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David M. Neville Jr. 《In vitro cellular & developmental biology. Plant》1974,9(6):445-454
Summary This presentation is confined to a discussion of the binding of insulin to specific insulin receptors on isolated preparations
of purified plasma membranes from rodent livers. The system has been studied extensively and has yielded a large amount of
quantitative data. This study was a collaborative effort between my laboratory and the Section on Diabetes of the National
Institute of Arthritis, Metabolic, and Digestive Diseases. The studies were begun by Pierre Freychet, Jesse Roth, and myself.
The work on the obese mouse was initiated by Ronald Kahn and has been continued recently with Andrew Soll.
When these experiments were begun, we hoped that, by using highly purified plasma membranes and a biologically active labeled
hormone, [125I]-monoiodoinsulin, we could accurately quantitative receptor binding isotherms, or receptor concentration and affinity, to
such a degree that we could determine whether receptors were involved in the pathophysiology of insulin-resistant states.
As the work progressed, and we realized that such quantitation was possible, we began to think of the insulin and insulin-receptor
interaction as a general model for the interaction of a message from the external environment and a cell surface, which creates
a change in cell behavior. Because the plasma membrane separates the external environment of a cell from the internal environment,
there seemed to be good reason to believe that plasma membranes would be highly specialized for receiving many diverse types
of messages.
The most important finding of this work, if we may generalize from the insulin receptor, is that the specialization of the
plasma membrane for receiving messages from the external environment involves control mechanisms which can alter the concentration
of plasma membrane receptor. The response of a cell to an external message is therefore not uniquely determined by the concentration
of the message, but also by the concentration of the receptor which, in the case of the insulin receptor, can be varied by
metabolic and hormonal factors.
Presented in the Symposium on Cell Membranes at the Twenty-fourth Annual Meeting of the Tissue Culture Association, June 1973. 相似文献
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Effect of guanine nucleotides on polyphosphoinositide synthesis in rat liver plasma membranes. 总被引:1,自引:0,他引:1
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The subcellular distributions of endogenous ADP-ribosylation products in hearts from 1-day-old neonatal and adult rats were investigated. In adult rat heart a 52 kDa mono-ADP-ribosylation product was identified in the plasma membrane fraction. In contrast, in neonatal rat heart a 130 kDa poly-ADP-ribosylation product was present in the nuclear fraction. The monomeric and polymeric nature of the two ADP-ribosylation products was determined by their sensitivity to thymidine and by analysis of their snake venom phosphodiesterase products. NADP+ enhanced both the mono- and polymeric reactions. The ADP-ribose-protein linkage of the adult 52 kDa product was stable to 1 h of treatment with hydroxylamine (0.5 M) and mercury ions, but was sensitive to alkali and a 12 h treatment with hydroxylamine (1 M). This is suggestive of an arginine linkage. The 130 kDa poly-ADP-ribosylation product from the neonatal rat heart was alkalilabile but stable to both hydroxylamine and HgCl2. This implies the presence of an unusual linkage in the 130 kDa product. The presence of these different ADP-ribosylation products in adult and neonatal rat hearts suggests the possible importance of these proteins and their ADP-ribosylation during cardiac development. 相似文献
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The objective of this study was to examine the effect of dietary Chol supplementation on SM metabolism in rat liver plasma membranes, as well as on membrane leaflet fluidity characteristics. The membrane Chol content increased significantly during the first 20 days of dietary feeding, but returned to the level of the control group when the diet was continued for another ten days. The initially more fluid outer leaflet of the membrane rigidified as a result of the diet, obliterating the natural asymmetry in the fluidity of the membrane bilayer. Changes in the neutral SMase activity were also observed. These changes were in strong negative correlation (r = -0.978) with the Chol/Pr ratio and are consistent with the in vitro inhibition of SMase activity reported earlier. In contrast, the SM synthesizing enzymes, PC:Cer-PCh and PE:Cer-PEt transferase, were stimulated in course of the dietary Chol feeding. The activity of PC:Cer-PCh transferase was more strongly affected. Our results support the concept that SM metabolism is regulated coordinately with that of Chol. The present work could contribute to the better understanding of the parallel accumulation of SM and Chol observed in a variety of pathological conditions such as atherosclerosis and Niemann-Pick disease. 相似文献
