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1.
The kinetics of the hydrolysis of -nitrophenyl trimethylacetate catalyzed by yeast carboxypeptidase have been measured under conditions of substrate in excess and indicate that the release of -nitrophenol in two discrete stages can be observed. A fast release of -nitrophenol in a concentration approximating that of the enzyme is seen initially, followed by a slow release, corresponding to the “turnover” reaction of the ester. These observations provide strong support for the postulation of a three step reaction sequence including the formation and decomposition of not only a Michaelis complex but also an acyl-enzyme species. 相似文献
2.
Addition of -nitroanisole to a reaction mixture containing phenobarbital-pretreated rabbit liver microsomes brings about an increase the reoxidation rate of NADH-reduced cytochrome b5. Addition of partially purified cytochrome b5 to a solution containing microsomes results in a marked increase in both NADH- and NADPH-dependent O-demethylation of -nitroanisole. -Nitroanisole also increases the rate of NADH mediated cytochrome P-450 reduction. From these and other results described in the Discussion section, we confirm that electrons required for NADH-dependent O-demethylation of -nitroanisole is transfered from NADH to cytochrome P-450 via cytochrome b5 and that cytochrome P-450 is the enzyme which catalyzes -nitroanisole O-demethylation. 相似文献
3.
S G Bhat M Ramanarayanan C S Vaidyanathan 《Biochemical and biophysical research communications》1973,52(3):834-842
A soluble fraction of catalyzed the hydroxylation of mandelic acid to -hydroxymandelic acid. The enzyme had a pH optimum of 5.4 and showed an absolute requirement for Fe2+, tetrahydropteridine, NADPH. -Hydroxymandelate, the product of the enzyme reaction was identified by paper chromatography, thin layer chromatography, UV and IR-spectra. 相似文献
4.
The disruption of erythyrocyte membrane cytoskeletons brought about by treatment with (PMBS) has been followed by measurements of turbidity and the binding of 203Hg-labelled PMBS. After pretreatment with to block readily reactive sulphydryl groups, incubation with [203Hg]PMBS showed incorporation of approximately 4 moles radiolabel per mole of spectrin and one per mole of actin. The incorporation of radiolabel paralleled the decrease in turbidity, and the labelling of spectrin paralleled that of actin. The kinetics were pseudo first order, and the pH dependence of the observed rate constant indicated a normal value for the sulphydryl group involved. The calculated second-order rate constant for the reaction of the sulphydryl anion with PMBS, however, was several orders of magnitude less than expected from model compound studies. The results suggest that association between spectrin and actin may result in the steric hindrance of reactivity of a limited number of sulphydryl groups in each protein. Disruption of the spectrin-actin association may then be linked to the modification of the sulphydryl groups. 相似文献
5.
The binding of the gamma labeled neuroleptic, 77Br--bromosprioperidol, in the rat brain was examined . This binding parallels the binding of 3H-spiroperidol, in that binding is especially high in dopaminergically innervated areas, is saturable, and is displaced by high doses of unlabeled spiroperidol (1–5). Thus, 77Br--bromospiroperidol is a suitable ligand for use in gamma ray imaging techniques for monitoring of receptor binding. 相似文献
6.
R W Furlanetto M Mochizuki E T Kaiser 《Biochemical and biophysical research communications》1974,58(1):192-196
-α-chloroacetophenone oxime has been found to inactivate papain rapidly at pH 7 and 25.0O in a 1:1 stoichiometric fashion as measured by rate assays with -nitrophenyl -benzyloxycarbonylglycinate and sulfhydryl group titrations with 5,5′-dithiobis-(2-nitrobenzoic acid). By the use of a 14C label in the halomethyl function a similar stoichiometric reaction with papain could be demonstrated for -α-bromoacetophenone oxime despite the rapidity of the competitive nonenzymatic solvolysis of the latter compound under the conditions employed. These results indicate that a new class of reactive modifying agents, α-haloalkyl oximes, can be used for the selective alkylation of catalytically essential functional groups in enzyme active sites. 相似文献
7.
Joseph J. Defrank Douglas W. Ribbons 《Biochemical and biophysical research communications》1976,70(4):1129-1135
A mutant strain (PL pT ) of PL, has been isolated for its inability to growth with -cymene as carbon source. The mutant oxidizes (and -cumate) to a compound (λmax 293 nm) which is readily converted to 3-hydroxy--cumate by acid. 4-Trifluoromethylbenzoate is oxidized by the mutant to an acid-stable intermediate (λmax 277nm) that has been crystallized. The spectral properties (u.v., i.r., NMR and mass) of this metabolite are consistent with those expected for a 2,3-dihydro-2,3-dihydroxy derivative of 4-trifluoromethylbenzoate. Further support of this structure was provided by elemental analysis and the properties of two derivatives of the metabolite, 4-trifluoromethyl-3-hydroxybenzoate and an acetonide formed with 2,2-dimethoxypropane. The stability of a product obtained by treatment of the dihydrodiol metabolite with triacetylosmate indicates that it is the . 相似文献
8.
