Distribution of enzymes in cell-free yeast extracts

Abstract

Cell-free yeast extracts were prepared by 10-, 30- and 90-sec. high-speed mechanical disintegration, and residual intact cells removed by centrifugation at 1000 g. The extracts were then fractionated into particulate fraction A (sedimented at 3500 g), particulate fraction B (sedimented at 10,000 g) and non-sedimentable supernatant. The following substrates, in decreasing order of activity, caused methylene-blue reduction in Thunberg tubes with whole unfractionated extracts: ethanol, L-malate, isocitrate, lactate, L-glutamate, citrate and succinate. Fumarase was about three times as active as aconitase. Increasing periods of mechanical disintegration caused a progressive migration of all enzymes tested from particles to supernatant. The enzyme content of both particulate fractions was so similar that they are probably not separate entities. Malic and ethanol dehydrogenase activites are maximal with DPN, "citric" and glutamic dehydrogenase activities with TPN. All four enzymes are activated to some extent by both coenzymes. The other enzymes require no added coenzymes. The particles of 10-sec. extracts are rich in all enzymes examined and contain bound coenzymes which are not removed by repeated washing, but are lost by 24 hr. storage at 2[degree] or by mechanical treatment. The particles of 30- and 90-sec. extracts contain less, if any, bound coenzymes. Several sets of findings point to masked fumarase and aconitase activities in unfractionated extracts, and the masking is associated with the particulate fraction. Various possible explanations are discussed. It is suggested that in the intact cell the microscopically visible granules, which some authors have called mitochondria on histological grounds, are active sites of enzyme activities concerned with respiration.