224 ^- ^VALD VOL. 4 (1950) 



of cozymase is nicotinamide, the anti-pellagra factor of the vitamin B complex. It 

 presents also the novel phenomenon of widely distinct vitamins not only interacting in 

 vitro, but of one of them paiticipating directly in the regeneration of the others. I do not 

 know a comparable i elation in the whole of biochemistry. 



STABILITY 



It has been known for some time that animal and certain plant tissues contain a 

 nucleotidase which cleaves cozymase and dihydrocozymase, and which is released into 

 homogenates and tissue breis by the breaking of the cells. Measurements made on various 

 tissues of the rat have shown this enzyme to be particularly active in brain, to which 

 of course retina is closely related (Mann and Quastel, 1941 ; Handler and Klein, 

 1942). The action of this enzyme makes a number of the preparations which we have 

 described unstable. 



It was noted above that solutions of rhodopsin, prepared by extracting fresh retinal 

 tissue with water solutions of digitonin, rapidly lose the power to reduce retinenCi. 

 Within 3-4 hours their ability to perform this process usually falls to very low levels. 

 The principal cause of this failure is the loss of cozymase. 



This is shown by the following experiment. A freshly prepared rhodopsin solution 

 was kept at about 23° C for 18 hours. At the end of this period it was divided into halves, 

 and to one half reduced cozymase was added (1.5 mg per ml). Both portions were 

 bleached in the light and were incubated for i hour. The control portion converted no 

 more than a trace of its retinenci to vitamin A^; that to which DPN-Ho was added 

 had completed this conversion. It is clear that the apoenzyme in such preparations is 

 relatively stable; their loss of activity is caused by the destruction of the coenzyme. 



Cozymase and reduced cozymase are protected from the action of the nucleotidase 

 by the presence of free nicotinamide (2-20 mg per ml) (Mann and Quastel, 1941; 

 Handler and Klein, 1942). It has recently been reported also that a-tocopheryl phos- 

 phate (about I mg per ml) similarly protects cozymase (Spaulding and Graham, 1947). 



The nucleotidase has been reported to be in general insoluble in water or dilute 

 salt solutions. Our experiments show that it does go into solution in the 2% digitonin 

 with which we extract rhodopsin. It also is active in all our retinal homogenates and 

 particulate preparations. Whether it enters the saline extracts which contain our 

 apoenzyme we have not yet determined. A number of our fish enzyme preparations 

 have definitely been unstable, but they also tend to be slightly turbid, and may contain 

 small amounts of very fine particles. 



In any case we have taken the precaution ordinarily to add nicotinamide to our 

 enzyme preparations; and to those from freshwater fish retinas, in which the nucleoti- 

 dase appears to be particularly active, we have added also a-tocopheryl phosphate. 



These adjustments extend still further the participation of vitamins in the retinene 

 reductase system. Nicotinamide acts not only as the key component of the cozymase 

 molecule, but in the free condition protects cozymase fiom destruction. In this action 

 it is aided by vitamin E phosphate. As many as three vitamins therefore interact with 

 one another in this single system. 



the state of the retinenes 

 With the first use of the synthetic retinenes as substrates there arose the problem 

 how, as typically fat-soluble substances, they were to be introduced into the aqueous 

 References p. 228. 



