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1.
C L Borders M L Woodall A L George 《Biochemical and biophysical research communications》1978,82(3):901-906
Yeast enolase is rapidly inactivated by butanedione in borate buffer, complete inactivation correlating with the modification of 1. 8 arginyl residues per subunit. Protection against inactivation is provided by either an equilibrium mixture of substrates or inorganic phosphate, a competitive inhibitor of the enzyme. Complete protection by substrates correlates with the shielding of 1. 3 arginyl residues per subunit, while phosphate protects 1. 0 arginyl residue per subunit from modification. 相似文献
2.
Phosphoglycerate mutase is inactivated by butanedione in borate buffer. Inactivation by 0.13 mM reagent correlates with the modification of one arginyl residue per subunit, and is prevented by either 2, 3-diphosphoglycerate or 3-phosphoglycerate. With 0.50 mM butanedione, inactivation is accompanied by the modification of three arginyl residues per subunit, two of which are protected by the combined presence of cofactor and substrate. 相似文献
3.
Yeast enolase (EC 4.2.1.11) is rapidly inactivated at pH 6.1 by three different water-soluble carbodiimides — 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, and 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide. Inactivation is most likely due to the modification of essential carboxyl residues at the enzyme active site. 相似文献
4.
Rabbit muscle aldolase (E.C. 4. 1. 2. 13) was guanidinated by reaction with O-methylisourea. Up to 60% of the lysine residues can be guanidinated without any dissociation of the tetramer but with a complete loss of enzymatic activity. Native and guanidinated aldolase can be dissociated into monomers in 2.4 m MgCl2 with only slight change in conformation of the subunit. Nitrotroponylation of guanidinated aldolase in dilute buffer gives no reaction whereas in 2.4 m MgCl2 nitrotroponlylation modifies another 8–12% of the lysine residues. Removal of MgCl2 by dialysis affords 100% recovery of activity and tetrameric structure for native aldolase and 100% recovery of tetrameric structure for guanidinated aldolase. In contrast nitrotroponylated and guanidinated aldolase remains monomeric before precipitating as the MgCl2 concentration is lowered. It is concluded that lysine may be involved in the protein-protein interaction of the subunit contact domains of muscle aldolase. 相似文献
5.
Interaction of manganese with fragments, complementary fragment recombinations, and whole molecules of yeast phenylalanine specific transfer RNA 总被引:3,自引:0,他引:3
Binding of Mn2+ to the whole molecule, fragments and complementary fragment recombinations of yeast tRNAPhe, and to synthetic polynucleotides was studied by equilibrium dialysis. The comparison of the binding patterns of the fragments, fragment recombinations and synthetic polynucleotides with that of intact tRNAPhe permits reasonable conclusions concerning the nature and location of the various classes of sites on tRNAPhe. Binding of Mn2+ to intact tRNAPhe consists of a co-operative and a non-co-operative phase. There are about 17 “strong” sites and several “weak” ones. Five of the 17 strong sites are associated with the co-operative phase. This phase is completely lacking in the binding of Mn2+ to tRNAPhe fragments (5′-, 3′-, 5′-, 3′-), poly-(A):poly(U) and poly(I):poly(C) helices, and single stranded poly(A) and poly(U). This argues that the co-operative sites arise from the tRNA tertiary structure. This conclusion is further strengthened by the observation that cooperativity is present in a tRNAPhe molecule which has been split in the anticodon loop, but it is absent in one which has been split in the extra loop. It is in the vicinity of the latter loop, but not the former, that tertiary interactions are seen in the crystal structure. The remaining 12 strong sites are “independent” and appear to be associated with cloverleaf helical sections. 相似文献
6.
L K Russell S A Kirkley T R Kleyman S H Chan 《Biochemical and biophysical research communications》1976,73(2):434-443
Oligomycin Sensitivity Conferral Protein (OSCP) and an F1-ATPase Binding Protein were isolated from F1-depleted rat liver mitochondrial membrane. Their molecular weights on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea were 22,500 and 8,500 respectively. When incubated with liver TUA (trypsin, urea and ammonia-treated) submitochondrial particles, the binding protein was effective in the binding of F1 to the particles with the resultant particle-bound ATPase activity not oligomycin sensitive. When OSCP was then incubated with the reconstituted membrane-bound ATPase, its activity became oligomycin sensitive. These results suggest that, first; the binding protein, but not OSCP, connects F1-ATPase to the membrane of rat liver mitochondria and maybe to the “stalk”, if indeed there is a stalk in mitochondrial membrane ATPase complex; and second; the function of OSCP is solely to render the ATPase activity sensitive to oligomycin and other similar inhibitors. 相似文献
7.
