Pyruvate kinase from the moderate halophile, Vibrio costicola

1977 ◽  
Vol 55 (8) ◽  
pp. 825-833 ◽  
Author(s):  
Eveline de Médicis ◽  
Bertrand Rossignol
Keyword(s):  
Molecular Weight ◽  
Potassium Chloride ◽  
Pyruvate Kinase ◽  
Specific Activity ◽  
Chloride Concentration ◽  
Maximum Velocity ◽  
Maximal Activity ◽  
Maximum Activity ◽  
Moderate Halophile ◽  
Exclusion Chromatography

Pyruvate kinase (EC 2.7.1.40)from Vibrio costicola was purified to homogeneity. The tetramer had a molecular weight of 270 000 measured by exclusion chromatography on Sepharose 4B and was dissociated into apparently identical subunits with a molecular weight of 65 000 measured by SDS–polyacrylamide gel.The kinetic parameters Vm, K0.5, and n were measured at variable KCl concentrations. Both affinity and activity were inhibited when potassium chloride concentration was at 0.025 M, a fact which indicates that low potassium concentration is required for activity. Maximal activity and affinity were obtained at 0.125 M KCl. Potassium chloride appeared as an allosteric inhibitor in the range of 0.125 M to 1.025 M. Above 1.025 M KCl, both maximum velocity and affinity were decreased. NaCl had the same effect as KCl. The pH profiles of K0.5 and Vm of the phosphoenolpyruvate dependence revealed two main pK values: at 6.4 which controls affinity, and at 8.6, which controls activity.The activity of the enzyme showed a hysteretic response to salt. The specific activity measured under fixed standard conditions of temperature and salt concentrations showed a reversible variation which depended upon the preincubation conditions of temperature and salt concentration. The maximum activity tested was reached after low (0.1 M) or high (2 M) KCl preincubation at room temperature.

scholarly journals Partial purification of topoisomerase IB from serum of diabetic patients and study it's kinetic properties and molecular weight

2019 ◽  
Vol 23 (10) ◽  
pp. 46
Author(s):  
Saif M. Hasan ◽  
Firas T. Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Maximum Velocity ◽  
Partial Purification ◽  
Diabetic Patients ◽  
Kinetic Properties ◽  
Topoisomerase Ib ◽  
Electrophoresis Method

This study was done to partially purification of  topoisomerase IB from serum of diabetic patients using Gel filtration technique, by using Sephadex G 100 gel. A single peak in fraction four has been obtained, and the degree of purification (17.1) fold, enzyme yield (108.2%) and specific activity (0.189ng/mg). Kinetics studies for the partial purified enzyme were carried out which showed optimal concentration of  substrate which was (0.1ng/ml), Michael's - Menten constant (Km=0.033ng) and maximum velocity (Vmax=0.90 ng/ml), while optimum Temperature was (37C°) and optimum pH was (7.5). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method, in presence of polyacrylamide  gel and sodium dodecyl sulphate (SDS-PAGE) which showed that the approximated molecular weight was (66KD).   http://dx.doi.org/10.25130/tjps.23.2018.168 

scholarly journals The preparation and properties of pyruvate kinase from yeast

1974 ◽  
Vol 139 (3) ◽  
pp. 665-675 ◽  
Author(s):  
David A. Fell ◽  
Peter F. Liddle ◽  
Arthur R. Peacocke ◽  
Raymond A. Dwek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Extinction Coefficient ◽  
Specific Activity ◽  
Thiol Group ◽  
New Method ◽  
Saccharomyces Carlsbergensis ◽  
Binding Characteristics

A new method is described for the preparation of pyruvate kinase from yeast. This eliminates proteolysis during the preparation. The molecular weight of yeast pyruvate kinase is 215000, and it is composed of four subunits. Such properties of the enzyme as its extinction coefficient, cold-lability, thiol-group reactivity and binding of Mn2+ ions are compared with those previously reported for yeast pyruvate kinase prepared by different methods. The specific activity is significantly higher than previously observed, but otherwise the enzyme is similar, apart from its molecular weight and Mn2+-binding characteristics, to preparations from Saccharomyces cerevisiae obtained in this laboratory (e.g. Fell et al., 1972, and references therein) and that of C. H. Suelter (e.g. Kuczenski & Suelter, 1971, and references therein), and is different from the enzyme isolated from Saccharomyces carlsbergensis by B. Hess and his co-workers (e.g. Wieker & Hess, 1972, and references therein).

