scholarly journals Purification and properties of two extracellular β-lactamases from Bacillus cereus 569/H

1970 ◽  
Vol 118 (3) ◽  
pp. 457-465 ◽  
Author(s):  
S. Kuwabara
Keyword(s):  
Molecular Weight ◽  
Bacillus Cereus ◽  
Zinc Sulphate ◽  
Deae Cellulose ◽  
Casein Hydrolysate ◽  
Crystalline Form ◽  
Casamino Acid ◽  
Fractional Precipitation ◽  
Purification And Properties ◽  
Rapid Production

1. When Bacillus cereus 569/H was grown in a casamino acid (casein-hydrolysate) medium containing zinc sulphate rapid production of extracellular β-lactamase II preceded that of β-lactamase I. 2. β-Lactamase I was separated from β-lactamase II by fractional precipitation with ammonium sulphate. 3. β-Lactamase I was purified by a process involving chromatography on Celite and DEAE-cellulose and β-lactamase II by chromatography on DEAE-cellulose after denaturation of β-lactamase I by heat. Both enzymes were obtained in crystalline form. 4. β-Lactamase II prepared in this way appeared to have a higher molecular weight than β-lactamase I and required Zn2+ as a cofactor for both cephalosporinase and penicillinase activities.

scholarly journals Separation, purification and properties of β-lactamase I and β-lactamase II from Bacillus cereus 569/H/9

1974 ◽  
Vol 143 (1) ◽  
pp. 115-127 ◽  
Author(s):  
Richard B. Davies ◽  
E. P. Abraham ◽  
J. Melling
Keyword(s):  
Molecular Weight ◽  
Bacillus Cereus ◽  
Large Scale ◽  
Cysteine Residue ◽  
Peptide Fragment ◽  
Tryptic Digest ◽  
Purification And Properties ◽  
Carbohydrate Complex ◽  
Entire Sequence ◽  
Main Components

1. A procedure was devised which is suitable for the isolation of β-lactamase I and β-lactamase II from Bacillus cereus 569/H/9 on a large scale. After adsorption on to Celite both enzymes were eluted in good yield and separated by chromatography on Sephadex CM-50. 2. β-Lactamase I was separated into three main components by isoelectric focusing and into two components by chromatography. 3. The Zn2+-requiring β-lactamase II obtained by this procedure had a lower molecular weight (22000) than β-lactamase I (28000) and also differed from the latter in containing one cysteine residue. 4. The β-lactamase II contained no carbohydrate, but showed the thermostability of the enzyme isolated earlier as a protein–carbohydrate complex. 5. Amino acid analyses and tryptic-digest ‘maps’ indicate that some degree of homology between β-lactamase I and β-lactamase II is possible, but that β-lactamase I is not composed of the entire sequence of β-lactamase II together with an additional peptide fragment. 6. A 6-methylpenicillin and a 7-methylcephalosporin showed much lower affinities for both enzymes than did penicillins and cephalosporins themselves.

scholarly journals Deoxyribonucleic acid polymerases of Euglena gracilis. Purification and properties of two distinct deoxyribonucleic acid polymerases of high molecular weight

1975 ◽  
Vol 151 (2) ◽  
pp. 227-238 ◽  
Author(s):  
A G McLennan ◽  
H M Keir
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Euglena Gracilis ◽  
Deoxyribonucleic Acid ◽  
Deae Cellulose ◽  
Low Molecular Weight ◽  
Optimum Conditions ◽  
Electrophoretic Mobilities ◽  
Substrate Activation ◽  
Purification And Properties

Two DNA polymerases of high molecular weight, pol A (mol.wt. 190 000) and pol B (mol.wt. 240 ooo), have been purified 6300-fold and 1600-fold respectively from an extramitochondrial supernatant of a bleached strain of Euglena gracilis. They have very similar requirements when assayed with an ‘activated’-DNA primer-template [the optimum conditions of pH and ionic (K+ and Mn2+) composition being 7.2, 25 mM and 0.2 mM respectively]. 0.2 mM-Mn2+ was about 1.5-2-fold as effective as 2 mM-Mg2+, owing to substrate activation by deoxyribonucleoside 5′-triphosphates in the presence of Mn2+. Km values for the triphosphates in the absence of activation were about 10(-6)M with Mn2+ and 8 × 10(-6) M with Mg2+ for both enzymes. They were inhibited to the same extent by N-ethylmaleimide, novobiocin and o-phenanthroline, but differed in their chromatographic behaviour on DEAE-cellulose and in their electrophoretic mobilities on polyacrylamide gel. No evidence was found for the existence in these cells of a DNA polymerase of low molecular weight, but there were indications that a third enzyme of high molecular weight might exist.

