Effect of pH on the formation and decay of the metarhodopsins of the frog.

Abstract

SUMMARY1. Suspensions of membrane vesicles were prepared by sonication of dark-adapted frog rod outer segments. The pH was adjusted to one of twelve values between 5 0 and 9 8; the temperature was 15 'C. Each preparation was exposed to a single yellow flash of 120 Pts duration, and rapid and slow changes of absorbance were measured at 475 nm wavelength.2. Rapid changes consist of a transient rise followed by a diphasic decay to a new level of absorbance which is lower than that before the light exposure. The level of absorbance reached at the end of the rapid changes is lower at lower pH.3. Kinetic analysis reveals that three reactants take part in the rapid reactions, viz. metarhodopsin I and two isochromic forms of metarhodopsin II named metarhodopsin II' and metarhodopsin II". The kinetics of the reversible transition from metarhodopsin I to metarhodopsin II' are not measurably influenced by the pH of the medium. However, the reversible reaction of metarhodopsin II' with metarhodopsin II" is dependent on pH because metarhodopsin II" is encountered either as a protonated or as a deprotonated compound.4. Slow reactions are due to metarhodopsin I and to metarhodopsin III. A combined quantitative analysis of both rapid and slow reactions involves a reversible reaction between metarhodopsin II' and metarhodopsin III and a hydrolysis of metarhodopsin II' to retinal and opsin. The scheme employed accounts for the pH dependence of all equilibria although only one of the metarhodopsins reacts directly with protons.

The identity of metarhodopsin III

“…The concentration of the long-wave product that we identify as meta III is also pH dependent, increasing in high pH (cf. Donner & Reuter, 1967;Gyllenberg et al, 1974;Reuter, 1976;Baumann & Zeppenfeld, 1981). The peak at 480 nm reaches its maximum at 100-200 s after bleaching ( Fig.…”

“…Big amphibian rods allow quite accurate measurements of their absorbance spectra, with sufficiently high signal-to-noise ratio. Rods of R. temporaria (and closely related R. esculenta) were used in many previous studies of late photolysis products in vivo on transilluminated retina (Donner & Reuter, 1967;Baumann, 1972Baumann, , 1978Reuter, 1973Reuter, , 1976Gyllenberg et al, 1974;Donner & Hemilä, 1975Baumann & Zeppenfeld, 1981). On the other hand, toad (B. bufo and B. marinus) and recently R. ridibunda rods are the sample of choice for studying processes of photoreceptor dark adaptation (Leibrock et al, 1994(Leibrock et al, , 1998Leibrock & Lamb, 1997;Firsov et al, 2002 and unpublished).…”

“…Meta III was proposed to be an intermediate between meta II and the formation of free retinal and opsin (Baumann, 1972;Ernst et al, 1978) or free retinol and opsin (Ernst et al, 1978), or to be in an alternate shunt pathway (Baumann, 1972(Baumann, , 1978Baumann & Bender, 1973;Bowmaker, 1973;Reuter, 1973Reuter, , 1976Gyllenberg et al, 1974;Brin & Ripps, 1977;Donner & Hemilä, 1979;Kliger & Lewis, 1995). It has also been considered to be a byproduct which is in equilibrium with meta II but does not directly decay to retinal and opsin (Chabre & Breton, 1979;Baumann & Zeppenfeld, 1981), or as a secondary product of the interaction between released retinal and opsin (Ernst et al, 1978;Van Breugel et al, 1979;DeGrip & Rothschild, 2000;Bartl et al, 2001).…”

“…Furthermore, it is unclear whether investigators were always dealing with the same chemical substance. Meta III has been considered to be a specific conformational form of metarhodopsin(s), with all-trans retinal still attached to its native binding site on opsin (Baumann, 1972(Baumann, , 1978Baumann & Bender, 1973;Bowmaker, 1973;Reuter, 1973Reuter, , 1976Gyllenberg et al, 1974;Donner & Hemilä, 1975Brin & Ripps, 1977;Chabre & Breton, 1979;Baumann & Zeppenfeld, 1981;Kliger & Lewis, 1995;Heck et al, 2003). Alternatively, meta III was considered as a heterogeneous product where released retinal is reattached to nonspecific binding sites on opsin and0or phosphatidylethanolamine (Van Breugel et al, 1979;DeGrip & Rothschild, 2000).…”

Studies on Visual Transduction in the Retinal Rods of the Frog

“…Its formation is assumed to be instantaneous although some 50 pis may elapse before the photoexcited rhodopsin molecules (R) arrive at the M 1 stage [Baumann, 1976]. It can be demon strated [Baumann and Zeppenfeld, 1981] that the M I to M II transition proceeds in two phases which reflect two consecutive reversible reactions in which one metarhodopsin I (M I) and two isochromic meta rhodopsins II (M IT, Mil") are involved. Mil" is not a homogeneous compound but repre sents an acid-base pair.…”