共查询到20条相似文献,搜索用时 31 毫秒
1.
Alexander Vardanis 《Biochemical and biophysical research communications》1984,125(3):947-953
The search for an unusual cyclic nucleotide-dependent protein kinase in nematodes represented an attempt to gain some insight into the proposed homology of the cAMP and cGMP-dependent protein kinases. Two species of protein kinase were found in high speed supernatants of the mycophagous nematode . One of the two, bound to DEAE cellulose and was eluted from it in a manner characteristic of the type I cAMP kinase. The enzyme had high affinity for cAMP and dissociated upon binding to the cyclic nucleotide, as judged by the fact that catalytic activity did not bind to a cAMP affinity column. The second enzyme did not bind to DEAE. Unexpectedly, it too had high affinity for cAMP and much lower affinity for cGMP (unlike the kinase from insects). The holoenzyme bound tightly to the cAMP affinity column and required a high concentration of the cyclic nucleotide for elution. This latter enzyme is the only example of a cAMP-dependent protein kinase that does not dissociate upon activation. 相似文献
2.
Controls of citrate synthase activity 总被引:3,自引:0,他引:3
P A Srere 《Life sciences》1974,15(10):1695-1710
The inhibition of citrate synthase by a variety of nucleotides and polycarboxylate compounds is not unexpected since many of the compounds are substrate analogs of citrate synthase. These effectors are interesting by virtue of the fact that many of them are intermediates and/or end products in the metabolic path of which citrate synthase can be considered the first committed step. As a consequence, it is possible to propose regulation of citrate synthase by ATP (or phosphorylation potential) by acyl CoA (acylation level) and NADH (redox potential). Aside from these putative controls, it is possible that the major control of citrate synthase activity is by changes in the concentration of its substrates acetyl CoA and oxalacetate.I discuss in this review the many factors that must be considered before one can decide whether or not interactions between metabolites and enzymes observed in an catalytic situation have metabolic relevance. These factors include 1) the concentrations of substrates at the enzyme site, 2) the concentrations of effectors at the enzyme site, 3) the presence of modifying substances, and 4) the difference in behavior of an enzyme at its concentration compared to its concentration . In the case of citrate synthase as is generally true for other enzymes, no accurate knowledge of these factors are available so that little can be said concerning the control of citrate synthase, which may be the result of all the factors acting in concert. The studies of effectors on enzymes can only serve as a guideline for parameters to study when techniques are available to study control of enzymes . 相似文献
3.
Charles D. Boyer Jack Preiss 《Biochemical and biophysical research communications》1978,80(1):169-175
Purification of starch branching enzymes from kernels of two nonlinked mutants of maize, and , showed the basis of the two mutations to be associated with branching enzymes I and IIb, respectively. Branching enzyme I from kernels purified as nonmutant branching enzyme I, but had an altered pattern of activity when amylose was used as a substrate. In addition to the typical fall in absorbance at high wavelengths (550–700 nm) of the amylose-iodine complex, branching of amylose by branching enzyme I caused an increase in absorbance at low wavelengths (400–550 nm). Branching enzyme IIb was undetected in extracts of kernels, while branching enzymes I and IIa appeared unaltered. Low umprimed starch synthase activity was also observed in DEAE-cellulose fractions of maize, but this activity was regenerated by the addition of any branching enzyme. 相似文献
4.
When retinol was incubated in the presence of ATP, UDP-galactose, magnesium ions and Triton X-100, with a whole homogenate of rat thyroid as an enzyme source the formation of a retinol containing phosphate compound was observed besides retinol galactoside described before. This phosphate compound is soluble in chloroform-methanol (3:2, ) and can be separated by chromatography on a column prepared with butanol-washed cellulose powder. The isolated compound was retinol pyrophosphate since it contained retinol and phosphate in a molar ratio of 1:2 shown by double isotopic labelling techniques and was found to be free of galactose. 相似文献
5.
G.Prem veer Reddy 《Biochemical and biophysical research communications》1982,109(3):908-915
Catalytic activity of thymidylate synthase, as measured , is tightly linked to S phase of the cell cycle in Chinese hamster embryo fibroblast cells. This activity, as measured , is found in all parts of the cell cycle. Thymidylate synthase activity in nuclear (karyoplast) extracts increased as the cells progressed from to S phase. This enzymatic activity in the nuclei of S phase cells is associated with the multienzyme complex (replitase) that also contained DNA polymerase and other enzymes of DNA replication and precursor synthesis. The degree of association of thymidylate synthase with replitase, which increased co-ordinately as the cells progressed from phase to S phase, coincided strongly with the level of activity of the enzyme. 相似文献
6.
