Detection and characterization of a novel factor that stimulates DNA polymerase alpha

A novel factor that stimulates DNA polymerase alpha activity on poly(dA) X oligo(dT) has been identified and partially purified from mouse FM3A cells. The assay system for the factor contained poly(ethylene glycol) 6000. The activities of DNA polymerase alpha on poly(dA) X oligo(dT) in the presence and absence of the stimulating factor were increased greatly by the addition of poly(ethylene glycol). Stimulation by the factor was observed at all the primer to template ratios tested from 0.01 to 0.3. The highest activity was observed at the ratio of 0.05, corresponding to about 3.3 primers on one template in the presence of the factor. The concentration of DNA polymerase alpha used in the assay affected the stimulation by the factor, and the stimulation became more prominent at concentrations of the enzyme lower than 0.04 unit per assay. The stimulating factor lowered the Km value of DNA polymerase alpha for the template-primer, though they had no effect on the Km value for dTTP substrate. The results of product analysis suggested that the stimulation by the factor is mainly due to the increase in the initiation frequency of DNA synthesis from the primers. The stimulating factor specifically stimulated DNA polymerase alpha but not DNA polymerases beta and gamma. Furthermore, the factor formed a complex with DNA polymerase alpha under a certain condition.     

Isolation and chemical characterization of the phosphoproteins of chicken bone matrix: heterogeneity in molecular weight and composition

Ethylenediaminetetraacetic acid and HCl extracts of calcified chicken bone were fractionated by a variety of techniques, including molecular sieving in guanidinium chloride, ion-exchange chromatography on DEAE-cellulose, high-performance liquid chromatography (HPLC), reverse-phase HPLC, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using several different experimental schemas, we isolated 14 apparently homogeneous components varying in molecular weight from approximately 150K to approximately 4K-5K. The compositions of all of the phosphoproteins were characterized by high concentrations of Asp, Glu, Ser, Gly, and Ala. Seven of the components which were analyzed contained concentrations of carbohydrate varying from approximately 4% to approximately 17%. Three of the components containing O-phosphoserine which behaved as single bands on SDS-PAGE with molecular weights of approximately 150K, approximately 90K, and approximately 70K contained Hyp and Hyl or Hyl alone and may represent covalently bonded or strongly associated collagen-phosphoprotein complexes or hydroxylated Pro and/or Lys residues of the phosphoproteins. The findings that the amino acid compositions of several of the components were very similar and that N-terminal partial amino acid sequences of the approximately 90- and approximately 60-kilodalton (kDa) and of the approximately 150- and approximately 32-kDa components, respectively, were identical make it clear that some of the lower molecular weight components are derived by proteolysis from higher molecular weight species. In addition to proteolysis, we speculate that it is possible, from the N-terminal amino acid sequence data and preliminary cross-reaction studies of antibodies to four of the phosphoproteins, that the heterogeneity observed in the phosphoprotein components may also be due in part to there being more than one independent gene product for chicken bone phosphoproteins.     

Effects of norharman on the mutagenicity of chlorophenylhydroxylamine and its metabolism with rat liver S9

o,p-Chlorophenylhydroxylamines (CPHAs) (10468-16-3, 823-86-9) only demonstrated mutagenicity in the presence of S9 mix and norharman (NOH) (244-63-3), as well as chloronitrobenzenes. The mutagenic activity of o-CPHA was 30 times higher than that of p-CPHA. When o-CPHA was preincubated with S9 mix without NOH, the mutagenic activity disappeared rapidly. The decrease in activity during the preincubation was suppressed by addition of NOH. HPLC analysis revealed that o-CPHA was metabolized to o-chloroaniline (o-CA) (95-51-2) and that the metabolic reduction was inhibited by NOH. When microsomes containing NADPH were used instead of S9 mix, o-CPHA exhibited only very weak mutagenicity. The activity in the microsome system, however, was greatly enhanced by adding cytosol or ascorbic acid (50-81-7). These phenomena were only observed in the conventional plate incorporation method. In the case of the liquid incubation assay, in which test compound metabolism and tester strain mutation only occur in the liquid incubation medium, the mutagenic activity of o-CPHA in the microsome system with NOH was comparable to that in the S9 system, indicating that o-CPHA was activated by an enzyme in microsomes in the presence of NOH. Consequently, it was concluded that NOH not only affects the metabolic inactivation of o-CPHA to o-CA by S9, but also the metabolic activation of o-CPHA by microsomes. No appreciable enhancing effects of cytosol and ascorbic acid were observed in the liquid incubation assay, suggesting that these factors affect the stability of CPHA or an active metabolite. The microsome activation of o-CPHA was dependent on NADPH and oxygen; SKF-525A (62-68-0), metyrapone (54-36-4) and alpha-naphthoflavone (604-59-1) inhibited the mutagenic activity by about 50%, suggesting that cytochrome P-450 was involved in the metabolic activation.     

