PURIFICATION AND PARTIAL CHARACTERIZATION OF BOVINE KIDNEY ALDEHYDE DEHYDROGENASE ABLE TO OXIDIZE RETINAL TO RETINOIC ACID

A NAD-dependent enzyme that catalyzes the oxidation of retinal to reti noic acid has been purified to homogeneity from bovine kidney. The pro cedures used in the purification included ion-exchange chromatography on DEAE-Sepharose, affinity chromatography on Affi-gel blue and chroma tography on a Mono-Q anion-exchange column. On the Mono-Q column, the enzyme aldehyde dehydrogenase (ALDH) resolved into two activity peaks designated as ALDH(1) and ALDH(2). The enzymes ALDH(1) and ALDH(2) wer e purified about 114- and 65-fold, respectively. Gel filtration chroma tography of the partially purified native enzyme on Sephacryl S-200 HR exhibited a molecular mass of about 108 kDa. Electrophoresis of the p urified enzymes under nondenaturing conditions showed a single protein band. However, sodium dodecyl sulfate - polyacrylamide gel electropho rsis indicated three protein bands in the 55, 30, and 22 kDa molecular mass regions. Both enzymes exhibited a broad substrate specificity ox idizing a wide variety of aliphatic and aromatic aldehydes. The ALDH(1 ) enzyme had a pI of 7.45 and exhibited a low K-m(6.37 mu M) for retin al, while the ALDH(2) enzyme was found to have very low K-m for acetal dehyde (0.98 mu M). Based on its kinetic properties, it is suggested t hat the ALDH(1) enzyme may be the primary enzyme for oxidizing retinal to retinoic acid in bovine kidney.