Abstract
A direct correlation between rates of condensation and overall chain elongation has been observed with palmitoylCoA, 6,9-octadecadienoyl-CoA, and 6,9,12-octadecatrienoylCoA. Rates of condensation and overall chain elongation were all depressed when microsomes were used from rats raised on a balanced uersus a fat-free diet. This effect was more pronounced for palmitoyl-CoA than for the unsaturated substrates. The rates of the /?-hydroxyacyl-CoA dehydrase and 2-truns-enoyl-CoA reductase reactions were always much higher than for condensation and were not altered by dietary change. These findings show that condensation is rate-limiting and implicate it in overall control of chain elongation. In addition, a difference in the rate of Nethylmaleimide inhibition for condensation activity with palmitoyl-CoA versus unsaturated substrates provides direct evidence for the involvement of at least one different enzyme in the condensation systems using saturated versus polyunsaturated primers. Rates of both the condensation and 2-trans-enoyl-CoA reductase reactions were always elevated when bovine serum albumin was included in the incubations. In the absence of albumin, 20 to 30
Highlights
A direct correlation between rates of condensation and overall chain elongation has been observed with palmitoyl
In order to further examine these aspects of chain elongation, we have studied individual reactions with representative saturated and polyunsaturated substrates chosen on the basis of their overall chain elongation activity [6]
Elution of the free fatty acid zone (Band 2 of Fig. IA) followed by methylation and analysis by GLC showed that more than 98% of the radioactivity was in methyl palmitate, with only trace amounts of radioactivity in methyl stearate and methyl oleate and with no detectable radioactivity in methyl 2-trans-octadecenoate. These data serve to characterize the products produced during the condensation reaction, but establish that when NADPH is omitted from the incubation the reaction is confined to the initial condensation step
Summary
Materials - CoA(SH), NADPH, dicyclohexylcarbodiimide, glutathione, and bovine serum albumin containing less than 0.005% free fatty acids were purchased from the Sigma Chemical Co., St. All CoA esters were purified as described by Al-Arif and Blecher (111, dissolved in water (pH 3) and extracted repeatedly with ether to assure complete removal of residual fatty acids. For condensation reactions the residue was taken up in chloroform:methanol (2:l) and fractionated by TLC on Silica Gel G using petroleum ether For measuring overall chain elongation, dehydrase and reductase reactions, the lipids from the Folch extract were converted to methyl esters by refluxing with 5% anhydrous HCl in methanol. For dehydrase assays the methyl esters were applied to Silica Gel G thin layer plates and developed with standards in petroleum ether:ethyl ether:acetic acid (70:30:1). The rate of dehydrase reversal as well as of the reductase reaction was determined by calculating the per cent of radioactivity present in unreacted substrate, P-hydroxy fatty acid, and in the a,p-saturated product
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