Kinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin II.

We have found that the 48-kDa protein (or S-antigen 48k) of the rod photoreceptor enhances the light-induced formation of the photoproduct metarhodopsin II (MII) from prephosphorylated rhodopsin. The effect is analogous to the known enhancement of MII (extra-MII) that results from selective interaction of MII with G-protein. We have determined some parameters of the MII-48k interaction by measuring the extra-MII absorption change induced by the 48-kDa protein. The amplitude saturation yields a dissociation constant for the MII-48k complex on the order of 50 nM. At the technical limit of these measurements, 13.7 degrees C and 12 microM 48-kDa protein, we find a rate of 2.3 s-1 for formation of the 48k-MII complex. Extrapolation of these values to cellular conditions yields an occupation time of phosphorylated MII by 48k less than 200 ms. This is short compared to estimated rates of phosphorylation. The temperature dependence of the MII-48k formation rate is very high (Q10 for 5 degrees C/15 degrees C = 9-10). The related Arrhenius activation energy (165 kJ mol-1) is correspondingly high and indicates a considerable transient chemical change during the binding process.

[1]  K. Hofmann,et al.  Complex formation between metarhodopsin II and GTP‐binding protein in bovine protoreceptor membranes leads to a shift of the photoproduct equilibrium , 1982, FEBS letters.

[2]  H. Kühn.  Light-regulated binding of rhodopsin kinase and other proteins to cattle photoreceptor membranes. , 1978, Biochemistry.

[3]  K. Hofmann,et al.  Interplay between hydroxylamine, metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes. , 1983, Biochimica et biophysica acta.

[4]  P. Liebman,et al.  Phosphorylation of rhodopsin and quenching of cyclic GMP phosphodiesterase activation by ATP at weak bleaches. , 1983, The Journal of biological chemistry.

[5]  I. Weyand,et al.  Rapid affinity purification of retinal arrestin (48 kDa protein) via its light‐dependent binding to phosphorylated rhodopsin , 1986, FEBS letters.

[6]  K. Hofmann,et al.  Temperature dependence of G-protein activation in photoreceptor membranes. Transient extra metarhodopsin II on bovine disk membranes. , 1987, Biophysical journal.

[7]  W. Dreyer,et al.  Phosphorylation of rhodopsin in bovine photoreceptor membranes. A dark reaction after illumination. , 1973, Biochemistry.

[8]  S. W. Hall,et al.  Light‐induced binding of 48‐kDa protein to photoreceptor membranes is highly enhanced by phosphorylation of rhodopsin , 1984, FEBS letters.

[9]  H. G. Smith,et al.  The isolation and purification of osmotically intact discs from retinal rod outer segments. , 1975, Experimental eye research.

[10]  R. M. Broekhuyse,et al.  Light induced shift and binding of S-antigen in retinal rods. , 1985, Current eye research.

[11]  L. Stryer,et al.  Cyclic GMP cascade of vision. , 1986, Annual review of neuroscience.

[12]  N. Bennett,et al.  The G-protein of retinal rod outer segments (transducin). Mechanism of interaction with rhodopsin and nucleotides. , 1985, The Journal of biological chemistry.

[13]  K. Hofmann,et al.  Shift in the relation between flash‐induced metarhodopsin I and metarhodopsin II within the first 10% rhodopsin bleaching in bovine disc membranes , 1981, FEBS letters.

[14]  George Wald,et al.  Tautomeric Forms of Metarhodopsin , 1963, The Journal of general physiology.

[15]  J. Miller,et al.  Amplification of phosphodiesterase activation is greatly reduced by rhodopsin phosphorylation. , 1986, Biochemistry.

[16]  P. Liebman,et al.  Temperature and pH dependence of the metarhodopsin I-metarhodopsin II kinetics and equilibria in bovine rod disk membrane suspensions. , 1984, Biochemistry.

[17]  N. Bennett,et al.  Inactivation of photoexcited rhodopsin in retinal rods: the roles of rhodopsin kinase and 48-kDa protein (arrestin). , 1988, Biochemistry.

[18]  H. Hamm,et al.  Site of G protein binding to rhodopsin mapped with synthetic peptides from the alpha subunit. , 1988, Science.

[19]  H. Hamm,et al.  Protein complement of rod outer segments of frog retina. , 1986, Biochemistry.

[20]  K. Hofmann,et al.  Photic modulation of a highly sensitive, near-infrared light-scattering signal recorded from intact retinal photoreceptors. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[21]  Y. de Kozak,et al.  Retinal S antigen identified as the 48K protein regulating light-dependent phosphodiesterase in rods. , 1985, Science.

[22]  U Wilden,et al.  Light-dependent phosphorylation of rhodopsin: number of phosphorylation sites. , 1982, Biochemistry.

[23]  R. M. Broekhuyse,et al.  Effect of light-adaptation on the binding of 48-kDa protein (S-antigen) to photoreceptor cell membranes. , 1987, Current eye research.

[24]  A. Sitaramayya.  Rhodopsin kinase prepared from bovine rod disk membranes quenches light activation of cGMP phosphodiesterase in a reconstituted system. , 1986, Biochemistry.

[25]  L. Donoso,et al.  Primary and secondary structure of bovine retinal S antigen (48-kDa protein). , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[26]  S. W. Hall,et al.  Phosphodiesterase activation by photoexcited rhodopsin is quenched when rhodopsin is phosphorylated and binds the intrinsic 48-kDa protein of rod outer segments. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[27]  N. Bennett,et al.  Interactions between photoexcited rhodopsin and GTP-binding protein: kinetic and stoichiometric analyses from light-scattering changes. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[28]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[29]  R. Zuckerman,et al.  A 48 kDa protein arrests cGMP phosphodiesterase activation in retinal rod disk membranes , 1986, FEBS letters.