VOL. 4 (1950) RETINENES AND VITAMINS A 221 



hydrogen atoms from DPN-H^ to retinene,, reducing its aldehyde group to the primary 

 alcohol group of vitamin A^. We may assume that in this process an apoenzyme, retinene 

 reductase, still to be revealed, takes part. The reaction may be written: 



CijHj^CHO + DPX-H, retinene reductase ^ Q^^U^-CH^OU + DPN 

 retinene^ vitamin Aj 



In the rod outer limb this system works in conjunction with a second dehydrogenase 

 system which reduces DPN, using a derivative of fructose diphosphate as hydiogen 

 donor. The total process may be formulated: 



Rhodopsin 



71 \ 



\light 



Orange intermediates 

 \ 



- , . . . . , . . retinene reductase t-. , • , . ■ 



\ itamm Aj + protem < Retmenci + protem 



DPN-H2 <-- 



fructose 

 diphosphate 



+ 



dehydrogenase 



system 



^ DPN 



THE RETINENE REDUCTASE SYSTEM 



With the coenzyme, the first component of the retinene reductase system was 

 defined. Up to this point the apoenzyme had remained a matter of surmise, buried in 

 the structure of the rod outer limb. The substrate had been obtained by bleaching 

 rhodopsin, and was both equivocal in character and very limited in quantity. 



The nature of the substrate was resolved with the observation that for this one 

 could use pure synthetic retinene j prepared as described above by the chromatographic 

 oxidation of crystalline vitamin Aj on manganese dioxide. Retinenej is fat-soluble, 

 and was originally introduced into the system with the aid of digitonin, with which it 

 forms a water-soluble complex. Later the digitonin proved to be unnecessary, for reasons 

 to be discussed below. 



The apoenzyme was found to be readily extracted with dilute salt solutions from 

 homogenates of frog or cattle retinas, forming clear, almost colourless solutions. Though 

 the apoenzyme has not yet been isolated as a pure substance, it has been separated 

 from the other components of the system and some of its properties have been deter- 

 mined. It is precipitated by half-saturated ammonium sulphate and re-dissolves without 

 losing its activity. It is retained by a Visking membrane, and survives dialysis for 18 

 hours at 5° C against neutral phosphate buffer. It is destroyed by heating at 100° 

 within 30 seconds. Its pn optimum lies at about 6.5. 



The retinene reductase system can therefore now be assembled from its separate 

 components, all in true solution: the coenzyme, DPN-Hj, prepared by the method 



* A short account has been published of the experiments which follow (Wald, 1949). A more 

 complete description of these experiments will appear in the Journal of General Physiology. 



References p. 228. 



