Studies on the flash-photolysis of visual pigments-IV: Dark reactions following the flash-irradiation of frog rhodopsin in suspensions of isolated photoreceptors

Abstract

The dark reactions occurring after flash-irradiation of frog rhodopsin in 37.5 per cent wt./vol. sucrose suspensions of the isolated photoreceptors have been examined. The dark reactions observed in these suspensions are influenced by the composition of the suspending medium. Evidence is produced to show that isolated photoreceptors are permeable to hydrogen ions, hydroxylamine, DPNH 2 and p-chloromercuribenzoate ions. In fresh suspensions (pH 6·6–6·7) the principal extinction changes observed are due to the conversion of free retinene to vitamin A. These changes are characterized by falling extinction maximal at 387–395 mμ and rising extinction maximal at 327–330 mμ. This reaction (the “fresh reaction”) is abolished in the presence of sodium p-chloromercuribenzoate ( 0·001M) and hydroxylamine ( 0·02M). It is absent at pH 9·0. The fresh reaction is very considerably diminished by washing the isolated photoreceptors with pH 4·6 buffer, and it may then be partially restored by supplementing the suspending medium with DPNH 2 ( 0·001M). The early phase of the reaction in acid suspensions of fresh and washed photoreceptors is also characterized by extinction losses maximal at 490–505 mμ and by extinction gains (or minimum losses) maximal at 432–440 mμ. The later phase exhibits the extinction changes principally attributable to the conversion of retinene to vitamin A. The dark reaction in acid suspensions of washed or fresh photoreceptors in the presence of p-chloromercuribenzoate causes extinction losses maximal at 355–358 mμ and at 494–505 mμ, with simultaneous gains maximal at 415–420 mμ. The dark reaction in alkaline suspensions (pH 7·9–9·4) of fresh or washed photoreceptors produces extinction losses at 470–480 mμ and gains at 365–375 mμ, as in solutions. The extinction losses at 470–480 mμ in alkaline suspensions and at 490–505 mμ in acid suspensions are due to the decomposition of an orange intermediate pigment with λ max at 470–480 mμ. This intermediate is more stable at alkaline pH, and decomposes at a rate which is only about one-fifth of that observed in solutions. If a very dilute acid suspension of washed photoreceptors is flash-irradiated and then treated with digitonin, the resulting extract consists of a mixture of rhodopsin (80 per cent) and iso-rhodopsin (20 per cent). These results are compared with the findings of the preceding paper, where the dark reactions following flash-irradiation of frog rhodopsin in solution were investigated. It is concluded that, apart from the ready conversion of free retinene to vitamin A, the same reactions occur in suspensions as in solutions, but that these reactions proceed at a very different rate. This rate can also vary in different types of photoreceptor.