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1.
Biochemical and ultrastructural characterization of a high molecular weight soluble Mg2+ -ATPase from human erythrocytes 总被引:1,自引:0,他引:1
J R Harris 《Journal of molecular biology》1984,174(4):705-721
The isolation and purification of a 600,000 Mr cytosolic Mg2+ -ATPase from human erythrocytes is described. The electrophoretic properties of the native and sodium dodecyl sulphate-dissociated protein are presented and compared with those of the erythrocyte protein cylindrin . The Mg2+-ATPase has a single subunit of Mr 100,000 and it has an isoelectric point of 4.9. From transmission electron microscopy of negatively stained specimens, it is proposed that the Mg2+-ATPase is hexameric, containing two superimposed trimers of the 100,000 Mr subunit, which gives rise to a 13 nm pseudohexagonal particle with a central 3 nm cavity. Varying the orientation of the protein in the negative stain also produces images that are not hexagonal. When orientated on-edge, the protein produces a double-disc image, which is most clearly defined under acidic negative staining conditions with uranyl acetate, when some aggregation of the protein is produced. The ultrastructure of the Mg2+-ATPase is shown to be distinctly different from that of cylindrin . A comparative discussion of the negatively stained transmission electron microscopical images of the Mg2+-ATPase, mitochondrial F1-ATPase and several other oligomeric proteins and enzymes is presented. 相似文献
2.
Human placental microsomes exhibit uptake of d-[3H]glucose which is sensitive to inhibition by cytochalasin B (apparent Ki = 0.78 /gm M). Characterization of [3H]cytochalasin B binding to these membranes reveals a glucose-sensitive site, inhibited by d-glucose with an ED50 = 40 mM. The glucose-sensitive cytochalasin B binding site is found to have a Kd = 0.15μM by analysis according to Scatchard. Solubilization with octylglucoside extracts 60–70% of the glucose-sensitive binding component. Equilibrium dialysis binding of [3H]cytochalasin B to the soluble protein displays a pattern of inhibition by d-glucose similar to that observed for intact membranes, and the measurement of an ED50 = 37.5 mM d-glucose confirms the presence of the cytochalasin B binding component, putatively assigned as the glucose transporter. Further evidence is attained by photoaffinity labelling; ultraviolet-sensitive [3H]cytochalasin B incorporation into soluble protein (Mr range 42 000-68 000) is prevented by the presence of d-glucose. An identical photolabelling pattern is observed for incorporation of [3H]cytochalasin B into intact membrane protein, confirming the usefulness of this approach as a means of identifying the presence of the glucose transport protein under several conditions. 相似文献
3.
Intact crude synaptosomes from bovine cerebellum contain, in addition to an externally accessible (postsynaptic) adenylate cyclase, an enzyme with its catalytic center oriented towards the inside of the synaptosome (presynaptic adenylate cyclase). This is demonstrated by the unmasking of latent adenylate cyclase activity by Triton X-100. Furthermore, intact crude synaptosomes can synthesize cyclic AMP from adenine. This synthesis takes place inside the synaptosome as the postsynaptic adenylate cyclase is inactive in the Krebs-Ringer buffer. Presynaptic adenylate cyclase activity is not influenced by depolarization, as shown by [3H]adenine pulse-labeling, but is stimulated by (?)-norepinephrine and (?)-isoproterenol. (±)-Propranolol inhibits this stimulation whereas phentolamine has no effect, suggesting the presence of a β-adrenergic receptor-coupled presynaptic adenylate cyclase. 相似文献
4.
Wolf-Jürgen Buhl Thomas F. Sarre Kurt Hilse 《Biochemical and biophysical research communications》1980,93(3):979-987
From a rabbit reticulocyte postpolysomal supernatant a fraction has been isolated which is enriched in ribosomal particles sedimenting at 50S. This fraction is efficiently in vitro translated predominantly into α-globin. Besides the RNAs and proteins of the small ribosomal subunit the 50S particle contains α-globin mRNA and additional high molecular weight proteins, most of which correspond to polypeptides of the initiation factors eIF-2 and eIF-3. The 50S particle may represent a native [mRNA·40S·eIF′s·Met-tRNAf·GTP] complex which may occur in vivo as a translatable intermediate in the initiation sequence. 相似文献
5.
Purified cytochrome from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, was less stable than in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal . Liposomal required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal was subjected to treatment. This reagent destroyed the liposomal . These results suggest that the heme is located in the proximity of the reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane. 相似文献