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1.
Isolation of a kaurene synthetase inhibitor from castor bean seedlings and cell suspension cultures 总被引:2,自引:2,他引:0
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Biosynthesis of ent-kaurene was investigated in extracts of cell suspension cultures and seedlings of castor bean. Both cell-free extracts contain an inhibitor of kaurene synthetase. The inhibition affects mainly the cyclization of geranylgeranyl pyrophosphate to copalyl pyrophosphate (activity A) and has little or no effect on the further cyclization of copalyl pyrophosphate to ent-kaurene (activity B) in both castor bean and Fusarium moniliforme cell-free enzyme preparations. In castor bean cell suspension cultures, the inhibitor diffuses out of the cells to the growth medium. The inhibitor is stable to 100 C heat treatment for 10 minutes and exposure to pH values of 2.0 or 13.0, and it diffuses through a dialysis bag (104-dalton cutoff). Gel filtration chromatography of the inhibitor on a calibrated Bio-Gel P-10 column indicated a molecular weight of 7,500. Kinetic studies indicate that the inhibition of activity of A of kaurene synthetase is noncompetitive and reversible. 相似文献
2.
Kaurene synthetases catalyse the biosynthesis of ent-kaurene, a precursor of the gibberellins. In 4-day-old dark- or light-grown Helianthus annuus seedlings, the cotyledons contained over 90% of the synthetase activity. The low enzyme activity in the seedling hypocotyls and roots is not a consequence of inhibitory factors in these tissues. The cotyledons not only have the highest kaurene synthetase activity, but also have the highest inhibitory activity. The differences in kaurene synthetase activities in the different tissues cannot be explained on the basis of the levels of inhibitor(s) in the extracts. The mature perennial root of Marah macrocarpus has very low kaurene synthetase activity, in contrast to the liquid endosperm of immature seeds of the same plant which is a rich source of the enzyme. 相似文献
3.
Properties of Kaurene Synthetase from Marah macrocarpus Endosperm: Evidence for the Participation of Separate but Interacting Enzymes 总被引:5,自引:3,他引:2
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Ent-kaurene is synthesized from geranylgeranyl pyrophosphate in a two step sequence catalyzed by kaurene synthetase; the first step (A activity) involves the conversion of geranylgeranyl pyrophosphate into the intermediate ent-trans labda-8(17), 13-dien-15-yl pyrophosphate (copalyl pyrophosphate) which is further cyclized to ent-kaurene in the second step (B activity). The resolution of enzyme fractions which catalyze each step independent of the other has been accomplished for the first time by means of QAE Sephadex A-50 chromatography and polyacrylamide gel electrophoresis of kaurene synthetase preparations from endosperm tissue of immature seed of Marah macrocarpus. Molecular weights for the A and B enzymes were each estimated as approximately 82,000 by means of gel filtration chromatography and sedimentation velocity determinations. 相似文献
4.
Kaurene synthetase B activity (conversion of copalyl pyrophosphate to ent-kaurene) is readily detectable in crude cell-free extracts of 3- to 4-day old dark-grown sunflower (Helianthus annuus cv. Mammoth) seedlings, whereas little or no kaurene synthetase AB activity (conversion of geranylgeranyl pyrophosphate to ent-kaurene) can be found in these extracts under comparable assay conditions. A low amount of AB activity is evident only if an extensively dialyzed extract is used in low concentrations as the enzyme source. One factor which may contribute to the low apparent levels of AB activity is the presence of inhibitory factors in the crude sunflower extract since these extracts can be shown to act as a potent inhibitor of Marah macrocarpus endosperm kaurene synthetase AB activity. Heat treatment (100°C) or dialysis of the sunflower extract reduces the amount of its inhibitory activity. Also, it was observed that low concentrations of extensively dialyzed sunflower extracts act to stimulate M. macrocarpus AB activity. There is no evidence for the presence of an inhibitory factor for M. macrocarpus kaurene synthetase B activity in sunflower extracts. However, there does appear to be present in the crude preparation of sunflower extract a dialyzable factor(s) that impedes its own B activity. There is little information to date on the nature of these inhibitory and stimulatory factors for kaurene synthetase activity or their possible roles in physiological regulation. The possible presence of such factors should be considered, however, when attempting to evaluate kaurene synthetase activities in extracts of vegetative plants. 相似文献
5.
