DNA polymerases from Chlamydomonas reinhardii. Purification and properties.

Three DNA polymerase activities, A, B and C, were identified in extracts of exponentially growing synchronous cultures of Chlamydomonas reinhardii, and DNA polymerases A and B were characterized in detail. Both enzymes have the same binding affinity for DEAE-cellulose at pH 7.8, but can be distinguished from each other by their behaviour on phosphocellulose and DNA-agarose. 'Activated' calf thymus DNA was used as template, and the pH, K+ and bivalent-cation optima were measured. DNA polymerase A sediments at 5.3 S in glycerol gradients, with an apparent mol.wt. of 90000-100000. Polymerase B sediments between 8S and 10S in 100mM-KCl, the predominant species having an apparent mol.wt. of 200000. In 200mM-KCl, polymerase B dissociates to a single species, which sediments at 5.8S. A 3S species was found in aged preparations of both enzymes. The activity of polymerase B from cells harvested during nuclear DNA synthesis is twice that found in Chlamydomonas at other times during the cell cycle.     

Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species.

Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate. The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.     

DNA polymerases of anucleated cells. Isolation and characterization of two DNA polymerases from human platelets.

Two different DNA polymerases have been purified and characterized from human platelets. In the mitochondrial fraction a unique activity of the polymerase gamma type has been found. The same enzyme is found in the extramitochondrial supernatant. A second DNA polymerase, called 'cytoplasmic' DNA polymerase has been found in the 10000 x g supernatant of human platelets. The following properties of the latter DNA polymerase from human platelets are identical to those of DNA polymerase alpha from normal cells: DEAE-cellulose and phosphocellulose chromatography, size, thermal stability, phosphonoacetic acid and ethidium bromide inhibition. However, some of its properties, like high resistance to N-ethylmaleimide and the lack of DNA polymerization using synthetic RNA primers, are those of DNA polymerase beta.     

Purification and characterization of an endonuclease specific for single-stranded DNA from Bacillus subtilis Marburg.

Bacillus subtilis Marburg TI (thy,trpC2) has at least four endonuclease activities as assayed by measuring the conversion of single-stranded circular f1 DNA to the linear form by agarose gel electrophoresis. One of them, which is specific for single-stranded DNA (named endonuclease MII), was purified about 320 times by two chromatographic steps and gel filtration, thereby eliminating exonuclease and phosphomonoesterase activities. This activity requires divalent cations but does not require ATP. The molecular weight estimated by gel filtration was about 57,000 daltons. The cleavage products have 5'-phosphoryl termini. At low concentrations, double-stranded DNA is not split to any detectable extent. At high concentrations, however, double-stranded superhelical DNA is attacked to yield open-circular and linear DNA's. The activity of the enzyme towards single-stranded circular DNA relative to that towards double-stranded linear DNA was calculated to be approximately 5,000:1 by comparing the initial rates of introducing single-strand breaks into the DNA's.     

Purification and partial characterization of a DNA polymerase alpha species from calf thymus.

We have purified a DNA polymerase alpha species from calf thymus to near homogeneity. The enzyme sediments at 5.7 S and contains two polypeptides of 123000 and 134000 daltons in about equimolar ratio. The enzyme is inhibited by aphidicolin and N-ethylmaleimide, and retains its activity in buffers containing moderate salt conditions. Activated DNA is a better substrate than poly-(dA) . (dT) 10.     

High-molecular-weight DNA polymerases from mouse myeloma. Purification and properties of three enzymes.

Cytoplasmic (high-molecular-weight) DNA polymerase was partially purified from mouse myeloma. Upon chromatography on DEAE-Sephadex, following fractionation on phosphocellulose, the enzyme was resolved into three species named CI, CII, and CIII. The species CI and CII have equal sedimentation coefficients (10.5 S) in sucrose gradients without salt. In the presence of 125 mM ammonium sulfate the sedimentation coefficients are reduced to 8.6 S. The species CIII shows sedimentation coefficients of 5.7 S and 5.2 S without salt and in the presence of 125 mM ammonium sulfate, respectively. This species is assumed to be an artifact arising from either CI or to a minor extent from CII. The optima for pH, KCl and Mg2+ concentration, and the extent of inhibition by N-ethylmaleimide are the same. However, the enzymes differ in their responses to Mn2+ (substituting for Mg-2+), and to addition of ethanol, dimethylsulfoxide, and various phospholipids in the assay mixture. The enzymes prefer poly[d(A-T - d(A-T)] or partially degraded (activated) DNA as template rather than double-stranded or single-stranded DNA. The activity on activated DNA relative to that on poly[d(A-T) - D(A-T)] was found to be 93, 66, and 29% for DNA polymerases CI, CII, and CIII, respectively.     