Liliana Gianfreda Anna Maria Livolsi Maria Rosaria Scarfì Guido Greco 《Enzyme and microbial technology》1982,4(5):322-326
Cellobiose hydrolysis by β-d-glucosidase (β-d-glucoside glucohydrolase EC 3.2.1.21) can become the rate-limiting step in the hydrolysis of cellulosic wastes because of inhibition phenomena involving other enzymes of the cellulase complex. Enhancement of the overall rate can therefore be obtained by increasing the amount of β-d-glucosidase present in the reactor. Unfortunately, the thermal stability of β-d-glucosidase is rather poor compared to and cellobiohydrolase. A novel stabilization method is proposed that exploits the polarization phenomena that take place in an unstirred ultrafiltration membrane enzymatic reactor. As much as a 20-fold increase in half-life compared to the native enzyme is obtained by injecting small amounts of hydroxyethyl cellulose into the system. No reduction in enzyme activity levels is observed. 相似文献
7.
The shikimate pathway is essential for the biosynthesis of aromatic compounds. The seventh and last step is catalysed by chorismate synthase, which has an absolute requirement for reduced FMN in its active site. There are two classes of this enzyme, which are distinguished according to the origin of the reduced cofactor. Monofunctional chorismate synthases sequester it from the cellular environment whereas bifunctional enzymes can generate reduced FMN at the expense of NADPH. These bifunctional enzymes are found in fungi and the ciliated protozoan Euglena gracilis while all bacterial and plant enzymes are monofunctional. In this study, we introduce an in vivo screen, which is based on a chorismate synthase-deficient Saccharomyces cerevisiae strain, allowing the classification of hitherto uncharacterized chorismate synthases. This analysis revealed that bifunctionality is present in the enzymes of protozoan species. In contrast, all bacterial and plant enzymes tested are monofunctional. In addition, we demonstrate that a monofunctional chorismate synthase confers prototrophy in conjunction with a NADPH : FMN oxidoreductase indicating that bifunctionality is required due to the lack of free reduced FMN in fungal and possibly protozoan species. Interestingly, the distribution of bifunctional chorismate synthase concurs with the presence of a pentafunctional enzyme complex. 相似文献
8.
P M Yuan R W Gracy F C Hartman 《Biochemical and biophysical research communications》1977,74(3):1007-1013
3-Bromo- and 3-iodoacetol phosphates irreversibly inactivate methylglyoxal synthase. The substrate, dihydroxyacetone phosphate, and inorganic phosphate protect against the inhibition. Although the 3-chloro derivative does not inactivate the enzyme, it is a competitive inhibitor. Reduction of the enzyme-inactivator complex with []-NaBH4 indicates the incorporation of four haloacetol phosphates per mole of enzyme. These studies suggest the bromo- and iodoacetol phosphates inactivate the enzyme by reacting with a nucleophilic group located in the active center. 相似文献
9.
A new method is described for the isolation of glycogen from rat liver using centrifugation, gentle heating, and gel chromatography. The prepared polysaccharide was judged by both sucrose density gradient centrifugation and the absorbance spectrum of an I2-glycogen complex to be highly branched, polydisperse, and of an unusually high molecular weight upon comparison to other glycogens. Using adult fasted rats, this glycogen was shown to be better than high-molecular-weight cold water-ethanol extracted glycogen for the binding of glycogen metabolizing enzymes. Further, the addition of 0.5% () of the glycogen to a crude liver extract from newborn rats facilitated the isolation of an almost 700-fold purified glycogen synthase with 40% recovery. It is suggested that this glycogen could also be used to study the role of enzyme binding in the regulation of carbohydrate metabolism. 相似文献
10.
H P Wahl U Matern H Grisebach 《Biochemical and biophysical research communications》1975,64(3):1041-1045
The biosynthesis of dTDP-L-dihydrostreptose from dTDP-6-deoxy-D--4-hexosulose requires two enzymes: dTDP-4-keto-L-rhamnose-3,5-epimerase and a NADPH-dependent dTDP-“dihydrostreptose synthase”. These enzymes could be separated on a Sephadex G-100 column. 相似文献
11.
Purification of estradiol-17 beta dehydrogenase from human placenta by affinity chromatography 总被引:1,自引:0,他引:1
Estriol 16-hemisuccinate has been synthesized and covalently attached to Sepharose through 1,5-diaminopentane. A crude preparation of estradiol-17β dehydrogenase from human placenta was adsorbed on the gel. After extensive washing, the enzyme was eluted by hydroxylamine in 0.1 potassium phosphate buffer (20–50% glycerol), pH 7, at room temperature. An apparently homogeneous enzyme with a specific activity of 7.2 U/mg (82% recovery) was obtained. It is stable for weeks in the eluting buffer. The hydroxylamine can be removed by passing the enzyme solution over a Sephadex G-100 column or by dialyzing it against 0.1 potassium phosphate buffer containing 20% glycerol. This one-step process makes purification of the enzyme simple and easy. 相似文献
12.
13.