Kaurene Synthetase Activity in Helianthus annuus L. : Increases in Enzyme Activity after Storage of Seedlings in Liquid Nitrogen
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In previous studies, the conversion of geranylgeranyl pyrophosphate to ent-kaurene (kaurene synthetase AB activity) could not be detected readily in crude extracts of sunflower (Helianthus annuus L.) seedlings (Shen-Miller, West 1982 Plant Physiol 69: 637-641). These investigations also revealed the presence of inhibitors for Marah macrocarpus kaurene synthetase AB activity in crude extracts of sunflower seedlings. It has now been found that crude extracts prepared from intact sunflower seedlings stored in liquid N2 for several days have greatly enhanced AB activity in comparison with frozen, but not stored, controls. The levels of activity for the conversion of copalyl pyrophosphate to ent-kaurene (kaurene synthetase B activity) are affected only slightly by storage of intact seedlings in liquid N2. Extracts from intact seedlings that had been stored in liquid N2 also showed less inhibitory activity for Marah macrocarpus endosperm kaurene synthetase AB activity. 相似文献
6.
Lysed chloroplasts from several higher plants synthesized ent-kaurene from copalyl pyrophosphate but not from geranylgeranyl pyrophosphate. The copalyl pyrophosphate transforming activity (so-called B activity of kaurene synthetase) was relatively stable in plastid lysates from Pisum sativum but remarkably unstable in similar preparations of Hordeum vulgare. The bulk of the B activity of kaurene synthetase appeared to reside in the stroma of plastids from P. sativum but required the presence of plastid membranes for maximum activity. 相似文献
7.
Previous studies with thyme (Thymus vulgaris L.) leaf slices indicated that γ-terpinene (1,4-p-menthadiene) is the precursor of the aromatic monoterpenes p-cymene (4-isopropyl toluene) and thymol (5-methyl-2-isopropyl phenol) (Poulose, A. and Croteau, R. (1978) Arch. Biochem. Biophys.187, 307–314). A 105,000g supernatant obtained from an extract of young thyme leaves catalyzed the cyclization of both [1-3H]neryl pyrophosphate and [1-3H]geranyl pyrophosphate to γ-[3-3H]terpinene. No evidence for the interconversion of the acyclic precursors was obtained, and isotopic dilution experiments suggested that γ-terpinene was synthesized directly from these acyclic precursors without the involvement of any free intermediates. Competing phosphatase activity in the soluble preparation was removed by ammonium sulfate fractionation followed by gel filtration on Sephadex G-150. In these fractionation steps, γ-terpinene synthetase activity co-purified with small amounts of α-thujene (1-isopropyl-4-methylbicyclo[3.1.0]-hex-3-ene) and α-terpineol (p-menth-1-en-8-ol) synthetase activities, and these three activities could not be resolved by subsequent hydroxylapatite chromatography, anion exchange chromatography on QAE-Sephadex, or affinity chromatography on neroic acid-substituted agarose. All the enzymatic products were identified by radio chromatography and by the synthesis of derivatives followed by radio chromatography or crystallization to constant specific activity. γ-Terpinene synthetase has an apparent molecular weight of 96,000, shows a pH optimum at about 6.8, and requires Mg2+ for catalytic activity. Mn2+ can partially substitute for Mg2+, but other divalent cations are ineffective. Estimated values of V and Km are 3.5 nmol/h/mg and 9 μm, respectively, for neryl pyrophosphate, and 3.0 nmol/h/mg and 14 μm, respectively, for geranyl pyrophosphate. Enzymic activity is inhibited by sulfhydryl-directed reagents and inorganic pyrophosphate, but not by γ-terpinene, p-cymene, or thymol. Based on the specific location of tritium in the product, a mechanism is proposed which involves the cyclization of the acyclic precursor, loss of a proton from C5 to form the Δ4 double bond, and a 1,2-hydride shift from C4 to C8 to give γ-terpinene. A similar mechanism, but with loss of the proton from C6 and the formation of a cyclopropane ring, would yield α-thujene. 相似文献
8.
