Analysis of protein kinase activities in rabbit ciliary processes: Identification and characterization using exogenous substrates

Nagahisa Yoshimura, Thomas W. Mittag, Steven M. Podos

Research output: Contribution to journalArticlepeer-review

8 Scopus citations

Abstract

Protein-kinase activities in rabbit ciliary process tissue were characterized and quantitated using histone, casein, and myosin light chain as substrates. At least four different protein-kinase activities were separated and identified in the supernatant (soluble) and in the particulate fraction using DEAE-cellulose ion-exchange chromatography. Typical activities of the protein kinases in ciliary processes dissected from one eye were as follows: in the supernatant fraction; protein kinase C, 185·0 pmol min-1; cyclic AMP-dependent protein kinase type II, 34·0 pmol min-1; casein kinase type II, 85·1 pmol min-1; protein kinase M, 9·8 pmol min-1: in the particulate fraction; protein kinase C, 55·1 pmol min-1; cyclic AMP-dependent protein kinase type II, 12·5 pmol min-1; casein kinase type II, 13·4 pmol min-1, and protein kinase M, 5·5 pmol min-1. No cyclic GMP-dependent and no calmodulin-dependent protein-kinase activities were detectable using histone, casein or myosin light chain as substrates. The apparent molecular weight of protein kinase C as estimated by exclusion chromatography on a column of Sephadex G-200 was about 90000. Inhibitory and stimulatory effects of recently synthesized isoquinolinesulfonamide derivatives (H-7 and H-8), heparin, and polylysine were studied on ciliary process protein kinases. H-7 and H-8 were potent inhibitors of cyclic AMP-dependent protein kinase, protein kinase C and protein kinase M, (IC50 < 10 μm) but had no inhibitory effects on casein kinase. Heparin at 4 μg ml-1 inhibited casein kinase activity almost completely without affecting cyclic AMP-dependent or protein kinase C activities. Poly d- or l-lysine were both found to activate (∼double) casein kinase activity at 40 μg ml-1, but did not significantly activate cyclic AMP-dependent protein kinase or protein kinase C. These results provide basic information on the protein kinase enzymes in the ciliary process and show that protein kinase C is the major kinase in this tissue. This suggests a possible role of the Ca2+ and protein kinase C system in transport functions of ciliary processes and in the regulatory mechanism of aqueous-humor formation additional to the already established importance of the cyclic AMP-dependent protein-kinase enzyme.

Original languageEnglish
Pages (from-to)45-56
Number of pages12
JournalExperimental Eye Research
Volume45
Issue number1
DOIs
StatePublished - Jul 1987
Externally publishedYes

Keywords

  • ciliary processes
  • exogenous substrates
  • identification
  • protein kinase
  • rabbit

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