Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, , to DDT was studied. The CaMg ATPase found in SR with the high Ca2+ affinity is sensitive to DDT while the portion of ATPase related to the low Ca2+ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and poisoning symptoms of the nervous system is discussed. 相似文献
The effects of quinine and 9-aminoacridine, two blockers of potassium conductance in islet cells, on 45Ca efflux and insulin release from perifused islets were investigated in order to elucidate the mechanisms by which glucose initially reduces 45Ca efflux and later stimulates calcium inflow in islet cells. In the absence of glucose, 100 μM quinine stimulated 45Ca net uptake, 45Ca outflow rate and insulin release. Quinine also dramatically enhanced the cationic and the secretory response to intermediate concentrations of glucose, but had little effect on 45Ca net uptake, 45Ca fractional outflow rate and insulin release at a high glucose concentration (16.7 mM). The ability of quinine to stimulate 45Ca efflux depended on the presence of extracellular calcium, suggesting that it reflects a stimulation of calcium entry in the islet cells. In the absence of extracellular calcium, quinine provoked a sustained decrease in 45Ca efflux. Such an inhibitory effect was not additive to that of glucose, and was reduced at low extracellular Na+ concentration. At a low concentration (5 μM), quinine, although reducing 86Rb efflux from the islets to the same extent as a non-insulinotropic glucose concentration (4.4 mM), failed to inhibit 45Ca efflux. In the presence of extracellular calcium, 9-aminoacridine produced an important but transient increase in 45Ca outflow rate and insulin release from islets perifused in the absence of glucose. In the absence of extracellular calcium, 9-aminoacridine, however, failed to reduced 45Ca efflux from perifused islets. It is concluded that quinine, by reducing K+ conductance, reproduces the effect of glucose to activate voltage-sensitive calcium channels and to stimulate the entry of calcium into the B-cell. However, the glucose-induced inhibition of calcium outflow rate, which may also participate in the intracellular accumulation of calcium, does not appear to be mediated by changes in K+ conductance. 相似文献
Polyacrylamide gradient gel electrophoresis was carried out in micellar solutions of various detergents which differ in degree of potency to denature proteins. From the application of this method to band 3 protein from erythrocyte membranes, it was suggested that the procedure was useful in studying the molecular state of membrane proteins.The electrophoretic behaviors of human and bovine band 3 protein did not show any species specificity in either a denature state and a state resembling the native state. As well as in nonionic detergent solutions, the dimeric and tetrameric structures of bovine band 3 protein were preserved in sodium deoxycholate solution, in which protein complexes maintained in nonionic detergent solutions are frequently dissociated. Even in dodecyltrimethylammonium bromide solution, which is a denaturant for water-soluble proteins, part of the band 3 protein was still present as the oligomer. The results suggest that the oligomeric form of band 3 protein is the stable structure and that the dimer and tetramer possibly coexist in membranes. 相似文献
To achieve specific cross-linking between the active sites of the non-identical subunits tryptophan synthase from E. coli was modified by a novel method. After reaction with bifunctional reagents of the isolated subunits at their active sites, the tetrameric complex was formed and the free ends of the reagent molecules reacted with each other forming a covalent bridge between the subunits. The distance between the amino acid side chains involved in the cross-linking should not exceed approx. 1.8 nm. A distance much shorter than that is unlikely since all attempts to cross-link the active sites with different shorter bifunctional reagents failed. The implications of these results in the mechanism of action of the enzyme are discussed. 相似文献
Low potential quinones are mediators of cyclic phosphorylation in washed spinach thylakoid membranes if they are prereduced to provide the proper redox poise. Cyclic phosphorylation catalyzed by different quinols varies in its sensitivity to the electron transfer inhibitor 2-iodo-6-isopropyl-3-methyl-2′,4,4′-trinitrodiphenyl ether (DNPINT), which is thought to inhibit electron flux from the bound plastoquinone (B) to the plastoquinone pool (Trebst, A., Wietoska, H., Draber, W. and Knops, H.J. (1978) Z. Naturforsch. 33c, 919–927). Cyclic phosphorylation catalyzed by uncharged quinols is extremely sensitive to DNPINT, whereas cyclic phosphorylation catalyzed by negatively charged quinols is approximately two orders of magnitude less sensitive. Many quinols have pK1 values in the physiological range (pH 7–9). Increasing the concentration of the deprotonated quinol either by raising the assay pH, increasing the mediator concentration, or increasing the fractional reduction of the quinone results in a decrease in the sensitivity of cyclic phosphorylation to DNPINT. At very high DNPINT concentrations, cyclic phosphorylation catalyzed by all quinols (and ferredoxin) is inhibited, but not phenazine methosulfate catalyzed cyclic phosphorylation.These data suggest that the deprotonated form of the quinol can donate electrons directly to the plastoquinone pool, whereas the uncharged quinol most obligately transfer electrons through the bound plastoquinone ‘B’. A second site of DNPINT action after the plastoquinone pool is also observed, which requires much higher DNPINT concentrations for inhibition of phosphorylation. 相似文献
The phosphorylation of myelin (basic protein) purified from rabbit brain was markedly stimulated by exogenously added calmodulin in the presence of calcium and inhibited by W-7(N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), a calmodulin interacting agent, in a dose-dependent fashion. However, exogenously added myelin basic protein free from protein kinase activity could not serve as a substrate of this calmodulin dependent protein kinase, suggesting that this kinase catalyzes the phosphorylation of the enzyme-substrate complex. These results suggest that a calmodulin-dependent protein kinase complex with the substrate (basic protein) is located in the myelin membrane of the central nervous system. 相似文献
Both NADPH- and ascorbic acid-dependent lipid peroxidations were inhibited by spermine, the degree of inhibition being greater with the former peroxidation. The effective concentration of spermine required for inhibition was higher when larger amounts of microsomes were used. However, the activities of NADPH-cytochrome c reductase and NADPH-peroxidase were not influenced by spermine. These results suggest that spermine inhibits lipid peroxidation by binding to phospholipids in the microsomes. 相似文献
A Ca2+-binding protein (TCBP), which was isolated from , enhanced about 20-fold particulate-bound guanylate cyclase activity in cells in the presence of a low concentration of Ca2+, while the adenylate cyclase activity was not increased. The enhancement was eliminated by ethylene glycol-bis (β-aminoethyl ether)-N,N′-tetraacetic acid. The enzyme activity was not stimulated by rabbit skeletal muscle troponin-C, the Ca2+-binding component of troponin, or other some proteins. In the presence of TCBP, stimulating effect of calcium ion on the enzyme activity was observed within the range of pCa 6.0 to 4.6, and was immediate and reversible. 相似文献
1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min.
2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP.
3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site.
4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.
5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism.
6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way.
7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD+ by succinate.
8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.