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).

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 Properties of pig heart aconitase

1974 ◽  
Vol 143 (3) ◽  
pp. 717-722 ◽  
Author(s):  
Oscar Gawron ◽  
Mary C. Kennedy ◽  
Richard A. Rauner
Keyword(s):  
Molecular Weight ◽  
Electron Spin Resonance ◽  
Resonance Spectrum ◽  
Specific Activity ◽  
Thiol Group ◽  
Molar Ratio ◽  
Candida Lipolytica ◽  
Molar Ratios ◽  
Acid Titration ◽  
Spin Resonance

Comparison of pig heart aconitase (Kennedy et al., 1972) with yeast (Candida lipolytica) aconitase (Suzuki et al., 1973) reveals similarities in molecular weight and iron content but not in sulphide content. Comparison with the Mildvan & Villafranca (1971) pig heart aconitase preparation reveals differences in iron ligands, specific activity and other properties; these differences possibly arise from protein association as pig heart protein associates under a variety of conditions. The electron spin resonance spectrum, g 4.25, and the low molar relaxivity, 473m−1·s−1, of water H+ suggest the presence of high-spin Fe(III) unco-ordinated to water in the enzyme. The iron chromophore on acid titration at 320nm gives a curve with an inflexion at pH4.2. Ten of 16 expected thiol equivalents are titrated with p-hydroxymercuribenzoate suggesting the presence of cystine as well as cysteine residues. Inhibition of the activation of inactive (activatable) enzyme is sigmoidally related to the molar ratio, p-hydroxymercuribenzoate/enzyme with 10–11mol of mercurial compound causing complete inhibition. Active enzyme, free from activating reagents, requires high molar ratios of mercurial compound for rapid inhibition. In terms of p-hydroxymercuribenzoate the enzyme then lacks an essential thiol group.

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.

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.

scholarly journals Development and Characterization of Two Types of Surface Displayed Levansucrases for Levan Biosynthesis

Catalysts ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 757
Author(s):  
Huiyi Shang ◽  
Danni Yang ◽  
Dairong Qiao ◽  
Hui Xu ◽  
Yi Cao
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Large Scale ◽  
Surface Display ◽  
Yeast Surface Display ◽  
Fusion Partner ◽  
Whole Cell ◽  
Food Industries ◽  
Yeast Surface ◽  
The Stability

Levan has wide applications in chemical, cosmetic, pharmaceutical and food industries. The free levansucrase is usually used in the biosynthesis of levan, but the poor reusability and low stability of free levansucrase have limited its large-scale use. To address this problem, the surface-displayed levansucrase in Saccharomyces cerevisiae were generated and evaluated in this study. The levansucrase from Zymomonas mobilis was displayed on the cell surface of Saccharomyces cerevisiae EBY100 using a various yeast surface display platform. The N-terminal fusion partner is based on a-agglutinin, and the C-terminal one is Flo1p. The yield of levan produced by these two whole-cell biocatalysts reaches 26 g/L and 34 g/L in 24 h, respectively. Meanwhile, the stability of the surface-displayed levansucrases is significantly enhanced. After six reuses, these two biocatalysts retained over 50% and 60% of their initial activities, respectively. Furthermore, the molecular weight and polydispersity test of the products suggested that the whole-cell biocatalyst of levansucrase displayed by Flo1p has more potentials in the production of levan with low molecular weight which is critical in certain applications. In conclusion, our method not only enable the possibility to reuse the enzyme, but also improves the stability of the enzyme.

Protease B of Saccharomyces cerevisiae: Isolation and Regulation of the PRB1 Structural Gene

Genetics ◽  
1987 ◽  
Vol 115 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Charles M Moehle ◽  
Martha W Aynardi ◽  
Michael R Kolodny ◽  
Frances J Park ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Structural Gene ◽  
Genomic Library ◽  
Time Lag ◽  
Proteolytic Processing ◽  
Coding Region ◽  
Protease B ◽  
Endonuclease Mapping ◽  
Protein Molecular Weight

ABSTRACT We have isolated the structural gene, PRB1, for the vacuolar protease B of Saccharomyces cerevisiae from a genomic library by complementation of the prb1-1122 mutation. Deletion analysis localized the complementing activity to a 3.2-kilobase pair XhoI-HindIII restriction enzyme fragment. The fragment was used to identify a 2.3-kilobase mRNA. S1 endonuclease mapping indicated that the mRNA and the gene were colinear. No introns were detected. The mRNA is of sufficient size to encode a protein of about 69,000 molecular weight, a number much larger than either the mature enzyme (≃30,000 protein molecular weight) or the sole reported precursor (≃39,000 protein molecular weight). These results suggest that proteolytic processing steps beyond that thought to be catalyzed by protease A may be required to convert the initial glycosylated translation product into mature protease B. The PRB1 mRNA is made in substantial amounts only when the cells have exhausted the glucose supply and enter the diauxic plateau. There is an extended time lag between PRB1 transcription and expression of protease B activity. A deletion that removes about 83% of the coding region was constructed as a diploid heterozygote. Spores bearing the deletion germinate, grow at normal rates into colonies, and have no obvious phenotype beyond protease B deficiency.

