scholarly journals A Study of Glutathione S-Aryltransferase from Costelytra Zealandica

2021 ◽  
Author(s):  
◽  
Graeme Lyall Dick
Keyword(s):  
Deae Cellulose ◽  
Effect Of Temperature ◽  
Endogenous Inhibitor ◽  
Catalysed Reaction ◽  
Purification And Properties ◽  
Isoelectric Points ◽  
Costelytra Zealandica ◽  
Optimum Ph ◽  
Effects Of Ph ◽  
The Stability

<p>An investigation has been made of the stability, purification and properties of Glutathione S-aryltransferase (Ec 2.5.1.13) from the grass-grub, Costelytra zealandica. The enzyme was found to be extremely unstable in crude homogenates of grass-grubs that had been stored frozen at -2O degrees C, but was considerably more stable in homogenates of live grass-grubs. The instability increased with increase of pH. Glutathione gave some protection against inactivation. Selective fractionation of crude homogenates with (NH4)2SO4 provided some evidence for the presence of an endogenous inhibitor of the enzyme. DEAE-cellulose chromatography and isoelectric focusing studies showed the presence of two major GSH S-aryltransferases with isoelectric points of 4.6 and 8.7. Both enzymes were present in the homogenate from a single, live, grass-grub. The molecular weight and optimum pH of each enzyme was identical within experimental error. A brief comparative study of GSH S-transferases showed the presence of GSH S-alkyl- and GSH s-alkene-transferase, but in only very small amounts compared with GSH S-aryltransferase. Differences in stability were demonstrated and some cross-specificity was indicated. Several inhibitor-substituted Sepharoses were prepared in an attempt to purify GSH s-aryltransferase by affinity chromatography. Although columns of the inhibitors removed the enzyme from solution an active enzyme could not be recovered. The effects of pH and temperature on the enzyme-catalysed reaction of GSH and 1, 2-dichloro-4-nitrobenzene (DCNB) were investigated in detail. Analysis of the variation of pKGSH with pH showed the presence of active site groups with pK approximately 9 involved in GSH binding. Calculation of the heat of ionization of these groups in the pI 8.7 enzyme, from the effect of temperature on their pK, suggested that the groups may be Lysine epsilon-NH2. Values for the enthalpy, free energy and entropy of GSH-binding to the pI 8.7 enzyme and of DCNB-binding to the enzyme-GSH complex were also obtained.</p>

scholarly journals A Study of Glutathione S-Aryltransferase from Costelytra Zealandica

2021 ◽  
Author(s):  
◽  
Graeme Lyall Dick
Keyword(s):  
Deae Cellulose ◽  
Effect Of Temperature ◽  
Endogenous Inhibitor ◽  
Catalysed Reaction ◽  
Purification And Properties ◽  
Isoelectric Points ◽  
Costelytra Zealandica ◽  
Optimum Ph ◽  
Effects Of Ph ◽  
The Stability

<p>An investigation has been made of the stability, purification and properties of Glutathione S-aryltransferase (Ec 2.5.1.13) from the grass-grub, Costelytra zealandica. The enzyme was found to be extremely unstable in crude homogenates of grass-grubs that had been stored frozen at -2O degrees C, but was considerably more stable in homogenates of live grass-grubs. The instability increased with increase of pH. Glutathione gave some protection against inactivation. Selective fractionation of crude homogenates with (NH4)2SO4 provided some evidence for the presence of an endogenous inhibitor of the enzyme. DEAE-cellulose chromatography and isoelectric focusing studies showed the presence of two major GSH S-aryltransferases with isoelectric points of 4.6 and 8.7. Both enzymes were present in the homogenate from a single, live, grass-grub. The molecular weight and optimum pH of each enzyme was identical within experimental error. A brief comparative study of GSH S-transferases showed the presence of GSH S-alkyl- and GSH s-alkene-transferase, but in only very small amounts compared with GSH S-aryltransferase. Differences in stability were demonstrated and some cross-specificity was indicated. Several inhibitor-substituted Sepharoses were prepared in an attempt to purify GSH s-aryltransferase by affinity chromatography. Although columns of the inhibitors removed the enzyme from solution an active enzyme could not be recovered. The effects of pH and temperature on the enzyme-catalysed reaction of GSH and 1, 2-dichloro-4-nitrobenzene (DCNB) were investigated in detail. Analysis of the variation of pKGSH with pH showed the presence of active site groups with pK approximately 9 involved in GSH binding. Calculation of the heat of ionization of these groups in the pI 8.7 enzyme, from the effect of temperature on their pK, suggested that the groups may be Lysine epsilon-NH2. Values for the enthalpy, free energy and entropy of GSH-binding to the pI 8.7 enzyme and of DCNB-binding to the enzyme-GSH complex were also obtained.</p>

