Studies on bacterial amino-acid decarboxylases: 3. Distribution and preparation of codecarboxylase.

METHODS Figures have been published showing the variation of velocity of decarboxylation with' the concentration of codecarboxylase for a given amount of apo-enzyme in the cases of l(+)-lysine decarboxylase (Gale & Epps, 1944) and of 1( )-tyrosine decarboxylase (Epps, 1944). In either case the velocity of decarboxylation bears an approximately linear relation to the amount of codecarboxylase added as long as this amount is considerably less than the saturation value for the apo-enzyme used. Consequently this velocity can be used as a measure of the amount of coenzyme present during test, and comparative measurements can be made as long as other conditions remain constant and the same amount of the same preparation of apo-enzyme be used for any one series of comparisons. We have used this as a basis for the investigation of distribution and purification of codecarboxylase. The rate of decarboxylation is measured manometrically as previously'described. In these studies we have made measurements with the apo-enzymes of both lysine decarboxylase and tyrosine decarboxylase, so that a double check should be provided on the results and also on evidence as to the identity of the coenzyme for these two enzymes. Sufficient enzyme was prepared in both cases to cover each full series of results; the apo-enzyme portions were prepared and made up in solutions of such strength that 1 ml. should contain 8 units of-enzyme when saturated with coenzyme. 0-5 ml. of this preparation was used per test and measured portions of the coenzyme preparations were taken, such that the C0 evolution/5 min. was less than 200p1., thus ensuring that the rate fell on the linear part of the velocitycoenzyme concentration curve. Tests were carried out at 300 and at the optimum pH of 6-0 for lysine, or 5-5 for tyrosine, decarboxylase. An arbitrary unit of coenzyme is defined as that amount of coenzyme which increases the rate ofdecarboxylation by the apo-enzyme by 100p4. /5 min. As a measure of the amount of substance in preparations, the C content was estimated by the wet combustion method of Van Slyke & Folch (1940). The activity, P, of coenzyme preparations is defined as p4. CO2 liberated from substrate, due to presence of coenzyme/hr./mg. C of coenzyme. P is calculated from the formula P = 12 (R r)/ W, where r =ju1. CO2 liberated/5 min. in presence of apo-enzyme alone; and R=_1. CO2 liberated/5 min. in presence of apo-enzyme plus W mg. C of coenzyme preparation, provided that the value ofR falls within the limits defined above. The values ofP, and number of units, can be used only for comparative purposes, for tests carried out with a standard amount of a given apo-enzyme preparation.