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- Hjalmarsson, Karin J., et al.
(författare)
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Purification and characterization of transfer RNA (guanine-1)methyltransferase from Escherichia coli
- 1983
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 258:2, s. 1343-1351
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Tidskriftsartikel (refereegranskat)abstract
- The tRNA modifying enzyme, tRNA (guanine-1)methyltransferase has been purified to near homogeneity from an overproducing Escherichia coli strain harboring a multicopy plasmid carrying the structural gene of the enzyme. The preparation gives a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is probably a single polypeptide chain of molecular weight 32,000. The amino acid composition is presented and the NH2-terminal amino acid sequence was established to be H2N-Met-Trp-Ile-Gly-Ile-Ile-Ser-Leu-Phe-Pro. The enzyme has a pI of 5.2. The tRNA (guanine-1)-methyltransferase has a pH optimum of 8.0-8.5, an apparent Km of 5 microM for S-adenosylmethionine. S-adenosylhomocysteine is a competitive inhibitor for the enzyme with an apparent Ki of 6 microM. Spermidine or putrescine are not required for activity, but they stimulate the rate of methylation 1.2-fold with optima at 2 and 6 mM, respectively. Ammonium ion is not required and is inhibitory at concentrations above 0.15 M. Magnesium ion inhibited the activity at a concentration as low as 2 mM. Sodium and potassium ions were inhibitory at concentrations above 0.1 M. The molecular activity of tRNA (guanine-1)-methyltransferase was calculated to 10.0 min-1. It was estimated that the enzyme is present at 80 molecules/genome in cells growing with a specific growth rate of 1.0.
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| 5. |
- Persson, Bengt L., et al.
(författare)
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Energy-linked nicotinamide nucleotide transhydrogenase : Properties of proton-translocating mitochondrial transhydrogenase from beef heart purified by fast protein liquid chromatography
- 1984
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 259, s. 8626-8632
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Tidskriftsartikel (refereegranskat)abstract
- Mitochondrial nicotinamide nucleotide transhydrogenasefrom beef heart was purified by a novel procedureinvolving fast protein liquid chromatography andcharacterized with respect to molecular and catalyticproperties. The method is reproducible, gives highlypure transhydrogenase as judged by silver staining,and can be modified to produce large amounts of puretranshydrogenase protein suitable for e.g. sequencingand other protein chemical studies.Transhydrogenase purified by fast protein liquidchromatography is reconstitutively active and pumpsprotons as indicated by an extensive quenching of 9-aminoacridine fluorescence. Under conditions whichgenerate a proton gradient in the absence of a membranepotential the activity of reconstituted transhydrogenaseis close to zero indicating a complete andproper incorporation in the membrane and a preferentialregulation of the enzyme by a proton gradientrather than a membrane potential.Treatment of reconstituted transhydrogenase withN,N-dicyclohexylcarbodiimide results in an inhibitionof proton pump activity without an effect on uncoupledcatalytic activity, suggestingt hat proton translocationand catalytic activities are not obligatory linked orthat this agent separates proton pumping from thecatalytic activity.
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