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Four rat lipoprotein classes [lymph chylomicrons, VLD (very-low-density), LD (low-density) and HD (high-density) lipoproteins] were tested for their ability to affect basal adenylate cyclase (EC 4.6.1.1) activity of rat liver plasma membranes. All the lipoproteins, with the exception of lymph chylomicrons, effectively increase the enzyme activity. VLD lipoproteins are the most active class (67% maximal increase), followed by HD lipoproteins (33%) and LD lipoproteins (23%). The effect of VLD lipoproteins is additive to that elicited by GTP or GTP plus glucagon (at least within a certain concentration range). VLD lipoproteins affect only the Vmax. of the enzyme, not the Km. 相似文献
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1. Plasma membranes were treated with phospholipase A2, phospholipase C or phospholipase D. The phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was deactivated by phospholipase C treatment, whereas phospholipase A2 and phospholipase D did not affect the enzyme. 2. Incorporation of phosphatidylethanolamine and phosphatidylglycerol into partially delipidated plasma membranes resulted in significant stimulation of the transferase, whereas inclusion of sphingomyelin and phosphatidylserine suppressed the enzyme activity. Our results suggest that phosphatidylserine is a regulator of sphingomyelin level in membranes. 3. The activity of phosphatidylethanolamine:ceramide-phosphorylethanolamine transferase was not influenced by the fluidity of its lipid environment. 相似文献
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Characterization of rat liver cell plasma membranes. 总被引:8,自引:0,他引:8
M M Fisher D L Bloxam M Oda M J Phillips I M Yousef 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1975,150(1):177-184
A method is described by which bile canalicular membranes (BCM) can be prepared, together with canaliculus-free plasma membrane (PM), both essentially free of contamination. The recovery of both fractions together was estimated to be 46%. The concentrations of total lipid, total phospholipid and cholesterol were substantially greater in the BCM, and polyacrylamide gel electrophoresis revealed differences in protein composition. The differences in lipid and protein composition of these two plasma membrane fractions are presumably related to their very different physiological functions. 相似文献
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T D Nguyen M S Wolfe G G Heintz D C Whitcomb I L Taylor 《The Journal of biological chemistry》1992,267(13):9416-9421
We report here the identification on rat liver plasma membranes and microsomes of proteins that bind pancreatic polypeptide (PP) with high affinity and specificity (plasma membranes: KD = 4.6 nM, Bmax = 3.28 pmol/mg protein; microsomes: KD = 3.45 nM, Bmax = 18.7 pmol/mg protein). These binding proteins appeared coupled to a G-protein, since 0.1 mM guanosine 5'-O-(3-thiotriphosphate) decreased the affinity by half. When 125I-labeled PP-binding protein complexes covalently cross-linked with disuccinimido suberate were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two radioactive bands with M(r) values of 52,000 and 38,000 were demonstrated. Both bands were inhibited by unlabeled PP with an IC50 of approximately 5 nM (but not by neuropeptide Y or peptide YY). After the cross-linked complexes were solubilized from liver microsomes with 0.2% Triton X-100 and gel-filtered, they did not interact with the lectins wheat germ agglutinin, Ulex europaeus agglutinin, Ricinus communis agglutinin, and soy bean agglutinin. That these binding proteins may not be glycosylated was further supported by the failure of either peptide N-glycosidase F and endo-beta-N-acetylglucosaminidase F to alter the size of the PP-binding protein complexes on gel electrophoresis. These PP-binding proteins may serve as receptors and mediate a hepatic effect of PP. 相似文献
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K Muraki Y Nishi M Arai N Kubo K Ueda H Shikata Y Nakata T Segawa N Yanaihara H Yajima 《Biochemical and biophysical research communications》1987,145(3):1071-1079