Purification and characterization of L-pyrrolidonecarboxylate peptidase from Bacillus amyloiliquefaciens.

Microorganisms capable of producing L-pyrrolidonecarboxylate peptidase [L-pyrrolidonyl peptidase, EC 3.4.11.8] were screened and a strain of Bacillus amyloliquefaciens was chosen as one of the most potent producers of the enzyme. The enzyme was purified from lysozyme-lysate of the bacterial cells by salting out with ammonium sulfate, adsorption on DEAE-cellulose, covalent chromatography on PCMB-Sepharose and by gel filtration on Sephadex G-150. By these procedures, the enzyme was purified about 800-fold with an activity recovery of 9%, and the preparation was electrophoretically homogenous. The enzyme was most active and stable at pH 7-8. The presence of 2-mercaptoethanol and EDTA was effective for stabilizing the enzyme. The molecular weight was estimated to be 72,000 by the gel filtration method and to be 24,000 by SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a subunit oligomer, presumably trimer. The enzyme was inactivated by the addition of PCMB, sodium tetrathionate, Hg2+ and Cu2+, but the activity lost was restored by the addition of 2-mercaptoethanol and EDTA. The purified enzyme split amide and ester linkages in L-pyroglutamyl derivatives of L-alanine, beta-naphthylamine, alpha-naphthol, and 4-methylumbelliferone, but was completely inert towards various peptides and esters used as substrates for usual amino- and carboxy-peptidases, and for endopeptidases such as trypsin, subtilisin and alpha-chymotrypsin.     

Purification and characterization of inorganic pyrophosphatase from Bacillus stearothermophilus.

Inorganic pyrophosphatase [EC 3.6.1.1] was purified from Bacillus stearothermophilus to a homogeneous state both ultracentrifugally and electrophoretically. Ultracentrifugal analysis revealed that the molecular weight of the enzyme is 122,000 and the sedimentation coefficient (S0.34%/20, W) is 5.2S. The enzyme molecule in 0.1% sodium dodecylsulfate solution containing 1 mM 2-mercaptoethanol had an estimated molecular weight of 70,000 on the basis of SDS-polyacrylamide gel electrophoresis results, which indicates that the enzyme may consist of two subunits. Divalent cations such as Mg2+, Mn2+, and Co2+ are required for the enzymatic activity. Pyrophosphate is the only substrate for the enzyme. ATP and p-chloromercuribenzoate inhibit the enzyme reaction markedly.     

Purification and characterization of alpha-L-fucosidase from Streptomyces species.

Streptomyces sp. 142, isolated from a soil sample, produced alpha-fucosidase when cultured in the presence of L-fucose. The enzyme was purified 700-fold with an overall recovery of 17% from a cell-free extract by cation exchange chromatography and gel filtration chromatography. The apparent molecular weight of the purified enzyme was 40,000 by gel filtration chromatography. The enzyme had a pH optimum of 6.0 and was stable at pH 4.5-7.0. Substrate specificity studies with oligosaccharides labeled with 2-aminopyridine as the substrate showed that the enzyme specifically hydrolyzed terminal alpha 1-3 and alpha 1-4 fucosidic linkages in the oligosaccharides but did not hydrolyze alpha 1-2 or alpha 1-6 fucosidic linkages or a synthetic substrate, p-nitro-phenyl alpha-L-fucoside. The purified enzyme released L-fucose from a fucosylated glycoprotein, alpha 1-acid glycoprotein. Thus, the substrate specificities of the Streptomyces alpha-fucosidase resembled those of alpha-fucosidases I and III isolated from almond emulsin rather than those of other microbial alpha-fucosidases.     

Purification and some properties of Candida albicans DNA polymerases.

DNA polymerases of Candida albicans were purified to near homogeneity. Three well distinguished peaks of DNA polymerase activity (Enzyme I, II and III respectively) were obtained by DEAE-Sephacel chromatography. This purification step was followed by column chromatographies on Sepharose 6B and denatured DNA-cellulose. The enzymes' molecular mass and biochemical properties, including their inhibition by aphidicolin, were studied. Molecular mass was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and was found to be 110 kDa for Enzyme I, 80 kDa for Enzyme II and 50 kDa for Enzyme III.     