Previous studies with soluble enzyme preparations from sage (Salvia officinalis) demonstrated that the monoterpene ketone (+)-camphor was synthesized by the cyclization of neryl pyrophosphate to (+)-bornyl pyrophosphate followed by hydrolysis of this unusual intermediate to (+)-borneol and then oxidation of the alcohol to camphor (R. Croteau, and F. Karp, 1977, Arch. Biochem. Biophys.184, 77–86). Preliminary investigation of the (+)-bornyl pyrophosphate synthetase in crude preparations indicated that both neryl pyrophosphate and geranyl pyrophosphate could be cyclized to (+)-bornyl pyrophosphate, but the presence of high levels of phosphatases in the extract prevented an accurate assessment of substrate specificity. The competing phosphatases were removed by combination of gel filtration on Sephadex G-150, chromatography on hydroxylapatite, and chromatography on O-(diethylaminoethyl)-cellulose. In these fractionation steps, activities for the cyclization of neryl pyrophosphate and geranyl pyrophosphate to bornyl pyrophosphate were coincident, and on the removal of competing phosphatases, the synthetase was shown to prefer geranyl pyrophosphate as substrate ( for geranyl pyrophosphate was 20-fold that of neryl pyrophosphate). No interconversion of geranyl and neryl pyrophosphates was detected. The partially purified bornyl pyrophosphate synthetase had an apparent molecular weight of 95,000, and required Mg2+ for catalytic activity (Km for Mg2+ ~ 3.5 mm). Mn2+ and other divalent cations were ineffective in promoting the formation of bornyl pyrophosphate. The enzyme exhibited a pH optimum at 6.2 and was strongly inhibited by both p-hydroxymercuribenzoate and diisopropylfluorophosphate. Bornyl pyrophosphate synthetase is the first monoterpene synthetase to be isolated free from competing phosphatases, and the first to show a strong preference for geranyl pyrophosphate as substrate. A mechanism for the cyclization of geranyl pyrophosphate to bornyl pyrophosphate is proposed. 相似文献
9.
Rodney Croteau Mark Felton Robert C. Ronald 《Archives of biochemistry and biophysics》1980,200(2):534-546
A soluble enzyme preparation from the leaves of fennel (Foeniculum vulgare M.) has been shown to catalyze the cation-dependent cyclization of both geranyl pyrophosphate and neryl pyrophosphate to the bicyclic rearranged monoterpene l-endo-fenchol (R. Croteau, M. Felton, and R. Ronald, 1980 Arch. Biochem. Biophys.200, 524–533). To examine the possible presence of free intermediates between the acyclic precursors and fenchol, and to remove competing cyclase and pyrophosphatase activities, the soluble preparation was partially purified by ammonium sulfate fractionation followed by gel filtration on Sephadex G-150 and ion exchange chromatography on O-diethylaminoethyl-cellulose. Activities for the cyclization of geranyl pyrophosphate and neryl pyrophosphate to fenchol were coincident on Chromatographic fractionation suggesting that the same enzyme was capable of cyclizing both acyclic substrates. No interconversion of the acyclic precursors was detected. Although bornyl pyrophosphate is a free intermediate in the biosynthesis of the related bicyclic monoterpenol borneol, both protein fractionation and isotopic dilution experiments ruled out endo-fenchyl pyrophosphate as a free intermediate in fenchol biosynthesis. Similarly, while construction of the fenchane skeleton was demonstrated to involve the rearrangement of an intermediate pinane skeleton, isotopic dilution experiments ruled out both optical antipodes of α-pinene, β-pinene, cis-2-pinanol, trans-2-pinanol, and the corresponding 2-pinyl pyrophosphates as free intermediates of the enzyme-catalyzed reaction. Furthermore, exhaustive search of the enzymatic reaction products provided no evidence to suggest the involvement of any free intermediate between the acyclic precursor and fenchol. The endo-fenchol synthetase has an apparent molecular weight of 60,000, shows a pH optimum near 7.0, and requires Mn2+ (1 mm) for catalytic activity. Co2+ can partially substitute for Mn2+, but other divalent cations are ineffective. The partially purified synthetase is inhibited by p-hydroxymercuribenzoate and by phenylglyoxal, and it exhibits a preference for geranyl pyrophosphate over neryl pyrophosphate as substrate. An integrated scheme is proposed for the cyclization and rearrangement catalyzed by fenchol synthetase. 相似文献
10.
