Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus.

A stable deoxyribonucleic acid (DNA) polymerase (EC 2.7.7.7) with a temperature optimum of 80 degrees C has been purified from the extreme thermophile Thermus aquaticus. The enzyme is free from phosphomonoesterase, phosphodiesterase and single-stranded exonuclease activities. Maximal activity of the enzyme requires all four deoxyribonucleotides and activated calf thymus DNA. An absolute requirement for divalent cation cofactor was satisfied by Mg2+ or to a lesser extent by Mn2+. Monovalent cations at concentrations as high as 0.1 M did not show a significant inhibitory effect. The pH optimum was 8.0 in tris(hydroxymethyl)aminomethane-hydrochloride buffer. The molecular weight of the enzyme was estimated by sucrose gradient centrifugation and gel filtrations on Sephadex G-100 to be approximately 63,000 to 68,000. The elevated temperature requirement, small size, and lack of nuclease activity distinguish this polymerase from the DNA polymerase of Escherichia coli.     

D-glyceraldehyde-3-phosphate dehydrogenase. Amino-acid sequence of the enzyme from the extreme thermophile Thermus aquaticus

1. The amino acid sequence of D-glyceraldehyde-3-phosphate dehydrogenase from the extreme thermophile Thermus aquaticus has been elucidated. 2. The polypeptide contains 332 amino acids and its sequence is 70% identical with that of the enzyme from the moderate thermophile Bacillus stearothermophilus. 3. In contrast to less thermostable forms of the enzymes from B. stearothermophilus, pig, lobster and yeast, the T. aquaticus enzyme has only one cysteine residue, namely cysteine-149 which is required for catalysis.     

Purification and characterization of the extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius

The extracellular alpha-amylase activity of the yeast Schwanniomyces alluvius has been purified by anion-exchange chromatography on DEAE-cellulose and gel-filtration chromatography on Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid analysis of the purified sample indicated that the enzyme preparation was homogeneous. The enzyme is a glycoprotein having a molecular mass of 52 kilodaltons (kDa) estimated by SDS-PAGE and 39 kDa by gel filtration on Sephadex G-100. Chromatofocusing shows that it is an acidic protein. It is resistant to trypsin but sensitive to proteinase K. Its activity is inhibited by the divalent cation chelators EDTA and EGTA and it is insensitive to sulfhydryl-blocking agents. Exogenous divalent cations are inhibitory as are high concentrations of monovalent salts. The enzyme has a pH optimum between 3.75 and 5.5 and displays maximum stability in the pH range of 4.0-7.0. Under the conditions tested, the activity is maximal between 45 and 50 degrees C and is very thermolabile. Analysis of its amino acid composition supports its acidic nature.     

Properties and substrate specificity of proteinase from buckwheat seeds]

A thiol proteinase was isolated from buckwheat seeds and purified 300-fold, using ammonium sulfate, acetone fractionation ion-exchange chromatography on Sephadex CM-50 and electrofocussing. The proteinase preparation obtained was found homogenous after polyacrylamide gel electrophoresis at pH 4.5. The molecular weight of the enzyme (75.000) was determined by gel-filtration through Sephadex G-100. The activation of proteinase by cysteine, 2-mercaptoethanol and dithiothreitol, its inhibition by p-chloromercurybenzoate and the absence of inhibition by diisopropyl fluorophosphate and EDTA suggest that the enzyme isolated is a thiol proteinase. The enzyme hydrolyzed many peptide bonds in the B-chain of insulin, showing high substrate specificity. The glutelin and globulin fractions of buckwheat seed proteins were hydrolyzed by the enzyme. It is assumed that the hydrolysis of reserve proteins of buckwheat seeds is the main function of the proteinase isolated.     

Deoxyribose 5-phosphate aldolase of Bacillus cereus: purification and properties.

Deoxyribose 5-phosphate aldolase was purified 41 times from Bacillus cereus induced by growth on deoxyribonucleosides. The purification procedure includes ammonium sulphate fractionation, gel filtration on Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel and preparative electrophoresis on 10% polyacrylamide gel. The enzyme is stable above pH 6.5, but is rapidly inactivated by sulfhydryl reagents. Being insensitive to EDTA, it may be considered as a Class I aldolase. Among a number of compounds tested (including some carboxylic acids, free and phosphorylated pentoses, nucleotides and nucleosides), none has been found to affect the enzyme activity. The enzyme appears to be dimeric, with a subunit Mr of 23,600. A Km of 4.4 x 10(-4) M was calculated for dRib 5-P.     