Purification and characterization of organellar DNA polymerases in the red alga Cyanidioschyzon merolae

DNA polymerase gamma, a mitochondrial replication enzyme of yeasts and animals, is not present in photosynthetic eukaryotes. Recently, DNA polymerases with distant homology to bacterial DNA polymerase I were reported in rice, Arabidopsis, and tobacco, and they were localized to both plastids and mitochondria. We call them plant organellar DNA polymerases (POPs). However, POPs have never been purified in the native form from plant tissues. The unicellular thermotrophic red alga Cyanidioschyzon merolae contains two genes encoding proteins related to Escherichia coli DNA polymerase I (PolA and PolB). Phylogenetic analysis revealed that PolB is an ortholog of POPs. Nonphotosynthetic eukaryotes also have POPs, which suggested that POPs have an ancient origin before eukaryotic photosynthesis. PolA is a homolog of bacterial DNA polymerase I and is distinct from POPs. PolB was purified from the C. merolae cells by a series of column chromatography steps. Recombinant protein of PolA was also purified. Sensitivity to inhibitors of DNA synthesis was different in PolA, PolB, and E. coli DNA polymerase I. Immunoblot analysis and targeting studies with green fluorescent protein fusion proteins demonstrated that PolA was localized in the plastids, whereas PolB was present in both plastids and mitochondria. The expression of PolB was regulated by the cell cycle. The available results suggest that PolB is involved in the replication of plastids and mitochondria.     

Purification and properties of nuclear and cytoplasmic DNA polymerases from JLS-V9 cells.

Four DNA polymerases, two enzymes from the nucleus and two from the cytoplasm, were purified 2000- to 7000-fold from continuous mouse cell-line (JLS-V9), by sequential column chromatography. Each of these polymerases require all the deoxynucleoside-5′-triphosphates in order to synthesize DNA, using activated DNA as a primer-template, and can copy the ribonucleotide strand of hybrid templates, but their rate of efficiency varies. The molecular weights of these DNA polymerases range from 35,000 to 160,000, as estimated by Sephadex column chromatography. Three out of the four DNA polymerases are probably a single polypeptide chain, since they have a single major band in polyacrylamide gel electrophoresis as well as one enzymatically active peak in guanidine hydrochloride gel filtration. The highly purified preparation of the high molecular weight cytoplasmic DNA polymerase contains two major bands in sodium dodecyl sulfate polyacrylamide gel electrophoresis and two enzymatically active peaks in guanidine hydrochloride gel filtration.     

Purification and characterization of alpha-L-arabinofuranosidase from Bacillus stearothermophilus T-6.

Bacillus stearothermophilus T-6 produced an alpha-L-arabinofuranosidase when grown in the presence of L-arabinose, sugar beet arabinan, or oat spelt xylan. At the end of a fermentation, about 40% of the activity was extracellular, and enzyme activity in the cell-free supernatant could reach 25 U/ml. The enzymatic activity in the supernatant was concentrated against polyethylene glycol 20000, and the enzyme was purified eightfold by anion-exchange and hydrophobic interaction chromatographies. The molecular weight of T-6 alpha-L-arabinofuranosidase was 256,000, and it consisted of four identical subunits as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The native enzyme had a pI of 6.5 and was most active at 70 degrees C and at pH 5.5 to 6.0. Its thermostability at pH 7.0 was characterized by half-lives of 53, 15, and 1 h at 60, 65, and 70 degrees C, respectively. Kinetic experiments at 60 degrees C with p-nitrophenyl alpha-L-arabinofuranoside as a substrate gave a Vmax, a Km, and an activation energy of 749 U/mg, 0.42 mM, and 16.6 kcal/mol, (ca. 69.5 kJ/mol), respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by 1 mM Hg2+. T-6 alpha-L-arabinofuranosidase released L-arabinose from arabinan and had low activity on oat spelt xylan. The enzyme acted cooperatively with T-6 xylanase in hydrolyzing oat spelt xylan, and L-arabinose, xylose, and xylobiose were detected as the end reaction products.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partial purification and characterization of two cytoplasmic DNA polymerases from ungerminated wheat.

Two DNA polymerases have been purified from the 105,000 x g supernatant of ungerminated wheat. The purification stages included: high speed centrifugation, salt fractionation, DEAE-cellulose chromatography, Sephadex G-150 filtration and phosphocellulose chromatography. Several properties of the two enzyme (called A and B according to the order of elution from the phosphocellulose column) have been studied. Enzyme A has a sedimentation coefficient of about 7 S, utilizes activated DNA and synthetic polydeoxynucleotides as well as poly rA-dT12, while B has a sedimentation coefficient of about 6.2 and uses only activated DNA and synthetic polydeoxynucleotides as templates. Other parameters like KCl effect, MnCl2 effect, optimum pH, etc. Allow us to distinguish clearly between both DNA polymerases.