A new method for monitoring the redox potential of fuel salt based on the deposition of 95Nb on Hastelloy C276

2021 ◽  
Vol 109 (5) ◽  
pp. 357-365
Author(s):  
Zhiqiang Cheng ◽  
Zhongqi Zhao ◽  
Junxia Geng ◽  
Xiaohe Wang ◽  
Jifeng Hu ◽  
...  
Keyword(s):  
Redox Potential ◽  
Molten Salt ◽  
Specific Activity ◽  
Chemical Reduction ◽  
Quantitative Approach ◽  
Experimental Results ◽  
New Method ◽  
Molten Salt Reactor ◽  
Fuel Salt ◽  
Well Correlation

Abstract To develop the application of 95Nb as an indicator of redox potential for fuel salt in molten salt reactor (MSR), the specific activity of 95Nb in FLiBe salt and its deposition of 95Nb on Hastelloy C276 have been studied. Experimental results indicated that the amount of 95Nb deposited on Hastelloy C276 resulted from its chemical reduction exhibited a positive correlation with the decrease of 95Nb activity in FLiBe salt and the relative deposition coefficient of 95Nb to 103Ru appeared a well correlation with 95Nb activity in FLiBe salt. Both correlations implied that the measurement of 95Nb activity deposited on Hastelloy C276 specimen might provide a quantitative approach for monitoring the redox potential of fuel salt in MSR.

Existence of lipid vesicles containing platelet-activating factor in endothelial cell lysate

1992 ◽  
Vol 12 (1) ◽  
pp. 15-21
Author(s):  
S. Kojima ◽  
K. Nara ◽  
Y. Inada ◽  
S. Hirose ◽  
Y. Saito
Keyword(s):  
Molecular Weight ◽  
Endothelial Cell ◽  
Specific Activity ◽  
Gel Filtration ◽  
Platelet Activating Factor ◽  
Lipid Vesicles ◽  
Specific Factor ◽  
Cell Lysate ◽  
Aggregation Activity ◽  
Molecular Weight Fractions

Platelet aggregation activity due to platelet-activating factor (PAF) was detected at high molecular weight (HMW) and low molecular weight fractions after gel-filtration chromatography of cell lysate of endothelial cells. [3H]PAF added to the cell lysate was similarly distributed after chromatography. The radioactivity associated with HMW fraction was not reduced by digesting the lysate with trypsin, suggesting that PAF was not making complexes with proteins but was included in lipid vesicles in cell lysate. Further evidence showed that an unknown specific factor(s) was needed to form these PAF-containing lipid vesicles. Radioactivity was not found in HMW fraction when [3H]PAF was mixed with cell lysate of vascular smooth muscle cells. When monomeric PAF was added to endothelial cell lysate, the specific activity of aggregation decreased to the level exerted by endogenous PAF-containing lipid vesicles due to incorporation into lipid vesicles. PAF in the form of lipid vesicles was more stable in plasma than monomeric form.

scholarly journals CONCENTRATION AND PURIFICATION OF BACTERIOPHAGE

1938 ◽  
Vol 21 (3) ◽  
pp. 335-366 ◽  
Author(s):  
John H. Northrop
Keyword(s):  
Molecular Weight ◽  
Small Molecules ◽  
Specific Activity ◽  
Active Agent ◽  
Pure Substance ◽  
Solubility Curve ◽  
Denatured Protein ◽  
Sedimentation Constant ◽  
Rate Of Sedimentation ◽  
Living Organisms

1. A method for isolating a nucleoprotein from lysed staphylococci culture is described. 2. It is homogeneous in the ultracentrifuge and has a sedimentation constant of 650 x 10–13 cm. dyne–1 sec.–1, corresponding to a molecular weight of about 300,000,000. 3. The diffusion coefficient varies from about 0.001 cm.2/day in solutions containing more than 0.1 mg. protein/ml. to 0.02 in solutions containing less than 0.001 mg. protein/ml. The rate of sedimentation also decreases as the concentration decreases. It is suggested, therefore, that this protein exists in various sized molecules of from 500,000–300,000,000 molecular weight, the proportion of small molecules increasing as the concentration decreases. 4. This protein is very unstable and is denatured by acidity greater than pH 5.0, by temperature over 50°C. for 5 minutes. It is digested by chymo-trypsin but not by trypsin. 5. The loss in activity by heat, acid, and chymo-trypsin digestion is roughly proportional to the amount of denatured protein formed under these conditions. 6. The rate of diffusion of the protein is the same as that of the active agent. 7. The rate of sedimentation of the protein is the same as that of the active agent. 8. The loss in activity when susceptible living or dead bacteria are added to a solution of the protein is proportional to the loss in protein from the solution. Non-susceptible bacteria remove neither protein nor activity. 9. The relative ultraviolet light absorption, as determined directly, agrees with that calculated from Gates' inactivation experiments in the range of 2500–3000 Å. u. but is somewhat greater in the range of 2000–2500 Å. u. 10. Solubility determinations showed that most of the preparations contained at least two proteins, one being probably the denatured form of the other. Two preparations were obtained, however, which had about twice the specific activity of the earlier ones and which gave a solubility curve approximating that of a pure substance. 11. It is suggested that the formation of phage may be more simply explained by analogy with the autocatalytic formation of pepsin and trypsin than by analogy with the far more complicated system of living organisms.

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