Partial purification and characterization of type I DNA topoisomerase from Bacillus stearothermophilus

Type I DNA topoisomerase was partially purified from Bacillus stearothermophilus by ammonium sulfate precipitation and column chromatographies on phosphocellulose, DEAE-cellulose and heparin-agarose. On heparin-agarose chromatography, topoisomerase I activity was separated into three fractions (designated Fractions A, B, and C). Each fraction was further subjected to gel filtration on Sephacryl S-200. From electrophoretic analysis on polyacrylamide gel, Fraction A was found to contain two enzyme species having molecular weights of 110,000 and 100,000, and Fraction B one enzyme species with a molecular weight of 80,000. The molecular weight of the enzyme in Fraction C was estimated to be around 150,000 by gel filtration. The enzymes in Fractions A and B exhibited little activity in the presence of Mg2+, while the activity was increased remarkably by NaCl with Mg2+. No activity was observed in the presence of NaCl alone. The enzyme in Fraction C required only Mg2+ for full activity. With Fraction A, the topoisomerase I-induced cleavage sites on tetracycline-resistant plasmid pNS1 (2.55 megadaltons) were mapped. Fraction A cleaved the DNA at ten specific sites. These sites were compared to those of the Haemophilus gallinarum enzyme, which have already been mapped (Shishido et al. (1983) Biochem. Biophys. Acta 740, 108). The results showed that there is a remarkably coincidence between the cleavage sites induced by the B. stearothermophilus and H. gallinarum enzymes.     

Induction of lesions in deoxyribonucleic acid by low molecular weight substances from normal and colicin E2-treated cells of Escherichia coli.

A fraction containing a variety of low molecular weight substances was extracted into 80% aqueous acetone from both a colicin E2-treated cell culture of Escherichia coli and an untreated one. The extract was divided into five fractions by Sephadex G15 chromatography. One of them, Fraction B, was separated into three subfractions by Sephadex G10 chromatography. Two subfractions, Fraction BI and Fraction BII, were further fractionated by several chromatographic systems. DNA was incubated with an aliquot from each of these fractions and was then analyzed by sedimentation in an alkaline sucrose density gradient. The activity which caused a decrease in the sedimentation coefficient of the DNA was found in some of these fractions. The activity from colicin E2-treated cells was compared with that from untreated ones. It was revealed that colicin E2 induces some increases in the activity toward DNA in one of the subfractions, Fraction BI, and also causes the appearance of a new species in another fraction, Fraction BII, which potentiates the activity in Fraction BI. These colicin E2-induced changes appeared at 5 min after the addition of colicin E2. The possible significance of such reactions for the action of colicin E2 are discussed.     

DNA polymerases in mouse spermatogenic cells separated by sedimentation velocity.

Activity levels of DNA polymerase alpha and DNA polymerase beta have been measured in mouse spermatogenic cells separated by sedimentation velocity. Testes from prepuberal (17 day old) and sexually mature mice were dissociated and separated by unit gravity sedimentation into 6 populations of cells. Phase contrast microscopy and [3H]thymidine labeling kinetics revealed that at least 85% of the cells in fraction A were pachytene-stage primary spermatocytes, fraction B was enriched for primary spermatocytes and round spermatids, fraction C contained spermatogonia and/or pre-leptotene primary spermatocytes and later stages of spermatids (no spermatids were present in fraction C from the testes of 17 day old mice) and fractions D to F contained mixed populations of cells, many in later stages of spermiogenesis. When expressed as activity in 10(6) cells or as a specific activity, fractions A, B, and C from mature animals population initially loaded onto the gradient while fractions D, E and F had activity levels similar to or below the population of dissociated cells. The ratio of activity between the DNA polymerases was constant in fractions A, B, and C, but in fractions D, E, and F, the ratio decreased due to a more rapid decline of activity of polymerase alpha. A comparison of activity levels in fraction C from prepuberal and sexually mature mice revealed an increase in DNA polymerase alpha activity and a decrease in the activity of DNA polymerase beta in the cells from the 17 day old animals.     