Nicola J. Blackmore Ali Reza Nazmi Richard D. Hutton Melissa N. Webby Edward N. Baker Geoffrey B. Jameson Emily J. Parker 《The Journal of biological chemistry》2015,290(29):18187-18198
Allostery, where remote ligand binding alters protein function, is essential for the control of metabolism. Here, we have identified a highly sophisticated allosteric response that allows complex control of the pathway for aromatic amino acid biosynthesis in the pathogen Mycobacterium tuberculosis. This response is mediated by an enzyme complex formed by two pathway enzymes: chorismate mutase (CM) and 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Whereas both enzymes are active in isolation, the catalytic activity of both enzymes is enhanced, and in particular that of the much smaller CM is greatly enhanced (by 120-fold), by formation of a hetero-octameric complex between CM and DAH7PS. Moreover, on complex formation M. tuberculosis CM, which has no allosteric response on its own, acquires allosteric behavior to facilitate its own regulatory needs by directly appropriating and partly reconfiguring the allosteric machinery that provides a synergistic allosteric response in DAH7PS. Kinetic and analytical ultracentrifugation experiments demonstrate that allosteric binding of phenylalanine specifically promotes hetero-octameric complex dissociation, with concomitant reduction of CM activity. Together, DAH7PS and CM from M. tuberculosis provide exquisite control of aromatic amino acid biosynthesis, not only controlling flux into the start of the pathway, but also directing the pathway intermediate chorismate into either Phe/Tyr or Trp biosynthesis. 相似文献
14.
C.H. Reynolds 《Biochemical and biophysical research communications》1975,67(2):538-543
NAD-linked α-glycerophosphate dehydrogenase plays a key role in the α-glycerophosphate cycle of . The activity in cell lysates was ample for this role. The enzyme was activated by salts (e.g. MgCl2 or NaCl); it had a broad pH-optimum for the reduction of dihydroxyacetone phosphate centred at pH 7.4, with an apparent Km of 0.5 mM; and it was weakly bound to particulate components of cell lysates. The enzyme from was similar to that of . These trypanosomal enzymes resemble that of the trypanosomatid , but are rather different from the enzymes of mammals, birds and insects. 相似文献
15.
Coenzyme dissociation, a possible determinant of short half-life of inducible enzymes in mammalian liver 总被引:8,自引:0,他引:8
Corticosteroid inducible, rapidly turning-over ( min to 3 hrs) enzymes of rat liver cytosol are complex enzymes with dissociable coenzymes. Enzymatic activity can be used to measure the relative rate of coenzyme dissociation. A comparison of rapidly inducible compared to relatively uninducible complex enzymes shows that the relative rate of coenzyme dissociation aligns with the shortness of the of the enzyme suggesting that coenzyme dissociation may be a limiting step in the degradation process of these enzymes. 相似文献
16.
Isolation and properties of the cytochrome B-C1 complex from Rhodopseudomonas sphaeroides 总被引:2,自引:0,他引:2
A highly purified and active cytochrome -1 complex has been isolated from the chromatophores of the photosynthetic bacteria R-26, through steps of Triton X-100 solubilization, salt fractionation and calcium phosphate column chromatography. The isolated enzyme complex catalyzes fully antimycin A sensitive oxidation of ubiquinol by cytochrome with a turnover number of 1500 per minute at 23° based on cytochrome 1. It contains 8.3 nmoles of cytochromes and 1 per mg protein and shows four polypeptides in the sodium dodecylsulfate polyacrylamide gel electrophoresis. 相似文献
17.
Dissociation by elastase digestion of enzyme complex catalyzing the initial steps of pyrimidine biosynthesis in rat liver 总被引:4,自引:0,他引:4
Glutamine-dependent carbamyl phosphate synthetase of rat liver, purified about 2,100-fold, existed as a complex with aspartate transcarbamylase and dihydroorotase, the second and third enzymes of pyrimidine biosynthesis, with a sedimentation coefficient of 27 S. Treatment of this complex with pancreatic elastase caused a selective inactivation of the transcarbamylase with concomitant dissociation of the complex. The dissociated synthetase was as sensitive to allosteric effectors as the enzyme within the complex, but had a 5 times higher apparent for MgATP2?. This change appears to be intimately related to the release of the enzyme from the complex. 相似文献
18.
γ-Aminobutyric acid-α-ketoglutarate transaminase from is irreversibly inhibited by 4-aminohex-5-yhoic acid, a new structural analog of GABA. The fact that this inhibition requires the pyridoxal form of the holoenzyme, and the formation of a Michaelis complex is in support of a catalytic mechanism. The compound is also active and on the same enzyme from mammalian brain. 相似文献
19.
Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity. 相似文献
20.
J Wiegel 《Biochemical and biophysical research communications》1978,82(3):907-912
The α-isopropylmalate synthase (EC 4.1.3.12) from was inactivated by EDTA in a time-dependent reaction. Only the addition of Mn++ plus dithiothreitol could restore the activity. The substrate, α-ketoisovalerate, prevented the inactivation; the feedback inhibitor, leucine, and it's antagonist, valine, increased the rate of inactivation. Except for α,α′-bipyridyl, chelating reagents, other than EDTA had no effect on the enzyme stability. It is suggested that the α-isopropylmalate synthase is a metallo enzyme - the evidence points to Mn++ as the metal ion - and that this enzyme uses a mechanism of catalysis which differs from that of the analogous malate synthase (EC 4.1.3.2) and citrate synthase (EC 4.1.3.4). 相似文献