Nucleotide sequence of the streptomycinphosphotransferase and amidinotransferase genes from Streptomyces griseus.
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Genes for streptomycin phosphotransferase and inosamine-P-amidinotransferase from a streptomycin-producing Streptomyces griseus were cloned on a 3.8kb BamHI-SphI fragment in S. lividans using the multicopy cloning vector pIJ702. The nucleotide sequence of this 3.8kb fragment was determined and the coding sequences for the two genes were identified by comparison with the amino-terminal sequences of the two enzymes purified from S. lividans clones. (+info)
Multiple forms of rat kidney L-arginine:glycine amidinotransferase.
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The relative amount of L-arginine:glycine amidinotransferase (transamidinase) protein in kidneys from rats fed a complete purified diet with and without the addition of creatine and/or glycine was determined by a monoclonal antibody-immunosorbent inhibition assay. Kidneys from the creatine-fed rats had 10% of the transamidinase activities and 78% of the monoclonal antibody immunoreactive transamidinase protein as kidneys from the control rats. An excellent correlation between transamidinase activities and protein was reported previously when the amounts of enzyme protein were determined by immunotitration with polyclonal antibodies. One possible explanation for the contrasting results was that multiple forms of transamidinase are present in rat kidneys. If so, the monoclonal antibody may have recognized forms of the enzyme that were not decreased in amounts commensurate with the decrease in enzyme activities as a result of creatine feeding. Evidence is presented in this report that multiple forms of transamidinase are present in rat kidneys. The distribution of the isoelectric points of the individual forms of transamidinase in kidneys of the control rats appeared to be dissimilar from that in the creatine-fed rats. Therefore, an alteration in the distribution of the individual forms of the enzyme may be a factor in the alteration of transamidinase activities in creatine-fed rats. (+info)
Creatine metabolism in the pyridoxine-deficient rat.
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Creatine metabolism was studied in rats fed a pyridoxine-deficient diet for 6-7 wk. Indirect evidence had suggested that creatine levels may be low in these animals, since their livers accumulate a competitive inhibitor of creatine biosynthesis (S-adenosylhomocysteine), and they develop muscle signs suggestive of abnormal creatine metabolism. In contrast, however, the concentration of creatine in both liver and skeletal muscle of pyridoxine-deficient rats was higher than in pair-fed control rats. Pair-fed control rats also had higher creatine levels in liver and skeletal muscle than ad libitum-fed control rats, whose feed intake was about double that of the pair-fed control rats. Thus, feed restriction also increased the tissue creatine levels, and this effect was augmented slightly by pyridoxine deficiency. These changes could not be explained by the results of in vitro measurement of the enzymes of creatine biosynthesis. The activity of guanidoacetate methyltransferase in liver did not differ significantly among the three groups of animals. Kidney arginine-glycine transamidinase activity was noticeably lower in the pyridoxine-deficient rats than in pair-fed control rats and lower in pair-fed control rats than in ad libitum-fed control rats. Since creatinine excretion did not differ significantly among the three animal groups, the results suggest that decreased turnover of creatine may occur during pyridoxine deficiency above and beyond that of an apparent feed restriction alone. (+info)
Isolation of streptomycin-nonproducing mutants deficient in biosynthesis of the streptidine moiety or linkage between streptidine 6-phosphate and dihydrostreptose.
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Eight streptidine idiotrophic mutants (SD20, SD81, SD141, SD189, SD245, SD261, SD263, and SD274) which required streptidine to produce streptomycin were derived from Streptomyces griseus ATCC 10137 by UV mutagenesis. By both the characterization of intermediates accumulated by the idiotrophs and the assay of enzymes involved in streptidine biosynthesis, the biochemical lesions of the mutants were deduced as follows: SD20 and SD263, transamination; SD81, SD261, and SD274, phosphorylation; SD141, transamidination; SD189, dehydrogenation; SD245, linkage between streptidine 6-phosphate and dihydrostreptose. An accumulation of streptidine 6-phosphate was found in SD245 to impair its aminotransferase activity. This finding suggests that aminotransferase activity might have been negatively controlled by the end product, streptidine 6-phosphate, of the streptidine biosynthetic pathway. (+info)
Higher homolog and N-ethyl analog of creatine as synthetic phosphagen precursors in brain, heart, and muscle, repressors of liver amidinotransferase, and substrates for creatine catabolic enzymes.
