Three forms of rat brain Ca2+-activated and phospholipid-dependent protein kinase (EC 2.7.1.37) were separated by hydroxylapatite column chromatography. These enzymes, designated type I, II, and m protein kinase C, all have a molecular weight of 82,000, undergo autophosphorylation in the presence of Ca2', phosphatidylserine, and diolein, and bind [3H]phorbol 12,13-dibutyrate. Autophosphorylation of these kinases resulted in an incorporation of 1-1.5 mol of 32p per mol of enzyme. Two-dimensional peptide mapping analysis revealed that these kinases had different sites of autophosphorylation. Phosphoamino acid analysis showed that type I and type m protein kinase C primarily autophosphorylated at a serine residue, whereas type II kinase autophosphorylated at both serine and threonine residues. In addition, polyconal antibodies raised against a mixture of three types of the kinase preferentially inhibited type I and type II enzymes. Monoclonal antibodies against type I and type II kinase only recognized their respective enzymes but not the type m enzyme. These results demonstrate the presence of isozymic forms of protein kinase C in rat brain.The Ca2+-activated and phospholipid-dependent protein kinase (protein kinase C) was first found in rat brain as a proteolytically activated protein kinase (1, 2). This enzyme was later shown to require Ca2+ and phospholipid (3,4) and to be further activated by diacylglycerol (DAG) (5), which markedly increased the affinity of the kinase for both Ca2+ and phospholipid. Protein kinase C has been purified to near homogeneity from many sources (6-12); however, the presence of isozymic forms of the enzyme has not been reported. This kinase is a monomeric polypeptide of molecular weight 82,000 that appears to be composed of two functionally different domains: one is a hydrophobic domain that may bind to membranes, and the other is a hydrophilic domain that carries the catalytically active center. These two domains are cleaved by Ca2+-dependent neutral proteases to produce a Ca2+-and phospholipid-independent active enzyme fragment (13). Intracellular stimulation of protein kinase C can be effected by the DAG generated from the signal-induced breakdown of inositol phospholipids or by the addition of synthetic DAG or tumor-promoting phorbol esters (14,15).Protein kinase C is an ubiquitous enzyme found in a variety of mammalian tissues (16,17). This enzyme has attracted particular attention because it plays a pivotal role in controlling many cellular functions. In addition, this enzyme has been identified as a putative receptor for tumor-promoting phorbol esters (18, 19). Recently we have prepared polyclonal and monoclonal antibodies against rat brain protein kinase C. In the process of characterizing these antibodies we noticed that some purified kinase preparations were less sensitive to inhibition by these antibodies. Therefore, we refined our purification procedure to separate the different forms ofprotein kinase C. In this report, we provide evidence to demonstrate the exist...

Recently, we isolated 3 protein kinase C (PKC) isozymes from rat brain (Huang et al., 1986a). Using isozyme-specific antibodies for immunoblot, we have determined the relative levels of each isozyme in various regions of the rat brain (Huang et al., 1987b). The present paper describes the cellular distributions of PKC isozymes in rat brain as determined by light microscopic immunocytochemistry. Staining with PKC antibodies revealed strong immunoreactivities in neuronal somata and their dendrites and weak to no reaction in axon and the astroglial structures. In the cerebellum, the type I PKC antibodies stained the Purkinje cell bodies and dendrites; the type II PKC antibodies stained the granule cells; and the type III PKC antibody stained both Purkinje and granule cells. In the cerebral cortex, all antibodies stained neurons resembling pyramidal cells and their apical dendrites in layers II to VI, while layer I was nearly devoid of staining. However, the various isozyme-specific antibodies revealed distinct laminar distribution patterns of the positively stained neurons, and the type III PKC-positive neurons exhibited a higher density than those of type I or II PKC-positive ones, especially in layer II of cingulate (retrosplenial) and piriform cortices. In the hippocampal formation, both pyramidal cells of the hippocampus and granule cells of the dentate gyrus were stained by all PKC antibodies. Subcellularly, type III PKC appeared mostly in the cytoplasm of these neurons, whereas type I and II PKC seemed to associate with the nucleus as well. In the olfactory bulb, both type II and III PKC antibodies stained the periglomerular and granular cells, and the latter also stained the mitral cells. The distinct cellular and subcellular distribution of PKC isozymes suggests that each isozyme plays a unique role in the various neural functions.

Carbamoylphosphate synthetase I deficiency (CPS1D) is a urea-cycle disorder characterized by episodes of life-threatening hyperammonemia. Correct diagnosis is crucial for patient management, but is difficult to make from clinical presentation and conventional laboratory tests alone. Enzymatic or genetic diagnoses have also been hampered by difficult access to the appropriate organ and the large size of the gene (38 exons). In this study, in order to address this diagnostic dilemma, we performed the largest 349-354 DOI 10.1007/s10038-007-0122-9 mutational and clinical analyses of this disorder to date in Japan. Mutations in CPS1 were identified in 16 of 18 patients with a clinical diagnosis of CPS1D. In total, 25 different mutations were identified, of which 19 were novel. Interestingly, in contrast to previous reports suggesting an extremely diverse mutational spectrum, 31.8% of the mutations identified in Japanese were common to more than one family. We also identified two common polymorphisms that might be useful for simple linkage analysis in prenatal diagnosis. The accumulated clinical data will also help to reveal the clinical presentation of this rare disorder in Japan.

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