A rapid purification procedure for pyruvate kinase from the hyphal fungus Aspergillus nidulans

1988 ◽  
Vol 34 (10) ◽  
pp. 1154-1158 ◽  
Author(s):  
Harry C. M. Kester ◽  
Jos H. A. A. Uitzetter ◽  
Leo H. de Graaff ◽  
Jaap Visser
Keyword(s):  
Molecular Weight ◽  
Liquid Chromatography ◽  
Aspergillus Nidulans ◽  
Pyruvate Kinase ◽  
Purification Procedure ◽  
Subunit Molecular Weight ◽  
Highly Active ◽  
A Subunit ◽  
Rapid Purification ◽  
Dye Affinity Chromatography

Pyruvate kinase was purified from the filamentous fungus Aspergillus nidulans with a 45–55% yield. The procedure involved dye-affinity chromatography and fast protein liquid chromatography, resulting in highly active and pure enzyme in milligram quantities within 2 days. The purified enzyme, a tetramer with a subunit molecular weight of 65 000 and an isoelectric point of 4.7, was used to determine the amino acid composition.

scholarly journals Purification and Characterization of TrzF: Biuret Hydrolysis by Allophanate Hydrolase Supports Growth

2006 ◽  
Vol 72 (4) ◽  
pp. 2491-2495 ◽  
Author(s):  
Nir Shapir ◽  
Gang Cheng ◽  
Michael J. Sadowsky ◽  
Lawrence P. Wackett
Keyword(s):  
Cyanuric Acid ◽  
Subunit Molecular Weight ◽  
Highly Active ◽  
Subunit Stoichiometry ◽  
Dependent Inhibition ◽  
A Subunit ◽  
Phenyl Phosphorodiamidate ◽  
Native Strains ◽  
Time Dependent Inhibition

ABSTRACT TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of α2 and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret.

scholarly journals Purification and properties of pantohenase from Pseudomonas fluorescens

1976 ◽  
Vol 157 (2) ◽  
pp. 409-413 ◽  
Author(s):  
R K Airas ◽  
E A Hietanen ◽  
V T Nurmikko
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Fluorescens ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Purification Procedure ◽  
Subunit Molecular Weight ◽  
Purification And Properties

Pantothenase (EC 3.5.1.22) from Pseudomonas fluorescens UK-1 was purified to homogeneity as judged by disc-gel electrophoresis and isoelectric focusing. The purification procedure consisted of four steps: DEAE-Sephadex chromatography, (NH4)2SO4 precipitation, hydroxyapatite chromatography and preparative polyacrylamide-gel electrophoresis. Gel filtration on Ultrogel AcA 34 was used to determine the molecular weight, and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis to study the subunit molecular weight. The enzyme appeared to be composed of two subunits with mol.wts. of approx. 50000 each. The total mol.wt. of the enzyme was thus about 100000. The isoelectric point was 4.7 at 10 degrees C.

Yeast pyruvate kinase. Native and subunit molecular weight

Biochemistry ◽  
1970 ◽  
Vol 9 (10) ◽  
pp. 2043-2047 ◽  
Author(s):  
R. T. Kuczenski ◽  
C. H. Suelter
Keyword(s):  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Subunit Molecular Weight

Temperature and the Regulation of Enzyme Activity in the Hibernator. Isoenzymes of Liver Pyruvate Kinase from the Hibernating and Non-hibernating Arctic Ground Squirrel

1974 ◽  
Vol 52 (10) ◽  
pp. 894-902 ◽  
Author(s):  
Hans W. Behrisch
Keyword(s):  
Molecular Weight ◽  
Pyruvate Kinase ◽  
Wide Temperature Range ◽  
Ground Squirrel ◽  
Energy Charge ◽  
High Energy ◽  
Ground Squirrels ◽  
The Arctic ◽  
Arctic Ground Squirrel ◽  
Temperature Range

