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1.
The turnover of dihydrofolate reductase has been studied in rapidly dividing cells of mouse lymphoma L1210 and Lactobacillus casei. Cells in culture were exposed to [14C]leucine for 24 hr and the subsequent decrease in radioactivity of the enzyme was followed as a function of time. The L1210 enzyme was isolated in pure form by subjecting the cell sonicate to affinity chromatography on amethopterin-Sepharose. The L. casei cells were processed by a multistep procedure which yielded the pure enzyme in both of its principal forms: (I), without TPNH; and (II), containing an equimolal amount of noncovalently bound TPNH. The half-lives (t12>) of dihydrofolate reductase in the amethopterin-sensitive L1210 cells (L1210/S) and in the cells of a partially resistant subline (L1210/R2), characterized by an 8-fold increase in enzyme level, were 18 and 19 hr. When these cells were grown in the presence of sublethal concentrations of amethopterin, t12 values were increased to 39 and 90 hr. These results suggest that the transient increase in dihydrofolate reductase activity, observed when cells are exposed to amethopterin, is due largely to a decreased susceptibility of the enzyme-inhibitor complex to degradation. Bound TPNH also increases the half-life of dihydrofolate reductase as shown by the fact that forms (I) and (II) of the L. casei enzyme had tx12 values of approximately 3 and 9 hr.  相似文献   

2.
A simple and rapid method for the purification to homogeneity of ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) from the nitrogen-fixing filamentous cyanobacterium Anabaena sp. strain 7119 is described. A crude extract prepared by solubilizing the cells with a detergent was first partially purified on a DEAE-cellulose column and then chromatographed on 2′,5′-ADP-Sepharose 4B. Ligand-bound ferredoxin-NADP+ oxidoreductase was eluted by a linear gradient of NaCl. The overall procedure provided an enzyme purified about 400-fold with a yield of 60 to 70%. The final enzyme preparation exhibited a specific activity of 120 units/mg protein and an absorbance ratio A280A458 of 8.26. The enzyme protein migrated as a single band when subjected to polyacrylamide gel electrophoresis and chromatographed as a single isoelectric species under chromatofocusing.  相似文献   

3.
Isolation and characterization of isocitrate lyase of castor endosperm   总被引:1,自引:0,他引:1  
Isocitrate lyase (threo-DS-isocitrate glyoxylate-lyase, EC 4.1.3.1) has been purified to homogeneity from castor endosperm. The enzyme is a tetrameric protein (molecular weight about 140,000; gel filtration) made up of apparently identical monomers (subunit molecular weight about 35,000; gel electrophoresis in the presence of sodium dodecyl sulfate). Thermal inactivation of purified enzyme at 40 and 45 °C shows a fast and a slow phase, each accounting for half of the intitial activity, consistent with the equation: At = A02 · e?k1t + A02 · e?k2t, where A0 and At are activities at time zero at t, and k1 and k2 are first-order rate constants for the fast and slow phases, respectively. The enzyme shows optimum activity at pH 7.2–7.3. Effect of [S]on enzyme activity at different pH values (6.0–7.5) suggests that the proton behaves formally as an “uncompetitive inhibitor.” A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.9. Successive dialysis against EDTA and phosphate buffer, pH 7.0, at 0 °C gives an enzymatically inactive protein. This protein shows kinetics of thermal inactivation identical to the untreated (native) enzyme. Full activity is restored on adding Mg2+ (5.0 mm) to a solution of this protein. Addition of Ba2+ or Mn2+ brings about partial recovery. Other metal ions are not effective.  相似文献   

