Premium
Purification and Some Characteristics of an ACTH‐Sensitive Protein Kinase and Its Substrate Protein in Rat Brain Membranes
Author(s) -
Zwiers H.,
Schotman P.,
Gispen W. H.
Publication year - 1980
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1980.tb11262.x
Subject(s) - isoelectric point , protein kinase a , cgmp dependent protein kinase , biochemistry , protein kinase c , chemistry , membrane , kinase , chromatography , mitogen activated protein kinase kinase , enzyme
ACTH inhibits the phosphorylation of a rat brain membrane‐bound protein (B‐50). Both the protein kinase and the substrate protein could be extracted from the membranes by means of treatment with Triton X‐100 in 75 mM‐KCl. Using column chromatography over DEAE‐cellulose and ammonium sulphate precipitation a protein fraction (ASP 55–80) enriched in endogenous B‐50 phosphorylating activity was obtained. The time course of the endogenous phosphorylation of B‐50 in this fraction showed a linear incorporation with time for at least 10 min and reached an estimated maximal incorporation of 0.65 mol P/mol B‐50 after 60 min. The inhibition by ACTH 1_24 of the B‐50 protein kinase in ASP 55–80 was dose‐dependent; the half‐maximal effective concentration was 5 × 10 −6 M, being 10 to 50 times lower as compared with intact synaptic plasma membranes (SPM). cAMP, cGMP and various endor‐phins had no effect on the B‐50 protein kinase. The B‐50 protein kinase required both magnesium and calcium for optimal activity. Using two‐dimensional electrophoresis on polyacrylamide slab gels the B‐50 protein kinase and the B‐50 protein could be identified and purified. The isoelectric point (IEP) of the kinase is 5.5 and the apparent molecular weight 70,000, whereas the IEP of the substrate protein B‐50 is 4.5 and the apparent molecular weight 48,000. Amino acid analysis on microgram quantities of purified kinase and B‐50 protein revealed basic/acidic amino acid ratios in agreement with the respective lEP's. It is speculated that the inhibition of B‐50 protein kinase may be related to known modulatory effects of ACTH and related peptides on certain types of neurotransmission and behaviour.