Purification and Properties of Rat α2 Acute-Phase Macroglobulin (α2M)

1979 ◽  
Author(s):  
W. Nieuwenhuizen ◽  
J. J. Emeis ◽  
J. Hemmink
Keyword(s):  
Molecular Weight ◽  
Acute Phase ◽  
Deae Cellulose ◽  
Rat Plasma ◽  
Low Molecular Weight ◽  
Purification Procedure ◽  
Purification And Properties ◽  
Purification Scheme ◽  
Pure Amino Acid ◽  
Proteolytic Activities

For our studies on the relation between blood fibrinolytic activity and repair of mechanically damaged arteries in our rat model we need a specific and sensitive assay for α2M. in the rat α2M is an acute-phase protein of which the level in blood is normally near zero but increases as a result of the damage. Moreover α2M is known to inhibit proteases involved in the fibrinolytic system. We developed a new purification procedure in which, conditions known to be harmful to the functionality of α2M were avoided. α2M was purified from plasma of turpentine-treated rats and proteolytic activities were suppressed throughout the purification procedure. The purification scheme successively involves: rivanol precipitation, Con A-Sepharose chromatography and DEAE-cellulose chromatography. Thus 48 mg of α2M was obtained from 100 ml rat plasma i.e. 20% recovery. The preparations were biochemically and immunologically pure. Amino acid and carbohydrate compositions were determined. The molecular weight is 760.000. The molecule consists of 4 subunits, M.W. = 190.000. A 1%1cm = 8.8 and p1 = 4.8. It binds 1 mole of trypsin or plasmin per mole. Bound proteases were only active on low molecular weight substrates such as BAEE and BOC-L-val-gly-L-arg βNA. Kinetic data of the bound enzymes (pH-optimas, Km and Vmax) indicate that factors other than steric hindrance are involved in the inhibitory action of α2M.

scholarly journals PREPARATION, PURIFICATION AND PROPERTIES OF LIPASE FROM HEPATOPANCREAS OF TRA (PANGASIUS) CATFISH

2011 ◽  
Vol 14 (3) ◽  
pp. 5-11
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Heavy Metals ◽  
Molecular Weight ◽  
Enzyme Activity ◽  
Crude Extract ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Purification And Properties ◽  
Temperature Optima

Lipase from the hepatopancreas of Tra (Pangasius) catfish was purified by ammonium sulfate fractionation, followed by ion-exhange chromatography on DEAE Cellulose and gel filtration Sephadex G-75. The preparation was homogeneous on polyacrylamide disc gel electrophoresis. The specific activity of the purified enzyme was 37.95 times higher than that of the crude extract. The enzyme showed a molecular weight of 57000 Da. The pH and temperature optima of purified lipase were 8 and 500C respectively. Enzyme activity was enhanced by Ca2+ but inhibited by heavy metals Zn2+, Cd2+, Mg2+.

Purification and properties of phosphoenolpyruvate carboxylase from green leaves of maize

1979 ◽  
Vol 44 (6) ◽  
pp. 1835-1840 ◽  
Author(s):  
Jaroslav Mareš ◽  
Jana Barthová ◽  
Sylva Leblová
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Deae Cellulose ◽  
Chloride Ions ◽  
Magnesium Ions ◽  
Ph Optimum ◽  
Green Leaves ◽  
Purification And Properties ◽  
Fructose 1,6 Bisphosphate ◽  
No Activation

Phosphoenolpyruvate carboxylate was isolated from green leaves of maize (Zea mays L.) by a procedure including fractionation with ammonium sulphate, chromatography on DEAE-cellulose and preparative electrophoresis on polyacrylamide gel. The specific activity of the electrophoretically homogeneous enzyme was 23 U/mg. Its molecular weight was about 405 000, pH optimum was within the range 7.9 to 8.3, Km for phosphoenolpyruvate was 1.05 . 10-3 and the apparent Km for the magnesium ions was 8.0 . 10-4M. The enzyme was inhibited by malate, aspartate, citrate, pyruvate, ATP and ADP and chloride ions. It was strongly activated by glycine and glucose 6-phosphate and to a lesser degree by glucose 1-phosphate and fructose 1,6-bisphosphate; no activation by orthophosphate and 3-phosphoglycerate was observed.

