A simple technique of purification of the soluble pig heart mitochondrial f1-ATPase is described. It consists of removal of extrinsic proteins from mitochondrial membranes before extraction with chloroform and ammoniu...
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A simple technique of purification of the soluble pig heart mitochondrial f1-ATPase is described. It consists of removal of extrinsic proteins from mitochondrial membranes before extraction with chloroform and ammonium sulfate fractionation. A high degree of purity, an excellent stability and a good yield are attained after gel filtration through an Ultrogel ACA 34 column equilibrated in the presence of 50% glycerol. The tested properties of the f1-ATPase prepared by this method are similar to those of the same enzyme extracted by sonication. The enzyme is virtually devoid of tightly bound nucleotides. Some characteristics of the behavior of the .beta. subunit are shown.
The latent coupling factor (f1)-ATPase of M. lysodeikticus [luteus] was purified to homogeneity as determined by a number of criteria including non-denaturing polyacrylamide gel electrophoresis, crossed immunoelectrop...
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The latent coupling factor (f1)-ATPase of M. lysodeikticus [luteus] was purified to homogeneity as determined by a number of criteria including non-denaturing polyacrylamide gel electrophoresis, crossed immunoelectrophoresis and analytical ultracentrifugation. By inclusion of 1 mM phenylmethyl sulfonyl fluoride, a serine protease inhibitor, in the shock-wash step of release off1 from the membranes, the spontaneous activation of both crude and purified ATPase by endogenous membrane protease(s) could be prevented, thereby yielding a highly latent ATPase preparation. Equilibrium ultracentrifugation of the latent ATPase gave a MW of 400,000. The ATPase contained 5 different subunits .alpha., .beta., .gamma., .delta. and .epsilon. and their MW determined by SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis were 60,000, 54,000, 37,000, 27,000 and 9000, respectively. The subunit composition was determined with 14C-labeled, f1-ATPase prepared from cells grown on medium containing [U-14C]-labeled algal protein hydrolysate. Within the limitations of this method the results tentatively suggest a subunit composition of 3:3:1:1:3.
Isolation of ATPase from rat liver submitochondrial particles by chloroform treatment requires the presence of ATP or ADP during enzyme solubilization. In the absence of adenine nucleotides the enzyme activity is very...
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Isolation of ATPase from rat liver submitochondrial particles by chloroform treatment requires the presence of ATP or ADP during enzyme solubilization. In the absence of adenine nucleotides the enzyme activity is very low although all protein components off1-ATPase are released. The low concentrations of ATP or ADP required (5 .mu.M) indicate that the high affinity nucleotide-binding sites are involved in enzyme stabilization. Other nucleotides tested (ITP, GTP, UTP, CTP) were less effective. Polyacrylamide gel electrophoresis and immunodiffusion in agar plates revealed that in the absence of adenine nucleotides a fraction off1-ATPase released by chloroform treatment is split into fragments. The part of the dissociated enzyme molecule has a MW identical with that of a .beta.-subunit off1-ATPase. Dissociation of the f1-ATPase molecule was prevented by aurovertin. Crude f1-ATPase solubilized by chloroform treatment was further purified by Sepharose 6B gel filtration. Specific ATPase activity of the purified enzyme was 90 .*** Pi/min per mg protein and the enzyme was composed of 5 protein subunits (.alpha., .beta., .gamma., .delta., .epsilon.) and MW 58,000, 55,000, 28,000, 13,000 and 8000, respectively. Chloroform-released f1-ATPase from rat liver mitochondria displayed immunochemical cross-reactivity with that isolated from beef heart mitochondria.
Rat splenic lymphocytes contain specific, saturable glucocorticoid receptors. When suspensions of these cells were incubated at ***. C in the presence of [3H]-triamcinolone acetonide ([3H]-TA), 80% of receptor-bound s...