Identification of multiple cellular factors required for SV40 replication in vitro

The replication of simian virus 40 has been studied by using cell-free extracts derived from human 293 cells. Fractionation of this extract has led to the identification of three fractions that are required for efficient DNA synthesis. Initial fractionation of the crude extract by phosphocellulose chromatography has produced two fractions, I and II, neither of which is able to support replication separately, but when they are combined, efficient synthesis is restored. Both fractions are required, with SV40 T antigen, for the formation of a presynthesis complex at the SV40 origin. The major replication enzymes, DNA polymerase, DNA primase and the topoisomerases I and II all reside in fraction II. Fraction I has been subdivided into two subfractions (A and B) by DEAE-cellulose chromatography. Fraction A is essential for replication and is required for presynthesis complex formation. Fraction B stimulates DNA replication and is only required at the elongation stage. This multicomponent system has provided the foundation for identification of individual components that are required for DNA replication in vitro.     

Purification and characterization of the neutral endopeptidase from human kidney

The presence of an endopeptidase hydrolyzing succinyl trialanine-p-nitroanilide [Suc(Ala)3-pNA] to Suc(Ala)2 and Ala-pNA in human kidney and its partial characterization have been reported (Ishida et al. (1981) Biochem. Int. 3, 239-246). This neutral metallo-endopeptidase was separated into two fractions (A and B) on Sephacryl S-300 and fraction B was further purified to an electrophoretically pure state. The fraction B enzyme had a molecular weight of 100,000 and was inhibited by metal chelators such as EDTA, o-phenanthroline and phosphoramidon, but not by serine protease inhibitors. The enzyme was found to hydrolyze peptide bonds preferentially at the amino sides of hydrophobic amino acids such as Leu and Phe, when its specificity was studied using insulin B chain and angiotensin I. Fraction A seems to be a tetramer of fraction B, judging from its molecular weight, pI, substrate specificity and immunological properties.     

Characterisation of secreted polysaccharides and (glyco)proteins from suspension cultures of Pyrus communis

High molecular weight material recovered from the culture filtrate of cell suspension cultured Pyrus communis was composed of 81% carbohydrate, 13% protein and 5% inorganic material. This material was separated into three fractions (one neutral (Fraction A) and two acidic (Fractions B and C)), by anion-exchange chromatography on DEAE-Sepharose CL-6B using a gradient of imidazole-HCl at pH 7.0. The monosaccharide and linkage composition of each fraction was determined after carboxyl reduction of uronic acid residues. From the combined results of the carbohydrate analyses, we conclude that the high molecular weight extracellular material consists of three major and two minor polysaccharides: a (fucogalacto)xyloglucan (36%) in the unbound neutral Fraction A; a type II arabinogalactan (as an arabinogalactan-protein, 29%) and an acidic (glucurono)arabinoxylan (2%) in Fraction B; and a galacturonan (33%) and a trace of heteromannan in Fraction C. The main amino acids in the proteins were Glx, Thr, Ser, Hyp/Pro and Gly. Further separation of Fraction B by solvent partition, SDS-PAGE and analysis by LC-MS/MS identified the major proteins as two chitanases, two thaumatin-like proteins, a beta-1,3-glucanase, an extracellular dermal glycoprotein and a pathogenesis-related protein.     

Plastein Reaction

α-Glucosidase (α-d-glucoside glucohydrolasc; EC: 3.2.1.20) has been extracted from bovine spleen and separated into two fractions (Fractions A and B) by gel filtration on Sephadex G-200. Fraction B which was retarded on Sephadcx columns contained only an acid α-glucosidase. This enzyme catalyzed hydrolysis of maltose and glycogen at comparable rates. Glycogen was hydrolyzed almost completely to glucose. The results of heat-treatment and of inhibition bv turanose demonstrated that Fraction A contained both acid if and neutral α-glucosidases. The neutral enzyme in Fraction A was further separated into four different components on preparative polyacrylamide gel electrophoresis. All of these neutral enzymes showed similar catalytic properties each other, and hydrolyzed maltose much more rapidly than glycogen. The acid enzyme in Fraction A was inactivated on the electrophoresis and was not further characterized.     

Characteristics of 3,4-benzopyrene-induced cytochrome P-448 forms isolated from mouse liver microsomes

Two cytochrome P-448 fractions, B1 and B2, were isolated from liver microsomes of 3,4-benzpyrene-induced inbred C57Bl/6 mice, using chromatography on octyl-Sepharose CL-4B and on Whatman 52E. During subsequent chromatography on hydroxylapatite fraction B1 was separated into 2 subfractions, G1 and G2. Cytochrome fractions B1, G1 and G2 have similar "peptide maps" differing from that of fraction B2. Cytochrome fraction B1 is immunologically identical to G2, partly to fraction B2 but is distinct from fraction G1. Fraction G2 is identified as the form of cytochrome P-448 catalyzing the hydroxylation of 3,4-benzpyrene and 7-ethoxyresorufin and existing in a low spin form. Cytochrome fraction G1 is apparently identical to the form P3-450. Fraction B2 was not yet described in current literature, since cytochrome P-448 (Mr = 53,000 Da) was identified only after the induction of mice with 3,4-benzpyrene but not with other inducers, e.g., polycyclic aromatic hydrocarbons.     

