Purification and partial characterization of rat kidney histamine-N-methyltransferase |
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Authors: | Rauno J Harvima E Olavi Kajander Ilkka T Harvima Jorma E Fraki |
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Institution: | 1. Department of Dermatology, University of Kuopio, SF-70211 Kuopio 21 Finland;2. Department of Biochemistry, University of Kuopio, SF-70211 Kuopio 21, Finland |
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Abstract: | Histamine-N-methyltransferase (EC 2.1.1.8) was purified 1700-fold with a yield of 9% from rat kidney. Purification included ammonium sulfate precipitation, linear gradient DEAE-cellulose chromotography and S-adenosylhomocysteine affinity chromotography. The purified enzyme preparation showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of 35 000. The isoelectric point of the enzyme was at pH 5.2. The purified enzyme preparation did not contain detectable amounts of histamine. The purified enzyme was totally inhibited in 100 μM parahydroxymercuric benzoate and in 10 μM iodoacetamide, and it was found to be stabilized with dithiothreitol (1 mM), suggesting that the enzyme has an SH-group in the active center. The Km values for histamine and S-adenosylmethionine were 6.0 and 7.1 μM, respectively. 50% inhibition of histamine-N-methyltransferase was obtained at 28 μM S-adenosylhomocysteine and 100 μM methylhistamine. The purified enzyme was slightly inhibited in 1 mM methylthioadenosine. Histamine in concentrations higher than 25 μM caused substrate inhibition. |
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Keywords: | Inhibition kinetics (Rat kidney) Hepes 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid |
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