NOVEL USE OF AN IODO-MYRISTYL-COA ANALOG IDENTIFIES A SEMIALDEHYDE DEHYDROGENASE IN BOVINE LIVER

We describe here the identification, purification, and characterizatio n of a semialdehyde dehydrogenase with a novel fatty acid binding func tion. The coenzyme A derivative of an I-125-labeled long chain saturat ed fatty acid (13-iodo-tridecanoate) was used to tag proteins which bi nd myristoyl-CoA. A prominent 57 kDa band was identified, which was is olated from bovine liver by a high salt extraction followed by ammoniu m sulfate precipitation. Sequential chromatographic separation using p henyl-Sepharose, hydroxyapatite, DEAE-Sepharose, Mono Q, and Fast Flow S resins resulted in a purified protein that migrated as a single ban d of 57 kDa on denaturing gels. Sephacryl-200 gel filtration provided a native molecular mass estimation of 118 kDa suggesting that this pro tein exists as a dimer. Two-dimensional gel analysis resolved three is oform variants with pl values of 7.4, 7.7, and 7.9, respectively, and established that the pI = 7.9 form has the highest propensity for fatt y acid binding. We proceeded to generate tryptic peptides from the pur ified protein and subjected several peptides to microchemical sequenci ng. Degenerate oligonucleotide probes were designed and polymerase cha in reaction was used to generate a unique nucleotide sequence. Subsequ ent screening of a bovine liver cDNA library yielded a 1.7-kilobase cl one which encodes a protein of 537 amino acids (58 kDa) with 95% ident ity to mammalian methylmalonate semialdehyde dehydrogenase (MMSDH). In vitro assays confirmed that the purified 57-kDa protein exhibited MMS DH activity, and that preincubation of the enzyme with fatty acyl-CoA inhibited its dehydrogenase activity. The myristyl-CoA analog therefor e serves as an affinity label for MMSDH. We propose that fatty acyl Co As may have the potential to function as enzyme regulators in vivo.