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1.
Carlos E. Argaraña Hector S. Barra Ranwel Caputto 《Molecular and cellular biochemistry》1978,19(1):17-21
Summary The carboxypeptidase previously described3 that releases tyrosine from tubulinyl-tyrosine was obtained from rat brain preparation free of tubulin-tyrosine ligase. The enzyme was purified 24-fold. Its activity was increased by 2 mm MgCl2 or 30 mm KCl. Mercaptoethanol (50 mm), colchicine (0.2 mm) and tyrosine (0.2 mm) showed practically no effect on the release of tyrosine whereas iodoacetate (2 mm), deoxycholate (0.5%), CuCl2 (0.1 mm), ZnC12 (0.1 mm) and NaCl or KCI (240 mm) had a strong inhibitory effect. The optimal pH of this enzyme. was 6.3–7.A preparation containing tubulin-tyrosine ligase free of carboxypeptidase was also obtained. This preparation catalyzed the release of tyrosine from tyrosinated tubulin in the presence of ADP, Mg2+, K– and Pi and the incorporation of tyrosine into tubulin. For the releasing activity the optimal concentration of MgCl2 was 3–20 mm and of KCl was 10–30 mm. For ADP the maximal activity was at 0.3 mm or higher.An important difference between the activities of the carboxypeptidase and the ligase was that the former was active on denatured tubulin whereas the latter was not. 相似文献
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Preferential action of a brain detyrosinolating carboxypeptidase on polymerized tubulin 总被引：22，自引：0，他引：22
A carboxypeptidase purified from brain catalyzes the release of COOH-terminal tyrosine without further digesting tubulin. It is distinct from previously described carboxypeptidases, and appears to have specificity for tubulin as it is not inhibited by peptides and proteins with COOH-terminal tyrosine, and because, unlike carboxypeptidase A (which by removing tyrosine from aldolase causes its inactivation), this enzyme does not decrease aldolase activity. The enzyme detyrosinolates both self-assembly-competent (cycle-purified) and -incompetent (phosphocellulose-purified) tubulin. However, under assembly conditions the rate was 2-3-fold higher for competent tubulin. Preincubation of assembly-competent tubulin with podophyllotoxin or colchicine resulted in a parallel concentration-dependent inhibition of tubulin polymerization and detyrosinolation. Similarly, when incompetent tubulin was induced to polymerize by preincubation with purified microtubule-associated protein 2 (an assembly-promoting protein) or taxol, the initial rate of its detyrosinolation increased 3-5-fold, and this increase was blocked if podophyllotoxin was also added along with microtubule-associated protein 2 or taxol during the preincubation. Oligomers induced by adding vinblastine to incompetent tubulin were also detyrosinolated more rapidly, and the stimulation was abolished by maytansine, which has been shown to disperse the vinblastine-induced oligomers. When polymerized and subunit fractions were separated after a steady state mixture had been partially digested with the carboxypeptidase, the former was found to have lost 2-3 times more COOH-terminal tyrosine. Although both polymer and monomer can be detyrosinolated by the enzyme, polymeric and oligomeric forms are the preferred substrates. Carboxypeptidase appeared to release tyrosine at the same rate from populations of short and long microtubules. 相似文献
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Thermophilic Anaerobic Biodegradation of [14C]Lignin, [14C]Cellulose, and [14C]Lignocellulose Preparations 总被引：2，自引：0，他引：2 
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下载免费PDF全文 Thermophilic (55°C) anaerobic enrichment cultures were incubated with [14C-lignin]lignocellulose, [14C-polysaccharide]lignocellulose, and kraft [14C]lignin prepared from slash pine, Pinus elliottii, and 14C-labeled preparations of synthetic lignin and purified cellulose. Significant but low percentages (2 to 4%) of synthetic and natural pine lignin were recovered as labeled methane and carbon dioxide during 60-day incubations, whereas much greater percentages (13 to 23%) of kraft lignin were recovered as gaseous end products. Percentages of label recovered from lignin-labeled substrates as dissolved degradation products were approximately equal to percentages recovered as gaseous end products. High-pressure liquid chromatographic analyses of CuO oxidation products of sound and degraded pine lignin indicated that no substantial chemical modifications of the remaining lignin polymer, such as demethoxylation and dearomatization, occurred during biodegradation. The polysaccharide components of pine lignocellulose and purified cellulose were relatively rapidly mineralized to methane and carbon dioxide; 31 to 37% of the pine polysaccharides and 56 to 63% of the purified cellulose were recovered as labeled gaseous end products. An additional 10 to 20% of the polysaccharide substrates was recovered as dissolved degradation products. Overall, these results indicate that elevated temperatures can greatly enhance rates of anaerobic degradation of lignin and lignified substrates to methane and low-molecular-weight aromatic compounds. 相似文献
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Stearoyl[1-14C]sulfogalactosylsphingosine ([14C]sulfatide) as substrate for cerebroside sulfatase assay 总被引：3，自引：0，他引：3
A convenient method for large scale preparation of stearoyl[1-14C]sulfogalactosylsphingosine has been developed. The first step consists in preparing the lysosulfatide intermediate with a good yield, which we have successfully performed. In the second step, the lysoderivative is coupled to [1-14C]stearic acid through the acyl chloride procedure. The labeled substrate thus synthetized appears to be particularly convenient for cerebroside sulfatase determination, because of the stability of the 14C isotope, compared to the other isotopes (tritium or 35S) which have been previously used for the same purpose. 相似文献
5.
