Aspartate transport in Bacterium cadaveris with special reference to the phosphate aging effect.

Few bacterial enzymes have been the subject of as much contention with regard to cofactor requirements as has aspartase. Since Gale's (1938) proposal of the two aspartases, it has become increasingly clear that much of the difficulty has arisen from the extrapolation of resting cell effects, based on ammonia data, to cell-free enzyme. The demonstration by Smith and Lichstein (1954) and by Trudinger (1951, 1954, 1955) that a miscellany of cofactors including glucose, adenosine, and inorganic salts were producing membrane permeability effects emphasized the need for more definitive studies on resting cells. Conspicuously absent have been experiments in which uptake of substrate was measured, or attempts made to examine an uptake system divorced from the deaminase. It has been assumed unjustifiably that ammonia production by resting cells was a true index of intracellular aspartase activity. This fallacy has been pointed out by Ellfolk (1956) and emphasized by recent experimental findings (Williams, 1957). Clarification of the role of aspartase cofactors of indeterminate status would be significantly advanced by thoroughly investigating transport in resting cells. Of these remaining substances, biotin is by far the most important and most intriguing. The acid-hydrolyzed glucose factor reported by Christman and Williams (1952) and Williams and Christman (1953) was found on analysis to be a mixture of succinic and formic acids, which did not stimulate the cell-free enzyme. The case for biotin rests almost entirely on the phosphate aging technique as developed by Lichstein and Umbreit (1947), although Smith and Lichstein (1954) have also presented some data for dried cells. An unequivocal demonstration of a cofactor role for biotin with cell-free enzyme, either crude or purified, has not been made despite intensive effort in this laboratory (Williams and McIntyre, 1955a) and others. Phosphate aging is an empirical treatment of washed cells with molar primary phosphate which appears to render certain of their enzymes sensitive to specific stimulants. It has been proposed that phosphate aging effects the resolution of certain intracellular enzymes but this has never been proved for aspartase. The purpose of the present communication was to (a) investigate the nature of phosphate aging in the resting cell, (b) study the transport of aspartate by resting cell preparations, and (c) discuss the interpretation of existing data in the light of this information.