Biosynthesis of unsaturated sphingolipid bases by microsomal preparations from oysters.
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Biomedical subjects
Publications and source records attributed to R K Hammond.
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The carotenoid pigments of Staphylococcus aureus U-71 were identified as phytoene; zeta-carotene; delta-carotene; phytofluenol; a phytofluenol-like carotenoid, rubixanthin; and three rubixanthin-like carotenoids after extraction, saponification, chromatographic separation, and determination of their absorption spectra. There was no evidence of carotenoid esters or glycoside ethers in the extract before saponification. During the aerobic growth cycle the total carotenoids increased from 45 to 1,000 nmoles per g (dry weight), with the greatest increases in the polar, hydroxylated carotenoids. During the anaerobic growth cycle, the total carotenoids increased from 20 nmoles per g (dry weight) to 80 nmoles per g (dry weight), and only traces of the polar carotenoids were formed. Light had no effect on carotenoid synthesis. About 0.14% of the mevalonate-2-(14)C added to the culture was incorporated into the carotenoids during each bacterial doubling. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The total carotenoids did not lose radioactivity when grown in the absence of (14)C for 2.5 bacterial doublings. The incorporation and turnover of (14)C indicated the carotenes were sequentially desaturated and hydroxylated to form the polar carotenoids.
Compounds known to be inhibitors of mixed-function oxidase systems inhibited the aerobic synthesis of hydroxylated carotenoids in Staphylococcus aureus U-71. Growth of the cells in the presence of 2-diethylaminoethyl-2,2-diphenyl valerate, 2,4-dichloro-6-phenylphenoxyethylamine, 2,4-dichloro-6-phenylphenoxyethyldiethylamine, and piperonyl butoxide reduced the levels of the rubixanthins found in stationary-phase cells by 75 to 97%. In cells grown with mevalonate-2-(14)C, the turnover rate of phytoene was reduced and the turnover rate of phytofluenol was increased in the presence of these inhibitors. The zeta- and delta-carotenes, which turn over in the absence of the inhibitors, accumulated (14)C in the presence of the inhibitors. This suggested that a mixed-function oxidase was responsible for the aerobic hydroxylation of delta-carotene in S. aureus U-71.
Diphenylamine at concentrations which did not effect the growth rate inhibited the synthesis of vitamin K(2) in both anaerobic and aerobic cultures by about 50%. At this concentration, diphenylamine inhibited the synthesis of the cyclic carotenoids delta-carotene and the rubixanthins 25 to 35% anaerobically and 60 to 90% aerobically. The inhibition of synthesis of cyclic carotenoids and vitamin K(2) by diphenylamine had no detectable effect on the formation of the membrane-bound electron transport system.
Lowering the temperature of growth of Staphylococcus aureus from 37 to 25 C decreased the growth rate and induced changes in the composition of the membrane lipids. Changes in lipid composition also occur in the transition between exponential and stationary growth phases at one temperature. To isolate the effects of lowering the temperature, exponentially growing S. aureus was abruptly switched from 37 to 25 C by transfer to cooler medium. Exponential growth continued at 25 C without a lag period but with a threefold increase in doubling time. In the period of exponential growth at suboptimal temperature, there was essentially no change in the fatty acid composition of the lipids, little change in the vitamin K(2) composition with perhaps a slight increase in the total level, and essentially no change in the phospholipid composition, but a marked stimulation of the synthesis of the rubixanthins. Growth of cells at 25 C was much more sensitive to the inhibition of rubixanthin formation by mixed-function oxidase inhibitors than cells growing at 37 C, suggesting some function for the rubixanthins at suboptimal temperatures. The striking increases in the proportions of monoenoic fatty acids observed at lowered growth temperatures in many biological systems are not detected in S. aureus.
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In Staphylococcus aureus, vitamin K(2) isoprenologues can be labeled with (14)C-acetate, mevalonate, glycerol, alpha-naphthol, and menadione. All the radioactivity from alpha-naphthol is in the ring nucleus. Eighty-six per cent of the radioactivity from mevalonate is in the side chain. The labeled isoprenologues of vitamin K(2) can be separated and recovered quantitatively. The rates of incorporation and turnover after pulse-chase experiments indicate that the isoprenologues do not lose radioactivity once they are formed. The kinetics of incorporation of radioactivity into the ring nucleus of each isoprenologue are the same. The alkylation of the ring does not involve a simple sequential addition of isoprenoid units of the same specific activity. The incorporation of mevalonate shows that shorter side chain isoprenologues have a higher specific activity per five carbon unit than the longer side chain isoprenologues. Perhaps the alkyl precursors are from different pools since the kinetics of incorporation into all isoprenologues are different.