scholarly journals Extraction and Purification of Organophosphorus hydrolase Enzyme from Soil Microorganism Pseudomonas diminuta

2017 ◽  
Vol 2 (4) ◽  
pp. 416 ◽  
Author(s):  
Maheshwari D. T. ◽  
Thygaraj Varsha ◽  
N. S. Kumar
Keyword(s):  
Specific Activity ◽  
Organophosphorus Compounds ◽  
Sodium Dodecyl ◽  
Maximum Velocity ◽  
Maximum Activity ◽  
Soil Microorganism ◽  
Organophosphorus Hydrolase ◽  
Kinetic Properties ◽  
Extraction And Purification ◽  
Sds Page

<p class="p1">Synthetic organophosphorus compounds are highly toxic hence, widely used as pesticides, insecticides and chemical warfare agents. Organophosphorus hydrolase (OPH) is an organophosphotriester hydrolyzing enzyme; effectively hydrolyze a range of organophosphate esters. The objective of the present study was extraction and purification of OPH enzyme from Pseudomonas <em>diminuta </em>bacteria (soil microorganism) and to study kinetic properties of the purified enzyme. Enzyme was extracted and purified from bacteria by ammonium sulphate precipitation and ion exchange chromatography. Purity of an enzyme was determined by sodium dodecyl sulphate-polyacryamide gel electrophoresis (SDS-PAGE). Purified OPH enzyme specific activity was found to be 27.7 fold of 32.8U/mg protein, molecular weight of 72 Kda and it is a homodimer since it has shown a single band in SDS-PAGE separation. Maximum activity of the free OPH enzyme was found at Optimum pH 7.5 and temperature 35<span class="s2"><sup>o</sup></span>C with the incubation time of 10 min. Michaelis constant (K<span class="s3">m</span>) and maximum velocity (V<span class="s3">max</span>) values of free OPH enzyme for methyl parathion as substrate was found to be 286.2μM and 2.5 μM/min respectively.</p>

scholarly journals Purification and Characterization of the Glucose Oxidase from Penicillium notatum

2018 ◽  
Vol 9 (01) ◽  
Author(s):  
Baydaa A. Hassan ◽  
Mohammed A. Jebor ◽  
Zahra M. Ali
Keyword(s):  
Molecular Weight ◽  
Glucose Oxidase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Penicillium Notatum ◽  
Oxidase Enzyme ◽  
Filtration Technique

This study aims to purification and characterization of the glucose oxidase enzyme from Penicillium notatum, the enzyme was purified by ammonium sulfate precipitation (60%), dialysis and gel filtration chromatography using sephadex G-200, A trial for the purification of glucose oxidase using gel filtration technique resulted in one type of glucose oxidase with specific activity of (62.382 U/mg) with (7.385 folds) purification. the purified glucose oxidase had a maximum activity at pH = 5.5, 45 °C, glucose oxidase was stable with pH values ranging between (5 – 6) and the enzyme was maintained the activity when it incubated into (25 -35) °C for 15 minutes, analyses of the glucose oxidase for molecular weight was carried out by PAGE and SDS-PAGE electrophoresis, which revealed 78 KDa, also molecular weight of the glucose oxidase was achieved by gel filtration technique and was found 87 KDa this means that enzyme consisting of only one subunit, the Km and Vmax value of glucoamylase (B) were (19.6 mM, 7.5 mM/min ) respectively using different concentration of glucose.

