scholarly journals The partial purification and characterization of cytosol alcohol dehydrogenase from Astasia

1974 ◽  
Vol 141 (2) ◽  
pp. 469-475 ◽  
Author(s):  
Rolf Morosoli ◽  
Nicole Bégin-Heick
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Total Activity ◽  
Growth Conditions ◽  
Partial Purification ◽  
Heat Inactivation ◽  
Kinetic Properties ◽  
Ph Optimum

1. The cytosol alcohol dehydrogenase (alcohol–NAD oxidoreductase, EC 1.1.1.1) of Astasia longa was partially purified and characterized from cells grown in the presence of air+CO2 (95:5) or of O2+CO2 (95:5). 2. Under both these growth conditions, the cells contained a fraction, ADHII, which was characterized by its electrophoretic properties, by a high degree of resistance to heat inactivation, by a sharp pH optimum at 8.2 and by its kinetic properties. The estimated molecular weight of this fraction was approx. 150000, which is similar to that of yeast alcohol dehydrogenase. 3. Cells grown in air+CO2 (95:5) contain another fraction, ADHI, which can be further separated into two subfractions by polyacrylamide-gel electrophoresis and by DEAE-cellulose chromatography. This was termed fraction ‘ADHI-air’. 4. In addition to fraction ADHII, cells grown in the presence of O2 have a twofold increase in fraction ADHI-air activity as well as two new fractions that could not be demonstrated in air-grown cells. These new fractions which we have called fraction ‘ADHI-O2’, account for about 10% of the total activity. 5. The ADHI fractions (air) and (O2) have similar broad pH–activity curves and similar kinetic properties, both having a lower Km for ethanol and NAD than fraction ADHII. However, they differ from each other with respect to their activity with various substrates. The estimated molecular weight of these two ADHI fractions and their chromatographic behaviour on hydroxyapatite and on DEAE-cellulose also distinguish them.

scholarly journals Characterization of two acid proteinases found in rabbit skin homografts

1978 ◽  
Vol 169 (2) ◽  
pp. 287-295 ◽  
Author(s):  
B Jasani ◽  
M K Jasani ◽  
M D Talbot
Keyword(s):  
Molecular Weight ◽  
Cathepsin D ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Effect Of Temperature ◽  
Heat Inactivation ◽  
Acid Proteinase ◽  
Polymorphonuclear Leucocytes ◽  
Ph Optimum ◽  
Rabbit Skin

Two types of acid proteinase activity found in rabbit skin homografts were characterized by studying the effect of temperature, pH and polyacrylamide-gel electrophoresis. Their chromatographic behaviour was characterized on DEAE-cellulose, Sephadex G-75, G-100 and G-200, and their molecular weights were estimated by gel filtration. One of the acid proteinases in the homograft resembled cathepsin D (EC 3.4.23.5) of normal skin. The other acid proteinase differed from cathepsin D with respect to heat inactivation, pH optimum and molecular weight; it was not inactivated on heating at 60 degrees C for 60 min, its pH optimum was 2.5 and its molecular weight measured by Sephadex G-100 chromatography was 100 000. In all these respects, the heat-stable proteinase resembles cathepsin E (EC 3.4.23.5) of rabbit polymorphonuclear leucocytes.

Guanosine 3’:5’-Cyclic Monophosphate -Dependent Particulate Protein Kinase Activity from Yeast (Saccharomyces cerevisiae)

1999 ◽  
Vol 54 (1-2) ◽  
pp. 84-93 ◽  
Author(s):  
Hans Eckstein ◽  
Birgit Flügge
Keyword(s):  
Protein Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Temperature Tolerance ◽  
Maximum Activity ◽  
Partial Purification ◽  
Protein Kinase Activity ◽  
Ph Optimum ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Concentrations

