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G. WALD 



VOL. 4 (1950) 



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its reductase might well be restricted to this tissue. A coen- 

 zyme or substrate, however, would ordinarily be unspecilic, 

 and one would expect to find it widely distributed among 

 the tissues. This thought led us to try an extract of frog 

 muscle as a suspension medium for rod outer limbs. 



The preparation we used was the Muskelkochsaft — the 



Fig. 4. Boiled muscle juice activates isolated rod outer limbs. Equal 

 numbers of rod outer segments were suspended in phosphate buffer 

 and in a boiled juice of frog muscle. The suspensions were exposed 

 to light, left at room temperature for i hour, and the residues extracted 

 with petroleum ether. The spectra of the antimony chloride tests 

 with these extracts are shown. The outer limbs in buffer had failed 

 to convert their retinene^ to vitamin A^ (curve a) ; those suspended 

 in boiled muscle juice had done so completely (curve b). The relatively 

 low content of vitamin Aj shown in curve b is due to its destruction in 

 preparations of this type. (From Wald and Hubbard, 1948-49). 



boiled muscle juice — of Meyerhof (1918). Rod outer segments suspended in this 

 medium converted their retinene^ quantitatively to vitamin A^ (Fig. 4). 



Boiled muscle juice contains a number of substances which could donate hydrogen 

 for the reduction of retinencj. It also contains a major coenzyme of hydrogen transfer, 

 cozymase, Coenzyme I, or DPN. 



When rod outer limbs were suspended in a buffer solution to which DPN had been 

 added, they failed to transform theii retinenci to vitamin A^. But if they — or an inactive 

 preparation of washed retinal tissue — ^were provided with reduced cozymase, DPN-Hg, 

 they performed this conversion quantitatively (Fig. 5). 



Given a proper substrate, rod outer limbs can themselves reduce cozymase. We 

 have found a first such substrate in fructose diphosphate. Rod outer segments suspended 

 in a solution to which both DPN and fructose diphosphate were added converted their 

 retinencj completely to vitamin A^. The outer segments must therefore contain an 

 enzyme system for reducing DPN when a suitable hydrogen donor is made available. 

 It is highly improbable that fructose diphosphate itself is the source of hydrogen in 

 this reaction. More probably the outer limbs also possess the enzyme aldolase, which 

 cleaves fructose diphosphate to yield 3-glyceraldehyde phosphate, the normal substrate 

 for the reduction of DPN in the lactic acid fermentation. 



The convex sion of retinenej to vitamin A^ is there- 

 fore a coupled reduction in which DPN-Hg acts as 

 coenzyme. The essential process is the transfer of two 



S30 



Fig. 5. The action of reduced cozymase on washed retina. 

 Equal portions of a preparation of water-extracted frog retina 

 were suspended in a solution containing reduced DPN, and in 

 an otherwise identical solution lacking only the DPN-Hj. Both 

 suspensions were bleached in the light, incubated, and the 

 residues extracted with petroleum ether. Spectra of the anti- 

 mony chloride tests with these extracts are shown. The control 

 preparation yielded retincncj alone (curve a) ; while in the 

 washed retina to which reduced DPN had been added this had 

 been converted almost completely to vitamin Aj (curve b). 

 (From Wald and Hubbard, 1948-49). 



References p. 228. 



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 a, ' X. ^ 



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