共查询到20条相似文献,搜索用时 15 毫秒
1.
Tryptic digestion of a multifunctional enzyme from porcine liver containing methylenetetrahydrofolate dehydrogenase (5,10-methylenetetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.5), methenyltetrahydrofolate cyclohydrolase (5,10-methenyltetrahydrofolate 5-hydrolase, EC 3.5.4.9) and formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase, EC 6.3.4.3) activities destroys the synthetase. A fragment containing both dehydrogenase and cyclohydrolase activities has been isolated by affinity chromatography on an NADP+-Sepharose affinity column. The purified fragment is homogeneous on dodecyl sulfate-polyacrylamide gel electrophoresis where its molecular weight was determined as 33 000 +/- 1200 compared with 100 000 for the undigested protein. The cyclohydrolase activity retains sensitivity to inhibition by NADP+, MgATP and ATP. 相似文献
2.
Formyltetrahydrofolate synthetase (formate:tetrahydrofolate ligase (ADP-forming), EC 6.3.4.3) from Clostridium cylindrosporum catalyzes phosphate transfer from carbamyl phosphate to ADP. This activity is lost when monovalent cations are removed and is recovered when K+ is added back. Carbamyl phosphate is an inhibitor of the formyltetrahydrolfolate synthetase forward reaction, and formate as well as phosphate inhibit the ATP synthesis reaction. Acetyl phosphate and phosphonoacetate are inhibitors of both reactions. The results of kinetic studies support the concept that carbamyl phosphate is an analog of the putative intermediate of the formyltetrahydrofolate synthetase reaction, formyl phosphate. 相似文献
3.
When division synchronized cultures of Euglena gracilis Klebs (strain Z) were aerated with 5% CO2 in air the specific activity of glycollate dehydrogenase was only 13% of that in cultures receiving unsupplemented air. The concentrations of 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) and formylfolate derivatives were also lowered by this treatment. In contrast, the specific activity of serine hydroxymethyltransferase (EC 2.1.2.1) and the concentration of methylfolates were raised by supplying CO2-supplemented air. These effects on enzyme levels were reversed when air was supplied following a period of CO2 treatment. The levels of glycollate dehydrogenase, 10-formyl-tetrahydrofolate synthetase and formylfolate derivatives were decreased when cells were aerated in media containing 5 mM α-hydroxy-2-pyridinemethane sulphonate. Cell free extracts had the ability to decarboxylate glyoxylate, producing ca equal amounts of CO2 and formate from C-1 and C-2 respectively. Cells receiving 5% CO2 in air had a decreased ability to incorporate formate-[14C] into serine and methionine. It is concluded that during growth at low CO2 concentrations glycollate metabolism will provide substrate for the formyltetrahydrofolate synthetase reaction. 相似文献
4.
Nordihydroguaiaretic acid (NDGA) is a plant lignan produced by Larrea tridentata, the creosote bush of the American southwest. In this report we examine the mechanism underlying the ability of NDGA to inhibit TNF-induced apoptosis. Our results show that NDGA blocks many key indicators of apoptosis. Caspase cleavage, mitochondrial inactivation, externalization of phosphatidyl serine, and (51)Cr-release were all blocked by low micromolar concentrations of NDGA. NDGA also inhibited the cPLA(2)-dependent release of (3)H-arachidonic acid. We investigated this activity and found that NDGA prevented the rise in intracellular calcium necessary for the apoptotic activation of cPLA(2). On the other hand, NDGA did not interfere with the TNF-induced phosphorylation of cPLA(2), indicating that NDGA does not block all TNF-dependent signaling. Finally, we asked whether the anti-apoptotic effect of NDGA could be attributed to its anti-oxidant activity. Comparison with the effects of butylated hydroxyanisole (BHA) did not completely support this hypothesis. While BHA strongly inhibited caspase activation and partially blocked the release of (51)Cr, it was unable to significantly block the calcium response or the release of (3)H-arachidonic acid associated with TNF-induced apoptosis. The anti-oxidant activity of NDGA may, therefore, explain some but not all of its anti-apoptotic activity. 相似文献
5.