Growth and Aspartate Kinase Activity in Wheat Cell Suspension Culture: Effects of Lysine Analogs and Aspartate-Derived Amino Acids

The effects of lysine analogs and aspartate-derived amino acidson the growth of wheat cell suspension culture were studied.S-(2-Aminoethyl)-L-cysteine (AEC), -hydroxylysine (DHL) andtrans-lysene caused complete growth inhibition at 1.0 mM. Thegrowth inhibition of lysine analogs were, in the order of decreasingeffectiveness; AECDHL, trans-lysene>oxalysine, homolysineand lysyne. cis-Lysene and methyllysine were not inhibitoryeven at concentrations of 10 mM. Lysine effectively relievedgrowth inhibition induced by the lysine analogs. Lysine plusthreonine showed concerted inhibition, which was relieved bythe addition of methionine. Activity of aspartate kinase extracted from wheat cell suspensionculture was strongly inhibited by L-lysine; 0.75 to 1 mM oflysine was required for half-maximal inhibition. Threonine andmethionine, individually or in combination with lysine, showedno inhibitory effect on the enzyme activity. S-Adenosylmethionine,when added with lysine in equimolar concentrations, enhancedthe feedback inhibition by lysine, lowering the concentrationof lysine for half-maximal inhibition to 0.13 mM. The aspartatekinase isolated from the cells cultured in the presence of 5mM lysine did not differ in regulatory properties from the enzymefrom the cells cultured without lysine. AEC at 5 mM inhibitedthe enzyme activity by 50%. Other lysine analogs were not inhibitoryto the enzyme activity even at 10 mM. Growth inhibition of wheat suspension culture by aspartate-derivedamino acids and lysine analogs were discussed in relation totheir inhibitory effects on aspartate kinase activity. (Received October 25, 1985; Accepted February 26, 1986)     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

Ultrastructural studies on the effect of Triton WR-1339 on macrophage-mycobacteria interaction

Mycobacterium bovis (BCG organisms) suspended in saline or a 5% solution of a non-ionic detergent, Triton WR-1339, was injected intraperitoneally into mice. Electron-microscopic observation was carried out on peritoneal exudate cells harvested therefrom. Electron-lucent vacuoles limited by the membrane structure were found in macrophages of the mice injected with BCG suspended in the detergent, but not in polymorphonuclear leukocytes or lymphocytes. Mycobacterial cells were present within such vacuoles. Without the detergent, the ingested mycobacterial cells were in close contact with the phagosomal membrane. Within the electron-lucent vacuoles, however, such close contact was not present. These observations, together with other collateral findings, led us to a view that Triton WR-1339 may inhibit the interaction between mycobacteria and the phagosomal membrane by intervening between them thus making the progress of infection delayed.     