Neurotensin (NT) is now classified as a brain-gut peptide in the central nervous system and gastrointestinal tract. In the present study, we characterized the NT receptors on the rat liver plasma membranes. The specific binding of [3H]NT was time dependent, reversible, and saturable. Scatchard analysis of the specific binding data yielded two classes of binding sites, a high affinity site and a low affinity site. The average maximum number of binding sites (Bmax) amounted to 13.3 +/- 1.1 fmol/mg protein at high affinity site and 122.3 +/- 21.5 fmol/mg protein at low affinity site, respectively. The dissociation constant (Kd) had values of 0.39 +/- 0.01 nM at high affinity site and 8.1 +/- 1.1 nM at low affinity site, respectively. The amount of specifically bound [3H]NT was significantly reduced in the presence of mono and divalent cations, EDTA, EGTA and a peptidase inhibitor bacitracin, NT1-13 competed with [3H]NT for its binding site with an IC50 of 0.19 nM at high affinity site (0.2 nM concentration of [3H]NT) and 0.7 nM at low affinity site (4.0 nM concentration of [3H]NT). Xenopsin, a NT analogue separated from the skin of Xenopus laevis, was equipotent (IC50 0.75 nM) with NT1-13 at 4.0 nM concentration of [3H]NT. C-terminal sequence of NT contains the structure necessary for interaction with NT binding sites whereas N-terminal sequence had no binding activity. Since NT has a hyperglysemic and a hypercholesterolemic effects in rats, these NT receptors on the rat liver plasma membranes may be involved in the hyperglycemia and/or hypercholesteroremia induced by NT. 相似文献
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Detection and characterization of anionic polypeptide fraction binding sites in rat liver plasma membranes and cultured hepatocytes 总被引:1,自引:0,他引:1
M Martigne B Melin F Mahlberg N Domingo F Chanussot H Lafont J C Hauton 《Biochimica et biophysica acta》1989,979(3):341-346
The binding of human 125I-labeled 'anionic polypeptidic fraction' (APF) to purified rat liver plasma membranes was studied. The dissociation constant for this binding was 3.0 micrograms protein/mg membrane protein. Binding was competitively inhibited by unlabeled human APF, but not by human LDL (low density lipoproteins). When unlabeled HDL3 was added, binding of labeled APF was competitively reduced to a level between that of unlabeled APF and unlabeled LDL. Experiments with cultured rat hepatocytes confirmed those obtained with liver membranes and suggested the presence in rat liver of saturable APF-binding sites which seem to be specific for APF. The physiologic significance of these APF binding sites is discussed in relation to the fate of cholesterol in the liver. 相似文献
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D H Petkova A B Momchilova T T Markovska K S Koumanov 《The International journal of biochemistry》1987,19(3):289-291
The influence of D-galactosamine administration on rat liver plasma membranes lipid composition, fluidity and some enzyme activities was investigated. D-Galactosamine was found to induce an increase of the total phospholipids, the cholesterol level and membrane rigidity. In liver plasma membranes of D-galactosamine-treated rats the exogenous phospholipase A2 activity was enhanced about 2 fold, whereas the endogenous activity was slightly decreased. No alteration of the neutral sphingomyelinase activity was observed. 相似文献
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Highly purified bile-canalicular vesicles and lateral plasma membranes isolated from rat liver on Nycodenz gradients. Biochemical and immunolocalization studies. 总被引:1,自引:1,他引:0
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1. A liver canalicular plasma-membrane fraction enriched 115-155-fold in five marker enzymes relative to the tissue homogenate was obtained by sonication of liver plasma membranes followed by fractionation in iso-osmotic Nycodenz gradients. 2. Two lateral-plasma membrane fractions were also collected by this procedure; the lighter-density fraction was still associated with canalicular membranes, as assessed by enzymic and polypeptide analysis. 3. The polypeptide composition of the domain-defined plasma-membrane fractions was evaluated. It was demonstrated by immunoblotting that the 41 kDa alpha-subunit of the inhibitory G-protein, associated in high relative amounts with canalicular plasma-membrane fractions, was partially lost in the last stage of purification; however, this subunit was retained by lateral plasma membranes. 4. Antibodies to the proteins of bile-canalicular vesicles were shown to localize to the hepatocyte surface in thin liver sections examined by immunofluorescent and immuno-gold electron microscopy. Two subsets of antigens were identified, one present on both sinusoidal and canalicular plasma-membrane domains and another, by using antisera pre-absorbed with sinusoidal plasma membranes, that was confined to the bile-canalicular domain. 相似文献
