Hiroyoshi Nohara

Enzymatic Acetylation of histones and some chemical characters of their acetyl groups

Abstract The enzymatic acetylation of histones by pigeon liver fractions and the chemical properties of acetyl groups accepted by histones were studied. The results are summarized as follows. 1. 1. Two pigeon liver fractions, which act as acetate activating and transferring enzyme respectively, catalyze the incorporation of [14C]acetate into histones in the presence of ATP, Mg2+ and CoA. 2. 2. Although all of histone fractions were labelled with [14C]acetate by the system, arginine-rich histone was found to be the most rapidly labelled fraction. 3. 3. The presence of both labile and stable acetyl groups for NH2OH treatment was observed in the labelled arginine-rich and slightly lysine-rich histone, whereas only the stable one was seen in the labelled lysine-rich histone.

Studies on the pH-5 enzyme. I. The effects of ribonuclease and some SH inhibitors on the amino hydroxamate formation by the pH-5 enzyme of rabbit liver.

Abstract The pH-5 enzyme was isolated from rabbit liver by the modified method of Hoagland et al. and the effects of some SH inhibitors, azide, and RNase on the amino hydroxamate formation by this enzyme were investigated. The following results were obtained: 1. 1. p -Chloromercuribenzoate, monoiodacetate and azide failed to inhibit the amino acid-dependent hydroxamate formation by the pH-5 enzyme. 2. 2. When the pH-5 enzyme, pretreated with RNase, was incubated with ATP and the amino acid mixture, the amount of hydroxamate formed by the amino acid decreased, as compared with the control experiment in which the pH-5 enzyme had not been pretreated with RNase. From these results it is concluded that the SH group is not involved in most of the amino hydroxamate formation by the pH-5 enzyme, and that, by contrast, the RNA of the enzyme plays an essential part in this reaction. The possible mechanism by which this RNA participates in the amino hydroxamate formation is discussed.

Separation and partial characterization of three forms of collagenase inhibitor from bovine gingiva

Three forms of collagenase inhibitor were isolated; one bound to Con A-Sepharose and the other two did not. The Con A-bound (Mr 38,000) and the two unbound (Mr 50,000 and 22,000) inhibitors contained about 20, 15 and 65% of the total inhibitory activity, respectively. The bound and one of the unbound (Mr 22,000) inhibitors were fairly specific for mammalian collagenase; the other unbound inhibitor was rather non-specific and also inhibited trypsin and thermolysin, but not bacterial collagenase. All the inhibitors were heat stable (90 degrees C, 30 min) and unaffected by 4-aminophenylmercuric acetate, but were inactivated by reduction and alkylation.

The effect of exonuclease I on the transcription of various DNA preparations by rat liver RNA polymerase I and II

Abstract The effect of exonuclease I on the template activity of various DNA preparations was studied with two rat liver RNA polymerases. Treatment of various DNA preparations, native, reassociated and sonicated DNAs, with optimal amounts of Escherichia coli exonuclease I, inhibited the synthesis of RNA by polymerase I and II by 25–75 %, suggesting that both enzymes prefer unpaired 3′-hydroxyl ends of DNA as the initiation site. When the frayed ends of various reassociated DNAs were eliminated, the synthesis of RNA by polymerase I increased as the C 0 t value of template DNA increased, while the synthesis by polymerase II was most prominent with C 0 t 200–1000 and 0–200 DNA.

Differential response of type I and type II cyclic AMP-dependent protein kinases in submandibular gland of isoproterenol-treated rats

The cytosolic protein kinase isozymes were studied in relation to the secretion of saliva and the proliferative process induced by isoproterenol. At the same time as salivary secretion began, cytosolic protein kinase was activated and, in the absence of cyclic AMP, increased to a maximum level at 10 min after administration of the drug. The active state of the enzyme was maintained for 1-2 h. Chromatographic analysis of the cytosol enzyme revealed that in an activated state type II kinase easily dissociates into the catalytic and regulatory subunits while type I kinase hardly dissociates. Thus we conclude that type I kinase, at least in submandibular gland, is less dissociable in the presence of cyclic AMP than type II kinase but highly active in a ternary complex (R2-4 cAMP-C2). The presence of cyclic AMP-independent protein kinase, which co-chromatographed with protein kinase II, was demonstrated by use of a heat-stable inhibitor from bovine muscle, which totally inhibited the cyclic AMP-dependent enzymes, but stimulated the cyclic AMP-independent protein kinase. The level of protein kinase I decreased to its lowest value, about half that of the control value, at 10 h after isoproterenol injection and then increased slightly at 15 h, whereas type II kinase remained virtually unchanged, suggesting that the two protein kinase isozymes are independently regulated in the proliferating cells.