Plastid Class I and Cytosol Class II Aldolase of Euglena gracilis (Purification and Characterization)

The plastidic class I and cytosolic class II aldolases of Euglena gracilis have been purified to apparent homogeneity. In autotrophically grown cells, up to 81% of the total activity is due to class I activity, whereas in heterotrophically grown cells, it is only 7%. The class I aldolase has been purified to a specific activity of 20 units/mg protein by anion-exchange chromatography, affinity chromatography, and gel filtration. The native enzyme (molecular mass 160 kD) consisted of four identical subunits of 40 kD. The class II aldolase was purified to a specific activity of 21 units/mg by (NH4)2SO4 fractionation, anion-exchange chromatography, chromatography on hydroxylapatite, and gel filtration. The native enzyme (molecular mass 80 kD) consisted of two identical subunits of 38 kD. The Km (fructose-1,6-bisphosphate) values were 12 [mu]M for the class I enzyme and 175 [mu]M for the class II enzyme. The class II aldolase was inhibited by 1 mM ethylenediaminetetraacetate (EDTA), 0.8 mM cysteine, 0.5 mM Zn2+, or 0.5 mM Cu2+. Na+, K+, Rb+, and NH4+ (but not Li+ or Cs+) enhanced the activity up to 7-fold. After inactivation by EDTA, the activity could be partially restored by Mn2+, Cu2+, or Co2+. A subclassification of class II aldolases is proposed based on (a) activation/inhibition by Cys and (b) activation or not by divalent ions.     

A novel nonhemorragic protease from the African puff adder (Bitis arietans) venom.

A nonhemorrhagic proteinase B-20 from the venom of Bitis arietans has been purified to apparent electrophoretic homogeneity by chromatography on Sephadex G-100, Q-Sepharose, and CM-cellulose. It has a molecular weight of 20 k Da as determined by size exclusion chromatography on Sephadex G-100 and migrated as a single 20-k Da band on SDS polyacrylamide. It has an optimum pH of 6-8 and is inactive at pH 4.0. EDTA and 1,10-phenanthroline strongly inhibited the enzyme suggesting it is a metalloenzyme. Also it is inhibited by antipain but is unaffected by trasylol, antitrypsin, and pepsptatin. Colombin, an identified active component of Aristolochia albida used in the treatment of snake poisoning, did not inhibit the protease activity. It lost over 90% of its activity in the presence of 0.5 microM Hg(2+) but the inhibition was completely blocked in the presence of 10 microM mercaptoethanol implicating sulfhydryl groups in the catalytic entity of the protein. The activity was also inhibited competitively by glutathione and cysteine with inhibition binding constants K(i) of 240 and 40 microM, respectively. The enzyme is unaffected by several divalent cations but activated by 1 mM Fe(3+). It had a prolyl endopeptidase and thermolysin-like activity. The enzyme displayed a fast acting alpha-fibrinolytic and delayed gamma-fibrinolytic activity when tested on human fibrinogen. The relevance of these findings is discussed.     

Characterization of the multiple forms and main component of dextransucrase from Leuconostoc mesenteroides NRRL B-512F

Multiple forms of dextransucrase (sucrose:1.6-alpha-D-glucan 6-alpha-D-glucosyltransferae EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F strain were shown by gel filtraton and electrophoretic analyses. Two components of enzyme, having different affinities for dextran gel, were separated by a column of Sephadex G-100. The major component voided from the Sephadex column was treated with dextranase and purified to an electrophoretically homogeneous state. The ]urified enzyme had a molecular weight of 64 000-65 000, pI value of 4.1, and 17% of carbohydrate in a molecule. EDTA showed a characteristic inhibition on the enzyme while stimulative effects were observed by the addition of exogenous dextran to the incubation mixture. The enzyme activity was stimulated by various dextrans and its Km value was decreased with increasing concentration of dextran. The purified enzyme showed no affinity for a Sephadex G-100 gel, and readily aggregated after the preservation at 4 degrees C in a concentrated solution.     

