Protein kinase C (PKC), a family of closely related enzymes, has been implicated in molecular processes involved in differentiation in a variety of cells, including neuronal cells. We studied the presence and distribution of four PKC isozymes immunocytochemically in primary neuronal cultures of the rat cerebellum. We employed four anti-PKC antisera raised against synthetic peptides predicted from the cDNA sequence of the C-terminal portion of four PKC isozymes, alpha, beta I, beta II, and gamma. The majority of neurons were PKC(beta II) immunoreactive both in the early and late (14 days) stage of culture, whereas PKC(alpha)-, (beta I)-, and (gamma)-immunoreactive neurons were most abundant in the late stage of culture. Immunoreactivity of each PKC was high in the cytoplasm, processes, and growth cones. Prominent nuclear staining was observed with anti-PKC(gamma) antibody. These results are in contrast with in vivo results where each PKC isozyme is localized in a distinct population of neurons and subcellular compartment, suggesting the presence of regulatory mechanisms for PKC expression and compartmentalization in vivo.

The effects of high pressure, up to 400 MPa, on the catalytic activity and the fluorescence and CD of carboxypeptidase Y (CPDY) were investigated. CPDY showed a pH-dependent Suc–Ala–Ala–Pro–Phe–pNA hydrolysis similar to other neutral substrates. The apparent second-order rate showed a gradual decrease with increasing pressure, which was related to an increase in Km and a decrease in kcat. The intrinsic fluorescence of CPDY showed a gradual decrease in the intensity and a red shift in the maximum wavelength with pressure. The transition curve did not follow a simple tow-state transition, but contained at least three states. The first transition occurred at around 100 MPa and the second one occurred at pressures higher than 200 MPa. After incubation at 300 MPa, both the peak intensity and the maximum wavelength did not show complete restoration; the pressure-induced change is substantially irreversible. The latter change corresponds to the increased binding of a fluorescent hydrophobic probe molecule (8-anilino-1-naphthalenesulfonic acid) to this protein; however, the CD spectrum showed practically no evidence of irreversible changes in the protein’s secondary structure.

The cytoplasmic leucyl-tRNA synthetases of Neurospora crassa wild type (grown at 37 "C) and mutant (grown at 28 "C) were purified approximately 1770-fold and 1440-fold respectively. Additional enzyme preparations were carried out with mutant cells grown for 24 h at 28°C and transferred then to 37°C for 10-70 h of growth. The mitochondrial leucyl-tRNA synthetase of the wild type was purified approximately 722-fold. The mitochondrial mutant enzyme was found only in traces. The cytoplasmic leucyl-tRNA synthetase from the mutant (grown at 37°C) in vivo is subject of a proteolytic degradation. This leads to an increased pyrophosphate exchange, without altering aminoacylation. Proteolysis in vitro by trypsin or subtilisin of isolated cytoplasmic wild-type and mutant leucyl-tRNA synthetases, however, did not establish any difference in the degradation products and in their catalytic properties. Comparing the cytoplasmic wild-type and mutant enzymes (grown at 28 "C) via steady-state kinetics did not show significant differences between these synthetases either. The rate-determining step appears to be after the transfer of the aminoacyl group to the tRNA, e.g. a conformational change or the release of the product. Besides leucine only isoleucine is activated by the enzymes with a discrimination of-1 : 600; however, no Ile-tRNALeu is released. Similarly these enzymes, when tested with eight ATP analogs, cannot be distinguished. For both enzymes six ATP analogs are neither substrates nor inhibitors. Two analogs are substrates with identical kinetic parameters. The mitochondrial wild-type leucyl-tRNA synthetase is different from the cytoplasmic enzyme, as particularly exhibited by aminoacylating Escherichia coli tRNALeu but not N. crassa cytoplasmic tRNALeu. The presence of traces of the analogous mitochondrial mutant enzyme could be demonstrated. Therefore, the difference between wild-type and mutant leu-5 does not rest in the catalytic properties of the cytoplasmic leucyl-tRNA synthetases. Differences in other properties of these enzymes are not excluded. In contrast the activity of the mitochondrial leucyl-tRNA synthetase of the mutant is approximately 1% of that of the wild-type enzyme. The mutant leu-5 of Neurospora crassa, which is temperature sensitive and leucine-auxotroph, has been reported earlier to have a mutation in the leucyl-tRNA synthetase gene, providing a cytoplasmic leucyl-tRNA synthetase with an increased K, for leucine, a protein biosynthesis with one or some inaccurate steps and very little mitochondrial leucyl-tRNA synthetase [ 11. These phenomena have been tentatively explained by Gross et al. in a proposal regarding the structure and function of the corresponding genes [2-41. Enzyme. Leucyl-tRNA synthetase (EC 6.1.1.4). However, the molecular biology of the mutation has not been established at the DNA level nor at the protein level, and this will be of interest regarding the structure and function of aminoacyl-tRNA synthetases. Moreover a leucyl-tRNA synthetase with reduced function may establish th...

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PC12h cells can be differentiated into sympathetic neuron‐like cells by various agents, including nerve growth factor, basic fibroblast growth factor, cyclic AMP analogues, and protein kinase C (PKC) activators. To study the involvement of PKC in the process of PC12h cell differentiation by cyclic AMP treatment, PKC isozymes (α, βI, βII, and γ) were analyzed using column chromatography and immunoblotting. Two PKC isozymes, PKC(α) and PKC(βII), were predominantly detected in PC12h cells. When stimulated by dibutyryl cyclic AMP, PKC(α) levels declined in the cytosolic fraction of the cells, whereas PKC(βII) levels increased. Increased PKC(βII) levels were also detected in the particulate fraction, whereas particulate PKC(α) levels did not change. The total PKC activity decreased in the cytosolic fraction following cyclic AMP stimulation of PC12h cells, whereas it stayed constant in the particulate fraction. Fractionation on a hydroxyapatite column showed a decreased level of PKC(α) activity and a transient increase followed by a decreased level of PKC(βII) activity. This discrepancy between increased PKC(βII) immunoreactivity and reduced PKC(βII) activity suggested the presence of nonactivatable PKC(βII) in cyclic AMP‐treated PC12h extract. These findings indicate that PKC(α) and PKC(βII) are differentially regulated during the differentiation of PC12h cells. In addition, the differentiation of PC12h cells triggered by cyclic AMP seems to involve characteristic alterations of PKC isozymes.