Ono K Hasegawa K Yoshiike Y Takashima A Yamada M Naiki H 《Journal of neurochemistry》2002,81(3):434-440
Inhibition of the accumulation of amyloid beta-peptide (Abeta) and the formation of beta-amyloid fibrils (fAbeta) from Abeta, as well as the degradation of pre-formed fAbeta in the CNS would be attractive therapeutic objectives for the treatment of Alzheimer's disease (AD). We previously reported that nordihydroguaiaretic acid (NDGA) inhibited fAbeta formation from Abeta(1-40) and Abeta(1-42) dose-dependently in the range of 10-30 micromin vitro. Utilizing fluorescence spectroscopic analysis with thioflavin T and electron microscopic study, we show here that NDGA dose-dependently breaks down fAbeta(1-40) and fAbeta(1-42) within a few hours at pH 7.5 at 37 degrees C. At 4 h, the fluorescence of fAbeta(1-40) and fAbeta(1-42) incubated with 50 microm NDGA was 5% and 10% of the initial fluorescence, respectively. The activity of NDGA to break down these fAbetas was observed even at a low concentration of 0.1 microm. At 1 h, many short, sheared fibrils were observed in the mixture incubated with 50 microm NDGA, and at 4 h, the number of fibrils reduced markedly, and small amorphous aggregates were observed. We next compared the activity of NDGA to break down fAbeta(1-40) and fAbeta(1-42), with other molecules reported to inhibit fAbeta formation from Abeta and/or to degrade pre-formed fAbeta both in vivo and in vitro. At a concentration of 50 microm, the overall activity of the molecules examined in this study was in the order of: NDGA > rifampicin = tetracycline > poly(vinylsulfonic acid, sodium salt) = 1,3-propanedisulfonic acid, disodium salt > beta-sheet breaker peptide (iAbeta5). In cell culture experiments, fAbeta disrupted by NDGA were less toxic than intact fAbeta, as demonstrated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Although the mechanisms by which NDGA inhibits fAbeta formation from Abeta, as well as breaking down pre-formed fAbetain vitro, are still unclear, NDGA could be a key molecule for the development of therapeutics for AD. 相似文献
6.
Regulation of asparaginase, glutamine synthetase, and glutamate dehydrogenase in response to medium nitrogen concentrations in a euryhaline chlamydomonas species
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The ammonium assimilatory enzymes glutamine synthetase (EC 6.3.1.2) and glutamate dehydrogenase (EC 1.4.1.3) were investigated for a possible role in the regulation of asparaginase (EC 3.5.1.1) in a Chlamydomonas species isolated from a marine environment. Cells grown under nitrogen limitation (0.1 millimolar NH(4) (+), NO(3) (-), or l-asparagine) possessed 6 times the asparaginase activity and approximately one-half the protein of cells grown at high nitrogen levels (1.5 to 2.5 millimolar). Biosynthetic glutamine synthetase activity was 1.5 to 1.8 times greater in nitrogen-limited cells than cells grown at high levels of the three nitrogen sources.Conversely, glutamate dehydrogenase (both NADH- and NADPH-dependent activities) was greatest in cells grown at high levels of asparagine or ammonium, while nitrate-grown cells possessed little activity at all concentrations employed. For all three nitrogen sources, glutamate dehydrogenase activity was correlated to the residual ammonium concentration of the media after growth (r = 0.88 and 0.94 for NADH- and NADPH-dependent activities, respectively).These results suggest that glutamate dehydrogenase is regulated in response to ambient ammonium levels via a mechanism distinct from asparaginase or glutamine synthetase. Glutamine synthetase and asparaginase, apparently repressed by high levels of all three nitrogen sources, are perhaps regulated by a common mechanism responding to intracellular nitrogen depletion, as evidenced by low cellular protein content. 相似文献
7.
An isolation procedure for phosphoribosyl succinocarboxamideaminoimidazole synthetase (SAICAR synthetase) (EC 6.3.2.6) has been developed. Pure SAICAR synthetase was found to be a monomeric protein with the apparent molecular weight of 36 kDa. The Michaelis constant for the three substrates of the reaction are 1.6 microM for CAIR, 14 microM for ATP and 960 microM for aspartic acid. The structural analogs of CAIR, 5-aminoimidazole ribotide and 5-aminoimidazole-4-carboxamide ribotide, act as competitive inhibitors of SAICAR synthetase. GTP and 2'-dATP can substitute for ATP in the reaction, while CTP and UTP inhibit the enzyme. No structural analogs of the aspartic acid were found to have affinity for SAICAR synthetase. The optimal reaction conditions for the enzyme were established to be at pH 8.0 and magnesium chloride concentration around 5 mM. 相似文献
8.
acid alpha-glucosidase (EC 3.2.1.20) was purified from fetal bovine muscle by affinity chromatography on concanavalin A and Sephadex G-100 and added to the culture medium of mature muscle cultures from animals affected by glycogenosis type II. The enzyme activity in homogenates of treated cultures was significantly increased within 4 h of the addition of enzyme, was maximal by 18 h and the internalised activity was stable for at least 48 h after the removal of the enzyme from the culture medium. The acid alpha-glucosidase activity was internalised with an uptake constant of 300 nM and a Vmax of uptake of 133 nmol/h per mg protein. The glycogen concentration in affected cultures treated with exogenous acid alpha-glucosidase for 24 h had decreased by 20% and after a further 24 h of culture was comparable to the concentration observed in cultures from non-affected animals. 相似文献
9.