Molecular cloning, expression, purification, and characterization of fructose-1,6-bisphosphate aldolase from Thermus aquaticus

Fructose-1,6-bisphosphate aldolase from the thermophilic eubacteria, Thermus aquaticus YT-1, was cloned and sequenced. Nucleotide-sequence analysis revealed an open reading frame coding for a 33-kDa protein of 305 amino acids having amino acid sequence typical of thermophilic adaptation. Multiple sequence alignment classifies the enzyme as a class II B aldolase that shares similarity with aldolases from other extremophiles: Thermotoga maritima, Aquifex aeolicus, and Helicobacter pylori (49--54% identity, 76--81% homology). Taq FBP aldolase was overexpressed under tac promoter control in Escherichia coli and purified to homogeneity using heat treatment followed by two chromatographic steps. Yields of 40--50 mg of monodisperse protein were obtained per liter of culture. The quaternary structure is that of a homotetramer stabilized by an apparent 21-amino-acid insertion sequence. The recombinant protein is thermostable for at least 45 min at 80 degrees C with little residual activity below 60 degrees C. Kinetic characterization at 70 degrees C, the optimal growth temperature for T. aquaticus, indicates extreme negative subunit cooperativity (h = 0.32) with a limiting K(m) of 305 microM. The maximal specific activity (V(max)) is 46 U/mg at 70 degrees C.     

烟草磷酸吡哆醛水解酶的分离纯化与表征

采用硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换、Sephadex G-100凝胶过滤和SP Sephadex C-25阳离子交换柱层析等步骤,对烟草磷酸吡哆醛水解酶进行了分离纯化。结果表明:该酶被纯化了119.6倍,得率为28.49%,经凝胶过滤和SDS-PAGE测得该酶的全分子量为49.6kDa,亚基分子量约为25kDa;该酶最适温度为50℃,最适反应pH为5.5;Mg2+、Ca2+、Mn2+等对该酶有激活作用,金属离子螯合剂EDTA对酶有抑制作用,加入Mg2+后抑制作用得到解除;在最适反应条件下,测得反应底物磷酸吡哆醛(PLP)和磷酸吡哆胺(PMP)的Km值分别为0.23mmol/L和0.56mmol/L。     

Purification of N-hydroxy-2-acetylaminofluorene reductase from rabbit liver cytosol

N-Hydroxy-2-acetylaminofluorene reductase was purified from rabbit liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose, Sephadex G-200 and hydroxylapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 34,000 by the electrophoresis and by gel filtration on Sephadex G-200. The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, NADPH or NADH as an electron donor. The enzyme activity was inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide, cupric sulfate or disulfiram, but little by oxygen.     

Purification and some properties of an alkaline proteinase from rat skeletal muscle.

1. Rat skeletal muscle was homogenized in 0.05M-Tris/HCl, pH 8.5, containing 1M-KCl. Myofibrillar proteins were precipitated by addition of (NH4)2SO4 (33% saturation). 2. The alkaline proteolytic activity that was precipitated with the myofibrillar proteins was solubilized with trypsin (conjugated to Sepharose) and further purified by affinity chromatography, ion-exchange chromatography and gel filtration. 3. The purified enzyme migrates as a single band in polyacrylamide-disc electrophoresis, and has optimum hydrolytic activity with azocasein and [14C]haemoglobin as substrates at pH 9.4 and 9.6 respectively. Its apparent molecular weight, as determined by gel filtration on Sephadex G-75, is 30800. 4. The purified alkaline proteinase is strongly inhibited by equimolar amounts of soya-bean trypsin inhibitor and ovomucoid, whereas di-isopropyl phosphorofluoidate and alpha-toluenesulphonyl fluoride have no effect. On the other hand N-ethylmaleimide and p-chloromercuribenzoate have inhibitory effects on the enzyme activity. 5. Bivalent metal ions (Fe2+, Co2+, Zn2+, Mg2+, Mn2+) diminish the proteolytic activity, at 1mM concentrations. Ca2+ ions and the metal-ion-chelating agent EDTA are without effect on enzyme activity. 6. The enzyme is part of the alkaline proteolytic activity that appears to be associated with myofibrillar proteins.     

