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A 10 min treatment of human neutrophils with phorbol 12-myristate 13-acetate (PMA) has been reported to induce accumulation of the proteolytically activated Ca2 +/phospholipid-independent catalytic fragment of protein kinase C in the cytosol of intact cells [(1986) J. Biol. Chem. 261, 4101-4105]. We investigated the proteolytic conversion of protein kinase C to the Ca2 +/phospholipid-independent form in the cytosol and membrane fractions of pig neutrophils. The activity of protein kinase C was measured with its specific oligopeptide substrate Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide designed previously. In our experiments the short-term treatment of neutrophils with PMA did not induce the accumulation of the proteolytically activated form of protein kinase C in the cytosol of intact cells. However, treatment of cells with PMA enhanced the limited proteolysis of protein kinase C during the preparation of cell extracts.

Recent data suggest different biological roles for type I and type II cyclic AMP-dependent protein kinases [ 11. The selective activation of type I protein kinase may represent a positive event in the process of activation of lymphocytes by a mitogenic signal, while the activation of type II enzyme may have an inhibitory effect on cell proliferation [2]. This hypothesis has been based on the assumption that in lymphocytes the dissociation of type I enzyme occurs at lower intracellular cyclic AMP concentration than that of type II enzyme. The differential activation of type I and type II kinases has been reported also in the liver of glucagon-treated rats [3 1. In the cytoplasmic extract of human tonsillar lymphocytes we have found the type II regulatory subunit predominantly in the dissociated state, while type I enzyme was present mainly as holoenzyme or in a partially dissociation form [4]. This finding was unexpected in view of the above potential regulatory mechanism. To get more information about the behaviour of cyclic AMP-dependent protein kinase isoenzymes in lymphocytes we have investigated the relative distribution of the subunits in the cytoplasmic and nuclear extracts of lymphocytes.

The activities of rat brain protein kinase C isoenzymic fractions separated by hydroxyapatite chromatography were measured with histone H1 or the oligopeptide Ala-Ala-Ala-Ser-Phe-Lys-Ala-Lys-Lys-amide as substrates. The oligopeptide was a better substrate than histone H1 for nearly all of the protein kinase C fractions. Two subtractions of type III isoenzyme were resolved (IIIa and IIIb); type IIIb was characterized by a very low histone kinase activity compared to its peptide kinase activity. In some brain extracts a phospholipid-dependent but Ca2+-inhibited protein kinase was also observed which was eluted from the hydroxyapatite column between type II and III isoenzymes of protein kinase C.

We compared the protein kinase C isoenzyme patterns of crude extracts of rabbit brain, cerebellum, spleen, thymus and human and pig granulocytes. The isoenzymes were fractionated by hydroxyapatite chromatography and the protein kinase C activity was determined with a synthetic oligopeptide substrate. In the extracts of several tissues we also observed an enzyme fraction which was activated by phosphatidylserine+diacylglycerol but inhibited by Ca2+.

The purification of ATP-dependent DNase from Bacillus cereus led to the isolation and characterization of a third DNA-dependent ATPase. The enzyme called ATPase III has been purified free of nuclease activity. None of the expected ATPases proved to be identical with ATP-dependent DNase-DNA-dependent ATPase. Separation of ATPase I, II and III and a DNase specific for single-stranded DNA from the same source excludes the possibility of ATP-dependent DNase being the action of a single enzyme molecule.

Galactosamine hepatitis [l] is accompanied by decreased incorporation of amino acid into protein [2,3] and of uridine into RNA in the liver [2]. Recently the inhibition of induction of rat liver tyrosine amino transferase by D-galactosamine has been reported [4]. The mechanism of liver damage is not known, and the involvement of unphysiological metabolites of galactosamine has been supposed [5-71. In this report we describe the in vitro inhibition of amino acid, uridine and orotate incorporation into mouse liver slices caused by D-galactosamine, and the enhancement of inhibition in the presence of uridine and its reversion by D-galactose. Galactosamine hepatitis is prevented by D-galactose in vivo.