4.
5.
《Insect Biochemistry》1985,15(1):35-44
Isolation of glutathione S-transferase from the New Zealand grass grub, is complicated by the marked loss of activity from crude homogenates. This loss may be due to proteolysis or to modification by endogenous chemicals. The effect may be minimized by immediate fractionation with ammonium sulphate and by inclusion of 5mM glutathione in homogenates.Two enzymes species, isoelectric at pH 8.7 and 5.9 respectively, could be isolated by ammonium sulphate fractionation, affinity chromatography, anion exchange chromatography and chromatography on hydroxyl apatite. They had different substrate specificities and had differing subunit structure. The pI 8.7 enzyme appeared to be a homodimer of subunits of Mr 23,700 and the pI 5.9 enzyme one of subunit Mr 22,500.A third major enzyme species, isoelectric at pH 4.3 differed from the other two enzymes in having low affinity for the affinity matrix. This preparation was heterogeneous. The enzymically active species in this preparation had the same molecular weight as that of the pI 8.7 enzyme, had a very similar substrate specificity to the basic enzyme species and was characterized by kinetic parameters almost identical to those of the pI 8.7 enzyme.  相似文献   

6.
NADH-cytochrome b5 reductase activities in hemolysates of young and old human erythrocytes, and in hemolysates of rabbit reticulocytes and erythrocytes were measured after the separation of the enzyme from the bulk of hemoglobin only by isoelectric focusing. In any cases, a single main peak of the enzyme activity was detected after the electrophoresis in the fraction with pH 6.8 and 8.3 for human and rabbit red cells, respectively. The rabbit enzyme showed more than 30 times higher enzyme activity than that of human erythrocytes under the standard assay conditions. Significant differences of Micahelis constants for cytochrome b5 of the enzyme were found between young and old human erythrocytes, and also between human and rabbit red cells.  相似文献   

7.
Luit Slooten  Adriaan Nuyten 《BBA》1981,638(2):305-312
(1) The ATPase enzyme in untreated chromatophores from Rhodospirillum rubrum is in a low-activity state (designated as E°). It can be activated by application of a transmembrane Δ\?gmH+ generated by light-induced electron transport, or by application of acid-base jumps. (2) After rapid dissipation of the light-induced Δ\?gmH+, the active state of the ATPase enzyme decays (in the absence of added substrates or products) to a low-activity state (designated as E′), with a half-time of the order of 2–4 min. This state differs from E° in that E′ (but not E°) can be rapidly reactivated by addition of substrate, but only when the Mg2+ concentration is kept below 20–30 μM. Since this is characteristic of an activated enzyme containing tightly bound ADP (Slooten, L. and Nuyten, A. (1981) Biochim. Biophys. Acta 638, 313–326), it is suggested that release of endogenous, tightly bound ADP is one of the factors involved in activation of the ATPase enzyme.  相似文献   

8.
Crystals of actinidin, a thiol protease from the fruit of Actinidia chinensis, which are suitable for high-resolution X-ray diffraction studies, have been obtained. The space-group is P212121, with a = 78.1 A?, 6 = 81.2 A? and c = 33.0 A?. The asymmetric unit contains one molecule, of molecular weight about 26,000.  相似文献   

9.
A protein which contains a cobalt porphyrin was isolated from the sulfate reducer Desulfovibrio gigas. This protein has a molecular weight of approximately 16,700 daltons and is acidic, having an iso-electric point at 3.7. The N-terminal residue was shown to be threonine, and a cobalt analysis gave 0.8 cobalt atoms/molecule, suggesting the presence of a single prosthetic group. The protein has a violet color with absorption bands typical of a metal porphyrin center with maxima at 420 nm, 580 nm with a shoulder at 550 nm. The ratio A420(γ)A588(α) is 2.1. The protein has no electron paramagnetic resonance (e.p.r.) spectrum, and as the visible spectrum suggests, it probably contains diamagnetic CoIII porphyrin. However the cobalt centre appears to be protected from reduction by sodium dithionite or sodium borohydride. Attempts at ligand substitution with strong nucleophiles such as CN, causes a slight spectral shift to higher wavelenghts. The cobalt porphyrin can be extracted from the protein with an acidified acetone solution, indicating that it is not covalently bound to the protein.  相似文献   