An enzyme system from Claviceps purpurea (Fr.) Tul. catalyzing the incorporation of l-phenylalanine into ergotamine - ergotamine synthetase - was purified 172-fold. This was done by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange chromatography on DEAE-Sepharose CL-6B, and hydroxyapatite chromatography. The activation of ergotamine specific amino acids as well as d-lysergic acid and dihydrolysergic acid via adenylates, as determined by the ATP-32PPi exchange, was investigated. Phenylalanyl-tRNA synthetase, catalyzing the same type of activation reaction, could not be separated from ergotamine synthetase by the purification procedure applied. Therefore, at the present stage of enzyme purification, phenylalanine-dependent ATP-32PPi exchange cannot be used to measure ergotamine synthetase activity specifically.Phenylalanyl-tRNA synthetase and leucyl-tRNA synthetase were separated into mitochondrial and cytoplasmic isoenzymes by hydroxyapatite chromatography. Their charging activities of procaryotic versus eucaryotic tRNA and their molecular masses were determined. 相似文献
11.
A cell-free system capable of converting [14C]geranylgeranyl diphosphate to ent-[14C]kaurene and to an unidentified acid-hydrolysable compound was obtained from the basal portions of 5-d-old shoots of wheat seedlings (Triticum aestivum L.). By means of marker enzyme activities, the synthesis of ent-kaurene and the unknown compound could be quantitatively assigned to a plastid fraction obtained by Percoll-gradient centrifugation of the homogenate. The enzyme activities were located within the plastids, probably in the stroma, because they withstood trypsin treatment of the intact plastids, and the plastids had to be broken to release the activity, which was then obtained in soluble form. Plastid membranes had no activity. Plastid stroma preparations obtained from pea (Pisum sativum L.) shoot tips and pumpkin (Cucurbita maxima L.) endosperm also yielded ent-kaurene synthetase activity, but did not form the unknown compound. The exact nature of the active plastids was not ascertained, but the use of methods for proplastid isolation was essential for full activity, and the active tissues are all known to contain high proportions of proplastids, developing chloroplasts or leucoplasts. We therefore believe that ent-kaurene synthesis may be limited to these categories. Mature chloroplasts from the wheat leaves did not contain ent-kaurene synthetase activity and did not yield the unknown component. Incorporation of [14C]geranylgeranyl diphosphate into ent-[14C]kaurene and the unknown component was assayed by high-performance liquid chromatography with on-line radiocounting. ent-[14C]Kaurene was identified by Kovats retention index and full mass spectra obtained by combined gas chromatography-mass spectrometry. The unknown component was first believed to be copalyl diphosphate, because it yielded a compound on acid hydrolysis, which migrated like copalol on high-performance liquid chromatography and gave a mass spectrum very similar to that of authentic copalol. However, differences in the mass spectrum and in retention time on capillary gas chromatography excluded identity with copalol. Furthermore, the unhydrolysed compound was not converted to ent-kaurene by a cell-free system from C. maxima endosperm as copalyl diphosphate would have been.Abbreviations ADH
alcohol dehydrogenase
- AMO 1618
2isopropyl-4-(trimethylammoniumchloride)-5-methylphenyl piperi-dine-1-carboxylate
- BSA
bovine serum albumin
- DTT
dithioth-reitol
- GAn
gibberellin An
- GAPDH
NADP+-glyceraldehyde 3-phosphate dehydrogenase
- GC-MS
combined gas chromatography-mass spectrometry
- GGPP
all trans-isomer of geranyl-geranyl diphosphate
- KS
ent-kaurene synthetase
- MDH
malate dehydrogenase
- MAA
mevalonate activating activity
- SOR
shikimate oxidoreductase
We thank Mrs. Gudrun Bodtke and Mrs. Dorothee Dasbach for able technical assistance, Prof. L.N. Mander (Australian National University, Canberra, Australia) for ent-[2H2]kaurene and Dr. Yuji Kamiya (RIKEN, Saitama, Japan) for geranylgeraniol and copalol. The work was supported by the Deutsche Forschungsgemeinschaft. 相似文献
12.