Continuing our studies on cGMP in growing yeast we detected a particulate cGMPdependent protein kinase (Pk-G), which was solubilized by detergents and NaCl. It achieves maximum activity at 25 °C and pH = 6.8, high concentrations of substrate proteins or cGMP produce saturation. Casein and histones are appropriate substrates, phosphatase-pretreated histone H-2a provokes outstandingly high activity. Pk-G differs from cAMP-dependent protein kinase (Pk-A) with respect to pH optimum, temperature tolerance above 50 °C, and stability. Partial purification is achieved by chromatography with DEAE-cellulose, Sepharose, and cGMP-substituted Sepharose. The latter step also markedly removes Pk-A. At least three proteins with Pk-G-activity and high cGMP-affinity are separated by polyacrylamide-gel-electrophoresis. Their apparent molecular masses, as deduced from comigrating marker proteins, differ considerably from those of other Pk-G’s, but also of Pk-A’s

scholarly journals Purification and properties of adenylyl sulphate:ammonia adenylyltransferase from Chlorella catalysing the formation of adenosine 5′-phosphoramidate from adenosine 5′-phosphosulphate and ammonia

1981 ◽  
Vol 195 (3) ◽  
pp. 545-560 ◽  
Author(s):  
Heinz Fankhauser ◽  
Jerome A. Schiff ◽  
Leonard J. Garber
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Low Ph ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Unknown Compound ◽  
Reactive Blue 2

Extracts of Chlorella pyrenoidosa, Euglena gracilis var. bacillaris, spinach, barley, Dictyostelium discoideum and Escherichia coli form an unknown compound enzymically from adenosine 5′-phosphosulphate in the presence of ammonia. This unknown compound shares the following properties with adenosine 5′-phosphoramidate: molar proportions of constituent parts (1 adenine:1 ribose:1 phosphate:1 ammonia released at low pH), co-electrophoresis in all buffers tested including borate, formation of AMP at low pH through release of ammonia, mass and i.r. spectra and conversion into 5′-AMP by phosphodiesterase. This unknown compound therefore appears to be identical with adenosine 5′-phosphoramidate. The enzyme that catalyses the formation of adenosine 5′-phosphoramidate from ammonia and adenosine 5′-phosphosulphate was purified 1800-fold (to homogeneity) from Chlorella by using (NH4)2SO4 precipitation and DEAE-cellulose, Sephadex and Reactive Blue 2–agarose chromatography. The purified enzyme shows one band of protein, coincident with activity, at a position corresponding to 60000–65000 molecular weight, on polyacrylamide-gel electrophoresis, and yields three subunits on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of 26000, 21000 and 17000 molecular weight, consistent with a molecular weight of 64000 for the native enzyme. Isoelectrofocusing yields one band of pI4.2. The pH optimum of the enzyme-catalysed reaction is 8.8. ATP, ADP or adenosine 3′-phosphate 5′-phosphosulphate will not replace adenosine 5′-phosphosulphate, and the apparent Km for the last-mentioned compound is 0.82mm. The apparent Km for ammonia (assuming NH3 to be the active species) is about 10mm. A large variety of primary, secondary and tertiary amines or amides will not replace ammonia. One mol.prop. of adenosine 5′-phosphosulphate reacts with 1 mol.prop. of ammonia to yield 1 mol.prop. each of adenosine 5′-phosphoramidate and sulphate; no AMP is found. The highly purified enzyme does not catalyse any of the known reactions of adenosine 5′-phosphosulphate, including those catalysed by ATP sulphurylase, adenosine 5′-phosphosulphate kinase, adenosine 5′-phosphosulphate sulphotransferase or ADP sulphurylase. Adenosine 5′-phosphoramidate is found in old samples of the ammonium salt of adenosine 5′-phosphosulphate and can be formed non-enzymically if adenosine 5′-phosphosulphate and ammonia are boiled. In the non-enzymic reaction both adenosine 5′-phosphoramidate and AMP are formed. Thus the enzyme forms adenosine 5′-phosphoramidate by selectively speeding up an already favoured reaction.

Properties and characteristics of partly purified glutamate dehydrogenase from sheep rumen mucosa

1981 ◽  
Vol 46 (11) ◽  
pp. 2766-2773
Author(s):  
Katarína Holovská ◽  
Viera Lenártová ◽  
Ivan Havassy
Keyword(s):  
Molecular Weight ◽  
Glutamate Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Enzymatic Reaction ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ph Optimum ◽  
Relative Molecular Weight ◽  
Sheep Rumen ◽  
The Individual

