Tissue and cellular distribution of the extended family of protein kinase C isoenzymes.

Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.

Genetically determined alcohol preference and cyclic AMP binding proteins in mouse brain.

Free-choice consumption of alcohol by mice with differing phenotypic alcohol preferences caused uniformly large decreases in brain cyclic AMP-dependent protein kinase activity toward an exogenous substrate (histone 2b) but the effect of alcohol on brain cyclic AMP binding activity was strain-specific. Furthermore, particulate kinase phosphorylating activity toward an endogenous protein (kinase regulatory subunit, RII) was altered by alcohol consumption in a strain-specific manner. The changes in cyclic AMP binding and phosphorylating activity appeared to result from phenotypic differences in the brain's response to alcohol. Thus, low preference animals were sensitive to alcohol and showed a large decrease in cyclic AMP binding and an increase in phosphorylation of regulatory subunit in response to alcohol. In contrast, high preference strain had only a small decrease in cyclic AMP binding and a decrease in phosphorylation, even though these animals consumed a significantly larger dose of alcohol. These data suggest that changes in cyclic AMP binding and/or phosphorylation of kinase regulatory subunit may be phenotypic markers of alcohol preference in inbred mice.