Purification and characterization of human seminal plasma aminopeptidase

A 50.4-fold purification of aminopeptidase is achieved by alcohol precipitation, DEAE-cellulose, CM-cellulose and finally Sephadex G-200 chromatography. On polyacrylamide gel electrophoresis of the purified enzyme after molecular sieving on Sephadex G-200, only one band was obtained, suggesting that the enzyme preparation was obtained almost homogeneous by three steps of column chromatography. Aminopeptidase showed highest activity at pH 7.0, using a buffer system, of 70 mM Na-phosphate. The enzyme was found to be active at 40 degrees C, even at 60 degrees C (80% activity), suggesting that the human seminal plasma enzyme is fairly thermostable. Amongst the various aminoacyl derivatives evaluated as substrates in the present study, L-alanine beta-naphthylamide hydrochloride was found to have the highest rate of hydrolysis. Ovalbumin showed effective cleavage in comparison to that of other natural substrates. The Km value for the purified seminal plasma aminopeptidase towards L-alanine beta-naphthylamide hydrochloride was 4 x 10(-4) M. Hg+2 showed highest inhibitory effect than other metal ions tested in the present study. Concentration causing 50% inhibition of the enzyme (I50) by Hg2+ was 4.7 x 10(-6) M. Inhibition by EDTA at 1 mM concentration in the incubation system was higher than by EGTA and sodium azide, suggesting that the enzyme contains a metallo group at the active site. A 50% inhibition of the enzyme by EDTA was obtained at 5.11 x 10(-3) M. The Ackerman and Potter plot for EDTA inhibition suggests that EDTA is a reversible inhibitor of seminal plasma aminopeptidase. A single molecular form of aminopeptidase was found to be present in human seminal plasma as shown by polyacrylamide activity gel electrophoresis.     

Properties of Leuconostoc mesenteroides B-512FMC constitutive dextransucrase

Leuconostoc mesenteroides B-512FMC, a constitutive mutant for dextransucrase, was grown on glucose, fructose, or sucrose. The amount of cell-associated dextransucrase was about the same for the three sugars at different concentrations (0.6% and 3%). Enzyme produced in glucose medium was adsorbed on Sephadex G-100 and G-200, but much less enzyme was adsorbed when it was produced in sucrose medium. Sephadex adsorption decreased when the glucose-produced enzyme was preincubated with dextrans of molecular size greater than 10 kDa. The release of dextransucrase activity from Sephadex by buffer (20 mM acetate, pH 5.2) was the highest at 28°–30°C. The addition of dextran to the enzyme stimulated dextran synthesis but had very little effect on the temperature or pH stability. Dextransucrase purified by ammonium sulfate precipitation, hydroxyapatite chromatography, and Sephadex G-200 adsorption did not contain any carbohydrate, and it synthesized dextran, showing that primers are not necessary to initiate dextran synthesis. The purified enzyme had a molecular size of 184 kDa on SDS-PAGE. On standing at 4°C for 30 days, the native enzyme was dissociated into three inactive proteins of 65, 62, and 57 kDa. However, two protein bands of 63 and 59 kDa were obtained on SDS-PAGE after heat denaturation of the 184-kDa active enzyme at 100°C. The amount of 63-kDa protein was about twice that of 59-kDa protein. The native enzyme is believed to be a trimer of two 63-kDa and one 59-kDa monomers.     

Pyruvate kinase in mussel foot (Mytilus edulis L.). Purification and general properties

The specific activity of pyruvate kinase in mussel foot is markedly higher than that from mantle and digestive gland. The foot enzyme shows maximum pH activity in the range between 7.0 and 7.5 and is stable (15 min, 37 degrees C) at pH values between 7.0 and 9.0. The activation energy value is 23 kJ/mol with a Q10 coefficient of 1.4. All of these experiments were carried out using partially purified extracts with (NH4)2SO4 treatment (30-60%) and posterior dialysis with EDTA 1.2 mM. No isoenzymatic forms could be detected using the column chromatography techniques with Sephadex G-150, DEAE Sephadex A-50 and polyacrylamide gel electrophoresis.     

Purification and characterization of pullulanase from Lactococcus lactis

This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co(2+) and completely inhibited by Hg(2+). The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.     

Oxytocinase-like enzyme in an ovarian dysgerminoma: a placenta-specific protein

Aminopeptidase from dysgerminoma was purified and characterized using L-leucine-beta-naphthylamide as substrate. The enzyme was resistant to puromycin, methionine, amastatin, bastatin, and EDTA, and it was heat labile at 60 degrees C. The enzyme showed the same electrophoretic mobility as pregnant-patient serum oxytocinase CAP1 on polyacrylamide gel electrophoresis. Km value against S-benzylcysteine-p-nitroanilide was 4.2 X 10(-4) M. Oxytocin and vasopressin competitively inhibited the enzyme activity. Molecular weight of the enzyme was estimated to be 80,000 by Sephadex G-200 column chromatography. These results suggest that aminopeptidase from dysgerminoma is an oxytocinase-like enzyme, a placenta-specific protein.     