Molecular characterization of an endo-type chitosanase from the fish pathogenRenibacteriumsp. QD1

2014 ◽  
Vol 94 (4) ◽  
pp. 681-686 ◽  
Author(s):  
Peichuan Xing ◽  
Dan Liu ◽  
Wen-Gong Yu ◽  
Xinzhi Lu
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Specific Activity ◽  
Fermentation Broth ◽  
Maximal Activity ◽  
Optimum Temperature ◽  
Sds Page ◽  
Ph Range ◽  
Tlc Analysis

Renibacteriumsp. QD1, a bacteria strain capable of hydrolysing chitosan, was isolated from the homogenate of small crabs. An extracellular chitosanase, Csn-A, was purified from the QD1 fermentation broth. The enzyme was purified to homogeneity, with a yield of eight-fold, 67% recovery and a specific activity of 1575 U/mg proteins. The molecular weight of Csn-A was estimated to be 26.1 kDa by SDS-PAGE. Unlike other chitosanases, the purified Csn-A displayed maximal activity at a pH range of 5.3–6.5, and it was stable in a broad pH range of 5.0–10.0. The optimum temperature for chitosanlytic activity was 55°C. The enzyme activity was strongly stimulated by Mn2+but inhibited by Fe3+, Cu2+, Al3+, Zn2+and SDS. TLC analysis demonstrated that Csn-A hydrolysed N-deacetylated polymeric glucosamines into chito-biose and -triose in an endo-type manner. The amino acid seuquence of Csn-A showed close identity with an uncharacterized chitosanase of strain ATCC33209.

scholarly journals Molecular weight dependent antistaphylococcal activities of oligomers/polymers synthesized from 3-aminopyridine

2010 ◽  
Vol 75 (9) ◽  
pp. 1203-1208 ◽  
Author(s):  
Cahit Akgul ◽  
Mehmet Yildirim
Keyword(s):  
Molecular Weight ◽  
Antibacterial Activities ◽  
Maximum Activity ◽  
Size Exclusion ◽  
Polycondensation Reaction ◽  
Reaction Conditions ◽  
Molecular Weights ◽  
Antistaphylococcal Activity ◽  
Exclusion Chromatography ◽  
The Relationship

The main aim of this study was to investigate the relationship between molecular weight and the antistaphylococcal activity of oligomers/polymers synthesized from 3-aminopyridine. Different oligomers/polymers were synthesized from 3-aminopyridine by changing the oxidative polycondensation reaction conditions. They were characterized by size exclusion chromatography and their antibacterial activities were compared by employing standardized susceptibility assays. The obtained experimental results demonstrated that 3-aminopyridine had no antistaphylococcal activity. However, as a result of polymerization, strong antistaphylococcal activity was obtained. Oligomers/polymers synthesized from 3-aminopyridine had varying degrees of antistaphylococcal activity and the maximum activity was obtained from relatively very short oligomers. It was therefore concluded that polymerization of 3-aminopyridine is required for antistaphylococcal activity and strength of this activity depends on the molecular weights of the synthesized molecules.

Allosteric pyruvate kinase from Phycomyces blakesleeanus: physicochemical and regulatory properties

1988 ◽  
Vol 66 (2) ◽  
pp. 148-157 ◽  
Author(s):  
Félix Busto ◽  
Pilar Del Valle ◽  
Joaquín Soler
Keyword(s):  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Specific Activity ◽  
Substrate Inhibition ◽  
Kinetic Properties ◽  
Saturation Curve ◽  
Phycomyces Blakesleeanus ◽  
Competitive Inhibitors ◽  
Fructose 1,6 Bisphosphate ◽  
Regulatory Properties

Pyruvate kinase from mycelium of Phycomyces blakesleeanus NRRL 1555 (−) was purified approximately 500-fold to a final specific activity of 25 U∙mg protein−1. The native enzyme has a molecular weight of 230 000. The enzyme appeared to be a tetramer with apparently identical subunits of 58 000 each. The enzyme from Phycomyces requires Mg2+ for activity, but not K+ or NH4. It showed a transition temperature at 36 °C. L-Alanine and ATP allosterically inhibited the enzyme by increasing the positive homotropic interactions for phosphoenolpyruvate and abolishing them for Mg2+ ions. Both effectors appeared to be competitive inhibitors with regard to ADP. Fructose 1,6-bisphosphate activates the Phycomyces pyruvate kinase allosterically by transforming the sigmoidal saturation curves to a hyperboolic form for phosphoenolpyruvate and Mg2+. Furthermore, fructose 1,6-bisphosphate relieved the inhibition caused by ATP and L-alanine. A lowering of the pH for reaction also activates the enzyme by abolishing the sigmoidal saturation curve for phosphoenolpyruvate, but produces substrate inhibition. The kinetic properties of Phycomyces pyruvate kinase are compatible to that of an allosteric K-type enzyme.