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Rat splenic lymphocytes contain specific, saturable glucocorticoid receptors. When suspensions of these cells were incubated at ***. C in the presence of [3H]-triamcinolone acetonide ([3H]-TA), 80% of receptor-bound steroid was found associated with the cytoplasm, whereas at ***. C, 80% of [3H]-TA binding was associated with the nucleus. The equilibrium dissociation constant for interaction of [3H]-TA with the cytoplasmic receptor at ***. C is 5.6 nM while that for nuclear binding at ***. C was 0.85 nM, suggesting an increase in receptor affinity for [3H]-TA at physiological temperatures. Specificity studies showed a high degree of glucocorticoid specificity at ***. C in cytoplasm and nucleus, with little competition observed for either progesterone (P) or cortexolone (S). At ***. C, P and S competed with [3H]-TA for receptor binding as effectively as dexamethasone. ***., testosterone, ***.-dihydrotestosterone and cortisone did not compete under any of these conditions. When cells were incubated at ***. C in the presence of 0.02 .mu.M [3H]-TA and concentrations of P and S ranging from 0.04-2.56 .mu.M, [3H]-TA binding was inhibited at steroid concentrations as low as 0.1 .mu.M. If these same cells were then warmed to ***. C [3H]-TA binding in cytoplasm and nucleus was inhibited only at P and S concentrations .gtoreq. 1 .mu.M. There is, concomitant with increasing temperature, an increase in affinity and specificity of the receptor for active glucocorticoids. This specificity change was regarded as evidence for a conformational change in the hormone-receptor complex coincident with activation.
Aurovertin forms a complex with soluble beef heart mitochondrial ATPase (f1 ) while exhibiting a biphasic fluorence enhancement. The effect of substrate, activators and inhibitors off1 1 of the fluorescence of the au...
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Aurovertin forms a complex with soluble beef heart mitochondrial ATPase (f1 ) while exhibiting a biphasic fluorence enhancement. The effect of substrate, activators and inhibitors off1 1 of the fluorescence of the aurovertin-f1 complex is reported. The aurovertin-f1 complex can exist in two different states, one showing low fluorescence and the other with high fluorescence. Transition into the low fluorescence state is induced by various nucleoside triphosphates (ATP ± Mg2+ , ITP ± Mg2+ , GIP + Gg2+ , and AMP-P(NH)P ± Mg2+ ). The rate and extent offluorescence decrease caused by nucleotide addition (except that caused by ATP) is dependent on the presence of added Mg2+ . The inhibitors of ATPase activity (AMP-P(NH)P, GMP-P(NH)P and EDTA) at concentrations that inhibit hydrolysis of ATP did not prevent the ATP induced decrease of aurovertin fluorescence. EDTA at high concentration (>0.4 mM) enhanced the effect of ADP. The complex of aurovertin with f1 that had previously been treated with butanedione loses sensitivity to ATP. Addition of ADP to the system containing butanedione-treated enzyme caused a 2-fold greater enhancement offluorescence than the addition of ADP to the control system. In contrast to the butanedione-treated enzyme, the complex of aurovertin with f1 previously treated at pH 5.6 loses sensitivity to ADP. Addition of ATP to this system lowered the fluorescence as in the system containing native enzyme. On the basis of the analyses of the aurovertin fluorescence changes and hydrolytic activity off1 , the existence of several types of ligand binding sties with varying degrees of specificity are proposed. It is further proposed that these sites are important in control of the conformation and the catalytic properties of the ATPase molecule.
An ATPase complex sensitive to the energy transfer inhibitors oligomycin, dicyclohexylcarbodiimide and venturicidin was solubilized from R. rubrum chromatophores with Triton X-100 and further purified by centrifugatio...
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An ATPase complex sensitive to the energy transfer inhibitors oligomycin, dicyclohexylcarbodiimide and venturicidin was solubilized from R. rubrum chromatophores with Triton X-100 and further purified by centrifugation on a glycerol gradient. The partially purified ***.f1 contains 13 distinct polypeptide subunits, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, including the subunits of the oligomycin-insensitive, water-soluble Rrf1 ATPase. The ATPase activity of ***.f1 as that of the membrane-bound enzyme complex depends on Ca2+ or Mg2+ and detailed kinetic studies showed that the divalent cation-ATP complex is the substrate for both ATPase complexes. free ATP and free Mg2+ act as competitive inhibitors, with Ki values of 1 mM and 7 .mu.M, respectively. The subunit composition of the purified ***.f1 and its similarity to the membrane-bound ATPase with respect to cation dependence and sensitivity to energy transfer inhibitors suggest that it contains all the subunits of the R. rubrum coupling factor-ATPase complex.
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