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J Bouhnik F Savoie A Michaud T Baussant F Alhenc-Gelas P Corvol 《Life sciences》1989,44(24):1859-1866
The influence of sex hormones on rat plasma T-kininogen concentration was examined. The level of T-kininogen in the post-pubertal female rat is about 3-times that of the male animal. Female rats castrated as adults or 15 days after birth, had low T-kininogen concentrations, near those of male rats. In contrast, castration of mature or immature male animals induced no change in T-kininogen. Treatment of castrated female or male rats with 17 alpha-ethinylestradiol significantly increased the T-kininogen level, whereas administration of testosterone or progesterone had no effect. The influence of estrogen was specific for T-kininogen, since plasma HMW kininogen concentration was the same in male and female rats and was not affected by castration or sex hormone treatment. T-kininogen concentration was not significantly changed in pregnant rat between the 12th and the 20th day of pregnancy, but increased after parturition. It was high in the newborn rat at birth and then decreased similarly over the next 3 weeks in males and females. It continued to decrease in the males, reaching the level of the adult rat, but it increased in the female from 3-4 weeks of age and reached the adult level at about 6-8 weeks. These data indicate that natural estrogens have a physiological influence on the plasma level of T-kininogen in female rats whereas testosterone had no effect on either male or castrated female rats. HMW kininogen is not physiologically dependent on sex hormones. 相似文献
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A method was developed to isolate renal basolateral membranes from cortical kidney tubule cells of single rats. The isolated membrane fraction was characterized by the measurement of marker enzyme activities and by electron microscopy. 1. After centrifugation of crude plasma membranes on a discontinuous sucrose density gradient the basolateral membranes accumulated at a sucrose density of p= 1.14-1.15 g/ml. The yield was 147 mug membrane protein/g kidney wet weight. Protein recovery was 0.1%. 2. (Na+ + K+)-ATPase was enriched 22-fold from the homogenate. The recovery was 2.6%. The (Na+ + K+)/Mg2+-ATPase ratio was 4.1. 3. The contamination by brush borders was small. Alkaline phosphatase was 1.6-fold enriched and 0.2% was recovered. Aminopeptidase was 1-fold enriched with a recovery of 0.1%. The contamination by mitochondria, lysosomes and endoplasmic reticulum was negligible. 4. In electron micrographs the basolateral membranes showed a typical triple layered profile and were characterized by the presence of junctional complexes, gap junctions or tight junctions. 相似文献
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D Blad-Holmberg 《Biochimica et biophysica acta》1979,553(1):25-39
Plasma membranes as well as mitochondrial and microsomal subfractions were subjected to zone electrophoresis. Treatment with neuraminidase, phospholipase A or C does not influence the movement of plasma membranes and smooth microsomes. Trypsin increases mobility of plasma membranes and smooth by about 20%, and further treatment with phospholipase C decreases mobility of plasma membranes, total smooth and smooth I microsomes, which, however, is not the case with smooth II microsomes. Low concentrations of trypsin also solubilize enzyme proteins of smooth microsomes from phenobarbital-treated rat liver, but electrophoretic mobility is not increased, indicating structural differences in induced membranes. The mobility of the outer and inner mitochondrial membranes is significantly higher than that of submitochondrial particles. For microsomes the negative surface charge density occurs in the decreasing order of: ribosomes--rough--smooth I--smooth II. A 10 mM CsCl gradient decreases the mobility of rough microsomes by 40% and of ribosomes by 20% but has no effect on total smooth micromes. On the other hand, 5mM MgCl2 decreased the mobility of all three fractions. EDTA-treated rough and EDTA-treated smooth microsomes have the same electrophoretic mobilities. However, the mobilities of non-treated rough and smooth microsomes differ significantly from each other. 相似文献
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