Studies of the cell walls of Schizophyllum commune

Mechanically isolated cell wall materials of eight strains of Schizophyllum commune were studied by chemical and enzymatic procedures. Isolated wall material of each strain was separated by chemical methods into three fractions: A (cold alkali-soluble, , amorphous), B (warm alkali-soluble, amorphous), and C (alkali-insoluble, retaining appearance of hyphal fragments). Chemical tests indicated the presence of chitin in Fraction C and the absence of cellulose, lignin and pectic substances from all fractions. Analyses of acid hydrolysates indicated the presence of glucose in Fractions A, B and C, of hexosamine in Fraction C and the absence of galactose, mannose, 6-deoxyhexoses, xylose and other pentoses from all fractions. Unfractionated material, Fraction A and Fraction B were slightly attacked by commercial cellulase whereas Fraction C was heavily attacked. Commercial chitinase by itself did not attack Fraction C or unfractionated material to a significant extent. In the presence of cellulase, it was active upon Fraction C. Qualitative differences in cell wall composition between strains were not detected; however, quantitative differences were observed in the proportion of Fraction A and Fraction C as well as in the amount of the various breakdown products in certain strains. It is visualized that the cell wall of this fungus consists of a core of chitin covered by or intermeshed with glucose-containing polymers.     

The deoxyribonucleic acid polymerases from the diatom Cylindrotheca fusiformis. Subcellular distribution, exonuclease activity and heterogeneity of the enzymes.

Four DNA polymerases from the marine diatom Cylindrotheca fusiformis, polymerases A, B, C and D, were further differentiated by their subcellular localization, presence of deoxyribonuclease activity, apparent heterogeneity and molecular weights. Polymerases A, B and D occur in significant amounts in the soluble fraction, suggesting that they were originally localized in the nuclei, whereas polymerase C predominates in the chloroplasts. A mitochondrial DNA polymerase was also isolated and characterized by ion-exchange chromatography. Polymerase D has an associated nuclease activity which prefers denatured DNA and Mg2+, and has a pH optimum higher than that for polymerase activity. Co-elution from a DEAE-Sephadex column and co-sedimentation in glycerol density gradients of deoxyribonuclease and polymerase D activity suggest a molecular association. Polymerases A, B and C are devoid of nuclease activity. Glycerol-gradient-sedimentation analysis showed that all DNA polymerase fractions are heterogeneous at low ionic strengths, with the appearance of a single homogeneous activity of 0.5M-KCl. Estimated molecular weights of 100000, 82000 and 120000 for polymerases A, B and C respectively were obtained from sedimentation analysis and gel filtration. Polymerase D was estimated to have a molecular weight of about 100000 as determined by sedimentation analysis alone.     

Isolation and Identification of Nigragillin as a Insecticidal Metabolite Produced by a Aspergillus niger

Arginine decarboxylase (E.C. 4.1.1) after purification from rice seedlings was separated into fractions A (MW 88000) and B (MW 174000) by gel chromatography. Fraction B was much more active than A. After DEAE cellulose chromatography, the active fraction of the enzyme (B) was purified to homogeneity, which appeared as a single band in gel electrophoresis. The optimal pH and temperature for the enzyme were 8.0 and 45°C, respectively. The enzyme followed typical Michaelis–Menten kinetics with a Km value of 0.28 mm. It had no dependence on a metal, and consisted of 16 amino acids of which proline was prominent. Pyridoxal-5-phosphate acted as a co-factor of the enzyme. The enzyme activity was inhibited by various amines and inhibitors, of which the highest inhibition was obtained with spermine and hydroxylamine. The plant hormones played a vital role in regulating the activity of the enzyme which was promoted by kinetin and inhibited by abscisic acid.     