Purification and Properties of Ribonuclease From Buffalo Milk Whey

1977 ◽  
Vol 40 (6) ◽  
pp. 375-377 ◽  
Author(s):  
AZZA A. ISMAIL ◽  
N. S. AHMED ◽  
M. A. KHORSHID
Keyword(s):  
Deae Cellulose ◽  
Ribonuclease A ◽  
Buffalo Milk ◽  
Optimum Temperature ◽  
Isolation And Identification ◽  
Milk Whey ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Ribonuclease B

A procedure was developed for isolation and identification of ribonuclease from buffalo milk whey. Ribonuclease was precipitated with (NH4)2SO4 between 65 and 90% saturation. The precipitate was dissolved, dialyzed, and fractionated on DEAE-cellulose. Two ribonuclease-rich fractions were collected, i.e. ribonuclease A and B. Ribonuclease A had an optimum pH of 7 .0, and ribonuclease B had an optimum pH of 8.6. Both had an optimum temperature at 38 C. The ribonucleases in the purified state were unstable to heat and their activity decreased as the time of exposure increased. Both enzyme fractions were sensitive to inhibitors. NaCl and NaN3 were stimulatory for ribonuclease A, while ribonuclease B was stimulated only by NaCl.

An extracellular proteolytic enzyme from Scopulariopsis brevicaulis. 1. Purification and properties

1971 ◽  
Vol 17 (8) ◽  
pp. 1029-1042 ◽  
Author(s):  
Kartar Singh ◽  
Claude Vézina
Keyword(s):  
Proteolytic Enzyme ◽  
Deae Cellulose ◽  
Alkaline Proteases ◽  
Casein Hydrolysis ◽  
Purification And Properties ◽  
Optimum Ph ◽  
Sh Reagents ◽  
Extracellular Proteolytic Enzyme ◽  
Scopulariopsis Brevicaulis ◽  
Insulin Chains

A proteolytic enzyme present in culture filtrates of Scopulariopsis brevicaulis was purified about 200-fold by (NH4)2SO4 and ethanol fractionations followed by chromatography on DEAE-cellulose, DEAE-Sephadex, and hydroxylapatite. Ultracentrifugation of the purified enzymes showed only one sedimenting component and its molecular weight was estimated to be about 24 000. The protease hydrolyzed casein, urea-denatured hemoglobin, gelatin, fibrinogen, fibrin, insulin chains A and B, but not human serum albumin or ovalbumin. It also coagulated milk. The enzyme had no action on the various peptides tested and showed low esterase activity. Optimum pH for casein hydrolysis was 10.5 to 11; for hemoglobin hydrolysis 7.0–9.5, and for gelatin hydrolysis, 6.0–8.0. The enzyme activity was unaffected by most metal ions, SH-reagents, and some natural trypsin inhibitors. The protease was strongly inhibited by diisopropylfluorophosphate and phenylmethanesulfonyl fluoride. Although similar in some respects to CA-7, the enzyme isolated from Aspergillus oryzae, and other alkaline proteases, the S. brevicaulis protease does not appear to be identical with any one of them.

Methods for the Concentration of Bovine Ac-Globulin (Factor V)

1961 ◽  
Vol 06 (03) ◽  
pp. 435-444 ◽  
Author(s):  
Ricardo H. Landaburu ◽  
Walter H. Seegers
Keyword(s):  
Room Temperature ◽  
Barium Carbonate ◽  
Deae Cellulose ◽  
Reducing Agents ◽  
Factor V ◽  
Isoelectric Precipitation ◽  
Stabilizing Agent ◽  
Bovine Plasma ◽  
The Stability

SummaryAn attempt was made to obtain Ac-globulin from bovine plasma. The concentrates contain mostly protein, and phosphorus is also present. The stability characteristics vary from one preparation to another, but in general there was no loss before 1 month in a deep freeze or before 1 week in an icebox, or before 5 hours at room temperature. Reducing agents destroy the activity rapidly. S-acetylmercaptosuccinic anhydride is an effective stabilizing agent. Greatest stability was at pH 6.0.In the purification bovine plasma is adsorbed with barium carbonate and diluted 6-fold with water. Protein is removed at pH 6.0 and the Ac-globulin is precipitated at pH 5.0. Rivanol and alcohol fractionation is followed by chromatography on Amberlite IRC-50 or DEAE-cellulose. The final product is obtained by isoelectric precipitation.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

scholarly journals STUDIES ON THE FUNCTION OF INTRACELLULAR RIBONUCLEASES

1966 ◽  
Vol 29 (3) ◽  
pp. 395-403 ◽  
Author(s):  
Takeshi Utsunomiya ◽  
Jay S. Roth
Keyword(s):  
Natural Products ◽  
Sodium Chloride ◽  
Rat Liver ◽  
Messenger Rna ◽  
Rnase Activity ◽  
Optimum Ph ◽  
Normal Rat ◽  
Pancreatic Rnase ◽  
The Stability ◽  
Normal Constituent