Cultures of Fusarium roseium 'Gibbosum' on rice were treated with [14C]zearalenone, alpha[14C]zearalenol, or beta-[14C]zearalenol to determine whether a precursor-product relationship exists among these closely related fungal metabolites. Culture extracts were purified by silica gel column chromatography and fractionated by high-pressure liquid chromatography, and the level of radioactivity was determined. Within 7 days, the beta-[14C]zearalenol was converted to zearalenone, and no residual beta-[14C]zearalenol was detectable. Most of the alpha-[14C]zearalenol added was also converted into zearalenone with 14 days. In cultures treated with [14C]zearalenone, no radioactivity was noted in any other components. 相似文献
6.
D A Redburn 《Journal of neurochemistry》1978,31(4):939-945
[14C]GABA is taken up by rat brain synaptosomes via a high affinity, Na+-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [14C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca2+, Mg2+, and Mn2+. However, the relatively smaller amount of [14C]GABA taken up by synaptosomes in the absence of Na+ is not released from synaptosomes by Ca2+ -dependent, K +-stimulation. [14C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na + -dependent process; and is subsequently released by Ca2+ -dependent, K+-stimulation. On the other hand, [14C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [14C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca2+ -dependent, K+-stimulation. These data suggest that entry of [14C]GABA into a releasable pool requires external Na+ ions and maximal evoked release of [14C]GABA from the synaptosomal pool requires external Ca2+ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na+-free medium) which inhibited uptake, had little effect on release. 相似文献
7.
Bioconversion of alpha-[14C]zearalenol and beta-[14C]zearalenol into [14C]zearalenone by Fusarium roseum 'Gibbosum'. 下载免费PDF全文
下载免费PDF全文 Cultures of Fusarium roseium 'Gibbosum' on rice were treated with [14C]zearalenone, alpha[14C]zearalenol, or beta-[14C]zearalenol to determine whether a precursor-product relationship exists among these closely related fungal metabolites. Culture extracts were purified by silica gel column chromatography and fractionated by high-pressure liquid chromatography, and the level of radioactivity was determined. Within 7 days, the beta-[14C]zearalenol was converted to zearalenone, and no residual beta-[14C]zearalenol was detectable. Most of the alpha-[14C]zearalenol added was also converted into zearalenone with 14 days. In cultures treated with [14C]zearalenone, no radioactivity was noted in any other components. 相似文献
8.
Kästner M Streibich S Beyrer M Richnow HH Fritsche W 《Applied and environmental microbiology》1999,65(5):1834-1842
Carbon partitioning and residue formation during microbial degradation of polycyclic aromatic hydrocarbons (PAH) in soil and soil-compost mixtures were examined by using [14C]anthracenes labeled at different positions. In native soil 43.8% of [9-14C]anthracene was mineralized by the autochthonous microflora and 45.4% was transformed into bound residues within 176 days. Addition of compost increased the metabolism (67.2% of the anthracene was mineralized) and decreased the residue formation (20. 7% of the anthracene was transformed). Thus, the higher organic carbon content after compost was added did not increase the level of residue formation. [14C]anthracene labeled at position 1,2,3,4,4a,5a was metabolized more rapidly and resulted in formation of higher levels of residues (28.5%) by the soil-compost mixture than [14C]anthracene radiolabeled at position C-9 (20.7%). Two phases of residue formation were observed in the experiments. In the first phase the original compound was sequestered in the soil, as indicated by its limited extractability. In the second phase metabolites were incorporated into humic substances after microbial degradation of the PAH (biogenic residue formation). PAH metabolites undergo oxidative coupling to phenolic compounds to form nonhydrolyzable humic substance-like macromolecules. We found indications that monomeric educts are coupled by C-C- or either bonds. Hydrolyzable ester bonds or sorption of the parent compounds plays a minor role in residue formation. Moreover, experiments performed with 14CO2 revealed that residues may arise from CO2 in the soil in amounts typical for anthracene biodegradation. The extent of residue formation depends on the metabolic capacity of the soil microflora and the characteristics of the soil. The position of the 14C label is another important factor which controls mineralization and residue formation from metabolized compounds. 相似文献
9.
S Ratner 《Analytical biochemistry》1983,135(2):479-488
A simple and sensitive radiochemical procedure to assay argininosuccinate synthetase activity in crude tissue homogenates and lysates of cultured cells is described. The new method depends on the location of 14C, uniformly, in the four carbons of aspartate. On incubation in the presence of excess of L-[U-14C]aspartate, L-citrulline, ATP, and an ATP-generating system, argininosuccinase and arginase, the [14C]fumarate formed is measured as the sum of malate and fumarate. After acidification the latter two acids are separated from [14C]aspartate on a small Dowex-50 column by elution with a few milliliters of water; the unutilized amino acid substrates remain on the column. With a specific radioactivity of 9 X 10(4) cpm, 1 to 2 nmol of product can be accurately measured under kinetically optimum conditions. 相似文献
10.
