COENZYMES DERIVED FROM B VITAMINS 179 



the two phosphates can be distinguished from each other and from their 

 nonphosphorylated derivatives. 208 This method, of course, is limited to 

 solutions of the pure substances and cannot be used with extracts of 

 crude material. 



Occurrence. Before the relationship of pyridoxal and codecarboxylase 

 was recognized, the coenzyme had been shown by enzymatic analyses to 

 occur in a variety of biological substances. 189 Since then, vitamin B 6 has 

 been shown by microbiological assays to occur primarily in bound forms, 

 presumably the phosphates of pyridoxal and pyridoxamine. On the basis 

 of distribution studies, using the differential analysis technique previously 

 mentioned, it appears that pyridoxal phosphate is the predominant form 

 of vitamin B 6 in most animal tissues. Liver appears to be an exception, 

 for in most samples of this tissue the phosphate of the amine accounts for 

 the greater part of the vitamin B 6 content. Yeast extract is also a rich 

 source of pyridoxamine phosphate. 207 



Biosynthesis. It has been observed that the synthesis of pyridoxal 

 phosphate is rapidly carried out by yeast, molds, and bacteria, but only 

 from the particular components of the vitamin B 6 group that serve to 

 satisfy the nutritional requirements of each particular organism. 212 It 

 has also been shown that the coenzyme content of rat tissues is directly 

 related to the dietary intake of pyridoxine. If the bacterial cells are rest- 

 ing, i.e., suspended in solutions lacking nutrients needed for growth, the 

 rate of phosphorylation of pyridoxal is decreased to one tenth that 

 observed in the metabolically active cells ; the conversion of pyridoxamine 

 to the coenzyme under resting conditions cannot be detected unless a 

 keto acid (pyruvic acid) is added during the incubation. In this organism 

 the route of biosynthesis would appear to be limited to the one in which 

 only pyridoxal can be phosphorylated, i.e., the amine must first undergo 

 a transamination (enzymatic?) with a keto acid to form the aldehyde. 

 Studies of this nature have been so limited in number that no general 

 statement can yet be made concerning the possible utilization by other 

 organisms of alternate routes of synthesis (via pyridoxamine phosphate, 

 for example) . 



One of the earliest methods of obtaining a "synthetic" coenzyme for 

 use with preparations of apoenzymes from treated cells was to add 

 pyridoxal and adenosine triphosphate to the protein preparation before 

 the substrate for the decarboxylation was introduced. 192 Whereas either 

 the vitamin or phosphorylating agent is inactive when used alone, together 

 they can effectively replace the missing coenzyme. It is assumed that 

 the phosphorylation is an enzymatic process catalyzed by a phosphorylat- 

 ing enzyme present in the crude decarboxylase preparations, although this 



