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A continuous fluorescence assay for protein kinase C by Brian K. McIlroy; John D. Walters; J.David Johnson is a Biochemistry, Genetics and Molecular Biology article available to read on EtoBox.

What is A continuous fluorescence assay for protein kinase C about?

A 6-acryloyl-2-dimethylaminonapthalene (acrylodan)-labeled 25-amino acid peptide (acrylodan-CKK-KKRFSFKKSFKLSGFSFKKNKK-COO-), containing the protein kinase C (PKC) phosphorylation sites of brain myristoylated alanine-rich kinase C substrate protein, undergoes a 20% fluorescence decrease when it is phosphorylated by phospholipid/calcium-dependent protein kinase (PKC). This fluorescence decrease is dependent on the presence of PKC, calcium (half-maximal stimulation at pCa = 6.2), phosphatidylserine, diacylglycerol, or phorbol-12-myristate-13-acetate (half-maximal stimulation at 2 nM) and ATP, and correlates well (r = 0.997) with [32P]phosphate incorporation into the peptide. This fluorescence assay allows detection of 0.02 nM PKC, while similar concentrations of cyclic AMP-dependent or type II calmodulin-dependent protein kinases produced no change in peptide fluorescence. The method can be used to assay purified PKC as well as activity in crude brain homogenates. Incubation of PKC with staurosporine inhibits the fluorescence decrease with an IC50 of 2 nM. Thus the fluorescence decrease that occurs in the acrylodan-peptide provides a continuous fluorescence assay for PKC activity.

Who reads A continuous fluorescence assay for protein kinase C?

Author

Brian K. McIlroy; John D. Walters; J.David Johnson

Publisher

Elsevier Science; Elsevier ; Elsevier Inc.; Elsevier BV (ISSN 0003-2697)

Published

1991

Language

EN

Field

Biochemistry, Genetics and Molecular Biology (Life Sciences)