8.
Rabbit muscle pyruvate kinase is inactivated by 2,3-butanedione in borate buffer. The inactivation follows pseudo-first-order kinetics with a calculated second-order rate constant of 4.6 m?1 min?1. The modification can be reversed with almost total recovery of activity by elimination of the butanedione and borate buffer, suggesting that only arginyl groups are modified; this result agrees with the loss of arginine detected by amino acid analysis of the modified enzyme. Using the kinetic data, it was estimated that the reaction of a single butanedione molecule per subunit of the enzyme is enough to completely inactivate the protein. The inactivation is partially prevented by phosphoenolpyruvate in the presence of K+ and Mg2+, but not by the competitive inhibitors lactate and bicarbonate. These findings point to an essential arginyl residue being located near the phosphate binding site of phosphoenolpyruvate. 相似文献
9.
S Takemori H Sato T Gomi K Suhara M Katagiri 《Biochemical and biophysical research communications》1975,67(3):1151-1157
A cytochrome P-450, which is functional in the steroid methylene 11β-hydroxylation (P-45011β), has been purified to a protein weight of 85 kg per heme from bovine adrenocortical mitochondria. The purification is accomplished in the presence of deoxycorticosterone as a substrate stabilizer. The procedure involved solubilization of sonicated mitochondrial pellets, ammonium sulfate fractionation, alumina Cγ gel treatment and aniline-substituted Sepharose 4B chromatography.The purified preparation when freed from deoxycorticosterone, has a low spin type absorption spectrum which can rapidly be converted into a typical high spin substrate-bound form by the addition of an 11β-hydroxylatable steroid, either deoxycorticosterone or testosterone. The preparation exhibits high 11β-hydroxylase activity and is free from the cholesterol side-chain cleavage cytochrome P-450 (P-450scc).The purified P-45011β, when submitted to SDS-polyacrylamide gel electrophoresis, exhibits a single protein band (molecular weight of 46 kilodaltons) which is clearly distinguished from P-450scc. As determined by the sedimentation equilibrium method, the molecular weight of the guanidine-treated P-45011β is estimated to be 43 kilodaltons. 相似文献
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11.
Defatted liver fatty acid binding protein (FABP) reverses the inhibitory effect of palmitoyl-CoA on adenine nucleotide transport in rat liver mitochondria; addition of titrating amounts of FABP to mitochondria pretreated with palmitoyl-CoA stimulates nucleotide transport and that activation parallels the removal of the inhibitor from mitochondria. This effect is specific only for FABP; all other cytosolic proteins which do not bind fatty acids do not influence nucleotide transport activity. Addition of free fatty acids (which can compete for ligand binding sites on FABP) to mitochondria pretreated with palmitoyl-CoA interferes with the reversal activity of FABP. Adding FABP alone to freshly isolated mitochondria also activates nucleotide transport activity suggesting that the originally submaximal activity is probably due to the presence of endogenous long-chain acyl-CoA esters in the mitochondrial preparation. Because FABP is present in relatively high concentration in most mammalian cells, these observations offer a likely explanation of why the potent inhibitory effects of long-chain acyl-CoA esters on adenine nucleotide transport in isolated mitochondria are not seen in the intact cell. 相似文献
12.
A simple and sensitive colorimetric method for the determination of long-chain free fatty acids in subcellular organelles 总被引:12,自引:0,他引:12
A colorimetric assay for the determination of long-chain free fatty acids (FFA) is described. The FFA were extracted from subcellular organelles with chloroform:heptane:methanol. The copper soaps of FFA were determined colorimetrically with diphenylcarbazide. There are three advantages to employing the present modified procedure. (a) The sensitivity has been increased approximately twofold over that of the previous procedure of K. Falholt, B. Lund, and W. Falholt (1973, Clin. Chim. Acta46, 105–111); (b) it takes less time to complete the assay compared to the tedious procedures currently available; and (c) the presence of bovine serum albumin, a known FAA-binding protein, does not interfere with the assay procedure. The assay shows a linear response over the range of 10 to 130 nmol of FFA. The recovery of free fatty acids from mitochondria is 99%. 相似文献
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14.
