Separation of Pinusbanksiana Lamb. glyceraldehyde 3-phosphate (NAD) dehydrogenase from phenolic-based pigments with affinity chromatography

1982 ◽  
Vol 12 (4) ◽  
pp. 1035-1038
Author(s):  
Bruce E. Haissig
Keyword(s):  
Enzyme Activity ◽  
Affinity Chromatography ◽  
Electrophoretic Mobility ◽  
Spectrophotometric Assay ◽  
Disc Electrophoresis ◽  
Enzyme Complex ◽  
Crude Extracts ◽  
Ungerminated Seed ◽  
Catalytically Active ◽  
The Individual

Glyceraldehyde 3-phosphate (NAD) dehydrogenase was extracted from Pinusbanksiana Lamb, seed and from seedlings up to 12 days old. Enzyme activity in crude extracts of seedlings increased markedly from days 2–8 and then decreased slightly, as indicated by spectrophotometric assay. In contrast, staining of acrylamide gels after disc electrophoresis indicated that catalytically active isozymes were not present in crude extracts at and after day 6. Phenolics greatly increased in extracts from days 0–10 and polymerization of these phenolics apparently lead to binding of the resulting phenolic-based pigment to enzyme. The pigment–enzyme complex demonstrated greatly enhanced electrophoretic movement, in comparison with the individual isozymes, which caused frontal migration of the collective isozymes. Affinity chromatography restored electrophoretic mobility of isozymes equal to that obtained with enzyme from ungerminated seed. Enzyme was inseparable from pigment by several other methods.

scholarly journals Limitations of commonly used spectrophotometric assay methods for phosphoenolypyruvate carboxykinase activity in crude extracts of muscle

1982 ◽  
Vol 206 (1) ◽  
pp. 147-152 ◽  
Author(s):  
D A Duff ◽  
K Snell
Keyword(s):  
Enzyme Activity ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Lactate Production ◽  
Spectrophotometric Assay ◽  
Nadh Oxidation ◽  
Crude Extracts ◽  
Na Ions ◽  
Assay Methods ◽  
Lactate Formation ◽  
Assay Conditions

Phosphoenolpyruvate carboxykinase activity in crude extracts of muscle has frequently been determined by using a continuous spectrophotometric method, which is shown to grossly overestimate enzyme activity. NADH oxidation attributed to phosphoenolpyruvate carboxykinase activity in the assay is due to lactate production. Under the normal assay conditions. Na+ ions stimulate pyruvate kinase, providing pyruvate for lactate formation by lactate dehydrogenase and sufficiently to account for most of the observed NADH oxidation.

scholarly journals Enzymic synthesis of sym-homospermidine in Lathyrus sativus (grass pea) seedlings

1980 ◽  
Vol 190 (2) ◽  
pp. 461-464 ◽  
Author(s):  
K S Srivenugopal ◽  
P R Adiga
Keyword(s):  
Enzyme Activity ◽  
Affinity Chromatography ◽  
Specific Activity ◽  
Lathyrus Sativus ◽  
Santalum Album ◽  
Grass Pea ◽  
Enzymic Synthesis ◽  
Crude Extracts ◽  
Pea Seedlings ◽  
Blue Sepharose

An enzyme catalysing the synthesis of sym-homospermidine from putrescine and NAD+ with concomitant liberation of NH3 was purified 100-fold from Lathyrus sativus (grass pea) seedlings by affinity chromatography on Blue Sepharose. This thiol enzyme had an apparent mol.wt. of 75000 and exhibited Michelis-Menten kinetics with Km 3.0mM for putrescine. The same enzyme activity could also be demonstrated in the crude extracts of sandal (Santalum album) leaves, but with a specific activity 15-fold greater than that in L. sativus seedlings.

scholarly journals Characterization of glyoxalase I purified from pig erythrocytes by affinity chromatography

1977 ◽  
Vol 165 (3) ◽  
pp. 503-509 ◽  
Author(s):  
Anne-Charlotte Aronsson ◽  
Bengt Mannervik
Keyword(s):  
Affinity Chromatography ◽  
Isoelectric Point ◽  
Reduced Glutathione ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Glyoxalase I ◽  
Active Components ◽  
Catalytically Active