The RNase activity and properties of ribosome and polysome preparations from normal rat liver and some hepatomas have been examined. Polysome and ribosome preparations from the Novikoff, McCoy MDAB, and Dunning hepatomas had considerably higher specific RNase activity than corresponding preparations from normal rat liver, Novikoff ascites, or Morris 5123 hepatomas. The optimum pH of the RNase was approximately 8.5 for all samples tested, and the samples showed no evidence of latent RNase activity when treated with 3 M sodium chloride, EDTA, urea, or p-chloromercuribenzenesulfonic acid. The RNase activity appeared to be associated principally with breakdown products and/or subunits smaller than 80S. In the presence of Mg++ ions, subunits could reaggregate to form monomer ribosomes indistinguishable from the natural products, but some of the reassociated ribosomes could contain RNase activity which had been bound to the smaller particles. Similar results were obtained with spermine. In the hepatomas, evidence was obtained for the preexistence of considerable amounts of the smaller, RNase-containing subunits in the cell. When a small amount of crystalline bovine pancreatic RNase was added to partly dissociated ribosomes, the RNase was found only in association with the smaller subunits, and little or no enzyme was taken up by ribosomes or polysomes. The results have led to the conclusion that RNase is not a normal constituent of the ribosome or polysome, but that RNase may become associated with these particulates if dissociation and reassociation take place. Some implications of these findings for the stability of messenger RNA and for the mechanism of its breakdown are discussed.

The effect of temperature and time on the properties of 2D Cs2ZnBr4 perovskite nanocrystals and its application in a Schottky barrier device

2021 ◽  
Author(s):  
Olusola Akinbami ◽  
Grace N Ngubeni ◽  
Francis Otieno ◽  
Rudo Kadzutu-Sithole ◽  
Cebisa Linganiso ◽  
...  
Keyword(s):  
Solar Cell ◽  
Schottky Barrier ◽  
Effect Of Temperature ◽  
Inorganic Perovskite ◽  
Perovskite Nanocrystals ◽  
The Stability ◽  
Barrier Device

2D hybrid perovskites are promising materials for solar cell applications, in particular, cesium based perovskite nanocrystals as they offer the stability that is absent in organic-inorganic perovskite. However, the most...

scholarly journals Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney

1989 ◽  
Vol 261 (3) ◽  
pp. 761-768 ◽  
Author(s):  
D R Deshmukh ◽  
S M Mungre
Keyword(s):  
Rat Kidney ◽  
Deae Cellulose ◽  
Dicarboxylic Acids ◽  
Structural Similarity ◽  
Appreciable Amount ◽  
Kinetic Properties ◽  
Bovine Kidney ◽  
Purification And Properties ◽  
Kidney Enzyme ◽  
Structural Differences

Previous studies with rat kidney preparations indicated that 2-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are properties of a single protein. We found that bovine kidney contains an appreciable amount of AadAT activity, but lacks KAT activity. AadAT from bovine and rat kidney extracts were purified to electrophoretic homogeneity. The purification procedure included fractionation with (NH1)2SO1, heat treatment, DEAE-cellulose chromatography and hydroxyapatite chromatography. Physical and kinetic properties, such as pH optima, Km for substrates, Mr, electrophoretic mobility and inhibition by dicarboxylic acids of bovine kidney AadAT, were similar to those of the rat kidney enzyme. However, bovine kidney AadAT differed from rat kidney AadAT in substrate specificity, amino acid composition and stability when stored. The titration curve of bovine kidney AadAT was also different from that of the rat kidney enzyme. The results suggest that bovine kidney AadAT may have some structural similarity to rat kidney AadAT and that the structural differences observed between the two enzymes may explain the absence of KAT activity in bovine kidney.

Effects of Stress and Plastic Deformation on the Corrosion of Steel

CORROSION ◽  
1970 ◽  
Vol 26 (5) ◽  
pp. 189-199 ◽  
Author(s):  
W. D. FRANCE
Keyword(s):  
Plastic Deformation ◽  
Electrochemical Techniques ◽  
Chloride Ions ◽  
Localized Corrosion ◽  
Dissolution Behavior ◽  
Potential Range ◽  
Chemical Corrosion ◽  
Effects Of Ph ◽  
Current Densities ◽  
The Stability

Abstract The rate and type of corrosion exhibited by mild steel in the annealed, stressed, and plastically deformed state have been investigated. Precise electrochemical techniques provided potential and polarization data to supplement the results of chemical corrosion tests. Experiments were conducted in 0.6M NH4NO3 solutions in which steel exhibits active-passive dissolution behavior as well as localized corrosion. At active potentials, the anodic polarization curves for annealed and deformed specimens were nearly identical, with only slight increases in current densities for the deformed steel. Results at passive potentials demonstrated that increased plastic deformation can markedly decrease the passive potential range, the stability of passivity, and the ability to passivate. At certain passive potentials, the deformed steel exhibited current densities that were 400 times greater than those for annealed steel. The effects of pH, chloride ions, and crevices on the corrosion of deformed steel were examined in detail. The differences between the dissolution behavior of annealed and deformed steel were most distinctive in the approximate pH range of 3 to 6. This work is relevant to the understanding of the initiation of localized corrosion and to anodic protection.

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