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Tissues of chicks fed 5% N-methyl-3-guanidinopropionate (N-amidino-N-methyl-beta-alanine) for 12 days accumulated the following amounts of free plus phosphorylated derivatives as mumol/g, wet weight: brain, 5.5; heart, 7.3; leg muscle, 21.0; and breast muscle, 24.4. Since total creatine levels remained nearly the same in brain, N-methyl-3-guanidinopropionate-P provided brain with a supplemental reservoir of high energy phosphate. Tissues of rats fed 2% N-ethylguanidinoacetate (N-amidino-N-ethylglycine) accumulated large amounts of N-ethylguanidinoacetate-P, which has thermodynamic properties similar to creatine-P and is the kinetically most reactive synthetic phosphagen yet described. N-Ethylguanidinoacetate derivatives replaced creatine derivatives mole-for-mole, and the fraction of synthetic to total phosphagen after 19 days was 60% in heart, 54% in slow oxidative muscle, 42% in fast glycolytic muscles, and 22% in brain. N-Ethylguanidinoacetate served as a false end product co-repressor of liver arginine:glycine amidinotransferase levels in both chicks and chick embryos; N-methyl-3-guanidinopropionate and N-propylguanidinoacetate were relatively inactive. Creatinine amidohydrolase reversibly cyclized both N-ethylguanidinoacetate and N-propylguanidinoacetate with even lower Km values than for creatine derivatives, but it did not react significantly with N-methyl-3-guanidinopropionate, 3-guanidinopropionate, or 1-carboxy-methyl-2-imino-imidazolidine (cyclocreatine). Creatine amidinohydrolase also hydrolyzed N-acetimidoylsarcosine, but was relatively unreactive toward N-ethylguanidinoacetate, N-methyl-3-guanidinopropionate, 3-guanidinopropionate, and cyclocreatine. Amidinohydrolase can therefore be used to remove interfering creatine in assays of tissues for coexisting N-ethylguanidinoacetate or N-methyl-3-guanidinopropionate. Assays are now available to follow changes during metabolic stresses of any combination or all of the following phosphagens accumulated by the same tissue: creatine-P, N-ethylguanidinoacetate-P, cyclocreatine-P, N-methyl-3-guanidinopropionate-P, and homocyclocreatine-P. (+info)
Partial purification and properties of a transamidinase from Lathyrus sativus seedlings. Involvement in homoarginine metabolism and amine interconversions.
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A transamidinase was purified 463-fold from Lathyrus sativus seedlings by affinity chromatography on homoarginine--Sepharose. The enzyme exhibited a wide substrate specificity, and catalysed the reversible transfer of the amidino groups from donors such as arginine, homoarginine and canavanine to acceptors such as lysine, putrescine, agmatine, cadaverine and hydroxylamine. The enzyme could not be detected in the seeds, and attained the highest specific activity in the embryo axis on day 10 after seed germination. Its thiol nature was established by strong inhibition by several thiol blockers and thiol compounds in the presence of ferricyanide. In the absence of an exogenous acceptor, it exhibited weak hydrolytic activity towards arginine. It had apparent mol.wt. 210000, and exhibited Michaelis--Menten kinetics with Km 3.0 mM for arginine. Ornithine competitively inhibited the enzyme, with Ki 1.0 mM in the arginine--hydroxylamine amidino-transfer reaction. Conversion experiments with labelled compounds suggest that the enzyme is involved in homoarginine catabolism during the development of plant embryo to give rise to important amino acids and amine metabolites. Presumptive evidence is also provided for its involvement in the biosynthesis of the guanidino amino acid during seed development. The natural occurrence of arcain in L. sativus and mediation of its synthesis in vitro from agmatine by the transamidinase are demonstrated. (+info)
Repression of rat kidney L-arginine:glycine amidinotransferase synthesis by creatine at a pretranslational level.
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The first committed reaction in the biosynthesis of creatine is catalyzed by the enzyme L-arginine:glycine amidinotransferase, commonly called transamidinase. Creatine, the end product of the biosynthetic pathway, is known to alter the levels of kidney transamidinase activity. Rats fed a diet containing 0.3% creatine had 26% of the kidney transamidinase activity of the rats fed a creatine-free diet. This reduction in transamidinase activity was correlated with a decrease in transamidinase protein in the creatine-fed rats. The relative synthetic rates and mRNA functional activities of transmidinase were measured in control and creatine-fed rats. The relative synthetic rate of transamidinase in creatine-fed rats was 21% of that found in the control animals. The functional transamidinase mRNA in creatine-fed rats was correspondingly reduced to 37% of the amount in the control animals. Thus, creatine affects transamidinase activity by altering its rate of synthesis at a pretranslational step and represents an example of end-product repression in a higher eukaryote. (+info)
The differential contribution of arginase and transamidinase to ornithine biosynthesis in two achromic human melanoma cell lines.
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Cellular ornithine biosynthesis could be expected to play a significant role in putrescine formation and hence in growth. Two enzymes are involved in ornithine biosynthesis: arginase and transamidinase. These enzyme activities were studied in two human melanoma cell lines differing in their Km of diamine oxidase for putrescine and in their tumorigenicity in nude mice. Arginase activity accounts for the majority of ornithine formed in the highly tumorigenic cell line, while the majority of ornithine is derived from transamidinase action in the poorly tumorigenic cell line, with concomitant formation of methyl guanidine, a potent inhibitor of diamine oxidase. (+info)