Some properties of RNA polymerase of rat submaxillary gland nuclei and effects of isoproterenol on the enzyme

Abstract RNA polymerase of rat submaxillary gland nuclei had a different affinity for three nucleoside triphosphate substrates from the enzyme from liver nuclei. The apparent K m values were 3.9 × 10 −5 mol/l for the enzyme of salivary gland nuclei in the presence of ammonium sulphate and 2.9 × 10 −4 mol/l for the liver enzyme in the presence and absence of ammonium sulphate. The administration of isoproterenol stimulated the enzyme activities of salivary gland nuclei in the presence and absence of ammonium sulphate and the maximum stimulation was about 100 per cent in both reactions 15 hr after the administration of the drug. After treatment with the drug, the apparent K m value of the enzyme increased to 1.7-fold of that of the untreated control in the presence of ammonium sulphate.

Production of chemotactic factor for lymphocyte by digestion of IgG with a highly purified polymorphonuclear leukocyte neutral thiol proteinase

In the present study, we purified a neutral thiol proteinase from dog PMN leukocytes and indicated that the proteinase elaborated the chemotactic factor for lymphocytes by cleavage of IgG. The neutral thiol proteinase was purified about 744-fold by ion-exchange chromatographies and affinity chromatography, and the final preparation was over 70% pure. After incubation of dog IgG with the proteinase, three distinct protein peaks were seen by the gel filtration on Sephadex G-200. Only the third peak, perhaps a dialyzable peptide, showed a significant chemotactic activity for dog lymphocytes.

Partial sequencing of two forms of concanavalin a-unbound collagenase inhibitor from bovine gingiva

Three forms of collagenase inhibitor, one ConA-bound and two ConA-unbound, were extensively purified from bovine gingiva by sequential column chromatography. Analysis by sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that inhibitory activity resides in proteins with M(r) of 26000-28000 and 22000 for ConA-bound and two ConA-unbound inhibitors, respectively. Of these, two ConA-unbound inhibitors were partially sequenced in the first 12 amino acids and found to have an identical sequence. The NH2-terminal sequence had 100% identity with TIMP-2 or MI.

Purification of a cytosolic factor from rat liver that stimulates transcription in isolated nuclei and its action on purified RNA polymerase II-DNA system.

A cytosolic factor that stimulates transcription in isolated nuclei was purified approximately 4000-fold to near homogeneity from rat liver. The molecular weight of the factor was determined as 47 000 by SDS-polyacrylamide gel electrophoresis. The factor had no detectable deoxyribonuclease and protease activity but showed ribonuclease inhibitor activity. The factor could stimulate transcription in isolated nuclei by 50% at about 3.0 ng and the maximal stimulation was about 100%. When [gamma-S]ATP and [gamma-S]GTP were included in the reaction, the factor stimulated the synthesis of RNA which was able to bind to a mercury-Sepharose column and about 80% of the bound RNA was sensitive to a low concentration of alpha-amanitin. When heparin was added before initiation to preincubation mixture containing RNA polymerases II and DNA, a small but definite incorporation of [14C]UTP was observed. The factor alone had no stimulatory effect on the heparin-resistant incorporation of [14C]UTP but, in the presence of two rat liver nuclear fractions, phosphocellulose 0.5 and 1 M KCl step fractions, could stimulate the incorporation above the level with the combination of the two nuclear fractions. Antibody raised against the factor inhibited accurate transcription from the adenovirus 2 major late promoter in a nuclear lysate from Ehrlich ascites tumor cells, and the inhibition was neutralized by the factor.

Insoluble immune complexes in experimental gingivitis of dogs

This study was undertaken to examine the levels of insoluble immune complexes in healthy and inflamed gingiva by measuring citrate buffer-elutable IgG. After establishment a clinically healthy gingiva by continuous toothbrushing for 6 weeks, the experimental gingivitis was induced by placement of silk floss ligature to accelerate plaque formation for 1 week, 1 and 3 months. The excised gingiva was prepared by the elution procedure with PBS and citrate buffer. The concentration of PBS-elutable IgG in gingiva was measured by SRID, besides the citrate bufferelutable IgG was determined by ELISA and RIA. As a results ; 1) The amount of PBS-elutable IgG had a tendenvy to increase in inflamed gingiva at each experimental period and the IgG level was significantly higher at 3 months experimental period than the healthy. 2) The citrate buffer-elutable IgG significantly increased in inflamed gingiva at each experimental period and the rate of increase was more prominent with advancing gingivitis. 3) There was an increase in ratio of citrate bufferto PBS-elutable IgG at each experimental period as compared with at the healthy. 4) As shown r=0. 945 in a coefficient of correlation, a close correlation between ELISA and RIA was found.