The control of phosphoprotein phosphatase by the second-site phosphorylation of a substrate. Studies with H2B histone as model substrate.

The phosphorylation of Ser-32, in addition to Ser-36 of H2B histone, stimulated the rate of Pi release from Ser-36 by the small form (Mr 31 000) of pig heart phosphoprotein phosphatase both in the absence and presence of 50 mM magnesium acetate. By phosphorylation at Ser-32, the Km value for Ser-36 phosphate in H2B histone was increased from 0.38 microM to 1.16 microM in the absence of magnesium acetate, but not significantly changed (from 37.4 microM to 26.2 microM) in the presence of magnesium acetate. With the large form (Mr 224000) of the phosphoprotein phosphatase, however, the phosphorylation at Ser-32 suppressed the rate of Pi release from Ser-36 both in the absence and presence of magnesium acetate. The Km value of the large form for Ser-36 phosphatase in H2B histone was nevertheless increased by phosphorylation at Ser-32, from 1.2 microM to 5.3 microM in the presence of magnesium acetate, but not changed (from 0.26 microM to 0.23 microM) in the absence of magnesium acetate.     

Ecological studies of collembolan populations in a pine forest soil IV. Comparison of distribution patterns

The distribution pattern of ten species of Collembola was studied during the four years period from July 1971 to May 1975 in a pine forest soil. The distribution patterns were analysed for two scales of distribution, i. e., the distribution over the plot of 10×10 m² and the micro-distribution within a block sample consisting of 36 contigious units each 2×2 cm² in area, by applying the -m regression method. The fundamental pattern which appeared was quite similar for the species examined and individuals were aggregated in response to the heterogeneity of habitat conditions. The causes of aggregations were discussed with regard to some environmental factors. The relative abundances of 10 species within the collembolan community was examined in relation to the habitat utilization and the relative abundance was not related to the degree of aggregation but rather to the area occupied by individuals. This suggests that the more numerically abundant species tend to occupy broader micro-habitat. Biological meaning of aggregation was discussed in connection with the population biology and community organization of collembola.     

Comparison of two forms of pig heart phosphoprotein phosphatase.

A phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) was partially purified from pig heart using as substrate H2B histone which had been phosphorylated at Ser-32 and Ser-36 by adenosine 3',5'-monophosphate-dependent protein kinase (EC 2.7.1.37). The enzyme had a molecular weight of approx. 250 000 and was converted to a smaller form with a molecular weight of approx. 30 000 upon treatment with ethanol. Phosphorylase alpha (EC 2.4.1.1) and phosphorylated H1 histone also served as substrates for both forms of the enzyme. The conversion of the large form of the enzyme to the small form decreased the phosphohistone phosphatase activity to 25-50% with a concomitant 7-fold increase in the phosphorylase alpha phosphatase activity. Ser-36 phosphate was removed 6- and 15-fold more rapidly than was Ser-32 phosphate by the large and small forms of the enzyme, respectively. Among Ser-36-containing tryptic phosphopeptides derived from phosphorylated H2B histone, Lys-Glu-Ser(P)-Tyr-Ser-Val-Tyr was the shortest phosphopeptide which was dephosphorylated at a significant reaction rate with the phosphoprotein phosphatase. The Km values for phosphorylated H2B histone and the tryptic phosphopeptide were 23.7 micron and 187.1 micron, respectively, with the large form, and 81.4 micron and 90.0 micron, respectively, with the small form of the enzyme.     

Studies on the sites in histones phosphorylated by adenosine 3':5'-monophosphate-dependent and guanosine 3':5'-monophosphate-dependent protein kinases.

Adenosine 3':5'-monophosphate-dependent protein kinase (protein kinase A) purified from silkworm pupae phosphorylated five major fractions of calf thymus histone, whereas guanosine 3':5'-monophosphate-dependent protein kinase (protein kinase G) purified from the same organism reacted preferentially with H1, H2A, and H2B histones. Amino acid analysis of the phosphopeptides which were obtained by proteolytic digestion revealed that both protein kinases A and G showed the abilities to phosphorylate the same serine hydroxyl groups in H1 and H2B histones. Both protein kinases reacted with Ser-38 in H1 histone. With H2B histone as substrate protein kinase A phosphorylated Ser-32 as well as Ser-36, whereas protein kinase G reacted preferentially with Ser-32 and the reaction with Ser-36 was very slow. H3 and H4 histones were practically inactive substrates for protein kinase G. Although H2A histone has not been analyzed, the evidence has raised a possibility that protein kinase G utilizes a portion of the substrate proteins for protein kinase A.     

Effect of DL-alpha-hydrazino-delta-aminovaleric acid, an inhibitor of ornithine decarboxylase, on polyamine metabolism in isoproterenol-stimulated mouse parotid glands.