Binding of nonsuppressible insulinlike activity to human serum. Evidence for a carrier protein. 总被引:5,自引:0,他引:5
When 125I-labeled nonsuppressible insulinlike activity—soluble in acid/ethanol (NSILA-S) is incubated with human serum between 10 and 20% of the radioactivity are bound to serum proteins and can be displaced specifically by cold NSILA-S. Chromatography of the incubation mixture on Sephadex G-200 at pH 7.5 reveals three peaks of radioactivity in the large molecular weight region and a fourth one corresponding to low molecular unbound labeled NSILA-S. An excess of cold NSILA-S during preincubation leads to the disappearance of the two major large molecular weight peaks and to a concomitant increase of the peak eluting in the low molecular weight range. Binding of 125I-labeled NSILA-S is highly sensitive to small concentrations of cold NSILA-S, whereas insulin, ACTH and human growth hormone are completely ineffective in displacing bound 125I-labeled NSILA-S. NSILA-S preparations of different purity show displacement according to their specific biological activities. Furthermore, binding of 125I-labeled NSILA-S to serum pH- and time-dependent and displays saturation characteristics. Chromatography of serum on Sephadex G-200 with 0.15 m acetic acid/0.15 m NaCl localizes the binding fraction in the 50,000–70,000 molecular weight range. Boiling of serum for 5 min abolishes binding completely.These studies help explain why the molecular weight of NSILA varied considerably from one group of investigators to the other. 相似文献
15.
The relationship between the molecular weight and the number of subunits in oligomeric globular proteins consisting of identical subunits has been analyzed. It has been shown that the molecular weights of the subunits are distributed about a mean value of 48,000 and consequently that the molecular weights of the native oligomeric proteins are distributed in clearly distinguishable molecular weight regions. This observation allows the probability of a particular oligomeric structure to be predicted from a measurement of the oligomer molecular weight alone, which is useful in a number of types of study of protein structure, particularly comparative studies. Calculations have been performed which suggest that there is no thermodynamic limitation, in terms of the subunit interactions themselves, to the size of an oligomeric protein with a given number of subunits. Rather, an individual polypeptide chain itself has inherent size limitations, which consequently limits the molecular weight of the corresponding oligomer. 相似文献
16.
B A Robbins S Y Wong Y Hatefi Y M Galante 《Archives of biochemistry and biophysics》1981,210(2):489-497
Antibody raised in rabbits against Complex V (miochondrial ATP synthetase complex) purified from beef heart mitochondria cross-reacted with Complex V and submitochondrial particles from beef heart, beef adrenals, and rat liver as shown by double-diffusion and rocket immunoelectrophoresis analysis. Of the various isolated and purified components of Complex V, only the oligomycin sensitivity-conferring protein showed strong reactivity with the anti-Complex V antibody, soluble F1-ATPase reacted very faintly, while F6 and ATPase inhibitor protein showed no precipitin lines. Crossed immunoelectrophoresis indicated that antigenic determinants recognized by the antibody were present on OSCP and possibly on the dicyclohexylcarbodiimide-binding protein. The components of Complex V could be precipitated from beef heart submitochondrial particles dissolved in Triton X-100 and pretreated with control IgG. When the composition of the immunoprecipitate was compared to that of purified Complex V, all the constituent polypeptides of the latter were present in the immunoprecipitate, except for one polypeptide in the low-molecular-weight region. Incubation of Complex V or submitochondrial particles with the anti-Complex V antibody in the absence of Triton X-100 caused inhibition of ATP-Pi exchange but not of ATPase activity. In the presence of Triton X-100, oligomycin sensitivity of Complex V was lost and the antibody was able to inhibit also the ATPase activity. The enzymic activity of soluble F1-ATPase was unaffected by the antibody in the absence or presence of Triton X-100. These results suggest that the anti-Complex V antibody might be a useful tool for identifying and probing the role of Complex V components involved in energy transduction. 相似文献
17.
