Acyl-CoA-ACP-transacylases of Mycobacterium smegmatis. An analytical study

Homogenates of Mycobacterium smegmatis have been prepared by sonication of bacteria in 0,1 M phosphate buffer, pH 7, containing 0,001 M EDTA and 0,01 M 2-mercaptoethanol. The 105 000 g supernatant of these homogenates has been fractionated by ammonium sulfate precipitation. The fraction precipitating between 25 and 65 per cent saturation in ammonium sulfate shows palmityl-CoA-ACP-transacylase, malonyl-CoA-ACP-transacylase and acetyl-CoA-ACP-transacylase activities. These activities are not associated, essentially, to a complexe of high molecular weight as they are retained on the column for 80 per cent or more upon gel filtration.By chromatographic procedures involving Sephadex G-150 gel filtration, and chromatography on DEAE-Sephadex A-50 the malonyl-CoA-ACP-transacylase activity is separated from the other two activities. The palmityl-CoA-ACP-transacylase activity and the acetyl-CoA-ACP-transacylase activity show two close but distinct major peaks either upon gel filtration on Sephadex G-150 or upon DEAE-Sephadex A-50 chromatography indicating thus that these two activities are attribuable to two distinct molecular entities. Besides the major peaks for these two activities, a supplementary minor peak for each activity is observed.An estimate of the approximate range of the apparent molecular weights for palmityl-CoA-ACP-transacylase, malonyl-CoA-ACP-transacylase and acetyl-CoA-ACP-transacylase has been made in using the curve of the K_av values of known proteins plotted against the logarithms of their molecular weights. The molecular weights thus estimated for these three enzymes are 100 000, 32 000 and 85 000 respectively.The void volume fractions of the chromatography of the homogenates on Sephadex G-150 present 20 per cent or less of the enzyme activity of the homogenates. Chromatography of the mixture of the void volume fractions on DEAE-Sephadex A-50 shows that the malonyl-CoA-ACP-transacylase activity associated to these fractions behaves differently from that of the fractions retained upon gel filtration; this may be taken as an indication for the presence of a complexe of the fatty acid synthetase present as a minor component of the homogenates.