The state of adenylylation of glutamine synthetase in Escherichia coli is regulated by the adenylyl transferase, the PII regulatory protein, uridylyl transferase (UTase), and the uridylyl removing enzyme (UR). The regulatory protein exists in an unmodified state (PII) which promotes adenylylation and in a uridylylated form (PII·UMP) which promotes deadenylylation of glutamine synthetase. The UR and UTase enzymes catalyze the interconversion of PII and PII·UMP. The UR and UTase have been partially purified by chromatography over DEAE-cellulose, AH-Sepharose 4B, Sephadex G-200, and gel electrophoresis. The two activities co-purify at all steps in the isolation although preparations containing different ratios of UTase:UR activities have been isolated. These UR·UTase activities have apparent molecular weight of 140,000. Both activities are inactivated by sulfhydryl reagents, both activities are heat inactivated, and both are stabilized by high salt concentrations. Both activities are inhibited in the crude extract by dialyzable inhibitors, but the UR is also inhibited by a nondialyzable inhibitor. This endogenous inhibitor is of molecular weight greater than 100,000 daltons, and binds CMP and UMP which are the apparent inhibitory agents. CMP and UMP are antagonistic in their effects on the UR activity. No effect of the CMP, UMP, or the large inhibitor on the other steps in the cascade could be demonstrated. The Mn2+-supported UR activity was also shown to be inhibited by a number of divalent cations, particularly Zn2+. 相似文献
13.
Glenn G. Preston Carolyn Zeiher Judy D. Wall David W. Emerich 《Applied microbiology》1989,55(1):165-170
Acetyl coenzyme A (acetyl-CoA) synthetase and acetate kinase were localized within the soluble portion of Bradyrhizobium japonicum bacteroids, and no appreciable activity was found elsewhere in the nodule. The presence of each acetate-activating enzyme was confirmed by separation of the two enzyme activities on a hydroxylapatite column, by substrate dependence of each enzyme in both the forward and reverse directions, by substrate specificity, by inhibition patterns, and also by identification of the reaction products by C18 reverse-phase high-pressure liquid chromatography. Phosphotransacetylase activity, found in the soluble portion of the bacteroid, was dependent on the presence of potassium and was inhibited by added sodium. The greatest acetyl-CoA hydrolase activity was found in the root nodule cytosol, although appreciable activity also was found within the bacteroids. The combined specific activities of acetyl-CoA synthetase and acetate kinase-phosphotransacetylase were approximate to that of the pyruvate dehydrogenase complex, thus providing B. japonicum with sufficient capacity to generate acetyl-CoA. 相似文献
14.
