28 NITROGEN METABOLISM 



respectively. Because of their specificity and the fact that 

 decarboxylation proceeds to completion, these enzymes 

 are invaluable for the quantitative determination of the 

 corresponding amino-acids. The analytical procedure origi- 

 nated by Gale is based on the use of a washed suspension 

 or an acetone powder of the appropriate organism and the 

 manometric determination of the CO 2 released. 



The growth conditions are especially important in deter- 

 mining whether the cells develop highly active decar- 

 boxylases, and owing to their adaptive nature, the medium 

 must contain the specific substrate. The enzymes are not 

 formed unless growth occurs in an acid environment, and 

 this is usually attained by allowing the organisms to meta- 

 bolize glucose. Temperature is also important: for example, 

 the decarboxylase activity of Esch. colt is better when cul- 

 tures are grown at 20-26° C. rather than at 37° C, but the 

 opposite is true of Strep, faecalis and CI. welchii. Decar- 

 boxylase activity becomes maximal at the cessation of active 

 cell division. Except for the histidine enzyme, there is 

 evidence that all the amino-acid decarboxylases possess a 

 prosthetic group, codecarboxylase, the existence of which 

 was first discovered by Gale during the purification of 

 the lysine decarboxylase. Fractionation with (NH4)2S04 in 

 alkaline conditions resulted in the precipitation of an in- 

 active apoenzyme to which decarboxylase activity could be 

 restored by the addition of extracts of bacteria, yeast or 

 animal tissues. Gunsalus and his co-workers later identified 

 codecarboxylase as being pyridoxal phosphate. It is there- 

 fore essential that the growth medium should contain ade- 

 quate amounts of pyridoxin, since even when the organism 

 is not exacting towards this factor, the rate of synthesis may 

 be insufficient to saturate the apoenzyme. The apoenzyme 

 of tyrosine decarboxylase can be prepared [6] by growing 

 Strep, faecalis in a pyridoxin-deficient medium containing 

 D-alanine, a substance which, according to the strain, 

 replaces or reduces the organism's pyridoxin requirements. 

 Washed cells of bacteria grown in this way exhibit no 

 activity unless the experimental system contains either 

 synthetic pyridoxal phosphate, pyridoxal or natural code- 