The purification of glutamate dehydrogenase from sheep rumen mucosa on DEAE-cellulose afforded two enzyme fractions with glutamate dehydrogenase activity. The enzyme fraction II (tissue glutamate dehydrogenase) was freed of contaminating proteins in the subsequent purification step on Sephadex G-200. The approximate relative molecular weight (260 000) of tissue glutamate dehydrogenase (fraction II) was determined by gel filtration on Sephadex G-200 and the approximate relative molecular weight of its polypeptide chain (48 000) was established by polyacrylamide gel electrophoresis in SDS. The pH-optimum of fraction II was 7.9. The effect of substrate concentration on the rate of the enzymatic reaction was examined and the following apparent Michaelis' constants were found for the individual substrates: NADH 6.25 . 10-5 mol/l, 2-oxoglutarate 4.5 . 10-3 mol/l, and NH4+ 77 . 10-3 mol/l.

scholarly journals Characterization of the multiple forms of hydroxymethylbilane synthase from rat spleen

1984 ◽  
Vol 217 (3) ◽  
pp. 675-683 ◽  
Author(s):  
D C Williams
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Kinetic Properties ◽  
Stable Form ◽  
Multiple Forms ◽  
Ph Optimum ◽  
Heat Stable ◽  
Decreasing Ph ◽  
Covalent Intermediate

Phenylhydrazine treatment induced hydroxymethylbilane synthase activity (EC 4.3.1.8) in rat spleen, erythrocytes and liver by 40-fold, 7.5-fold and 6-fold respectively. Five multiple forms of the enzyme were resolved by DEAE-cellulose chromatography. In the presence of phenylmethanesulphonyl fluoride only three forms, two major and one minor, were resolved by the fractionation, suggesting that two of the original forms arose by proteolytic modification. Heat treatment (70 degrees C) in the presence of proteinase inhibitor converted one of the major forms into the other major form. Product isomer analysis suggested that this heat-labile form represented an enzyme-substrate covalent intermediate and not a hydroxymethylbilane synthase-uroporphyrinogen III synthase complex. Identical elution profiles and kinetic properties of the enzymes from rat spleen and erythrocytes suggested that the enzyme isolated from spleen was possibly from stored erythrocytes. Sephadex G-75 chromatography of the heat-stable DEAE-cellulose-purified form yielded pure enzyme as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The Mr was found to be 43000 +/- 1500. Initial-velocity studies on all enzyme forms showed a hyperbolic dependence of velocity on substrate concentration, demonstrating the existence of a displacement-type mechanism. For the heat-stable form Vmax, varied with pH as a typical bell-shaped curve, indicating that two ionizable groups with pK values of 7.4 and 8.8 are important for catalysis. Km decreased with decreasing pH on the acid side of the pH optimum, suggesting the absence of ionization of a group with pK 7.4 in free enzyme or substrate.

Characterization of an enzyme from Rhizoctonia praticola which polymerizes phenolic compounds

1979 ◽  
Vol 25 (2) ◽  
pp. 229-233 ◽  
Author(s):  
Jean-Marc Bollag ◽  
Roy D. Sjoblad ◽  
Shu-Yen Liu
Keyword(s):  
Molecular Weight ◽  
Phenolic Compounds ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Phenol Oxidase ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Rhizoctonia Praticola

An extracellular phenol oxidase from the fungus Rhizoctonia praticola which polymerizes various xenobiotic phenols was isolated and characterized. The enzyme was purified by DEAE-cellulose and Sephadex G-200 chromatography followed by preparative polyacrylamide gel electrophoresis. Atomic absorption and EPR spectroscopy indicated the presence of copper, and SDS gel electrophoresis revealed a molecular weight of 78 000. With 2,6-dimethoxyphenol as substrate, the enzyme showed a pH optimum of 6.7–6.9, and a temperature optimum of 40 °C. According to these and additional characteristics it appears that the enzyme belongs to the class of laccases.

Enzymatic sulfation of steroids. V. Partial purification and some properties of sulfotransferase III, the major glucocorticoid sulfotransferase of liver cytosols from male rats

1978 ◽  
Vol 56 (11) ◽  
pp. 1028-1035 ◽  
Author(s):  
Sanford S. Singer ◽  
James Gebhart ◽  
Edward Hess
Keyword(s):  
Molecular Weight ◽  
Sulfhydryl Groups ◽  
Male Rats ◽  
Partial Purification ◽  
Ph Optimum ◽  
Fold Enrichment ◽  
Competitive Inhibitors ◽  
Sequential Mechanism ◽  
Inhibition Studies ◽  
Estradiol 17Β