The effects of DL-alpha-hydrazino-delta-aminovaleric acid (DL-HAVA) on polyamine metabolism in isoproterenol(IPR)-stimulated mouse parotid glands were investigated both in vitro and in vivo. Using partially enzyme preparations, it was found that DL-HAVA strongly inhibited ornithine decarboxylase (EC 4.1.1.17) by competing with L-ornithine. Other enzymes metabolizing ornithine and pyridoxal phosphate-dependent enzymes were at least 2-3 orders of magnitude less sensitive to DL-HAVA than ornithine decarboxylase. Administration of DL-HAVA greatly depressed the increases in both the putrescine level and putrescine formation from L-ornithine induced by IPR in the mouse parotid glands. Under the same conditions, the stimulation of DNA synthesis and subsequent cell proliferation in the glands were also suppressed. However, the IPR-dependent increases in S-adenosyl-L-methionine decarboxylase (EC 4.1.1.50) activity, synthesis and the tissue concentration of spermidine, and RNA synthesis in the parotid glands were not affected appreciably by DL-HAVA. The inhibition of DNA synthesis by DL-HAVA was effectively prevented by putrescine, but not by spermidine or 1,7-diaminoheptane, given at the same time when DL-HAVA inhibited stimulation of putrescine formation by IPR. From these results, it is proposed that putrescine is involved in cell proliferation besides being a precursor of spermidine. The effects of methylglyoxal bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl-L-methionine decarboxylase, on the metabolism of polyamines and nucleic acids in growing parotid glands were also examined.     

Effect of sodium dodecyl fulfate on the dissociation of bovine liver catalase.

Native bovine liver catalase [EC 1.11.1.6] and catalase acetylated with N-acetylimidazole (AI) both combined with sodium dodecyl sulfate (SDS) to form catalase-SDS complexes. The differences between native and acetylated catalase bound to SDS were investigated as regards enzymatic activity, absorption spectra, ORD and CD, sedimentation velocity and fluorescence spectra. It was found that the binding of SDS with both catalases depended on incubation time and SDS concentration, and that the acetylation of catalase had some protective effect on the denaturation of the molecule by SDS, which may be ascribed to a reduction of ionic interaction between SDS and the protein on acetylation. The native catalase was found to split into three smaller components on incubation with 1% SDS for 96 hr, whereas the acetylated catalase split into two smaller components. These smaller components were isolated by gel filtration through Sephadex G-100. The isolated components has estimated molecular weights of 60,000, 30,000, aide. It seemed likely that the modification occurred stepwise. Approximately 26% of the carboxyl groups of fibrinogen was modified finally. The modified fibrinogen had no interaction with cationic detergent, and did not form any complex with the detergent. In dilute acid, fibrinogen was observed to show only a slight interaction with cationic detergent. It is probable that the exposed and ionized carboxyl groups are essential for the formation of a complex between fibrinogen and cationic detergent.     

Two new iridoid glucosides from Ixora chinensis

From leaves and twigs of Ixora chinensis, two new iridoid glucosides, ixoroside (1) and ixoside (7,8-dehydroforsythide) (2) along with known geniposidic acid (3) have been isolated and their structures have been established.     

Identification of a multi-component berberine 11-hydroxylase from Burkholderia sp. strain CJ1

ABSTRACT

Berberine (BBR) is a protoberberine alkaloid extracted from plants such as Coptis japonica (Ranunculaceae). In a previous report, we demonstrated the existence of a 11-hydroxylation pathway employed by BBR-utilizing bacteria for metabolism of BBR. In the present study, we report the identification of the genes brhA, brhB, and brhC as encoding a multicomponent BBR 11-hydroxylase in Burkholderia sp. strain CJ1. BrhA is belonging to the Rieske non-heme iron oxygenase (RO) family, a class of enzymes known to catalyze the first step in bacterial aromatic-ring hydroxylation. We further demonstrate that BrhA activity requires BrhB (ferredoxin reductase) and BrhC (ferredoxin) as electron transport chain components. A BLAST search revealed that BrhA exhibits 38% and 33% sequence identity to dicamba O-demethylase (DdmC; AY786443) and chloroacetanilide herbicides N-dealkylase (CndA; KJ461679), respectively. To our knowledge, this work represents the first report of a bacterial oxygenase catalyzing the metabolism of a polycyclic aromatic-ring alkaloid.

Abbreviations: BBR: berberine; D-BBR: demethyleneberberine; H-BBR: 11-hydroxyberberine; HD-BBR: 11-hydroxydemethyleneberberine; HDBA: 2-hydroxy-3,4-dimethoxybenzeneacetic acid; PAL: palmatine; H-PAL: 11-hydroxypalmatine; BRU: berberrubine; Fd: ferredoxin; FdR: ferredoxin reductase; ETC: electron transport chain