Pea chloroplast carnitine acetyltransferase.
The purpose of this study was to resolve the controversy as to whether or not chloroplasts possess the enzyme carnitine acetyltransferase (CAT) and whether the activity of this enzyme is sufficient to support previously reported rates of fatty acid synthesis from acetylcarnitine. CAT catalyses the freely reversible reaction: carnitine + short-chain acylCoA <--> short-chain acylcarnitine + CoASH. CAT activity was detected in thc chloroplasts of Pisum sativum L. With membrane-impermeable acetyl CoA as a substrate. activity was only detected in ruptured chloroplasts and not with intact chloroplasts, indicating that the enzyme was located on the stromal side of the envelope. In crude preparations, CAT could only be detected using a sensitive radioenzymatic assay due to competing reactions from other enzymes using acetyl CoA and large amounts of ultraviolet-absorbing materials. After partial purification of the enzyme, CAT was detected in both the forward and reverse directions using spectrophotometric assays. Rates of 100 nmol of product formed per minute per milligram of protein were obtained, which is sufficient to support reported fatty acid synthesis rates from acetylcarnitine. Chloroplastic CAT showed optimal activity at pH 8.5 and had a high substrate specificity, handling C2-C4 acyl CoAs only. We believe that CAT has been satisfactorily demonstrated in pea chloroplasts.
Masterson
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Department of Biological and Nutritional Sciences, University of Newcastle, Newcastle upon Tyne, UK.
Wood
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D000105:Acetyl Coenzyme A / Q000378:metabolism; D000108:Acetylcarnitine / Q000378:metabolism; D015153:Blotting, Western; D002331:Carnitine / Q000378:metabolism; D002332:Carnitine O-Acetyltransferase / Q000378:metabolism*; D002736:Chloroplasts / Q000201:enzymology*; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D018532:Pisum sativum / Q000201:enzymology* / Q000502:physiology
D000108:Acetylcarnitine; D000105:Acetyl Coenzyme A; D002332:Carnitine O-Acetyltransferase; D002331:Carnitine