Liver of the hibernating (H) Arctic ground squirrel (Citellus undulatus) contains a single species of pyruvate kinase (PyK) that is distinct from the single isoenzyme of pyK observed in the non-hibernating (NH) ground squirrel, which has been previously described (Behrisch &Johnson (1974) Can. J. Biochem. 52, 547–559). The H-PyK has a pI value of 5.7 and a molecular weight of 241 000 – 243 000. Affinity of the H-PyK for the substrates phosphoenolpyruvate (PEP) and ADP is not affected by changing temperature. It is argued that this stability of the apparent Km's for substrate over a wide temperature range permits the hibernator to take advantage of the Q10 effect in maintaining a low rate of the PyK reaction. Similarly, affinity of H-PyK for the allosteric activator fructose-1,6-phosphate (FDP) and the inhibitor ATP is also conspicuously independent of temperature, suggesting a fine stoichiometry in the relative concentrations of the regulatory ligands in control of H-PyK over a wide temperature range. Further, affinity of H-PyK for the inhibitor ATP is about three- to fourfold lower than that of the NH-PyK, a condition that would favor the maintenance of a high energy charge in the hibernating liver cell. ATP apparently inhibits PyK by causing a dissociation of the enzyme molecule into two "halves" of about 110 000 molecular weight each. This dissociation is offset and reversed by FDP. Removal of the ATP by dialysis does not of itself result in a reassociation of the PyK "halves"; FDP and/or the substrates are required for the two subunits of PyK to reassociate. As the apparent Ki of H-PyK for ATP is higher than that of NH-PyK, substantially higher concentrations of ATP are required to effect the dissociation of H-PyK. Similarly, elevated concentrations of FDP are required to offset the ATP-caused dissociation of the H-PyK.Hibernating Arctic ground squirrels that are preparing to emerge finally from the hibernating state already possess substantial activities of the NH-PyK isoenzyme. This suggests that the animal "anticipates" its transition from one metabolic state from another. On the basis of these data a formal mechanism is proposed for the regulation of liver PyK in the Arctic ground squirrel in both the non-hibernating and hibernating states.

scholarly journals A chromatographic preparation of ox growth hormone

1966 ◽  
Vol 100 (3) ◽  
pp. 593-600 ◽  
Author(s):  
M Wallis ◽  
HBF Dixon
Keyword(s):  
Molecular Weight ◽  
Growth Hormone ◽  
Gel Electrophoresis ◽  
Anterior Pituitary ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Starch Gel Electrophoresis ◽  
Highly Active ◽  
Cross Linkage ◽  
Starch Gel

1. A method is described for the chromatographic preparation of ox growth hormone. It involves chromatography of an extract of anterior pituitary lobes on DEAE-cellulose, followed by rechromatography on a dextran gel of low cross-linkage (Sephadex G-100). 2. The product is highly active in growth-hormone assays, and is obtained in good yield. It was homogeneous by several criteria, but showed some heterogeneity on starch-gel electrophoresis. 3. The molecular weight of the hormone was estimated from its behaviour on gel-filtration columns under various conditions. Evidence that the hormone may dissociate into sub-units under some conditions is presented.

Acid proteases from species of Mucor. III. Interaction with concanavalin A and concanavalin A Sepharose

1976 ◽  
Vol 54 (2) ◽  
pp. 120-129 ◽  
Author(s):  
W. S. Rickert ◽  
P. A. McBride-Warren
Keyword(s):  
Molecular Weight ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Affinity Column ◽  
Mucor Miehei ◽  
Highly Active ◽  
Elution Buffer ◽  
Turbidimetric Assay ◽  
Ph Range

The reaction of Mucor miehei protease with concanavalin A was followed by a turbidimetric assay in the pH range 5–8. At pH 4.0, no turbidity developed but binding of the enzyme to concanavalin A could be demonstrated by gel filtration. Two fractions of apparent molecular weight 65 000 and 52 000 were isolated, the 65 000 molecular weight species apparently representing a protomer of concanavalin A (24 000) bound to the enzyme. An analysis of the circular dichroism spectrum of this complex suggested that protomer binding results in a conformational change in the enzyme which is associated with a 30% increase in proteolytic activity.At pH 6.0, the enzyme was strongly bound to columns of concanavalin A Sepharose but could be removed by including α-methyl D-glucoside and NaCl in the elution buffer. Some column degradation occurred at room temperature but was not detectable at 4 °C where rapid elution of the enzyme resulted in a greater than 90% yield of highly active protein. Periodate-oxidized Mucor miehei protease and Mucor rennin did not react with concanavalin A and were not bound to the affinity column.

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