Isolation and characterization of proteoglycans from growing antlers of wapiti (Cervus elaphus)

Proteoglycans were extracted with 4 M guanidine–HCl from the zone of maturing chondrocytes, the site of endochondral ossification of growing antlers of wapiti (Cervus elaphus). Proteoglycans were isolated by DEAE-Sephacel chromatography and separated by Sepharose CL-4B chromatography into three fractions. Fraction I contained a high molecular mass (>1000 kDa) chondroitin sulfate proteoglycan capable of interacting with hyaluronic acid. Its amino acid composition resembled that of the cartilage proteoglycan, aggrecan. Fraction II contained proteoglycans with intermediate molecular weight which were recognized by monoclonal antibodies specific to chondroitin sulfate and keratan sulfate. Fraction III contained a low molecular mass (<160 kDa) proteoglycan, decorin, with a glucuronate-rich glycosaminoglycan chain.     

Identification of aldosterone metabolites formed by A6 cells

The A6 cell line of the toad kidney is well known to form an Na+ transporting tight epithelium in culture and is often used as an experimental model for Na+ transport systems. Although it has been shown that A6 cells can convert aldosterone to polar metabolites, these metabolites have not been identified. Therefore, in this study, we tried to identify the metabolites of aldosterone formed by A6 cells in culture. A6 cells at confluence were incubated with serum-free culture media containing [3H]aldosterone. When radioactive compounds in incubation media were separated by reversed phase high-pressure liquid chromatography (HPLC), four fractions (fractions A-D) were obtained. Fraction A, a mixture of two components, comprised the majority of metabolites formed. The more polar material (fraction A-1) and the less polar material (fraction A-2) of fraction A contained 47-71 and 9-19% of total radioactivity, respectively. When incubated in cell-free media, fraction A-2 was found to be unstable and partially converted to fraction A-1. Fraction B, 0.7-1.5% of total radioactivity, and fraction C, 8-21% of total radioactivity, cochromatographed with iso-aldosterone and D-aldosterone, respectively. Fraction D, 4-8% of total radioactivity, was a mixture of two components, which cochromatographed with 3 beta,5 beta-tetrahydroaldosterone and 5 alpha-dihydroaldosterone, respectively. In order to identify fraction A-2 material, large-scale cultures were performed and fraction A-2 was separated and purified by reversed phase HPLC. The purified material was analyzed by fast atom bombardment mass spectrometry and nuclear magnetic resonance spectroscopy. These two procedures unambiguously revealed that this material was 6 beta-hydroxyaldosterone. These results demonstrate that aldosterone can be converted to at least four metabolites by the incubation with A6 cells, and that major metabolites are polar compounds, a portion of which is 6 beta-hydroxyaldosterone.     

Occurrence of 5SrRNA in high molecular weight complexes of aminoacyl-tRNA synthetases in a rat liver supernatant.

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new DNA polymerase species from Drosophila melanogaster: a probable mus308 gene product.

Harris et al. [P.V. Harris, O.M. Mazina, E.A. Leonhardt, R.B. Case, J.B. Boyd, K.C. Burtis, Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes, Mol. Cell. Biol., 16 (1996) 5764-5771.] reported the molecular cloning of Drosophila mus308 gene, and its nucleotide and protein sequences similar to DNA polymerase I. In the present study, we attempted to find and isolate the gene product by purifying a DNA polymerase fraction not present in mus308 flies. A new DNA polymerase with properties different from those of any known polymerase species was identified and partially purified from the wild-type fly embryos through ten column chromatographies. The enzyme was resistant to aphidicolin, but sensitive to ddTTP and NEM. Human proliferating cell nuclear antigen (PCNA) and Drosophila replication protein A (RP-A) did not affect the polymerase activity. It preferred poly(dA)/oligo(dT) as a template-primer. The molecular mass was about 230 kDa with a broad peak region of 200 to 300 kDa in HiPrep16/30 Sephacryl S-300 gel filtration. These properties a different from those of all reported Drosophila polymerase classes such as alpha, beta, gamma, delta, epsilon and zeta and closely resemble those of the gene product expected from the nucleotide sequence. The new polymerase species appears to have ATPase and 3'-5' exonuclease activities as shown by the chromatographies.     

DNA POLYMERASES IN FRACTIONATED RAT BRAIN NUCLEI

Abstract— —Adult rat brain nuclei were separated by discontinuous sucrose gradient centrifugation into astrocyte enriched, neuron enriched, and oligodendrocyte/microglia fractions. Nuclear fractions were subjected to velocity sucrose gradient centrifugation and gradient fractions assayed using relatively specific reaction mixtures for DNA polymerase-α, -β and TdT. NEM resistant DNA polymerase activity (DNA polymerase-β) was detected in equivalent amounts in all nuclear fractions. High molecular weight NEM sensitive activity (DNA polymerase-α) was found primarily in the neuron enriched fraction. The significance of the presence of DNA polymerase-α, an enzyme thought to be involved in DNA replication, in a cell incapable of cell division is unknown. TdT was detected in all fractions with increased activity in the neuron enriched fraction. The finding of TdT in thymocytes and neurons further supports the hypothesis that this enzyme is involved in the storage of noninherited information.     