A sensitive and simple radiochemical assay is described to measure argininosuccinase activity in crude tissue homogenates and cultured cells. The method depends on the use of argininosuccinate labeled uniformly with 14C in the six carbons of the arginine moiety. On incubation in the presence of excess arginase, the [U-14C]arginine formed is measured as the sum of radioactivity in [U-14C]ornithine and [14C]urea. Separation from the substrate is accomplished on a small Domex 1-acetate column eluted with 25 mm acetic acid; ornithine and urea emerge in the first few milliliters while unutilized substrate remains on the column. [14C]Argininosuccinate was synthesized enzymatically from l-[U-14C]arginine and fumarate and isolated and purified as the barium salt. Development of a new purification step has brought the amino acid to a purity of 97% as judged by chromatographic and barium analysis. With the present specific radioactivity, as little as 5 to 10 nmol of product can be accurately measured under kinetically optimum conditions. 相似文献
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Calf brain synaptic plasma membranes were saturated under extracellular conditions with [14C]glutamic acid and the resulting labelled membranes fractionated with 0.9% NaCl, distilled water,n-butanol-water, 0.05 mol/L NaOH and 0.5% Triton X-100 solutions in this order. Triton X-100 was the most effective solubilizer, liberating altogether about 24% of the membrane proteins, but only 4–7% of the label, while NaOH was the most potent solubilizer for the protein-bound label (50–70%). Slab gel electrophoresis showed significant qualitative differences in the banding patterns of the protein extracts, but only two fractions, a low-molecular weight (around 15 kd) and a high-molecular weight (>200 kd) fraction bound [14C]glutamate. 相似文献
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The microtubule protein, tubulin, was isolated from most other proteins of cell suspension cultures of Nicotiana tabacum L. by its copolymerization with cow-brain tubulin. Cow-brain tubulin was added to the soluble protein fraction of extract from 35S-labeled tobacco cells and subjected to two cycles of temperature-dependent assembly-disassembly (copolymerization). When analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) about 70% of the radioactivity in the twice copolymerized protein was found in a prominent doublet migrating close to the doublet of brain tubulin. When analyzed by two-dimensional isoelectric-focusing-SDS-PAGE the radioactive doublet behaved like the doublet of brain tubulin. Limited proteolysis of the individual polypeptides of the coublets showed that, while the peptide maps of the leading radioactive band and of the β-subunit of brain tubulin were virtually indistinguishable, the maps of the trailing radioactive band and of the α-subunit of brain tubulin, though similar, were not identical. Most of the copolymerized 35S-labeled protein also behaved like brain tubulin during gel filtration and ion-exchange chromatography. It is concluded that the doublet of radioactive polypeptides isolated by copolymerization with brain tubulin are tobacco tubulin polypeptides that have, in their native as well as denatured forms, properties very similar to, but not identical with, cow brain tubulin. Apparently, tubulin has been highly conserved during evolution. 相似文献
17.
A new enzymatic method for the synthesis of [14C]pyridoxal 5'-phosphate is presented. [14C]Pyridoxal 5'-phosphate was synthesized from [14C]pyridoxine through the successive actions of pyridoxal kinase and pyridoxamine 5'-phosphate oxidase in a reaction mixture containing ATP, [14C]pyridoxine, and both enzymes. [14C]Pyridoxal 5'-phosphate was isolated by omega-aminohexyl-Sepharose 6B column chromatography. The overall yield of the product was more than 60%, starting from 550 nmol of [14C]pyridoxine. The radiochemical purity of the products, as determined by thin-layer and ion-exchange chromatography, was greater than 98%. 相似文献
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G G Forstner 《Biochimica et biophysica acta》1968,150(4):736-738
20.
The distribution in rat brain of angiotensin converting enzyme (EC3.4.15.1) using hippuryl-His-Leu as substrate was identical to a dipeptidyl carboxypeptidase present in membranes assayed with Met-enkephalin as substrate. Highest activity occurred in pituitary, followed by cerebellum, corpus striatum, midbrain, pons-medulla, hypothalamus, cerebral cortex and spinal cord. The ratio of products His-Leu/Tyr-Gly-Gly was identical for all regions but differed from His-Leu/Tyr. Angiotensin converting enzyme purified by immunoaffinity chromatography gave a Km for hippuryl-His-Leu of 0.5mM and for Met-enkephalin of 0.1 mM. In the presence of the specific inhibitor of angiotensin converting enzyme, SQ 14,225, the Ki value was 10?7M. Present data point to the co-identity of brain angiotensin converting enzyme with the dipeptidyl carboxypeptidase inactivating enkephalin. 相似文献