The effects of adenine nucleotides on pea seed glutamine synthetase (EC 6.3.1.2) activity were examined as a part of our investigation of the regulation of this octameric plant enzyme. Saturation curves for glutamine synthetase activity versus ATP with ADP as the changing fixed inhibitor were not hyperbolic; greater apparent Vmax values were observed in the presence of added ADP than the Vmax observed in the absence of ADP. Hill plots of data with ADP present curved upward and crossed the plot with no added ADP. The stoichiometry of adenine nucleotide binding to glutamine synthetase was examined. Two molecules of [gamma-32P]ATP were bound per subunit in the presence of methionine sulfoximine. These ATP molecules were bound at an allosteric site and at the active site. One molecule of either [gamma-32P]ATP or [14C]ADP bound per subunit in the absence of methionine sulfoximine; this nucleotide was bound at an allosteric site. ADP and ATP compete for binding at the allosteric site, although ADP was preferred. ADP binding to the allosteric site proceeded in two kinetic phases. A Vmax value of 1.55 units/mg was measured for glutamine synthetase with one ADP tightly bound per enzyme subunit; a Vmax value of 0.8 unit/mg was measured for enzyme with no adenine nucleotide bound at the allosteric site. The enzyme activation caused by the binding of ADP to the allosteric sites was preceded by a lag phase, the length of which was dependent on the ADP concentration. Enzyme incubated in 10 mM ADP bound approximately 4 mol of ADP/mol of native enzyme before activation was observed; the activation was complete when 7-8 mol of ADP were bound per mol of the octameric, native enzyme. The Km for ATP (2 mM) was not changed by ADP binding to the allosteric sites. ADP was a simple competitive inhibitor (Ki = 0.05 mM) of ATP for glutamine synthetase with eight molecules of ADP tightly bound to the allosteric sites of the octamer. Binding of ATP to the allosteric sites led to marked inhibition. 相似文献
10.
Inactivation of Escherichia coli ADP-glucose synthetase (EC 2.7.2.27) by the arginine-specific reagents cyclohexanedione and phenylglyoxal resulted primarily from interference with normal allosteric activation. Partial modification by phenylglyoxal resulted in a lessened ability of fructose 1,6-bisphosphate (fructose-P2) to stimulate and of 5'-AMP (5'-adenylate) to inhibit enzymic activity. The apparent affinity for fructose-P2 and the Vmax at saturating fructose-P2 concentrations were decreased by the arginine modification. Fructose-P2, 5'-adenylate, and several other allosteric effectors were able to partially protect the enzyme from inactivation. However, catalytic activity was not decreased by arginine modification under conditions where the enzyme was assayed in the absence of fructose-P2. The two arginine-modifying reagents differed markedly in their reactivity with the enzyme. Cyclohexanedione inactivated the enzyme quite slowly and eventually reacted with at least 14 of the 32 arginines present per subunit. Phenylglyoxal was some 50-fold more effective in inactivation, but it modified only one arginine residue per subunit. 相似文献
11.
G G Chang F Pan P N Deng B Y Wu 《International journal of peptide and protein research》1979,13(3):267-273
The detailed pH and temperature kinetics of human term placenta cysteinyl-tRNA synthetase (EC 6.1.1.16) were studied. The ATP-PPi exchange reaction catalyzed by the cysteinyl-tRNA synthetase was highly dependent on temperature, pH, and ionic strength. The Arrhenius plot at temperatures between 5 degrees and 40 degrees was linear, giving an activation energy of 19 +/- 2.5 Kcal/mol. The pH dependence of the kinetic parameters Km and Vmax was investigated. Apparent pKa value of 6.4 was observed in the pH-dependence of Vmax/Km plot. The pH versus Vmax plot showed two apparent pKa values of about 5.8 and 7.8. Van't Hoff's enthalpies were used to differentiate the nature of the possible groups responsible for the ionization. These results are valuable for the selection of chemical modifying reagents in characterizing the amino acid residues involved in substrate (nucleotide) binding or catalysis. 相似文献
12.