This manuscript describes purification of sulfotransferase III (STIII), the major hepatic glucocorticoid sulfotransferase of male rats, 77.8 ± 16 fold from cytosol. This represents a probable 250–345 fold enrichment, compared with homogenates. Purified STIII has a molecular weight of 61 500 ± 2500 from Sephadex G-100 chromatography. It is markedly activated by 5 mM divalent Ba, Ca, Co, Cr, Mg, Mn, and Ni salts; inhibited strongly by 5 mM divalent Zn and Cd; and unaffected by 8 mM ADP, ATP, and AMP. Comparison of the ability of purified STIII to sulfate equimolar Cortisol, estradiol-17β, testosterone, and dehydroepiandrosterone suggests that the enzyme may sulfate glucocorticoids preferentially. However, its Cortisol sulfotransferase activity is inhibited by a variety of steroids. Of these, dehydroepiandrosterone, dexamethasone, and progesterone were tested extensively. They were found to be competitive inhibitors. STIII has a sharp pH optimum at pH 6.0 ± 0.1. However, it is routinely assayed at pH 6.8, as explained in the text. It exhibits a sequential mechanism and Km values of 6.82 ± 1.2 and 6.28 ± 0.64 μM for Cortisol and 3′-phosphoadenosine-5′-phosphosulfate, respectively. It also possesses essential sulfhydryl groups, as shown by p-hydroxymercuribenzoate inhibition studies.

Characterization and distribution of arginine kinase in the tissues of the scorpion, Palamneus phipsoni

1985 ◽  
Vol 63 (10) ◽  
pp. 2262-2266 ◽  
Author(s):  
A. V. Arjunwadkar ◽  
S. Raghupathi Rami Reddy
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Alimentary Canal ◽  
Polyacrylamide Gel Electrophoresis ◽  
Arginine Kinase ◽  
High Specificity ◽  
Ph Optimum ◽  
High Concentrations ◽  
Claw Muscle ◽  
Slight Inhibition

Arginine kinase in claw muscle extracts of the scorpion, Palamneus phipsoni, was characterized. The enzyme, with a pH optimum of 8.5 in the direction of phosphoarginine synthesis, showed activation by Mg2+, high specificity towards L-arginine as the guanidino substrate, slight inhibition by high concentrations of L-arginine and ATP, and a molecular weight of 33 500. On polyacrylamide gel electrophoresis at pH 8.3 the enzyme migrated to the anode as a single molecular species. In addition to the claw muscle, the enzyme activity was also found to be present in the heart, alimentary canal, hepatopancreas, and nervous system. In general, scorpion muscle arginine kinase appears to be similar in its properties to the enzyme from other arthropods.

scholarly journals Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties

1978 ◽  
Vol 175 (2) ◽  
pp. 391-406 ◽  
Author(s):  
R Jones ◽  
M B Wilkins ◽  
J R Coggins ◽  
C A Fewson ◽  
A D B Malcolm
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Maximum Velocity ◽  
Single Band ◽  
Kinetic Properties ◽  
Subunit Structure

Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.

Partial purification and properties of an L-arabinose dehydrogenase from Azospirillum brasiliense

1983 ◽  
Vol 29 (2) ◽  
pp. 242-246 ◽  
Author(s):  
Norman J. Novick ◽  
Max E. Tyler
Keyword(s):  
Molecular Weight ◽  
Divalent Cation ◽  
Gel Filtration ◽  
Reducing Agent ◽  
Partial Purification ◽  
Ph Optimum ◽  
Glycine Buffer ◽  
Purification And Properties ◽  
Filtration Technique

An L-arabino-aldose dehydrogenase responsible for the oxidation of L-arabinose to L-arabino-γ-lactone has been purified 59-fold from L-arabinose grown cells of Azospirillum brasiliense. The dehydrogenase was found to be specific for substrates with the L-arabino-configuration at carbons 2, 3, and 4. Km values for L-arabinose of 75 and 140 μM were found with NADP and NAD as coenzymes, respectively. The enzyme had a pH optimum of 9.5 in glycine buffer and was stable when heated to 55 °C for 5 min. No enhancement of activity in the presence of any divalent cation or reducing agent tested was found. L-Arabinose dehydrogenase had a molecular weight of 175 000 as measured by the gel filtration technique.

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