Roger A. Freedman Julia A. MacLaughlin Milton M. Weiser 《Archives of biochemistry and biophysics》1981,206(2):233-241
A previous study of energy-independent in vitro Ca2+ uptake by rat intestinal epithelial membrane vesicles demonstrated that uptake by Golgi membrane vesicles was greater than that by microvillus or lateral-basal membrane vesicles, was markedly decreased in vitamin D-deficient rats, and responded specifically to 1,25-(OH)2D3 repletion (R. A. Freedman, M. M. Weiser, and K. J. Isselbacher, 1977, Proc. Nat. Acad. Sci. USA74, 3612–3616; J. A. MacLaughlin, M. M. Weiser, and R. A. Freedman, 1980, Gastroenterology78, 325–332). In the present study, properties of Ca2+ uptake and release by intestinal Golgi membrane vesicles have been investigated. The initial rate of uptake was found to be saturable, suggesting carrier-mediated uptake. Uptake was markedly inhibited by Mg2+ and Sr2+, but not by Na+ or K+. Lowering the external [H+] or raising the internal [H+] resulted in enhancement of the initial rate of uptake; the intial rate was found to correlate with the internal-to-external [H+] gradient. The initial rate of uptake could be enhanced by preloading the vesicles with MgCl2 or SrCl2 but not CaCl2, NaCl, or KCl. Vesicles preloaded with K2SO4 failed to show enhanced uptake in the presence of valinomycin, suggesting that enhancement in uptake by vesicles preloaded with MgCl2 was not due to transmembrane potentials. The internal volume of the Golgi membrane vesicles was determined and found to be 9 μl/mg protein; this volume could accomodate less than 1% of the Ca2+ uptake maintained at equilibrium. Therefore, the remainder of the Ca2+ taken up was presumably bound to the Golgi membranes. A dissociation constant of 3.8 × 10?6m was found for this binding. The bound Ca2+ could be rapidly released by external Mg2+ or Sr2+, but not Ca2+, Na+, or K+. Release of bound Ca2+ could also be induced by raising the [H+] of the external medium. Failure of external Ca2+ to release bound Ca2+ suggested that the release induced by external Mg2+, Sr2+, or H+ was not due to competitive displacement of Ca2+ from its binding sites. These results indicated that Ca2+ uptake by intestinal Golgi membrane vesicles consists of carrier-mediated transport followed by binding of Ca2+ to the vesicle. The effects of H+, Mg2+, and Sr2+ on Ca2+ uptake and release suggest the existence of cation countertransport in the Golgi membrane vesicles. 相似文献
18.
Extracellular polysaccharides from Rhizobium trifolii, U226, Coryn and Bart A; Rhizobium phaseoli, U453; Rhizobium leguminosarum, U331; and Rhizobium meliloti, U27, after chemical modification, become substrates for certain β-d-glucan hydrolases. The Streptomyces (1 → 4)-β-d-glucan endohydrolase (EC 3.2.1.4) hydrolyses reduced and deacetylated rhizobial polysaccharides, both before and after removal of carboxyethylidene substituents, to produce a series of oligosaccharides. The Rhizopus arrhizus (1 → 3)-β-d-glucan endohydrolase (EC 3.2.1.6) hydrolyses only fully modified polysaccharides to yield, in the case of R. meliloti U27, laminarabiose, and, in all other instances, a disaccharide identified β-d-Gal-(1 → 3)-D-Glc. The same disaccharides are released by the Rhizopus enzyme from oligosaccharides produced by the action of the Streptomyces enzyme on fully modified polysaccharides. The results are discussed in relation to the available data for the structure of the polysaccharides and the specificity of the enzymes. 相似文献
19.
Mechanism of stimulation of Ca2+ plus Mg2+ -dependent phospodiesterase from rat cerebral cortex by the modulator protein and Ca2+ 总被引:2,自引:0,他引:2
The activity of phosphodiesterase (“Ca2+ plus Mg2+-dependent” phosphodiesterase) of a preparation from brain was found to depend on the presence of both Ca2+ and a protein factor called modulator. It was shown by gel filtration that the active enzyme-modulator complex (MW, about 200,000) was formed from the modulator (MW, 28,000) and an inactive enzyme (MW, about 150,000) in the presence of Ca2+. When EGTA was added, this active enzyme-modulator complex dissociated into inactive enzyme and modulator. These results, together with the finding of Teo and Wang that Ca2+ binds to the modulator, could explain the stimulatory effect of Ca2+ on this enzyme as follows: The “Ca2+ plus Mg2+-dependent” phosphodiesterase may exist as the inactive free form in equilibrium with the active enzymemodulator (Ca2+) complex, and Ca2+, through binding to the modulator, may shift the equilibrium towards formation of the active enzyme-modulator (Ca2+) complex, thereby increasing the activity of the mixture. On decreasing the concentration of Ca2+, the process is reversible. 相似文献
20.
John M. Waud Ellen Feldman Keith J. Schray 《Archives of biochemistry and biophysics》1981,206(2):291-295
Substrate analogs xylulose 1,5-bisphosphate, glucitol 1,6-bisphosphate, α-2,5-anhydroglucitol 1,6-bisphosphate, α-, β-methyl fructofuranoside 1,6-bisphosphate, ribulose 1,5-bisphosphate, ribulose 5-phosphate, and ribose 5-phosphate and inactivating agents 1-chloro-2, 4-dinitrobenzene, 4-hydroxymercuribenzoate, and pyridoxal phosphate were examined for their effects on liver aldolase. These studies support the use of the β-anomer and acyclic form as substrate. They also suggest that the liver enzyme active site is similar to the muscle enzyme but with a much weaker 6-phosphate binding site. 相似文献