Changes in proteoglycan composition during development of rat skin. The occurrence in fetal skin of a chondroitin sulfate proteoglycan with high turnover rate

Extraction of the skin of newborn rat yielded two populations of galactosaminoglycan-containing proteoglycan: a Mr = 111,000-200,000 dermatan sulfate proteoglycan (DS-PG) with a Mr congruent to 55,000 core glycoprotein and a Mr congruent to 10(6) chondroitin sulfate proteoglycan (CS-PGs) composed of two subpopulations with different size core-glycoproteins (Mr congruent to 480,000 and 520,000). Tryptic peptide mapping of chondroitinase-treated DS-PG and CS-PGs indicated that the peptide patterns observed with the two core molecules from CS-PGs were identical with each other but distinct from the peptide pattern of the DS-PG core molecule. It is likely therefore that the two forms of CS-PGs are derived from the same gene product by post-translational modification or partial degradation, but DS-PG is derived from a distinct gene product. Comparison of the concentration (hexuronate/DNA) of the proteoglycans in newborn and fetal rat skin showed an age-related change in proteoglycan composition; at 4 days before birth, the uronic acid proportions, DS-PG:CS-PGs, were about 14:1 and during the next 4 days, DS-PG increased 2.2-fold whereas CS-PGs decreased 4-fold. On a per DNA basis, the rate of [3H]serine incorporation into CS-PGs was 2.5 times the rate for DS-PG at 4 days before birth but decreased by 95% during the next 4 days. The rate for DS-PG also decreased but to a much lesser extent, so that by 2 days before birth, it began to exceed the rate for CS-PGs. The striking change in the concentration and labeling rate of CS-PGs can be interpreted either as a decrease of CS-PGs synthesis, or as an increase of CS-PGs breakdown, or both, a process which might be involved in the transition of extracellular matrix from a fetal type to a newborn or adult type.     

Immunological characterization of sn-1,2-diacylglycerol and sn-2-monoacylglycerol kinase from pig brain

Rabbit antisera were raised against diacylglycerol kinase purified from pig brain cytosol. Upon immunoblot analysis, the antibody was specifically reactive with the kinase (Mr = 79,000-80,000). Pig brain cytosol, microsomal, and synaptosomal fractions all contained the immunoreactive Mr = 80,000 polypeptide, thus showing that the same enzyme is present in the soluble as well as membrane fractions of the brain. The antibody could precipitate only 60% of the kinase activity present in the crude cytosol. Further, the antibody exhibited very little or no cross-reactivity toward liver cytosolic enzymes obtained from different animals including pigs. Immunostaining of brain tissues demonstrated that neurons, in particular, their nuclei, were positively stained, whereas glial cells were not stained. It is likely that there exists a tissue-and/or cell-dependent immunological multiplicity of diacylglycerol kinase. The enzyme activities phosphorylating sn-1 and sn-2 monoacylglycerols were co-precipitated by the antibody, indicating their identity with diacylglycerol kinase. The enzyme activity toward sn-1 monoolein was much lower than that obtained with sn-2 monoolein. Enzymic as well as chemical analyses of acyl isomers of the reaction products showed that even tested with pure (greater than 95%) sn-1 monoolein, about 70% of the formed lysophosphatidate was of the sn-2 acyl type. The results show that diacylglycerol kinase phosphorylates almost exclusively the sn-2 acyl type of monoacyl-glycerol.     

In vitro poly(ADP-ribosyl)ation of seminal ribonuclease

The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase.     

Streptococcal cytoplasmic pH is regulated by changes in amount and activity of a proton-translocating ATPase

The Streptococcus faecalis H+-ATPase (F1 X F0 complex) level was elevated when the cytoplasmic pH was shifted below 7.5. The elevated level was attained by the increase in functional unit (F1 X F0 complex) in membranes, but not by the activation of the enzyme. Our data strongly suggested that the increase in enzyme arises from stimulation of enzyme biosynthesis. When calls growing at pH 7.6 were transferred to an acid medium with a pH below 7, the amount of H+-ATPase increased. The amount of H+-ATPase decreased to the basal level when the medium was alkalized again. Cytoplasmic pH was not controlled normally in cells where a change in the amount of H+-ATPase was inhibited. Based on these findings and previous data (Kobayashi, H. (1985) J. Biol. Chem. 260, 72-76), we propose a model for the regulatory mechanism of streptococcal cytoplasmic pH: the pH is regulated by changes in amount and activity of the H+-ATPase, which are dependent on the cytoplasmic pH.