The ionization of fatty acids, fatty amines and acids incorporated in phosphatidylcholine single-walled vesicles has been measured. The guest molecules have been specifically enriched with 13C and titrated by using NMR spectroscopy. The apparent p of fatty acids in phosphatidylcholine bilayers is 7.2–7.4 and those of fatty amines are approx. 9.5. These p values depend on many different parameters related to the structure of the lipid/ solution interface, to the composition of the aqueous medium and to the localization of the ionizable groups. A special sensitivity to the ionic strength and to the surface charge has been found. A positive surface charge decreases the p value whereas a negative one increases it, the total range of variation being 2.5–3 units. In a qualitative macroscopic interpretation, it is proposed that p is essentially determined by the low polarity of the lipidic matrix. 相似文献
9.
Four ethyl -nitrophenyl alkylphosphonates were studied for the inhibition of elastase. A pH-dependence study using the assay substrate BOC-Ala-ONp or the phosphonate inhibitors showed the participation of an ionizing group with an apparent pKa of 6.9 and a decrease of reaction or inhibition at higher pH. Out of the four compounds investigated ethyl -nitrophenyl pentylphosphonate was found to be the best inhibitor of elastase as judged from the value of . This value, which is the measure of inhibitory capacity, is the highest reported so far for the inhibition of elastase. 相似文献
10.
Earlier work has shown that streptokinase and human plasminogen form a stoichiometric complex in which the presence of a functional active center can be detected by reaction with the active center-specific reagent, -nitrophenyl--guanidinobenzoate. The complex possesses activator activity, i.e. it catalyzes the conversion of plasminogen to plasmin. Evidence is presented to show that pancreatic trypsin inhibitor abolishes both the activator activity and the ability to react with the active center-specific reagent. This is accomplished, not by displacement of streptokinase, but by the formation of a ternary complex with streptokinase-plasminogen. 相似文献
11.
12.
A major fraction of the protein sulfhydryl groups of human erythrocyte membranes can be oxidized to disulfide bonds by the lipid soluble reagent, diamide, and the hydrophilic reagent, tetrathionate. Furthermore, the same fraction also reacts with the monofunctional reagent, . About 20% of the SH groups, however, do not react with any of these agents even upon prolonged treatment and increased concentrations.These ‘non-reacting’ SH groups were now localized by a procedure involving blockage of the accessible SH groups by non-labelled or by diamide, subsequent isolation and solubilization of the membranes in SDS and labelling of the now accessible, residual SH groups with .The distribution of the radioactivity over the peptide fractions shows that the non-reacting SH groups are mainly localized in the intrinsic proteins, while essentially all of the SH groups of the extrinsic protein, spectrin, are reactive.After solubilization of the membranes with Triton X-100 the non-reacting SH groups became reactive towards . It is proposed that lack of reaction of SH groups in the native membranes is due to their localization within the hydrophobic core of the membrane. 相似文献
13.
The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca2+, Mg2+-activated ATPase of . In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an mutant of was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked. 相似文献
14.
S H de Bruin H S Rollema L H Janssen G A van Os 《Biochemical and biophysical research communications》1974,58(1):210-215
The immobilization of glucose oxidase, a glycoenzyme from consisting of 16% carbohydrate, has been achieved by oxidizing its carbohydrate residues with periodic acid followed by coupling the activated enzyme to water-insoluble -aminostyrene. At pH 5.6 and 25°, approximately 60% of the carbohydrate residues are oxidized, but the enzyme retains full activity. No oxidation of any amino acid residue is evident. The enzyme-polymer conjugate derived from this activated enzyme retains full activity and even shows a slightly enhanced thermal stability at 60° compared with the soluble native and oxidized glucose oxidases. 相似文献
15.
H. Fredrick Vernay Eliezer S. Rachaman Ronald Eby Conrad Schuerch 《Carbohydrate research》1980,78(2):267-273
Several 1-O-sulfonyl derivatives of d-galactopyranose having a participating benzoyl or p-methoxybenzoyl group at O-2 were synthesized from the corresponding d-galactopyranosyl chloride derivatives by use of silver p-toluenesulfonate or trifluoroethanesulfonate in acetonitrile. The reaction of the derivatives with several alcohols in various solvents at different times and temperatures served as model reactions to determine the best conditions for synthesizing stereoselectively β-d-galactopyranosides in high yields. This method was used to prepare, in good yield, several β-d-galactopyranosyl-containing disaccharides. 相似文献
16.
