Purification and properties of an α‐methylacyl‐CoA racemase from rat liver

Abstract

The (R)-and (S)-isomers of a-methyl-branched fatty acids were shown to be rapidly interconverted as coenzyme A thioesters, by an a-methylacyl-CoA racemase. The enzyme was purified some 5600-fold from rat liver, to apparent homogeneity. It is a monomer of 45 kDa with an isolectric point of pH 6.1 and is optimally active between pH 6 and pH 7. It acts only on coenzyme A thioesters, not on free fatty acids, and accepts as substrates a wide range of a-methylacyl-CoAs, including pristanoyl-CoA and trihydroxycoprostanoyl-CoA (an intermediate in bile acid synthesis), but neither 3-methyl-branched nor linear-chain acyl-CoAs. The racemase catalyzes a rapid exchange of the H atom in the a-position of the fatty acid against a proton from water, indicating that the mechanism involves abstraction of a proton. Based on this observation, a very sensitive and convenient radiometric assay, with 2-methy1[2-3H]acyl-CoAs as substrates, was developed. The enzyme was inactivated by micromolar concentrations of Hg2' and to a lesser extent by Cu2+ but not by iodoacetamide and only slightly by N-ethylmaleimide and thimerosal.

Use of novel immunohistochemical markers expressed in colonic adenocarcinoma to distinguish primary ovarian tumors from metastatic colorectal carcinoma

“…17,30,31 Overexpression of P504S has been observed in several tumors, most notably prostate and colorectal carcinoma and in one study, 92% of colorectal carcinomas were found to overexpress this protein. 16,32 The expression of this protein in cancer precursor lesions such as high-grade prostatic intraepithelial neoplasia and colorectal adenoma suggests that it has a role in early carcinogenesis.…”

Stereochemistry of Peroxisomal and Mitochondrial β‐oxidation of α‐methylacyl‐CoAs

“…Purification of rat liver a-methylacyl-CoA racemase and preparation of an antiserum in rabbits were described before (Schmitz et al, 1994(Schmitz et al, , 1995.…”

“…and purified by column chromatography on 20 ml silica gel, with 100 ml of the same solvent mixture. Wittig reaction of the aldehyde with (l-ethoxycarbonylethyliden)-triphenylphosphoran to pristenic acid ethyl ester and hydrolysis of the ester were done essentially as described for 2-methyltetradec-2-enoic acid (Schmitz et al, 1994).…”

“…Since a-oxidation does not affect the hydrogen atom in P-position (Avigan et al., 1966;Fingerhut et al, 1993), the resulting product, pristanic acid, is likewise a mixture of diastereomers (Ackman and Hansen, 1967). We have previously shown that, in both rat liver (Schmitz et al, 1994) and human tissues (Schmitz et al, 1995), a-methyl-branched fatty acids are racemized as coenzyme A thioesters by a specific a-methylacyl-CoA racemase. It was not known, however, whether this racemase is indeed required for catabolism of branched-chain fatty acids or whether both enantiomers can be degraded directly.…”

“…The chemicals used were purchased from the following companies: farnesol, (I-ethoxycarbonylethy1iden)-tri- phenylphosphoran, ferrocene, NaPF,, trifluoroacetic acid anhydride, diisobutylaluminium hydride, Pd on charcoal, H,O, (30 % solution), sucrose, Tris, heptadecanoic acid, pentadecanoic acid and (R)-1 -phenylethylamine from Aldrich; dichloromethane, diethyleneglycol dimethyl ether (diglyme), acetic acid ethyl ester and TiO(S0,) from Riedel-de Haen; phenazine methosulfate, 2,6-dichloroindophenol, iodonitrotetrazolium violet, coenzyme A (Li, salt), Nycodenz and protein-A-agarose from Sigma; ATP from Boehringer (Mannheim) ; Coomassie brilliant blue G-250 from Roth; reverse-phase silica gel (RP-18) from ICN Chemicals; silica gel 60 and all other solvents from Merck. All other chemicals were of analytical grade or of the highest purity available.GC columns were purchased from CS-Chromatographie Service (SE-30) and from Macherey & Nagel (PB-OV-1701).Purification of rat liver a-methylacyl-CoA racemase and preparation of an antiserum in rabbits were described before (Schmitz et al, 1994(Schmitz et al, , 1995.Chemical syntheses. Ferricenium hexufluorophosphate (Neuse, 1985).…”