Induction and characterization of -galactosidase in an extreme thermophile

A thermostable beta-galactosidase (EC 3.2.1.23; beta-dgalactoside galactohydrolase) was found to be inducible in an extreme thermophile resembling Thermus aquaticus. Enzyme induction was achieved by the addition of lactose, galactose, or the alpha-galactoside, melibiose, to growing cultures. The addition of glucose to induced cultures had a repressive effect on further enzyme synthesis. The enzyme was purified 78-fold, and the optimum temperature and pH for activity were determined to be 80 C and pH 5.0, respectively. The enzyme was activated by both manganese and ferrous iron. Sulfhydryl activation and thermal stabilization indicate that the thermophilic beta-galactosidase is a sulfhydryl enzyme. Kinetic determinations at 80 C established a K(m) of 2.0 x 10(-3)m for the chromogenic substrate o-nitrophenyl beta-d-galactopyranoside (ONPG) and a K(1) of 7.5 x 10(-3)m for lactose. The Arrhenius energy of activation (for the hydrolysis of ONPG) was calculated to be 13.7 kcal/mole. A molecular weight of 5.7 x 10(5) daltons was estimated by elution of the enzyme from Sephadex 4B.     

A cysteine metalloproteinase from mouse liver cytosol

A cysteine metalloproteinase that degrades 125I-insulin B chain at neutral pH values was isolated from C3H mouse liver. The enzyme was partially purified from the 100,000g supernatant fraction by ammonium sulfate precipitation, DEAE-cellulose chromatography, and fast protein liquid chromatography. The molecular weight of the proteinase was estimated to be 190,000 by gel filtration on Sephadex G-200. Degradation of 125I-insulin B chain by the proteinase was inhibited by p-hydroxymercuribenzoate (PHMB) and iodoacetate (cysteine proteinase inhibitors) and by ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline (metalloproteinase inhibitors). The proteinase also degraded 125I-glucagon but did not hydrolyze 125I-insulin, leucine-2-naphthylamide, or several large proteins. Equivalent levels of EDTA- and PHMB-inhibitable 125I-insulin B chain-degrading activity were observed in the 100,000g supernatant fractions of brain, liver, lung, kidney, heart, and spleen from four mouse strains (C3H/HeN, CBA/J, ICR, and C57BL/6). High levels of 125I-insulin B chain-degrading activity were found in the particulate fraction of kidneys and lungs from these four mouse strains; these activities were inhibited by EDTA but not by PHMB. The activity of the soluble liver cysteine metalloproteinase was not altered in C3H mice treated ip with metal chelators, bacterial endotoxin, phenobarbital, dexamethasone, or insulin. Starvation for 24 or 48 hr and alloxan-induced diabetes diminished total activity of this enzyme in liver by about 50 and 30%, respectively. This soluble polypeptide-degrading enzyme appears to be ubiquitous in mice and to be regulated by nutritional conditions.     

The nature of the collagenolytic cathepsin of rat liver and its distribution in other rat tissues

1. An enzyme present in rat liver extracts degraded insoluble collagen maximally at pH3.5. Collagenolytic activity was more abundant in kidney, spleen and bone marrow and was also present in decreasing concentrations in ileum, lung, heart, skin and muscle. 2. The crude collagenolytic cathepsin was activated by cysteine and dithiothreitol, but not by 2-mercaptoethanol. Iodoacetamide, p-chloromercuribenzoate and 7-amino-1-chloro-3-l-tosylamidoheptan-2-one hydrochloride inhibited the enzyme. Zn(2+), Fe(3+) and Hg(2+) ions were strongly inhibitory, but Ca(2+), Co(2+), Mg(2+) and Fe(2+) ions had little or no effect. EDTA was an activator of the enzyme. Inhibitors of cathepsin B were found to enhance collagenolysis, but phenylpyruvic acid, a cathepsin D inhibitor, inhibited the enzyme. Di-isopropyl phosphorofluoridate had no effect. 3. Collagenolysis at pH3.5 and 28 degrees C was restricted to cleavage of the telopeptide region in insoluble collagen, and the material that was solubilized consisted mostly of alpha-chains. 4. The collagenolytic cathepsin was separated from cathepsins B2 and D by fractionation on Sephadex G-100 and a partial separation from cathepsin B1 was obtained by chromatography on DEAE-Sephadex. 5. The function of the collagenolytic cathepsin in the catabolism of collagen is discussed in relation to the action of the other lysosomal proteinases and the neutral collagenase.