scholarly journals The purification and properties of l-histidine-2-oxoglutarate aminotransferase from Pseudomonas testosteroni

1975 ◽  
Vol 147 (2) ◽  
pp. 327-334 ◽  
Author(s):  
A J Hacking ◽  
H Hassall
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Purification And Properties ◽  
Ping Pong ◽  
Purification Scheme ◽  
Low Activity ◽  
Cellulose Column ◽  
Pseudomonas Testosteroni

1. Inducible L-histidine--2-oxoglutarate aminotransferase was purified some 170-fold from extracts of Pseudomonas testosteroni. 2. The preparation showed only one major component after electrophoresis on polyacrylamide gels, though additional minor bands were observed when samples concentrated on a DEAE-cellulose column were used. 3. The molecular weight of the enzyme was found to be approx. 70000 by chromatography on Sephadex G-200. 4. The purification scheme produced enzyme that was inactive in the absence of pyridoxal 5′-phosphate. 5. The equilibrium constant for the reaction L-histidine+2-oxoglutarate equilibrium imidazolylpyruvate+L-glutamate was 0.49. 6. The reaction mechanism was Ping Pong. 7. The enzyme was shown to have only low activity towards aromatic amino acids and was highly specific for 2-oxoglutarate.

Purification and Properties of Bovine Pituitary Renin

1982 ◽  
Vol 63 (s8) ◽  
pp. 179s-181s
Author(s):  
Tamiko Ohsawa ◽  
Shigehisa Hirose ◽  
Tadashi Inagami ◽  
Kazuo Murakami
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Anterior Pituitary ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Purification And Properties ◽  
Antigenic Properties ◽  
Hog Kidney

1. Renin was purified to homogeneity from bovine anterior pituitary by using batchwise DEAE-cellulose chromatography, pepstatin-aminohexyl-agarose affinity chromatography, Ultrogel AcA 44 gel filtration and DEAE-Sephacel and CM-cellulose ion exchange chromatography. 2. The enzyme has a molecular weight of 36 000 and an isoelectric point of 5.25, and exhibits optimum activity at a pH between 6.5 and 7.5. 3. The amino acid composition and antigenic properties of this purified renin are very similar to those of rat, dog and hog kidney renins.

Preparation and Properties of Human Prothrombin Complex

1970 ◽  
Vol 24 (03/04) ◽  
pp. 325-333 ◽  
Author(s):  
G. H Tishkoff ◽  
L. C Williams ◽  
D. M Brown
Keyword(s):  
Molecular Weight ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Specific Activity ◽  
Crystalline Form ◽  
Sedimentation Equilibrium ◽  
Crystalline Complex ◽  
Interaction Product ◽  
Proteolytic Enzyme Inhibitor ◽  
Purification Scheme

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

Biosynthesis of the Diguanosine Nucleotides. I. Purification and Properties of an Enzyme from Yolk Platelets of Brine Shrimp Embryos

1974 ◽  
Vol 52 (3) ◽  
pp. 231-240 ◽  
Author(s):  
A. H. Warner ◽  
P. C. Beers ◽  
F. L. Huang
Keyword(s):  
Molecular Weight ◽  
Brine Shrimp ◽  
Artemia Salina ◽  
Temperature Optimum ◽  
Small Molecular Weight ◽  
Ph Optimum ◽  
Optimal Activity ◽  
Purification And Properties

An enzyme that catalyzes the synthesis of P1P4-diguanosine 5′-tetraphosphate (Gp4G) has been isolated and purified from yolk platelets of encysted embryos of the brine shrimp, Artemia salina. The enzyme GTP:GTP guanylyltransferase (Gp4G synthetase) utilizes GTP as substrate, has a pH optimum of 5.9–6.0, a temperature optimum of 40–42 °C, and requires Mg2+ and dithiothreitol for optimal activity. The synthesis of Gp4G is inhibited markedly by pyrophosphate, whereas orthophosphate has no effect on the reaction. In the presence of GDP the enzyme also catalyzes the synthesis of P1,P3-diguanosine 5′-triphosphate (Gp3G), but the rate of synthesis is low compared with Gp4G synthesis and dependent upon other small molecular weight components of yolk platelets.

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