic matrix proteins in 7 M urea/1 mM Ca2+. Treatment of crude lens plasma membranes with proteinase to remove the matrix material yielded only low levels of crystalline gap junctions upon freeze-fracture. We discuss our results on the connexon order of isolated lens gap junctions with regard to the possible use of these junctions as a model system for the regulation of gap junctions in general.

Binding of an imidazolidine (clonidine), an oxazoloazepin (B-HT 933) and a thiazoloazepin (B-HT 920) to rat brain α-adrenoceptors and relation to cardiovascular effects

The specific binding to α-adrenoceptors in crude plasma membrane preparations of the rat brain was studied by means of 3H-clonidine (specific radioactivity 26.7 Ci/mmole). Equilibrium binding of 3H-clonidine could be described adequately according to a two-site model with a minor population of high affinity sites (K D1 = 0.4 nM) and a major population of low affinity sites (K D2 = 6.1 nM). The heterogeneity of 3H-clonidine binding was also indicated by a biphasic association rate in kinetic binding studies. In competition experiments with 3H-clonidine concentrations of 0.5 or 4.0 nM respectively, concentration-dependent displacement was observed with the non-radioactive compounds: clonidine, B-HT 920 (2-amino-6-ally--5,6,7,8-tetrahydro-4H-thiazolo-[5,4-d]-azepin-dihydrochloride) and B-HT 933 (2-amino-6-ethyl-4,5,7,8-tetrahydro-6H-oxozolo-[5,4-d]-azepinddihydrochloride). IC 50 values of 3, 21 and 160 nM or 10, 63 and 380 nM respectively were thereby evaluated. Cardiovascular effects were estimated in rats. The blood pressure increase in spinal animals was taken as parameter for α-adrenoceptor stimulation at peripheral vascular sites. The bradycardic effect in vagotomized animals was taken as parameter for central nervous sympathoinhibition. The ranking order of the potency of the three drugs was the same in both in vivo tests and parallels the in vitro binding affinities at both binding sites: clonidine > B-HT 920 > B-HT 933. These results indicate the similarity of the α-adrenoceptor structures in brain membrane preparations, at peripheral vascular sites and at central sympathoinhibitory sites.

Studies of a methotrexate binding protein fraction from L1210 lymphocyte plasma membranes

[ 14 C]- N-ethylmaleimide has been used to label binding sites for methotrexate in the plasma membranes of L 1210 cells previously ‘protected’ from reaction with unlabeled N-ethylmaleimide by the presence of methotrexate. A [ 14 C]- N-ethylmaleimide labeled protein of molecular weight 56,000 was subsequently identified by electrophoresis of crude plasma membranes in sodium dodecyl sulfate containing polyacrylamide gels. Sepharose—methotrexate affinity chromatography was used to isolate an active methotrexate binding protein fraction from L 1210 lymphoma cell plasma membranes. This protein fraction was isolated from crude plasma membrane preparations and from a detergent extract of intact cells. The latter method carried out in the presence of 0.01% TritonX- 100 permits the rapid extraction of plasma membrane proteins from intact L 1210 cells in the absence of extensive cellular destruction. The methotrexate binding capacity of the extracted protein material has been demonstrated. The protein fraction purified by affinity chromatography contains 3 major protein components of molecular weights 67,000, 63,000 and 56,000 as determined by sodium dodecyl sulfate gel electrophoresis.

ADENYL CYCLASE ACTIVITY IN HUMAN THYROID PLASMA MEMBRANES FROM NORMAL HUMAN THYROID TISSUE AND THYROID ADENOMAS

The adenylcyclase (AC) activity of crude human thyroid plasma membranes were studied in some detail and conditions for optimal cyclic AMP-production established. Membranes from eight "cold" and two "hot" thyroid adenomas were investigated and compared to membranes from corresponding normal, paranodular tissues. The investigated membranes were found to contain similar basal AC activities, which were stimulated three to five times with TSH and 20--30 times with fluoride. Further, no difference in the TSH-sensitivity of AC from normal and "cold" adenomas could be detected. Thyrotrophin 0.02 mU/ml caused a measurable and 0.15 mU/ml a half-maximal stimulation of the cAMP-production. The data indicate, that disturbances of thyroid hormone production characteristic of thyroid adenomas, are not caused by alterations of the TSH-AC system and suggest that in a thyroid cell the degree of thyroid hormone synthesis do not alter the amount of plasma membrane-bound adenylcyclase.

Chinese hamster ovary cell population density affects intracellular concentrations of calcium-dependent regulator and ability of regulator to inhibit adenylate cyclase activity

The adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of crude Chinese hamster ovary cell membranes was inhibited 30-40% by low concentrations (6-600 ng/ml) of calcium-dependent regulator (CDR). This inhibitory effect was lost at concentrations of CDR above 600 ng/ml. The adenylate cyclase activity of membranes prepared from low population density Chinese hamster ovary cells was not appreciably altered by CDR. However, with increasing cell population density there was a significant increase in the ability of CDR to inhibit cyclic AMP formation. Further, the intracellular levels of CDR determined in the 12,000 x g supernatant and particulate fractions varied inversely with increasing cell population density. As cell number increased from 2 x 10(6) to 10 x 10(6) cells per dish the CDR concentration present in the supernatant fraction increased from 0.4 to 0.8 mug of CDR per mg of protein, while the amount of endogenous CDR associated with the particulate fraction decreased from 0.6 to 0.4 mug of CDR per mg of protein. This suggests that possible changes in the distribution of CDR between the supernatant and membrane fractions might serve as a regulatory mechanism for activities under CDR control.