scholarly journals Purification of alcohol dehydrogenase from Drosophila by general-ligand affinity chromatography

1979 ◽  
Vol 179 (3) ◽  
pp. 479-482 ◽  
Author(s):  
A J Brown ◽  
C Y Lee
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Affinity Chromatography ◽  
Good Yield ◽  
Purification Procedure ◽  
Ligand Affinity ◽  
Novel Technique ◽  
Affinity Ligand ◽  
Sepharose 4B ◽  
Homogeneous Preparation

A method for the purification of alcohol dehydrogenase from Drosophila melanogaster is described. The method makes use of 8-(6-aminohexyl)amino-5′-AMP, immobilized on Sepharose 4B, as an affinity ligand. Since alcohol dehydrogenase from Drosophila shows weak affinity for this column, a novel technique was developed to separate alcohol dehydrogenase from both unbound proteins and more strongly bound enzymes. The purification procedure is simple to operate and give a homogeneous preparation in good yield after only three steps.

scholarly journals Affinity chromatography of nicotinamide nucleotide-dependent dehydrogenases on immobilized nucleotide derivatives

1974 ◽  
Vol 141 (3) ◽  
pp. 775-787 ◽  
Author(s):  
Ian P. Trayer ◽  
Hylary R. Trayer
Keyword(s):  
Affinity Chromatography ◽  
Glucose Dehydrogenase ◽  
Three Dimensional ◽  
Ligand Affinity ◽  
Nicotinamide Mononucleotide ◽  
Adenosine Phosphate ◽  
Three Dimensional Models ◽  
Separation Of Mixtures ◽  
Phosphate Ligands ◽  
Sepharose 4B

A series of chemically-defined adenosine phosphate ligands attached to Sepharose 4B were used as active-site probes in studying the interaction of enzymes with their coenzymes and substrates and to test the suitability of these matrices for ‘general ligand’ affinity chromatography. Nicotinamide nucleotide-dependent dehydrogenases were used as models to test this methodology. Elution from these columns by NAD+and/or AMP gradients (in the presence or the absence of substrates and/or nicotinamide mononucleotide) was consistent with: (1) the compulsory ordered addition of substrates to lactate and malate dehydrogenase; (2) the necessity for the NMN moiety of NAD+to bind to these enzymes before the substrate; and illustrated: (3) that the binding of these two hydrogenases to these columns compared very well with the published three-dimensional models for these enzymes and (4) that separation of mixtures of dehydrogenases depended on the choice of matrix and displacing ion and whether any additions (e.g. substrates) were made to the gradients used. These techniques were used to purify UDP-glucose dehydrogenase from a crude starting material on a phosphate-linked UDP (or ADP) matrix. The binding of this enzyme to these two columns was not consistent with either an ordered or random addition of substrates and suggested a more complex mechanism.

scholarly journals Purification and enzyme stability of alcohol dehydrogenase from Drosophila simulans, Drosophila virilis and Drosophila melanogaster adhS

1980 ◽  
Vol 189 (1) ◽  
pp. 105-110 ◽  
Author(s):  
E Juan ◽  
R González-Duarte
Keyword(s):  
Drosophila Melanogaster ◽  
Affinity Chromatography ◽  
Enzyme Stability ◽  
Gel Filtration ◽  
Drosophila Simulans ◽  
Drosophila Virilis ◽  
Purification Procedure ◽  
Alcohol Dehydrogenases ◽  
Isoelectric Points ◽  
Chromatography Step

Three alcohol dehydrogenases from Drosophila simulans, Drosophila virillis and Drosophila melanogaster adhS (which possesses an alloenzyme with slow electrophoretic mobility) were purified essentially to homogeneity. The purification procedure involves a new step of affinity chromatography, which efficiently lowers the amount of contaminants in the final preparation, producing a very stable enzyme. The purification procedure developed consists of a salmine sulphate precipitation, two CM-Sepharose CL-6B colume-chromatography steps, an affinity-chromatography step and a Sephacryl gel filtration. A minimum of 30-fold purification is obtained and the yield is not less than 34%. The isoelectric points and molar absorption coefficients were determined.