Changes in isoaccepting species of tRNALeu were determined in germinating pea seedlings and in developing pods. Leucine specific transfer ribonucleic acids of pea cotyledons can be fractionated into four isoaccepting species by reversed-phase chromatography (RPC-5) on a Plaskon column. In contrast, only two species of tRNALeu were observed in developing seed pods. Leucyl-tRNA synthetase purified by ammonium sulfate precipitation and DEAE cellulose column chromatography retained the full range of specificity towards all four tRNALeu species of pea cotyledons. This partially purified pea cotyledon enzyme could be further separated on a hydroxylapatite (HA) column into two peaks of leucyl-tRNA synthetase activity. Enzyme 1 is dominant in seed pods while 2 is predominant in cotyledons. Enzymes 1 and 2 from cotyledons were examined for the amino acid acceptor activity of twelve different amino acids. Both these fractions showed less than 3% acceptor activity for eleven other amino acids as compared to leucine-tRNA synthetase activity. Preliminary characterization of enzyme 2 from cotyledon, by isoelectric focusing and polyacrylamide gel electrophoresis indicates at least three subspecies. 相似文献
15.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1995,1254(1):56-65
We have previously reported that human B cell differentiation is accompanied by sequential changes in glycosphingolipid expression. Pre-B cells contain lacto-series type II chain-based glycolipids and GM3 ganglioside; mature/activated B cells do not synthesize lacto-series compounds but express GM3 and globo-series glycolipids (Gb3 and Gb4); terminally differentiated B cells, in addition to these compounds, also contain GM2 ganglioside. At the cell surface, Gb3, Gb4 and GM2 constitute stage-specific antigens. To elucidate the biosynthetic mechanism leading to these modifications we have compared activities of the glycosyltransferases involved in the core structure assembly and the first elongation steps of neo-lacto, ganglio- and globo-series glycolipids. These glycosyltransferase activities have been measured in B cell lines and normal B lymphocytes at various stages of differentiation. We first determined the optimal requirements of the four glycosyltransferases which wynthesize Lc3, GM3, Gb4 and GM2 glycolipids in B lymphocytes and then tested these enzymes and the Gb3 synthetase in the selected B cells. The following results were obtained: ß1 → 3N-Acetylglucosaminyltransferase (Lc3 synthetase) has a high activity in pro- and pre-B cells whereas it is undetectable in more differentiated cells; α2 → 3 sialyltransferase (GM3 synthetase) is activated from the pre-B cell stage to the terminally differentiated myeloma cells; α → 4 galactosyltransferase (Gb3 synthetase) is only detected in cells representing the late stages of B cell differentiation; ß1 → 3N-Acetylgalactosaminyltransferase (Gb4 synthetase) is only found in some lymphoblastoid cell lines, representative of activated B cells whereas the ß1 → 4 N-Acetylgalactosaminyltransferase (GM2 synthetase) has a high activity in these lymphoblastoid cell lines and in terminally differentiated myeloma cells. These results suggest that the sequential shifts in the three major glycosphingolipid series observed during B cell differentiation are mostly due to sequential activations of the corresponding glycosyltransferases. 相似文献
16.
A soluble enzyme preparation obtained from young sage (Salvia officinalis) leaves catalyzes the conversion of neryl pyrophosphate to (+)-borneol and the oxidation of (+)-borneol to (+)-camphor. Attempts to purify the borneol synthetase activity by gel permeation column chromatography resulted in the apparent loss of catalytic capability; however, subsequent recombination of column fractions demonstrated that two separable enzymatic activities were required for the conversion of neryl pyrophosphate to borneol. Several lines of evidence indicated that a water-soluble, dialyzable intermediate was involved in this transformation. The intermediate was isolated and subsequently identified as bornyl pyrophosphate by direct chromatographic analysis and by the preparation of derivatives and chromatographic analysis of both the hydrogenolysis (LiAlH4) and enzymatic hydrolysis products of bornyl pyrophosphate. The results presented indicate that borneol is derived by cyclization of neryl pyrophosphate to bornyl pyrophosphate, followed by hydrolysis. This is the first demonstration of a cyclic pyrophosphorylated intermediate in the biosynthesis of bicyclic monoterpenes. 相似文献
17.
Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures 总被引:2,自引:0,他引:2
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Squalene synthetase, an integral membrane protein and the first committed enzyme for sterol biosynthesis, was solubilized and partially purified from tobacco (Nicotiana tabacum) cell suspension cultures. Tobacco microsomes were prepared and the enzyme was solubilized from the lipid bilayer using a two-step procedure. Microsomes were initially treated with concentrations of octyl-β-d-thioglucopyranoside and glycodeoxycholate below their critical micelle concentration, 4.5 and 1.1 millimolar, respectively, to remove loosely associated proteins. Complete solubilization of the squalene synthetase enzyme activity was achieved after a second treatment at detergent concentrations above or at their critical micelle concentration, 18 and 2.2 millimolar, respectively. The detergent-solubilized enzyme was further purified by a combination of ultrafiltration, gel permeation, and Fast Protein Liquid Chromatography anion exchange. A 60-fold purification and 20% recovery of the enzyme activity was achieved. The partially purified squalene synthetase protein was used to generate polyclonal antibodies from mice that efficiently inhibited synthetase activity in an in vitro assay. The apparent molecular mass of the squalene synthetase protein as determined by immunoblot analysis of the partially purified squalene synthetase protein separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 47 kilodaltons. The partially purified squalene synthetase activity was optimal at pH 6.0, exhibited a Km for farnesyl diphosphate of 9.5 micromolar, and preferred NADPH as a reductant rather than NADH. 相似文献
18.