Secretion of Plasminogen Activators by Cultured Bovine Endothelial Cells: Partial Purification, Characterization and Evidence for Multiple Forms

1981 ◽  
Vol 45 (03) ◽  
pp. 219-224 ◽  
Author(s):  
W E Laug
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Plasminogen Activators ◽  
Maximum Activity ◽  
Partial Purification ◽  
Diisopropyl Fluorophosphate

SummaryEndothelial cells were obtained from the aortae of newborn calves and cloned. High plasminogen activator (PA) activity was detected in the supernatant medium and the cell lysates of confluent cultures. The PA activity in the growth medium increased steadily during 12 hrs of incubation, indicating active enzyme secretion by these cells. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the concentrated medium demonstrated the presence of four plasminogen activators with apparent molecular weights of 77,000 (±3000), 43,000 (±2000), 26,000 (±1500) and 14,500 (±1500) respectively. The 43,000, 26,000 and 14,500 molecular weight forms could be converted to radioactive derivates by active site labeling with 3H diisopropyl fluorophosphate (3H DFP) while the 77,000 Dalton form took up only traces of this radioactively labeled compound. The 43,000 molecular weight form was partially purified by means of salt precipitation and gel filtration. This enzyme preparation activated plasminogen by proteolytic cleavage with maximum activity at pH 7.5-8.5 and demonstrated a specific activity of 80,000 CTA (Committee on Thrombolytic Agents) units/mg protein when tested on 125I-fibrin in the presence of plasminogen. This PA was rapidly and irreversibly inhibited by diisopropyl fluorophosphate (DFP), suggesting that it was a serine protease. The partially purified enzyme was extremely labile at temperatures from 0-60° C, but could be stabilized by lowering the pH to 3 or by the addition of albumin.

Purification and molecular properties of glycogen phosphorylase b from trout white muscle

1993 ◽  
Vol 71 (5-6) ◽  
pp. 308-312 ◽  
Author(s):  
Hossein Mehrani ◽  
Kenneth B. Storey
Keyword(s):  
Molecular Weight ◽  
Oncorhynchus Mykiss ◽  
Glycogen Phosphorylase ◽  
White Muscle ◽  
Specific Activity ◽  
Fish Muscle ◽  
Maximum Activity ◽  
Substantial Contribution ◽  
Physiological Concentration ◽  
Glycogen Phosphorylase B

Glycogen phosphorylase b (EC 2.4.1.1) was isolated from white skeletal muscle of rainbow trout (Oncorhynchus mykiss) and purified 214-fold to a final specific activity of 135 U/mg protein (assayed in the direction of glycogen breakdown at 21 °C) by using glycogen – concanavalin A, DEAE-Sephadex, and 3′,5′-cAMP affinity chromatography. Purified phosphorylase b was a dimer with a native molecular weight of 193 000 and a subunit molecular weight of 87 000. Michaelis constants for glycogen, phosphate, and AMP were 128 μM, 31 mM and 142 μM, respectively, at pH 7.2; maximum activity of the enzyme was obtained at pH 7.5 and 25 °C. Glucose and ATP behaved as phosphorylase b inhibitors; glucose inhibition decreased at lower pH values. IMP did not affect the enzyme. The catalytic properties of trout phosphorylase b indicate that the enzyme would be virtually inactive at the physiological concentration of substrates and activators found in resting trout white muscle, but changes in cellular pH, ATP, Pi, and AMP levels during burst muscle work could allow phosphorylase b to augment phosphorylase a activity and make a substantial contribution to overall glycogenolysis in working trout white muscle.Key words: Oncorhynchus mykiss, regulation of glycogenolysis, fish muscle exercise, fish white muscle metabolism.

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.

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