The use of Spheron as a matrix for affinity chromatography of NAD-dependent dehydrogenases

1986 ◽  
Vol 51 (7) ◽  
pp. 1542-1549
Author(s):  
Jan Kovář ◽  
Alena Škodová ◽  
Jaroslav Turánek
Keyword(s):  
Lactate Dehydrogenase ◽  
Alcohol Dehydrogenase ◽  
Affinity Chromatography ◽  
Dehydrogenase Activity ◽  
Nitrous Acid ◽  
Separation Efficiency ◽  
Binding Capacity ◽  
The Stability ◽  
Sepharose 4B

The paper compares several methods of coupling common ligands of dehydrogenases, viz. N6-[(6-aminohexyl)carbamoylmethyl]-AMP and N6-[(6-aminohexyl)carbamoylmethyl]-NAD, to a hydrophilic macroporous glycolmethacrylate gel, Spheron. The affinants coupled best to a CNBr-activated gel and to a gel with hydrazine groups (after activation with nitrous acid). The affinity properties of gels based on Spheron and on Sepharose 4B were similar ( the stability and separation efficiency were almost identical, the binding capacity and the recovery of dehydrogenase activity were somewhat better with the Sepharose). The materials based on Spheron were used in several separation experiments, viz. separation of lactate dehydrogenase form albumin, separation of lactate dehydrogenase from alcohol dehydrogenase under different conditions and separation of isoenzymes of lactate dehydrogenase. Spheron 300 with a coupled affinant was also employed in an attempt to purify a crude alcohol dehydrogenase.

Separation of the S-Adenosylmethionine: 5-and 8-Hydroxyfuranocoumarin O-Methyltransferases of Ruta graveolens L. by General Ligand Affinity Chromatography

1979 ◽  
Vol 34 (5-6) ◽  
pp. 387-391 ◽  
Author(s):  
Satish K. Sharma ◽  
Jinnie M. Garrett ◽  
Stewart A. Brown
Keyword(s):  
Affinity Chromatography ◽  
Conclusive Evidence ◽  
Ruta Graveolens ◽  
Ligand Affinity ◽  
Affinity Ligand

Abstract Two S-adenosyl-ʟ-methionine:furanocoumarin O-methyltransferases of R . graveolens, acting at the 5-and 8-hydroxyls of the psoralen nucleus, were completely resolved by adsorption on a general affinity ligand, 5 -(3-carboxypropanamido) xanthotoxin, followed by specific desorption by bergaptol and xanthotoxol, respectively. The 5-O-methyltransferase was purified 450-fold by this procedure, the 8-O-methyltransferase 112-fold, and both enzyme fractions were electrophoretically homogeneous. No resolution could be achieved of the activity against two 5-hydroxypsoralens or of the activity against two 8-hydroxypsoralens, and conclusive evidence is presented for the existence of only one 5-O-methyltransferase and only one 8-O-methyltransferase acting on linear furanocoumarins.

Purification of Histone F3 by Covalent Chromatography

1977 ◽  
Vol 32 (1-2) ◽  
pp. 72-74 ◽  
Author(s):  
Oskar Oster ◽  
Gerhard Buchlow
Keyword(s):  
Affinity Chromatography ◽  
Purification Procedure ◽  
Pure Protein ◽  
Short Time ◽  
Sepharose 4B ◽  
Covalent Chromatography

Abstract The arginine-rich histone F3 has been purified by covalent affinity chromatography. By the use of activated Thiol-Sepharose 4B for the purification of cysteine containing histone F3 a highly pure protein was obtained. The simple purification procedure offers the opportunity to get larger amounts of pure histone F3 within short time.