Glyoxalase I (EC 4.4.1.5) was purified about 10000-fold from pig erythrocytes in a yield of approx. 20%. The purification included affinity chromatography on S-hexylglutathione coupled to Sepharose 4B. The purified enzyme normally contained two catalytically active components which were resolved by polyacrylamide-gel electrophoresis. After treatment with reduced glutathione only one component was found. The two components were also demonstrable after isoelectric focusing or DEAE-cellulose chromatography and could also in these cases be fused into one species by preincubation with reduced glutathione. It is proposed that the most acidic form of glyoxalase I is a mixed disulphide with glutathione. Except for these interconvertible forms, the purified enzyme was homogeneous, as judged by disc electrophoresis and sodium dodecyl sulphate/polyacrylamidegel electrophoresis. The molecule is a dimer (48000 daltons), composed of apparently identical subunits (24000 daltons). The isoelectric point was 4.8 at 4°C. The amino acid composition was consistent with the low isoelectric point. The enzyme contained about two thiol groups per enzyme molecule. EDTA inactivated the enzyme and bivalent metal ions could restore fully or partially the catalytic activity; Mg2+ and Mn2+ gave highest activity. It is proposed that a major biological function of glyoxalase I is the detoxification of methylglyoxal formed by enterobacteria in the alimentary canal.

Mixed Mode Chromatography: A Novel Way Toward New Selectivity

2018 ◽  
Vol 20 (1) ◽  
pp. 14-21 ◽  
Author(s):  
Xavier Santarelli ◽  
Charlotte Cabanne
Keyword(s):  
Salt Tolerance ◽  
Affinity Chromatography ◽  
Mixed Mode ◽  
Ionic Groups ◽  
Crude Extracts ◽  
The Way

Mixed mode chromatography offers a diversity of ligands, each providing a new selectivity. This allows the design of novel purification processes with reduced column steps. Structure of ligands is based on both hydrophobic and ionic groups. Thanks to its salt tolerance, crude extracts or post-IEX samples can be loaded directly without conditioning. The selectivity could be enhanced by modulating elution parameters or by using additives. More importantly, mixed mode chromatography could be as effective as affinity chromatography for mAb purification processes. Mixed mode chromatography opens the way to short and economical processes.

scholarly journals Fluxes and Metabolic Pools as Model Traits for Quantitative Genetics. I. The L-Shaped Distribution of Gene Effects

Genetics ◽  
1999 ◽  
Vol 153 (4) ◽  
pp. 2001-2012 ◽  
Author(s):  
Bruno Bost ◽  
Christine Dillmann ◽  
Dominique de Vienne
Keyword(s):  
Enzyme Activity ◽  
Metabolic Pathways ◽  
Quantitative Traits ◽  
Intrinsic Property ◽  
Gene Effects ◽  
Analytical Approximations ◽  
Mean And Variance ◽  
The Individual ◽  
Metabolic Control Theory ◽  
Control Coefficients

Abstract The fluxes through metabolic pathways can be considered as model quantitative traits, whose QTL are the polymorphic loci controlling the activity or quantity of the enzymes. Relying on metabolic control theory, we investigated the relationships between the variations of enzyme activity along metabolic pathways and the variations of the flux in a population with biallelic QTL. Two kinds of variations were taken into account, the variation of the average enzyme activity across the loci, and the variation of the activity of each enzyme of the pathway among the individuals of the population. We proposed analytical approximations for the flux mean and variance in the population as well as for the additive and dominance variances of the individual QTL. Monte Carlo simulations based on these approximations showed that an L-shaped distribution of the contributions of individual QTL to the flux variance (R2) is consistently expected in an F2 progeny. This result could partly account for the classically observed L-shaped distribution of QTL effects for quantitative traits. The high correlation we found between R2 value and flux control coefficients variance suggests that such a distribution is an intrinsic property of metabolic pathways due to the summation property of control coefficients.