Studies on Barley and Malt Amylases

From the 2 m urea extract of ground barley two zymogen β-amylase (Z-β-A) fractions (Fractions A and B) and one active β-amylase (A-β-A) fraction (Fraction C) were isolated by Sephadex G-75 gel filtration and purified by DEAE-Sephadex A-50 column chromatography. The molecular weights of Fractions A, B and C were estimated to be approximately 280,000, 160,000 and 56,000, respectively.

Both the Z-β-A fractions which were ultracentrifugally and electrophoretically homogeneous were found to be accompanied by a small amount of saccharogenic activities. From the estimation of Km values for these saccharogenic activities and the behavior in their activation with 2-mercaptoethanol and papain, it seems reasonable to conclude that Fraction A is a heteropolymer type of Z-β-A composed of both A-β-A and barley reserve proteins; that Fraction B is a homopolymer type of Z-β-A composed of A-β-A alone; and that two different activation mechanisms, proteolytic activation and disulfide bond cleaving activation, are necessary for full activation of Z-β-A in barley.     

Purification of DNA polymerase delta as an essential simian virus 40 DNA replication factor.

DNA replication from the SV40 origin can be reconstituted in vitro using purified SV40 large T antigen, cellular topoisomerases I and II, replication factor A (RF-A), proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and a phosphocellulose fraction (IIA) made from human cell extracts (S100). Fraction IIA contains all DNA polymerase activity required for replication in vitro in addition to other factors. A newly identified factor has been purified from fraction IIA. This factor is required for complete reconstitution of SV40 DNA replication and co-purifies with a PCNA-stimulated DNA polymerase activity. This DNA polymerase activity is sensitive to aphidicolin, but is not inhibited by butylanilinodeoxyadenosine triphosphate or by monoclonal antibodies which block synthesis by DNA polymerase alpha. The polymerase activity is synergistically stimulated by the combination of RF-A, PCNA, and RF-C in an ATP-dependent manner. Purified calf thymus polymerase delta can fully replace the purified factor in DNA replication assays. We conclude that this factor, required for reconstitution of SV40 DNA replication in vitro, corresponds to human DNA polymerase delta.     

Effect of Individual Fractions Released from Myofibrils by CAF (Ca2+-activated Factor) on Z-Disk Reconstitution

To investigate the exact protein constituents of 2-disk on the basis of the success or failure of reconstitution of Z-disk, proteins released from myofibrils by CAF(Ca²⁺-activated factor) were fractionated, the Z-disk was reconstituted by incubating individual fractions with Z-disk- extracted fiber bundles and the proteins in each fraction were analyzed by polyacrylamide gel electrophoresis.Released materials from myofibrils by CAF were divided into three fractions, A, B and C, in the order of elution from a Sepharose 6B column. The materials in Fractions A and B have been bound in the Z-disk region, and the Z-disk extracted from myofibrils in a low ionic strength solution has been reconstituted. The Z-disk reconstituted by incubating the materials in Fraction A with Z-disk-extracted myofibrils seems to have a structure similar to the intact Z-disk. Fraction A consisted principally of proteins having subunit molecular weights near 100,000 and 34,000 daltons.     

Mitochondrial DNA polymerase, deoxyribonuclease and ribonuclease H activities from brain of chick embryo

R-DNA polymerase, D-DNA polymerase, DNase and RNase H activities in mitochondria from chick embryonic brain were studied by ion-exchange chromatography. Two main fractions were separated according to their chromatographic behaviour: a fraction M Ib which is eluted with the washing buffer from two successive DEAE-cellulose columns and a fraction M IV which is eluted at 400 mM KC1 from a phosphocellulose column. Although the two fractions contain both the DNA polymerase and the degrading activities, all the specific activities are higher in fraction M IV than in fraction M Ib. Heat inactivation experiments have shown that R-DNA polymerase is inactivated in both fractions, whereas RNase H and DNase are not affected. Thus, degrading activities and R-DNA polymerase activity seem to be catalyzed by different molecular entities. However the fact that in most cases these activities co-chromatograph suggests that the corresponding molecules form rather stable complexes.