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991.
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
992.
993.
The NADP+ specific glutamate dehydrogenase from wild-type forms a stable binary complex with NADPH. This can combine with L-glutamate, α-ketoglutarate or the substrate analogue D-glutamate to form ternary complexes which can be distinguished by their different fluorescence properties. The affinity of the enzyme for NADPH diminishes with increases in pH or ionic strength of the solution. Experimental data obtained using modified glutamate dehydrogenases from mutant strains of suggest that the reduced-coenzyme binding sites observed fluorimetrically are the same as those observed by enzyme kinetics. 相似文献
994.
The enthalpy of the bioluminescent reaction has been studied by direct calorimetric methods. Bacterial luciferase, isolated from Beneckea harveyi (formerly strain MAV) has been used to catalyze the oxidation of reduced flavin mononucleotide (FMNH2) and a long chain aliphatic aldehyde (dodecanal, RCHO) by molecular oxygen to give the indicated products and blue-green light. The enthalpy measured for this process was found to be ΔHL = ?338.9 k.J (mol FMN)?1 (?81.0 kcal) at 25.00 °C and ?402.9 kJ (mol FMN)?1 (?96.3 kcal) at 7.00 °C. Calculations based on redox electrode potentials indicate a corresponding value of the free energy change, ΔGL = ?464.8 kJ (mol FMN)?1 (?111.1 kcal), at 25 °C. Measurements were performed in 0.15 m phosphate buffer, pH 7.0 and the values were arrived at by correcting the observed heats for the heat associated with the autoxidation process: FMNH2 + O2 ? FMN + H2O2; ΔHD = ?158.5 kJ (mol FMN)?1 (?37.8). These data and a detailed thermodynamic analysis have demonstrated the need for two parameters, referred to as the intrinsic free energy, ΔG1, and intrinsic enthalpy, ΔH1, which are functionally defined by the relations ΔGI = ΔGL ? uhvΔHI = ΔHL ? uhv, where u is the quantum yield of the reaction expressed in einsteins mole?1.These parameters reflect the thermochemistry of the bioluminescent reaction corrected for emitted photons. Thus, they are useful for comparing the thermochemistry of a chemiluminescent process. Their values for the bacterial luciferase system at 25 °C and pH 7.0 are ?391.6 and ?266.9 kJ (mol FMN)?1 (?93.6 and ?63.8 kcal), respectively, assuming a value of 0.3 for the quantum yield. The calorimetric data also suggest the existence of a long-lived species which persists after photon emission. 相似文献
995.
An exo-β-glucan hydrolase, present in the digestive juice of the snail, Helix pomatia, has been purified to homogeneity by chromatography on Bio-Gel P-60, Sephadex G-200, DEAE-cellulose, and DEAE-Sephadex. The enzyme degrades β-(1 → 3)-linked oligosaccharides and polysaccharides, rapidly and to completion, or near completion, yielding glucose as the major product of enzyme action. Mixed linkage (1→3; 1→4)-β-glucans are also extensively degraded and β-(1→6)- and β-(1→4)-linked glucose polymers are slowly degraded by the enzyme. This enzyme differs from other exo-β-glucanases, reported previously, in the broadness of its substrate specificity. The Km values for action on laminarin and lichenin are respectively 1.22 and 2.22 mg/ml; the maximum velocity of action on laminarin is approximately twice that on lichenin. The enzyme has a molecular weight of 82,000 as determined by polyacrylamide gel electrophoresis. Maximum activity is exhibited at pH 4.3 and at temperatures of 50–55 °C. 相似文献
996.
Possible effects of changes in net charge on protein hydrogen exchange rates were investigated by desalting hen egg-white lysozyme, which allowed its net charge to increase with decreasing pH in the acid region. Chloride ion-binding ratios, expressed as ratios of free to total Cl?, were measured with a chloride-specific electrode at pH 5 on a 2.4% solution of a five-time-desalted product. This ratio was used to show a 97% reduction of the 11% Cl? present in a commercial lysozyme preparation upon three passes of the enzyme through a column of ion-retardation resin. Net charges on the purified product were assigned from a combination of electrophoretic mobility and proton titration data gathered under minimal ionic strength conditions. The net charge on the desalted product increased by 1.64 units between pH 5.0 and 3.0. Hydrogendeuterium exchange studies on the purified lysozyme in D2O were obtained using the near-infrared region of a Cary 14R spectrophotometer. The rate-pD profile for k2, the rate constant for the intermediate class of exchanging hydrogens, showed a decrease in the apparent pD of minimum exchange rate of 0.3 units, when compared to that obtained earlier in 0.2 m added NaCl. However, the rate of exchange at pD minimum and the number of hydrogens in the class remained largely unaffected. A similar shift was observed for the rate-pD profile of the class 1 hydrogens. Thus, the effect of an increase in net positive charge is to shift the rate-pD profile to a lower pD. Moreover, the effect extended to the interior peptide hydrogens of this globular protein. Consequently, the exchange rates of all the observable hydrogens are altered by the net charge changes, and the effect appeared uniform. The shift can be accounted for quantitatively by applying electrostatic interaction terms to the acid and base catalytic constants characterizing the exchange process. The calculated electrostatic interaction factors in minimal salt and 0.2 m added NaCl were found to be 29 and 18% lower, respectively, than those obtained theoretically. Therefore, under conditions where changes in net charge may occur for a globular protein, the effect on hydrogen exchange rates can be estimated fairly well theoretically, especially at moderate ionic strengths. 相似文献
997.
998.
A permanent human lymphoblast culture was synchronized with repetitive thymidine blocks, and the changes in the levels of activity of four X-chromosome-linked enzymes were followed during the cell cycle. The four enzymes studied were phosphoglycerate kinase (PGK), α-galactosidase (α-Gal), hypoxanthine-guanine phosphoribosyltransferase (HGPRT), and glucose-6-phosphate dehydrogenase (G6PD). The levels of PGK and α-Gal activities increased simultaneously in G1 and in S, while HGPRT and G6PD increased close together in middle and late S. Therefore, different control mechanisms may be involved in the increases of the activities of these two sets of enzymes. 相似文献
999.
1000.
Twelve anthocyanins have been isolated from flax: the 3-glucosylrutinosides of pelargonidin, cyanidin and delphinidin; the 3-triglucosides of delphinid 相似文献