Elsevier

Methods in Enzymology

Volume 316, 2000, Pages 330-344
Methods in Enzymology

[21] Multienzyme analysis of visual cycle

https://doi.org/10.1016/S0076-6879(00)16733-3Get rights and content

Publisher Summary

The visual cycle is a fundamental chemical transformation that generates 11-cis-retinal from all-trans-retinal. The chromophore 11-cis-retinal recombines with apoprotein opsin to form rhodopsin, the fight-sensitive receptor of the visual transduction cascade. Several reactions are required for this transformation. 11-cis-Retinol is produced enzymatically by an isomerase, which still has defied purification and molecular characterization. To understand the production of 11-cis-retinal in retinal pigment epithelium cells (RPE), an assay system is required for the simultaneous detection of lecithin:retinol acyltransferase (LRAT), retinol isomerase, and retinyl ester hydrolase activities.This chapter describes techniques designed to study these enzymatic reactions in RPE. This reliable enzyme assay allows measurement of the kinetic parameters of these reactions, as well as the influence of different compounds on the activities of LRAT, retinyl ester hydrolase, and retinol isomerase. The enzymatic activities of all three enzymes were a function of the concentration of native RPE microsomes (Fig. 3A) or UV-treated RPE microsomes

References (17)

  • A. Polans et al.

    Trends Neurosci.

    (1996)
  • A. Simon et al.

    J. Biol. Chem.

    (1995)
  • H. Stecher et al.

    J. Biol. Chem.

    (1999)
  • A. Ruiz et al.

    J. Biol. Chem.

    (1999)
  • N.L. Mata et al.

    J. Biol. Chem.

    (1992)
  • J.C. Saari et al.

    Methods Enzymol.

    (1990)
  • M.M. Bradford

    Anal. Biochem.

    (1976)
  • G.M. Landers et al.

    J. Chromatogr.

    (1988)
There are more references available in the full text version of this article.

Cited by (0)

View full text