Novel use of an iodo-myristyl-CoA analog identifies a semialdehyde dehydrogenase in bovine liver.
We describe here the identification, purification, and characterization of a semialdehyde dehydrogenase with a novel fatty acid binding function. The coenzyme A derivative of an 125I-labeled long chain saturated fatty acid (13-iodo-tridecanoate) was used to tag proteins which bind myristoyl-CoA. A prominent 57 kDa band was identified, which was isolated from bovine liver by a high salt extraction followed by ammonium sulfate precipitation. Sequential chromatographic separation using phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, Mono Q, and Fast Flow S resins resulted in a purified protein that migrated as a single band of 57 kDa on denaturing gels. Sephacryl-200 gel filtration provided a native molecular mass estimation of 118 kDa suggesting that this protein exists as a dimer. Two-dimensional gel analysis resolved three isoform variants with pI values of 7.4, 7.7, and 7.9, respectively, and established that the pI = 7.9 form has the highest propensity for fatty acid binding. We proceeded to generate tryptic peptides from the purified protein and subjected several peptides to microchemical sequencing. Degenerate oligonucleotide probes were designed and polymerase chain reaction was used to generate a unique nucleotide sequence. Subsequent screening of a bovine liver cDNA library yielded a 1.7-kilobase clone which encodes a protein of 537 amino acids (58 kDa) with 95% identity to mammalian methylmalonate semialdehyde dehydrogenase (MMSDH). In vitro assays confirmed that the purified 57-kDa protein exhibited MMSDH activity, and that preincubation of the enzyme with fatty acyl-CoA inhibited its dehydrogenase activity. The myristyl-CoA analog therefore serves as an affinity label for MMSDH. We propose that fatty acyl CoAs may have the potential to function as enzyme regulators in vivo.
Deichaite
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I
Department of Cell Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Berthiaume
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L
Peseckis
S M
SM
Patton
W F
WF
Resh
M D
MD
D000214:Acyl Coenzyme A; D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004247:DNA; D004591:Electrophoresis, Polyacrylamide Gel; D005227:Fatty Acids / Q000378:metabolism; D007454:Iodides; D008099:Liver / Q000201:enzymology*; D050646:Methylmalonate-Semialdehyde Dehydrogenase (Acylating); D008969:Molecular Sequence Data; D051381:Rats; D017386:Sequence Homology, Amino Acid
D000214:Acyl Coenzyme A; D005227:Fatty Acids; D007454:Iodides; C040349:S-tetradecanoyl-coenzyme A; D004247:DNA; D000445:Aldehyde Oxidoreductases; D050646:Methylmalonate-Semialdehyde Dehydrogenase (Acylating)