10.
A method for calculating the rate constant (KA1A2) for the oxidation of the primary electron acceptor (A1) by the secondary one (A2) in the photosynthetic electron transport chain of purple bacteria is proposed.The method is based on the analysis of the dark recovery kinetics of reaction centre bacteriochlorophyll (P) following its oxidation by a short single laser pulse at a high oxidation-reduction potential of the medium. It is shown that in Ectothiorhodospira shaposhnikovii there is little difference in the value of KA1A2 obtained by this method from that measured by the method of Parson ((1969) Biochim. Biophys. Acta 189, 384–396), namely: (4.5±1.4) · 103s?1 and (6.9±1.2) · 103 s?1, respectively.The proposed method has also been used for the estimation of the KA1A2 value in chromatophores of Rhodospirillum rubrum deprived of constitutive electron donors which are capable of reducing P+ at a rate exceeding this for the transfer of electron from A1 to A2. The method of Parson cannot be used in this case. The value of KA1A2 has been found to be (2.7±0.8) · 103 s?1.The activation energies for the A1 to A2 electron transfer have also been determined. They are 12.4 kcal/mol and 9.9 kcal/mol for E. shaposhnikovii and R. rubrum, respectively.  相似文献   

11.
The isoelectric points of the blood group A1, A2 and B gene-associated glycosyltransferases in human ovarian cyst fluids were found by isoelectric focusing to be in the pH range 9.5–10. The A1 and B transferases in serum had isoelectric points similar to those of the enzymes in cyst fluids but A2 transferases in serum had considerably lower isoelectric points, in the pH range 6–7. The difference in the pI values of the A1 and A2 transferases in the serum of a donor of the genotype A1A2 enabled the two enzymes to be preparatively separated by the isoelectric focusing technique. The dissimilarity in the pI values of the A2 transferases in ovarian cyst fluids and serum samples indicates that the isoelectric point arises from a post-translational modification of the enzyme protein.  相似文献   

12.
13.
Quantitative structure-activity relationships have been formulated for the inhibition of dihydrofolate reductase from bovine and rat liver by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(3-X-phenyl)-s-triazines. The best correlations are: bovine, log1C = 1.05π3 ? 1.21 log (β·10π3 + 1) + 6.64, where log β = ?0.736 and r = 0.955 for 28 congeners; rat, log1C = 1.12π3 ? 1.34 log (β·10π3 + 1) + 6.29, where log β = ?0.978 and r = 0.977 for 18 congeners. In these expressons C is the molar concentration of inhibitor producing 50% inhibition of the enzyme, π3 is the octanol/water hydrophobic parameter for the 3-X-phenyl substituent, β is an iteratively derived coefficient, and r is the multiple least squares correlation coefficient. The implications of these bilinear models are discussed and compared with earlier work by B. R. Baker.  相似文献   

14.
Theory is presented relating to the reversible interaction of an f-valent acceptor, A, with a bivalent ligand, B, which leads to the formation of a series of complexes comprising networks of alternating A and B molecules. An explicit expression is derived for the overall extent of reaction in terms of the total molar concentrations of reactants (mA and mB), the valency of the acceptor and the site-binding constant, k, governing the equilibria. It is shown by differentiation of this expression holding mA (or mB) fixed that relations are available for the independent evaluation of f and k from a combination of precipitin and radioimmunoassay experiments. Moreover, it is established that dilution with solvent (mA/mB fixed) cannot lead to the appearance of a precipitate with this type of crosslinking system. The latter observation forms the background for the development of theory pertaining to the joint operation of ligand dimerization, 2B?B2, and crosslinking of the multivalent acceptor with bivalent B2. The theoretical examination of this system is developed in terms of site-probability functions and involves the delineation of unique solutions for the extent of crosslinking reaction aided by the definition of the extent of binding in defined limits. It is shown with the use of numerical examples that the system involving self-associating ligand may result in the appearance of a precipitate on dilution with solvent and the conditions for the operation of this phenomenon are elucidated. It is noted that other types of ligand self-interaction may lead to similar effects in crosslinking systems, and the general principles emerging from this study are discussed in terms of systems in which antibody ligands are known to be involved in association reactions or are suspected to be so involved on the basis of precipitation effects observed on dilution with solvent.  相似文献   

