scholarly journals Biosynthesis of proline in Pseudomonas aeruginosa. Partial purification and characterization of γ-glutamyl kinase

1979 ◽  
Vol 181 (1) ◽  
pp. 215-222 ◽  
Author(s):  
R V Krishna ◽  
T Leisinger
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Kinase Activity ◽  
Wild Type Strain ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Gamma Glutamyl ◽  
Hydrolysis Of ◽  
Molecular Sieving

A gamma-glutamyl kinase (ATP-L-glutamate 5-phosphotransferase) was purified about 85-fold from crude extracts of Pseudomonas aeruginosa strain PAO 1 by (NH4)2SO4 precipitation, molecular-sieving by Sephadex G-150 and DEAE-cellulose chromatography. The molecular weight of this enzyme was 84,000. The preparation catalysed formation of gamma-glutamyl hydroxamate from L-glutamate, ATP and Mg2+ or Mn2+ with concomitant hydrolysis of ATP to ADP + Pi. L-Proline inhibited the gamma-glutamyl kinase activity by 50% at 5 mM and almost completely at 30 mM. The inhibition of L-proline was non-competitive, wherease L-methionine-DL-sulphoximine inhibited the enzyme competitively. Proline was found to inhibit the gamma-glutamyl kinase activity of the wild-type strain and of representatives of two of the three transductional classes of proline-auxotrophic mutants. Strain PAO 879, a mutant representing the third transductional class of proline auxotrophs, lacked proline-inhibitible gamma-glutamyl kinase. Thiol-blocking reagents inhibited the gamma-glutamyl kinase and this effect was prevented by dithiothreitol.

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

1988 ◽  
Vol 55 (1) ◽  
pp. 97-107 ◽  
Author(s):  
Efstathios Alichanidis
Keyword(s):  
Aeromonas Hydrophila ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Partial Purification ◽  
Significant Activity ◽  
Metal Chelators ◽  
Purification And Characterization ◽  
Tris Maleate ◽  
Ph Range

SummaryAn extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatogrpahy on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43000 and contained 2 g atom Ca/mol. It was active over a pH range 4·8–9·5 and had optimum activity on casein at pH 7·0 with Km = 0·17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 °C; above 50 °C activity declined rapidly, but significant activity persisted at 4 °C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.

Post-proline endopeptidase. Partial purification and characterization of the enzyme from pig kidneys

1982 ◽  
Vol 47 (4) ◽  
pp. 1139-1148 ◽  
Author(s):  
Karel Hauzer ◽  
Linda Servítová ◽  
Tomislav Barth ◽  
Karel Jošt
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Peptide Bond ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Optimum Ph ◽  
Hydrolysis Of ◽  
Prolyl Leucyl Glycinamide

Post-proline endopeptidase was isolated from pig kidneys and partially purified. The procedure consisted of fractionation with ammonium sulphate, ion exchange chromatography on DEAE-Sephadex A-50, gel filtration on Sephadex G-200 and rechromatography on DEAE-Sephadex A-50. The preparation had 55 times higher specific activity than the crude extract and did not contain any contaminating enzymic activities. The enzyme cleaved a number of proline-containing peptides and was strictly specific in catalyzing the hydrolysis of the peptide bond on the carboxyl side of the proline residue. The optimum pH for the hydrolysis of the synthetic peptides benzyl-oxycarbonylglycyl-prolyl-leucyl-glycinamide and benzyloxycarbonyl-glycyl-proline β-naphtylamide was 7.8-8.0 and, in the case of benzyloxycarbonylglycyl-proline p-nitroanilide, 7.2 to 7.5. For the hydrolysis of the tetrapeptide benzyloxycarbonylglycyl-prolyl-leucyl-glycinamide, the Km value of 75 μ mol l-1 was obtained.

DEGRADATION OF RUTIN BY ASPERGILLUS FLAVUS. PURIFICATION AND CHARACTERIZATION OF RUTINASE

1961 ◽  
Vol 7 (6) ◽  
pp. 921-932 ◽  
Author(s):  
G. W. Hay ◽  
D. W. S. Westlake ◽  
F. J. Simpson
Keyword(s):  
Aspergillus Flavus ◽  
Partial Purification ◽  
Dihydroxybenzoic Acid ◽  
Purification And Characterization ◽  
Dihydroxyphenylacetic Acid ◽  
The Common ◽  
Hydrolysis Of

Aspergillus flavus produces an adaptive glycosidase (rutinase) that hydrolyzes rutin to quercetin and rutinose. Production of rutinase occurs when the mold is grown on the glycosides rutin, hyperosid, and naringin, and on the aglycones quercetin, kaempferol, rhamnetin, 2,4-dihydroxybenzoic acid, and 3,4-dihydroxyphenylacetic acid, but not when grown on glucose, galactose, rhamnose, or rutinose. Rutinase, after partial purification, is relatively stable when stored at −20 °C, and is most stable and most active at pH 5.6. The enzyme is quite specific, hydrolyzing the 5-glucoside of sakuranetin, the 3-rutinoside and 3-galactoside of quercetin, but not the 3-L-rhamnoside nor any of the common glycosides. The hydrolysis of rutin is carried to completion aided by the insolubility of the aglycone quercetin in water.

scholarly journals Partial purification and characterization of the soluble phosphatidate phosphohydrolase of rat liver

1984 ◽  
Vol 220 (3) ◽  
pp. 825-833 ◽  
Author(s):  
S C Butterwith ◽  
R Hopewell ◽  
D N Brindley
Keyword(s):  
Calcium Phosphate ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Thiol Groups ◽  
Purification And Characterization ◽  
Phosphatidate Phosphohydrolase ◽  
Arginine Residues

A method is described by which the Mg2+-stimulated phosphatidate phosphohydrolase can be purified from the soluble fraction of liver from ethanol-treated rats. The increase in specific activity was about 416-fold. This involved purification by adsorption on calcium phosphate, chromatography on DE-52 DEAE-cellulose, separation on Ultrogel AcA-34 and chromatography on CM-Sepharose 6B. The effects of phosphatidylcholine, phosphatidate and Mg2+, Mn2+ and Zn2+ on the activity are described. Inhibitor studies indicate that the phosphohydrolase contains functional thiol groups and arginine residues.

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