Partial purification and characterization of an acetylcarnitine hydrolase from bovine epididymal spermatozoa.
We previously reported that intact epididymal spermatozoa from bulls and hamsters oxidize [1-14C]acetyl-L-carnitine to 14CO2 at about the same rate as they oxidize [1-14C]acetate. In addition, we showed that acetylcarnitine is hydrolyzed by a hydrolase present in the plasma membrane and that the carnitine moiety does not enter the cell. Here we report the partial purification of the acetylcarnitine hydrolase from bovine spermatozoa and describe some of its properties. The detergent-extracted enzyme was purified by FPLC using an anion-exchange Mono-Q column. The hydrolase activity eluted from the column with the application of 0.22 to 0.30 M NaCl and was separated from acetylcholinesterase activity, which eluted with 0.35 to 0.40 M NaCl. Specific inhibitors of acetylcholinesterase had little effect on acetylcarnitine hydrolase but p-hydroxymercuriphenylsulfonate was a potent inhibitor of the hydrolase. Kinetic studies of the hydrolase yielded a K'm of 6-10 mM for acetylcarnitine and a V'max of 0.16 nmol min-1 mg protein-1. Similar studies with the acetylcholinesterase yielded a K'm for acetylcholine of about 300 microM and a V'max of 165 nmol min-1 mg protein-1. Acetylcarnitine was a poor substrate for the acetylcholinesterase. Several acyl-L-carnitines were tested as substrates for the hydrolase and the preferred substrate was acetylcarnitine. The role of acetylcarnitine hydrolase in the metabolism of acetylcarnitine by epididymal spermatozoa is discussed.
Bruns
K
K
Department of Chemistry, New Mexico State University, Las Cruces 88003.
Casillas
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D000110:Acetylcholinesterase / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002331:Carnitine / Q000031:analogs & derivatives / Q000138:chemical synthesis; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D004822:Epididymis; D007700:Kinetics; D008297:Male; D013094:Spermatozoa / Q000201:enzymology*; D013379:Substrate Specificity; D013869:Thiolester Hydrolases / Q000302:isolation & purification* / Q000378:metabolism
D000110:Acetylcholinesterase; D013869:Thiolester Hydrolases; C023749:acetylcarnitine hydrolase; D002331:Carnitine