Glutamine Involvement in Nitrogen Control of Gibberellic Acid Production in Gibberella fujikuroi 总被引:2,自引:0,他引:2
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When the fungus Gibberella fujikuroi ATCC 12616 was grown in fermentor cultures, both intracellular kaurene biosynthetic activities and extracellular GA3 accumulation reached high levels when exogenous nitrogen was depleted in the culture. Similar patterns were exhibited by several nonrelated enzymatic activities, such as formamidase and urease, suggesting that all are subject to nitrogen regulation. The behavior of the enzymes involved in nitrogen assimilation (glutamine synthetase, glutamate dehydrogenase, and glutamate synthase) during fungal growth in different nitrogen sources suggests that glutamine is the final product of nitrogen assimilation in G. fujikuroi. When ammonium or glutamine was added to hormone-producing cultures, extracellular GA3 did not accumulate. However, when the conversion of ammonium into glutamine was inhibited by L-methionine-DL-sulfoximine, only glutamine maintained this effect. These results suggest that glutamine may well be the metabolite effector in nitrogen repression of GA3 synthesis, as well as in other nonrelated enzymatic activities in G. fujikuroi. 相似文献
19.
Cysteine synthetase (O-acetylserine sulfhydrylase) was partially purified from cells of Bacillus subtilis by the use of ammonium sulfate fractionation technique and DEAE-Sephadex A–50 chromatography. The cysteine synthetase preparation was compared with cystathionase (cystathionine β-cleavage enzyme) of the same organism in regard to biochemical properties and to changes in activity during sporulation.The optimal pH and temperature for the cysteine synthetase were 8.5 and 25°C respectively. The enzyme was relatively stable at temperatures below 50°C and fairly resistant to proteases, in contrast to cystathionase. Production by B. subtilis of cysteine synthetase in sulfur-deficient synthetic medium was repressed by the addition of cysteine and derepressed by djenkolic acid. Activity of the enzyme was inhibited by methionine and increased by acetate. The cysteine synthetase activity was almost constant until the late sporulation stage commenced, but the specific activity of cystathionase (Fraction I) decreased rapidly in the course of sporulation and it could not be detected in the free spores. 相似文献
20.
Effects of Protein Synthesis Inhibitors on ent-Kaurene Biosynthesis during Photomorphogenesis of Etiolated Pea Seedlings 总被引:1,自引:1,他引:0
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Excised shoot tips from 10-day-old etiolated pea (Pisum sativum L. cv. Alaska) seedlings were incubated in solutions of chloramphenicol, cycloheximide, and lincomycin at different concentrations during periods of 0, 4, 8, and 12 hours of irradiation with high intensity white light. Enzyme extracts were prepared from the whole shoot tips and compared with extracts from nontreated shoot tips for their capacity to synthesize ent-kaurene from mevalonate. In control samples, kaurene synthesis increased during the first 8 hours of irradiation and decreased after 12 hours. Chlorophyll content increased steadily up to 12 hours of irradiation. Chloramphenicol and cycloheximide reduced both kaurene synthesis and chlorophyll formation to a similar extent during all periods of irradiation, the reduction being greatest after 8 hours of irradiation. Lincomycin, a specific inhibitor of the formation of chloroplast ribosomes in detached pea shoot tips, did not significantly affect kaurene synthesis activity but strongly inhibited chlorophyll formation. It is tentatively concluded that the increase in kaurene synthesis activity during normal photomorphogenesis in pea seedlings is due to photoinduction of de novo synthesis of one or more proteins involved in the biosynthetic pathway from mevalonate to kaurene. 相似文献