High-performance liquid chromatographic assay for S-adenosyl- l-methionine:macrocin O-methyltransferase

A high-performance liquid chromatographic (HPLC) procedure was developed to assay S-adenosyl- l-methionine:macrocin O-methyltransferase. This enzyme catalyses the rate-limiting terminal reaction of tylosin biosynthesis in Streptomyces fradiae. HPLC analysis was improved by resin treatment of cell-fr...

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Bibliographic Details
Published in:Journal of Chromatography A 1984-01, Vol.288 (1), p.157-165
Main Authors: Yeh, Wu-kuang, Bauer, Nancy J., Dotzlaf, Joe E.
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Enzymes and enzyme inhibitors
Fundamental and applied biological sciences. Psychology
Transferases
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Summary:A high-performance liquid chromatographic (HPLC) procedure was developed to assay S-adenosyl- l-methionine:macrocin O-methyltransferase. This enzyme catalyses the rate-limiting terminal reaction of tylosin biosynthesis in Streptomyces fradiae. HPLC analysis was improved by resin treatment of cell-free extracts to remove endogenous tylosin and related compounds. Relomycin was selected as an internal standard and the enzymatic reaction conditions were optimized. The reaction mixture was extracted with ethyl acetate to recover the substrate, product and the internal standard. Efficient separation of the macrolide antibiotics was provided by ion-pair reversed-phase HPLC. An average relomycin recovery was 90%. The O-methyltransferase activity could be routinely and reproducibly determined by monitoring tylosin formation at 285 nm.
ISSN:0021-9673
DOI:10.1016/S0021-9673(01)93690-8