Abstract
Abstract The enzymatic synthesis of tuberculostearic acid (10-methylstearic acid) was catalyzed by extracts of Mycobacterium phlei. This process involved two reactions of the olefinic fatty acid chain of phospholipids. The chain was first alkylenated at the 10-carbon to give a methylene group, which was subsequently reduced to a methyl group. The first reaction could be measured by using S-adenosylmethionine-methyl-14C. The enzyme was found in the supernatant fraction when extracts of cells broken by sonic oscillation were subjected to centrifugation at 100,000 x g. S-Adenosyl-l-methionine was the only effective donor of the 1-carbon unit. Phosphatidylglycerol, phosphatidylinositol, and phosphatidylethanolamine were substrates for the reaction, and both 16- and 18-carbon chains were alkylenated although only the Δ9-olefinic chains appeared to be converted. The enzyme acted upon chains at either position 1 or 2 of the glyceride molecule. Several detergents had little effect on the rate of the reaction.
Highlights
We have reported prelimmary evidence that these processes are described by the following reactions in crude, cellfree extracts [8] : (Olefinic fatty acyl) phospholipid + S-adenosylmethionine -+ [2]
The fractions were counted and the we used the procedure reported by Jaureguiberry et al [7] and Azerad, Bleiler-Hill, and Lederer [23] for preparing homogenates of M. phlei, we used much smaller volumes for incubations and S-adenosylmethionine instead of methionine
Further centrifugal fractionation showed that most of the enzymatic activity resided in the 100,000 X 9 supernatant fraction (Table I)
Summary
Preparation of Cell-free ExtractsWashed cells (0.2 g per ml) were suspended in 0.1 M phosphate buffer (pH 7.0) and disrupted at O-4” for 10 to 15 min in a sonic disintegrator (Branson Instruments, Inc., Stanford, Connecticut). The resulting supernatant, designated 20-S was further centrifuged at 105,000 X g for 60 min in the Spinco model L preparative centrifuge. This supernatant was adjusted to 10 mg of protein per ml with 0.1 M phosphate buffer (pH 7.0), and designated 100-S. Radioactive IO-methylene stearate was collected from the gas-liquid chromatography column and mixed with 1 mg of carrier lo-methylene stearate. This was dissolved in 1 ml of methanol and 2 mg of PtOz were added. The combined filtrate was reduced in volume and subjected to gas-liquid chromatography
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