The structural requirements for inhibition of bacterial RNA polymerase and rabbit liver formyltetrahydrofolate synthetase activity by a series of purine nucleoside analogs related to 6-chloro-8-aza-9-cyclopentylpurine (689) were investigated. To achieve an inhibitory effect, preincubation of the enzyme preparations with the purine analogs, prior to assay of enzyme activity, was required. The greatest inhibition was produced by analogs containing all three alterations of the purine nucleoside structure: the 6-halo, 8-aza, and 9-cyclopentyl groups. It is suggested that 689 inhibits the activity of enzymes involved in nucleic acid synthesis by a site-directed alkylation. 相似文献
13.
The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity. 相似文献
14.
1. The concentrations of folate derivatives in aerobic cultures of Saccharomyces cerevisiae (A.T.C.C. 9763) were determined by microbiological assay employing Lactobacillus casei (A.T.C.C. 7469) and Pediococcus cerevisiae (A.T.C.C. 8081). Cells cultured in media lacking l-methionine contained higher concentrations of folate derivatives than cells grown in the same media supplemented with 2.5mumol of l-methionine/ml. The concentrations of highly conjugated derivatives were also decreased by supplementing the growth medium with l-methionine. 2. DEAE-cellulose column chromatography of extracts prepared from cells grown under these conditions revealed that the concentrations of methylated tetrahydrofolates were drastically decreased by the methionine supplement. Smaller decreases were also observed in the concentrations of formylated and unsubstituted derivatives. 3. The concentrations of four enzymes of C(1) metabolism were compared after 6h of growth in the presence and in the absence of l-methionine (2.5mumol/ml). The specific activities of formyltetrahydrofolate synthetase, methylenetetrahydrofolate reductase and serine hydroxymethyltransferase were not altered by this treatment but that of 5-methyltetrahydrofolate-homocysteine methyltransferase was decreased by approx. 65% when l-methionine was supplied. The activities of 5-methyltetrahydrofolate-homocysteine methyltransferase, serine hydroxymethyltransferase and formyltetrahydrofolate synthetase were not appreciably altered by l-methionine in vitro. In contrast this amino acid was found to inhibit the activity of methylenetetrahydrofolate reductase. 4. Feeding experiments employing sodium [(14)C]formate indicated that cells grown in the presence of exogenous methionine, although having less ability to convert formate into methionine, readily incorporated (14)C into serine and the adenosyl moiety of S-adenosylmethionine. 5. It is suggested that exogenous l-methionine controls C(1) metabolism in Saccharomyces principally by regulation of methyl-group biogenesis within the folate pool. 相似文献
15.
Metabolic adaptations during lactogenesis. Fatty acid and lactose synthesis in cow mammary tissue 总被引:4,自引:2,他引:2
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
1. Mammary-tissue biopsies were obtained from multiparous cows at 30 and 7 days pre partum and 7 and 40 days post partum. Investigations of the effect of lactogenesis on fatty acid and lactose synthesis involved measurements of biosynthetic capacity (tissue-slice incubations in vitro) and activities of relevant enzymes. 2. Fatty acid synthesis from acetate increased over 20-fold from 30 days pre partum to 40 days post partum. Changes in the lipogenic capacity of mammary-tissue slices more closely paralleled increases in the activities of acetyl-CoA carboxylase (EC 6.4.1.2) and acetyl-CoA synthetase (EC 6.2.1.1) than of other enzymes involved in acetate incorporation into fatty acids or in NADPH generation. 3. Lactose biosynthesis by mammary-tissue slices, lactose synthetase activity (EC 2.4.1.22) and alpha-lactalbumin concentration were all negligible at 30 days pre partum but increased 2.5-4-fold between 7 days pre partum and 40 days post partum. Phosphoglucomutase (EC 2.7.5.1), UDP-glucose pyrophosphorylase (EC 2.7.7.9) and UDP-glucose 4-epimerase (EC 5.1.3.2) had substantial activities at 30 days pre partum and increased less dramatically during lactogenesis. 4. Results are consistent with acetyl-CoA carboxylase and perhaps acetyl-CoA synthetase representing the regulatory enzyme(s) in fatty acid synthesis, with lactose synthetase (alpha-lactalbumin) serving a similar function in lactose biosynthesis. 相似文献
16.