Heat stable proteinase fromPseudomonas fluorescensAH-70: purification by affinity chromatography on cyclopeptide antibiotics

1988 ◽  
Vol 55 (2) ◽  
pp. 217-226 ◽  
Author(s):  
Juan I. Azcona ◽  
Rosario Martín ◽  
Miguel A. Asensio ◽  
Pablo E. Hernández ◽  
Bernabé Sanz
Keyword(s):  
Affinity Chromatography ◽  
Specific Activity ◽  
Fold Increase ◽  
Total Activity ◽  
Purification Procedure ◽  
Extracellular Proteinase ◽  
Original Activity ◽  
Gramicidin S ◽  
Heat Stable ◽  
Sepharose 4B

SummaryA heat stable extracellular proteinase from the psychrotrophPseudomonas fluorescensAH-70 was purified to electrophoretic homogeneity by affinity chromatography on a gramicidin S–Sepharose-4B column. Bacitracin linked to Sepharose-4B was unable to retain any proteolytic activity, whereas the same antibiotic bound to AH-Sepharose-4B retained ~ 25% of the total activity. The purification procedure on the gramicidin S–Sepharose-4B column was easy to perform, fast and reproducible; it resulted in a 207-fold increase in the specific activity and a yield of 41% of the original activity. The purified enzyme was a monomer with a mol. wt of 33000. The enzyme hydrolysed whole casein and its fractions whereas no activity was observed against bovine serum albumin. The enzyme was a metalloproteinase. It was heat stable, havingD-values at 121, 135 and 150 °C of 3·8, 1·9 and 0·6 min respectively.

Quantitative Assessment of Soluble Fibrin in Plasma by Affinity Chromatography – A Comparative Study with desAA-Fibrin, desAABB-Fibrin and Fibrinogen

1985 ◽  
Vol 54 (02) ◽  
pp. 533-538 ◽  
Author(s):  
Wilfried Thiel ◽  
Ulrich Delvos ◽  
Gert Müller-Berghaus
Keyword(s):  
Affinity Chromatography ◽  
Calcium Ions ◽  
Quantitative Assessment ◽  
Fluid Phase ◽  
Soluble Fibrin ◽  
Binding Characteristics ◽  
Different Temperatures ◽  
Immobilized Proteins ◽  
Sepharose 4B

SummaryA quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20° C and 37° C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20° C as well as at 37° C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20° C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20° C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37° C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37° C (95%) but only 58% of the desAABB-fibrin were bound at 20° C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patients’ plasma.

Use of Dye-Ligand Affinity Chromatography for Separation of Toxin Components of Bacillus Anthracis

1983 ◽  
Author(s):  
Stephen F. Little ◽  
Kenneth W. Hedlund ◽  
Bruce E. Ivins ◽  
Joseph D. Ristroph ◽  
Dale W. Seburn
Keyword(s):  
Affinity Chromatography ◽  
Bacillus Anthracis ◽  
Ligand Affinity

scholarly journals The Alcohol Dehydrogenase Gene Is Nested in the outspread Locus of Drosophila melanogaster

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 897-911 ◽  
Author(s):  
S McNabb ◽  
S Greig ◽  
T Davis
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Pcr Amplification ◽  
Transcription Unit ◽  
Adjacent Gene ◽  
Dehydrogenase Gene ◽  
Chromosomal Breakpoints ◽  
Splicing Patterns ◽  
Somatic Musculature

Abstract This report describes the structure and expression of the outspread (osp) gene of Drosophila melanogaster. Previous work showed that chromosomal breakpoints associated with mutations of the osp locus map to both sides of the alcohol dehydrogenase gene (Adh), suggesting that Adh and the adjacent gene Adh' are nested in osp. We extended a chromosomal walk and mapped additional osp mutations to define the maximum molecular limit of osp as 119 kb. We identified a 6-kb transcript that hybridizes to osp region DNA and is altered or absent in osp mutants. Accumulation of this RNA peaks during embryonic and pupal periods. The osp cDNAs comprise two distinct classes based on alternative splicing patterns. The 5′ end of the longest cDNA was extended by PCR amplification. When hybridized to the osp walk, the 5′ extension verifies that Adh and Adh' are nested in osp and shows that osp has a transcription unit of ≥74 kb. In situ hybridization shows that osp is expressed both maternally and zygotically. In the ovary, osp is transcribed in nurse cells and localized in the oocyte. In embryos, expression is most abundant in the developing visceral and somatic musculature.

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