Purification and characterization of protein kinase C isozymes from rat heart

Abstract

A calcium-sensitive, phospholipid-dependent protein kinase (protein kinase C) and its three isozymes were purified from rat heart cytosolic fractions utilizing a rapid purification method. The purified protein kinase C enzyme showed a single polypeptide band of 80 KDa on SDS-polyacrylamide gel electrophoresis, and was totally dependent on the presence of Ca2+ and phospholipid for activity. Diacylglycerol was also found to stimulate enzymatic activity. Autophosphorylation of the purified PKC showed an 80 KDa polypeptide. The identity of the purified protein was also verified with monoclonal antibodies specific for PKC. Further fractionation of the purified PKC on a hydroxylapatite column yielded three distinct peaks of enzyme activity, corresponding to type I, II and III based on similar chromatographic behaviour as the rat brain enzyme. All three forms were entirely Ca2+ and phosphatidylserine dependent. Type II was found to be the most abundant. Type I was found to be highly unstable. PKC activity studies demonstrate that types II and III isozymic forms are different with respect to their sensitivity to Ca2+.

Transcriptional activation of the cardiac myosin light chain 2 and atrial natriuretic factor genes by protein kinase C in neonatal rat ventricular myocytes.

“…Rat heart has been shown to contain a, and y forms of PKC (55,56). In the present study, we find that either the activated a or the activated isoform of PKC stimulate transcription through the ANF and MLC-2 promoters.…”

Protein kinase C isoenzymes in rat and human cardiovascular tissues

“…While cardiac PKCα im‐munoreactivity at least partly comes from fibroblasts (Rybin & Steinberg, 1994), data showing whether it also exists in cardi‐omyocytes (Clerk et al , 1995) or not (Bogoyevitch et al , 1993; Rybin & Steinberg, 1994) are controversial. Results on the detection of cardiac PKCβ are inconsistent (Kosaka et al , 1988; Qu et al , 1991; Clerk et al , 1995), and the present study has also failed to find it in human or rat heart. When detected in whole heart, parallel detection in cardiac myocytes was not successful (Rybin & Steinberg, 1994) indicating a possible primary location of PKCβ in non‐myocytes.…”

“…PKC isoenzyme expression has been investigated either at the mRNA level by RNA protection assays (Seynaeve et al , 1994) or Northern blotting (Selbie et al , 1993; Akimoto et al , 1994) and at the protein level immunologically with PKC isoenzyme specific antibodies. While many studies have described the isoenzyme expression pattern in rat tissues (Kosaka et al , 1988; Qu et al , 1991; Wetsel et al , 1992; Selbie et al , 1993; Bogoyevitch et al , 1993; Church et al , 1993; Rybin & Steinberg, 1994; Clerk et al , 1995), little is known in man. Moreover, most studies have been performed with total protein extracts from tissues or cells (Wetsel et al , 1992; Bogoyevitch et al , 1993; Rybin & Steinberg, 1994).…”