Paula Maloberti Rocío Castilla Lozano Pablo G Mele Florencia Cano Cecilia Colonna Carlos F Mendez Cristina Paz Ernesto J Podestá 《European journal of biochemistry》2002,269(22):5599-5607
Although the role of arachidonic acid (AA) in the regulation of steroidogenesis is well documented, the mechanism for AA release is not clear. Therefore, the aim of this study was to characterize the role of an acyl-CoA thioesterase (ARTISt) and an acyl-CoA synthetase as members of an alternative pathway in the regulation of the intracellular levels of AA in steroidogenesis. Purified recombinant ARTISt releases AA from arachidonoyl-CoA (AA-CoA) with a Km of 2 micro m. Antibodies raised against recombinant acyl-CoA thioesterase recognize the endogenous protein in both adrenal tissue and Y1 adrenal tumor cells by immunohistochemistry and immunocytochemistry and Western blot. Stimulation of Y1 cells with ACTH significantly stimulated endogenous mitochondrial thioesterases activity (1.8-fold). Nordihydroguaiaretic acid (NDGA), an inhibitor of AA release known to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochondrial acyl-CoA thioesterases. ACTH-stimulated steroid synthesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C an inhibitor of the AA-CoA synthetase. The apparent IC50 for NDGA was reduced from 50 micro m to 25, 7.5 and 4.5 micro m in the presence of 0.1, 0.5 and 2 micro m triacsin C, respectively. Our results strongly support the existence of a new pathway of AA release that operates in the regulation of steroid synthesis in adrenal cells. 相似文献
17.
CDP-diacylglycerol(DAG) synthetase (EC 2.7.7.41) has been solubilized from bovine brain microsomes by the detergent CHAPS (3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate). Optimal solubilization with 1.5% CHAPS yielded 55-60% of the synthetase activity. The effect of CHAPS on the enzyme was biphasic inhibiting at 0.3% and giving maximal activity at 0.5% (the concentration used for all assays). The solubilized, but not the microsomal enzyme is activated by phosphatidylcholine (PC) and strongly inhibited by cardiolipin and lysoPC. Strong inhibition by N-ethylmaleimide, 5,5'-dithio-bis (2-nitrobenzoic acid) and p-chloromercuribenzoate supported a sulfhydryl requirement for the enzyme. Phosphatidic acid (PA) from egg lecithin and 1-stearoyl,2-arachidonoyl PA were preferred substrates for the microsomal synthetase. Solubilized synthetase showed selectivity for the latter PA which is consistent with this enzyme functioning to help form the preponderant 1-stearoyl,2-arachidonoyl species of phosphatidylinositol. Further attempts to purify the synthetase were unsuccessful. All findings suggested the enzyme exists as an unstable complex. 相似文献
18.
19.
Cerulenin, a specific inhibitor of beta-ketoacyl-(acyl carrier protein) synthetase [EC 2.3.1.41] and 3-hydroxy-3-methylglutaryl-CoA synthetase [EC 4.1.3.5], was studied to determine whether it inhibits 6-methylsalicylic acid synthesis, in which so-called "polyketide" formation, a condensation step similar to that in fatty acid synthesis, is involved. In fact, 100 mug/ml (4.5 X 10(-4) M) of cerulenin inhibited 60% of 6-methylsalicylic acid synthetase activity and 68% of fatty acid synthetase activity of Penicillium urticae. 相似文献
20.
Biosynthesis of Prostaglandins D2 and E2 in Chick Dorsal Root Ganglion During Development 总被引:1,自引:1,他引:0
Newly formed prostaglandins (PGs), which are assumed to act as modulators of afferent sensory messages, were studied in chick dorsal root ganglia (DRG) during development. [1-14C]Arachidonic acid was converted by DRG homogenates from 1-week-old chickens into two major 14C-PGs: PGE2 and PGD2. The enzymatic conversion of arachidonic acid was characterized as follows: (a) Boiled preparations were inactivated; (b) synthesis of PGs was inhibited by pretreatment with aspirin or indomethacin and enhanced by esculetin, a protector of cyclooxygenase; and (c) [14C]PGE2 and [14C]PGD2 accumulation was a protein dose-dependent process. Further fractionation of crude homogenates indicated that PG endoperoxide synthetase (EC 1.14.99.1) and PGE2 synthetase (EC 5.3.99.3) were membrane-bound enzymes, whereas PGD2 synthetase (EC 5.3.99.2) was recovered in the cytosol. During development, from embryonic day 10 to day 14 after hatching, PGD2 synthetase activity remained constant; in contrast, a sharp rise in [14C]PGE2 synthesis was observed from embryonic day 14 to 18. The time curves of PGD2 and PGE2 synthetase specific activity may be related to changes taking place in the cell population of developing DRG. It is therefore suggested that arachidonic acid would be enzymatically converted early into PGD2 by maturing ganglion cells and then later into PGE2 by proliferating fibroblasts. 相似文献