Phosphorylation and dephosphorylation of proteins play a pivotal role in the regulation of intracellular processes (reviewed in [l-3]). Beside several well-characterized protein kinases a number of protein phosphorylating enzymes have been found; however, their natural substrates and regulatory properties are not known. A possible way to identify these enzymes is the determination of their intrinsic substrate specificity; i.e., the most important residues of the amino acid sequence that they recognize in their substrate. A cyclic nucleotide-independent histone kinase (designated as HK II) was described in 1451. We have also reported a type of cyclic nucleotide-independent histone kinase found in the cytoplasm and nucleus of human tonsillar lymphocytes [6,7] and in the extract of bovine thymus [8]. This enzyme phosphorylates the Ser-32 residue of calf thymus H2b histone and a synthetic peptide containing the amino acid sequence of H2b from Gly-26-Lys-34, but it does not act on Ser-36 which is phosphorylated preferentially by the catalytic subunit of the cyclic AMP-dependent protein kinase 19-l 11. Separation of the cyclic nucleotide-independent histone kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase was carried out by DEAE-cellulose chromatography in the presence of cyclic AMP, as in 181. The histone kinase fraction obtained by this procedure from bovine thymus was dialyzed against 0.005 M potassium phosphate (pH 7.5) and it was applied onto a hydroxylapatite (Bio-Gel HTP) column. The column was washed by 5 vol. of each of 0.01 M, 0.07 M, 0.085 M, 0.1 M and 0.4 M potassium phosphate (pH 7.5). The histone kinase was eluted by the last volumes of 0.07 M and by the first volume of 0.085 M potassium phosphate. The specific activity of the enzyme preparation obtained was lo-12 nmol phosphate transferred mg protein -’ . min -‘, as measured in the presence of 1 mg histone Hi/ml and 2 x lo-’ M ATP. This preparation yielded a single (Mr 55 000) histone kinase peak on Sephadex G-100 gel-chromatography.

Parenteral administration of D-galactosamine causes acute hepatic injury in rodents, morphologically resembling viral hepatitis [ 11. An inhibition of hepatic RNA synthesis due to D-galactosamine has been reported on the basis of decreased incorporation of radioactive precursors [2-41, and it has been considered to be the consequence of the depletion of the hepatic UTP pool by D-galactosamine, from which excessive formation of UDP-N-acetyl hexosamines and UDP-hexosamines occurs [S]. Little attention has been paid to the simultaneous inhibition of protein synthesis also reflected by decreased amino acid incorporation [6-81. The reversal of the hepatotoxic action of D-galactosamine by D-galactose has been observed in this laboratory [8]. In the experiments presented here the in vitro incorporation of radioactive uridine and guanosine has been investigated, and a decrease of the specific radioactivity of UTP is demonstrated by a double labeling technique. D-Galactosamine is known to be incorporated into glycogen [9] and the formation of atypical polysaccharides has been demonstrated by electron microscopy [3]. It is known that in vivo treatment of mice with D-galactosamine and D-glucose simultaneously causes an alteration of mouse liver microsomal membranes, which can then be pelleted through a 2 M solution of sucrose in the ultracentrifuge. This change can be reversed by treating the membranes with cu-amylase. The effect of the combined treatment can be prevented by simultaneous administration of D-galactose. An interrelation between the synthesis of basic polysaccharide, damage of the endoplasmic

The inactivation of rec BC nuclease activity and simultaneously the separation of 3 DNA-dependent ATPases and an ATP-independent DNases specific for single-stranded DNA have been observed after DEAE-cellulose chromatography of cell extracts from Escherichia coli . Two of the ATPases catalyze the strand separation of duplex DNA. Reconstitution of ATP-dependent DNase activity has been carried out by the combination of the separation enzymes.