15.
Neutrophil myeloperoxidase from a patient with chronic myelocytic leukemia was isolated under conditions designed to minimize proteolysis. Those methods yielded an α2β2 form of myeloperoxidase from normal individuals. Purified enzyme from the patient had electronic absorbances (A430A280 = 0.85), enzymatic activity, and electrophoretic and Chromatographic behavior indistinguishable from that of normal myeloperoxidase. Edman degradation and physical studies after reduction and denaturation, however, showed that as compared to normal enzyme, one 55,000-dalton α subunit of the patient's myeloperoxidase was replaced by a 39,000-dalton peptide with a different amino-terminal sequence, a mixture of smaller peptides, and an heme derivative. Myeloperoxidase from the leukemic neutrophils appeared to have been partially degraded in vivo by lysosomal proteases.  相似文献   

16.
A-Side (4-R)-(4-2H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol-d6 to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4-S) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize to oxidize glucose-1-d in 40% dimethyl aulfoxide. Subsequent purifieation of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 m LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final A260A340 ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifieally deuterated substrates by coupled enzymatic synthesis. L-Malate-2-d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase.  相似文献   

17.
The kinetic α-secondary deuterium isotope effect, kHkD, for the pH-independent hydrolysis of nicotinamide riboside, yielding nicotinamide and ribose, in water at 25 ° is 1.14, establishing that this reaction proceeds with unimolecular substrate decomposition to yield a carboxonium ion, or related species, in the rate-determining step. Surprisingly, the corresponding isotope effect for the base-catalyzed decomposition of the same substrate is 1.12, a value indicating considerable sp2 character at the Cl′ position in the transition state for this reaction. A similar result, kHkD = 1.15, was obtained for base-catalyzed hydrolysis of NAD+. The kinetic alpha deuterium isotope effect for the pig brain NAD glycohydrolasecatalyzed hydrolysis of nicotinamide riboside is 1.08. This value suggests that CN bond cleavage to form an intermediate carboxonium ion, or structurally related species, is at least partially rate-determining. In contrast, the corresponding value for the hydrolysis of this substrate catalyzed by Escherichia coli nicotinamide ribonucleotide glycohydrolase is very near unity, a result consistent with several interpretations including a rate-determining enzyme isomerization reaction.  相似文献   

18.
Quantitative expressions have been developed for systems such as yeast reductions where competing enzymes act on one substrate to yield two enantiomeric products. These expressions relate the observed stereochemical variables, the extent of conversion (C), the optical purity expressed as enantiomeric excess (ee), and the initial substrate concentration (A0) to the kinetic parameters KR and KS (apparent Michaelis constants) and y (VRVS, the ratio of maximal velocities) of such competing enzymes. The expressions have been experimentally verified using a purified competing enzyme system of l- and d-lactic dehydrogenases. Furthermore, the enantioselective reduction of β-keto esters by intact yeast cells has been examined by means of this kinetic analysis.  相似文献   

19.
The cyclic AMP receptor protein (CRP) of Escherichia coli has been crystallized. The crystals are orthorhombic, space group P212121, a = 46.5 A?, b = 97.1 Å, c = 105.4 A?, with one dimeric CRP molecule per asymmetric unit.  相似文献   

20.
A two cluster (4Fe4S) ferredoxin and a rubredoxin have been isolated from the sulfur-reducing bacterium Desulfuromonas acetoxidans. Their amino acid compositions are reported and compared to those of other iron-sulfur proteins.The ferredoxin contains 8 cysteine residues, 8 atoms of iron and 8 atoms of labile sulfur per molecule; its minimum molecular weight is 6163. The protein exhibits an absorbance ratio of A385A283 = 0.74. Storage results in a bleaching of the chromophore; the denatured ferredoxin is reconstitutable with iron and sulfide. The instability temperature is 52°C.The rubredoxin does not differ markedly from rubredoxins from other anaerobic bacteria.  相似文献   

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