Julian G. Domszy George A.F. Roberts 《International journal of biological macromolecules》1985,7(1):45-48
The reaction between chitosan and 2,4-dinitrofluorobenzene has been studied and suitable conditions established for hydrolysis of the product prior to determination of the extent of reaction by u.v./visible spectroscopy. The chromophore system in , the final product from the acid hydrolysis of N-(2,4-dinitrophenyl)chitosan, is unstable to heating in solution in either water or aqueous acid. The temperature of hydrolysis should therefore not exceed 50°C and at this temperature the u.v./visible absorption spectrum of is constant for up to 50 h. Complete reaction of the amine groups is not achieved under heterogeneous or homogeneous conditions, only approximately 50% of the available amine groups undergoing reaction under homogeneous conditions. This restricted reactivity results from the bulky N-(2,4-dinitrophenyl) residues shielding adjacent unreacted amine groups on the same chain, thereby preventing their reaction with 2,4-dinitrofluorobenzene. Such intramolecular steric hindrance would be expected to increase with increase in the free amine group content of the sample, due to the increase in the fraction of amine groups occurring in sequence length of two or more, and an inverse relationship between the total initial free amine group content and the percentage of these that react with 2,4-dinitrofluorobenzene has been found 相似文献
17.
C H Seiter R Margalit R A Perreault 《Biochemical and biophysical research communications》1979,86(3):473-477
Cytochrome was chemically coupled to cytochrome oxidase using the reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) which couples amine groups to carboxyl residues. The products of this reaction were analyzed on 2.5–27% polyacrylamide gradient gels electrophoretically. Since cytochrome binds to cytochrome oxidase electrostatically in an attraction between certain of its lysine residues and carboxyl residues on the oxidase surface, EDC is an especially appropriate reagent probe for binding-subunit studies. Coupling of polylysine to cytochrome oxidase using EDC was also performed, and the products of this reaction indicate that polylysine, an inhibitor of the cytochrome reaction with oxidase, binds to the same oxidase subunit as does cytochrome , subunit IV in the gel system used. 相似文献
18.
Illumination of the chlorophyll light-harvesting complex in the presence of caused quenching of fluorescence emission at 685 nm (77 K) relative to 695 nm and covalent modification of light-harvesting complex polypeptides. Fluorescence quenching saturated with one p-nitrothiophenol bound per light-harvesting complex polypeptide (10–13 chlorophylls); maximal quenching occurred with one p-nitrothiophenol bound per light-harvesting complex polypeptides (190–247 chlorophylls). This result provides direct evidence for excitation energy transfer between light-harvesting complex subunits which contain 4–6 polypeptides plus 40–78 chlorophylls per complex.Illumination of chloroplasts or Photosystem II (PS II) particles in the presence of caused inhibition of PS II activity and labeling of several polypeptides including those of 42–48 kilodaltons previously identified as PS II reaction center polypeptides. In chloroplasts, inhibition of oxygen evolution accelerated p-nitrothiophenol modification reactions; DCMU or donors to PS II decreased p-nitrothiophenol modification. These results are consistent with the hypothesis that accumulation of oxidizing equivalents on the donor side of PS II creates a ‘reactive state’ in which polypeptides of PS II are susceptible to p-nitrothiophenol modification. 相似文献
19.
A series of 12α-hydroxy steroids with varying side chains was prepared, and their 24-hour acetylation yields were compared, l2α-Hydroxy-5β-pregnan-20-one () was prepared from 3α, 12α-diacetoxy-5β~pregnan-20-one () and also by side chain degradation of 12α-acetoxy-5β-cholanoic acid (). 21-Benzyl-5β-pregnan-12α-ol () was synthesized by hydrogenation of the 21-benzylidine derivative of ketone . 23-Pheny1-5β-norcholan-12α-ol () was obtained by the Grignard reaction of 2-phenyl-ethylmagnesium bromide and ketone , dehydration, hydrogenation and hydride reduction; a similar sequence produced 20-methyl-5β-pregnan-12α-ol (). The acetylation results (Table 11) imply that branching at C-20 may be more significant for 12α-hydroxyl reactivity than side chain length or type. An additional compound with an unbranched side chain, 21-nor-5β-cholan-12α-ol (), was synthesized by a Grignard reaction on the 21-bromo intermediate . Acetylation rates determined by glc indicate (Table 111) That compounds with unbranched side chains have 12α-hydroxyl groups about ten times as reactive as their analogs with 20-methyl groups. 相似文献
20.
The replication defective transducing phage λp3 carries a portion of the operon in the 2 region of the lambda phage. This operon segment contains the promoter, the operator, and the β-galactosidase gene, but does not contain the repressor gene. The gene can be expressed from both the inserted promoter and the phage promoter. When strain 594 (?, +) or JC6256 (Δ) is infected by λp3 in the absence of additional cyclic AMP, β-galactosidase synthesis is shown to be expressed from the phage promoter. When 594 (λ+) or JC6256 (λ+) is infected by λp3 in the presence of additional cyclic AMP and IPTG, β-galactosidase synthesis is shown to be expressed from the inserted promoter.The ability to separate the phage promoter from the inserted promoter for β-galactosidase expression will simplify the interpretation whenever λp5 is used. 相似文献