A Rapid Electrophoretic Method for the Determination of the Isoenzymes of Serum Lactate Dehydrogenase

1966 ◽  
Vol 12 (5) ◽  
pp. 308-313 ◽  
Author(s):  
Albert W Opher ◽  
Charles S Collier ◽  
Joseph M Miller
Keyword(s):  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Quantitative Determination ◽  
Serum Lactate ◽  
Serum Lactate Dehydrogenase ◽  
Electrophoretic Method ◽  
The Individual ◽  
Electrophoretic Procedure ◽  
Purple Formazan

Abstract A convenient electrophoretic procedure for the separation and quantitation of lactate dehydrogenase (LDH) isoenzymes is described. The system uses polyacetate Sepraphore III strips.* The areas of activity are shown by incubation with an LDH substrate combined with tetra-nitro-blue-tetrazolium. The reduction of the latter to the purple formazan is quantitatively related to the enzyme activity. Quantitative determination of the individual colored areas is performed by densitometry.

scholarly journals Topology and quaternary structure of pro-sucrase/isomaltase and final-form sucrase/isomaltase

1986 ◽  
Vol 237 (2) ◽  
pp. 455-461 ◽  
Author(s):  
G M Cowell ◽  
J Tranum-Jensen ◽  
H Sjöström ◽  
O Norén
Keyword(s):  
Electron Microscopy ◽  
Quaternary Structure ◽  
Functional Organization ◽  
Enzyme Complex ◽  
Single Chain ◽  
Pancreatic Duct Ligation ◽  
Duct Ligation ◽  
Catalytically Active ◽  
Identical Series ◽  
Dimeric Model

Pig sucrase/isomaltase (EC 3.2.1.48/10) was purified from intestinal microvillar vesicles prepared from animals with and without pancreatic-duct ligation to obtain the single-chain pro form and the proteolytically cleaved final form respectively. The purified enzymes were re-incorporated into phosphatidylcholine vesicles and analysed by electron microscopy after negative staining. The two forms of the enzyme were observed as identical series of characteristic projected views that could be unified in a single dimeric model, containing two sucrase and two isomaltase units. This shows a homodimeric functional organization similar to that of other microvillar hydrolases. The bulk of the dimer was separated from the membrane by a maximal gap of 3.5 nm, representing a junctional segment connecting the intramembrane section of the anchor to the catalytically active domain of sucrase/isomaltase. The enzyme complex protrudes from the membrane for a distance of up to 17 nm. From charge-shift immunoelectrophoresic studies of hydrophilic prosucrase/isomaltase and from electron microscopy of reconstituted pro-sucrase/isomaltase, there was no evidence to suggest the presence of anchoring sequences between the sucrase and isomaltase subunits.

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

scholarly journals The Influence of Heavy Metal Ions on the Viability and Metabolic Enzyme Activity of the Marbled Crayfish Procambarus virginalis (Lyko, 2017)

2018 ◽  
Vol 70 ◽  
pp. 11-23 ◽  
Author(s):  
Oleg Marenkov ◽  
Mykola V. Prychepa ◽  
Julia Kovalchuk
Keyword(s):  
Heavy Metals ◽  
Heavy Metal ◽  
Enzyme Activity ◽  
Metal Ions ◽  
Heavy Metal Ions ◽  
Physiological State ◽  
Metabolic Enzyme ◽  
Nickel Ions ◽  
The Individual ◽  
Marbled Crayfish

In the experiment with marbled crayfishProcambarusvirginalis(Lyko, 2017), chronic effects of various concentrations of heavy metal ions on the physiological state and enzyme activity were investigated. The obtained results showed that among the investigated heavy metals nickel ions influenced the weight indexes and mortality of crustaceans the most negatively. According to the results of the research, significant changes were noted in the individual biochemical parameters of marbled crayfish under the influence of manganese, lead and nickel ions. The most significant changes in the activity of lactate dehydrogenase were detected in muscle tissues affected by manganese and nickel ions. A significant decrease in the activity of succinate dehydrogenase in muscle of marbled crayfish was determined after the action of heavy metal ions. Investigation of changes in the activity of alkaline phosphatase under the influence of the ions of manganese, lead and nickel has its own characteristics, which indicates certain violations in the tissues of cell membranes. Changes in the activity of enzymes were also reflected in the overall protein content. Changes in these parameters may indicate a rapid biochemical response of crustaceans to the toxic effects of heavy metals.

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