α‐Hydroxylation of Fatty Acids in Brain: Characterization of Heat‐Labile Factor

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Abstract
The particulate fraction, heat‐labile factor, heat‐stable factor, and NADPH are essential for the conversion of lignoceric acid (tetracosanoic acid) to cerebronic acid (α‐hydroxylignoceric acid). The heat‐labile factor was extracted from calf cerebellum and partially purified in four steps: ammonium sulfate precipitation, hydroxylapatite chromatography, isoelectric focusing, and NAD‐Agarose affinity chromatography. The specific activity of the heat‐labile factor was increased 105‐fold during the last three steps, with a yield of 37% of the activity. One major and several minor bands were visible when the preparation was examined by SDS‐polyacrylamide gel electrophoresis with Coomassie blue staining. The major band corresponded to a protein of molecular weight 32,700, and the minor bands corresponded to proteins of molecular weights 62,000 and 67,000. The activity was lost when the heat‐labile factor was incubated with 1 mM‐N‐ethylmaleimide. This inhibition was prevented by preincubating the heat‐labile factor with 1 mM‐NADH. These observations indicate that the heat‐labile factor contains a sulfhydryl group which is essential for activity, and that it is located at or near the binding site for the pyridine nucleotide.