Characterization of alcohol dehydrogenase isolated from germinating bean Vicia faba seeds

1989 ◽  
Vol 54 (9) ◽  
pp. 2519-2527 ◽  
Author(s):  
Sylva Leblová ◽  
Mustafa El Ahmad
Keyword(s):  
Amino Acids ◽  
Alcohol Dehydrogenase ◽  
Pyridoxal Phosphate ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Carbon Chain ◽  
Substrate Oxidation ◽  
Gel Chromatography ◽  
Ph Optimum ◽  
Substrate Reduction

We have isolated alcohol dehydrogenase (ADH, E.C. 1.1.1.1) from beans germinating 3 days by ammonium sulfate precipitation of the sodium phosphate extract of the homogenate of seeds, followed by chromatography on DEAE-cellulose and gel chromatography on Sephadex G-200 and G-100; both chromatographic operations were repeated twice. The activity of ADH increased 960 times after these procedures. The enzyme whose Mr is 60 000 consists of two identical subunits of Mr = 30 000. Allyl alcohol is oxidized and acetaldehyde reduced at the highest rate of all the alcohols and aldehydes tested. The reaction rate decreases with the increasing length of the carbon chain of the substrate. In contrast, the rate of oxidation of alcohols with a double bond in their molecules increased. The pH-optimum of substrate oxidation (pH 8.5) is different from the pH-optimum of substrate reduction (pH 7.5). From kinetic studies of the effect of pH on Vmax and Km the pK-value of amino acids participating on substrate oxidation is 9.2 and 8.5 whereas amino acids with pK 8.3 and 6.8 participate on substrate reduction. We have verified the participation of the SH-groups in experiments with the inactivation of ADH by iodoacetate: the inactivation was weaker after the enzyme had been preincubated with NAD or AMP, adenosine or nicotinamide. Likewise pyridoxal phosphate inactivates bean ADH by modifying its ε-amino group of lysine. The degree of inactivation depends also on the pH and the ionic strength of the medium. The protective effect of NAD or its analogs shows that lysine is present in the active center of the enzyme in the coenzyme-binding site.

Pea alcohol dehydrogenase: Substrate specificity and binding of coenzyme to enzyme

1979 ◽  
Vol 44 (2) ◽  
pp. 631-636 ◽  
Author(s):  
Marie Stiborová ◽  
Roman Lapka ◽  
Noemi Nováková ◽  
Sylva Leblová
Keyword(s):  
Alcohol Dehydrogenase ◽  
Substrate Specificity ◽  
Competitive Inhibitor ◽  
Substrate Oxidation ◽  
Enzyme Protein ◽  
Ph Optimum ◽  
Substrate Reduction ◽  
Noncompetitive Inhibitor ◽  
Ph Dependent ◽  
Broad Specificity

Pea alcohol dehydrogenase (EC 1.1.1.1) shows a broad specificity with respect to aldehydes and alcohols. The pH-optimum of substrate oxidation is 8.7 and of substrate reduction 7.0. The enzyme is inhibited by ATP, adenosine, and adenine. The inhibition is competitive with respect to NAD. The inhibition by ATP is pH-dependent. The competitive character of the inhibition by adenine and its derivatives with respect to NAD indicates the importance of the adenine moiety of the coenzyme for its binding to the enzyme. Phenanthroline is a competitive inhibitor with respect to NAD, a mixed inhibitor with respect to ethanol and a noncompetitive inhibitor with respect to acetaldehyde. Experiments carried out simultaneously with ATP and phenanthroline show that the adenine moiety of NAD does not bind via the zinc atom to the enzyme protein.

GLUTAMIC CARBOXYLASE OF THE MATURE WHEAT LEAF

1952 ◽  
Vol 30 (6) ◽  
pp. 755-763 ◽  
Author(s):  
P. Weinberger ◽  
K. A. Clendenning
Keyword(s):  
Pyridoxal Phosphate ◽  
Kinetic Studies ◽  
Wheat Plant ◽  
Leaf Extracts ◽  
Ph Optimum ◽  
Carboxylase Activity ◽  
Mature Leaves ◽  
Heat Stable ◽  
Wheat Leaf ◽  
Weak Activity

A study of the distribution of glutamic carboxylase within the developing wheat plant revealed that it was absent in young plants, and was present only in traces in mature roots, and that it accumulated in mature leaves. Glutamic carboxylase was particularly abundant in the mature and senescent third leaf. Extracts of leaves of other cereals showed only weak activity, while extracts of roots, other than barley, were inactive. The high enzyme activity of the barley root extracts was exceeded only by that of mature wheat leaf extracts. A convenient method is described for enzyme storage at −40 °C. and a purification procedure was developed which effected a 500-fold concentration (nitrogen basis). The glutamic carboxylase activity of crude extracts was enhanced by preparatory exposure to phosphate buffer; after selective salt precipitation and lengthy dialysis, activity was reduced, but could be restored by the addition of pyridoxal phosphate. A heat-stable inhibitor of glutamic carboxylase at its pH optimum was found in the ether-soluble organic acid fraction of the cell sap of Kalanchoe leaves. Similar inhibiting effects were shown by malate, tartrate, and citrate, but not by succinate, fumarate, aspartate, and alanine. Kinetic studies indicated that the inhibition of plant glutamic carboxylase by cyanide is noncompetitive.

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.

Kinetic studies on the permanganic oxidation of amino acids. Effect of the length of amino acid carbon chain

1997 ◽  
Vol 29 (3) ◽  
pp. 181-185 ◽  
Author(s):  
A. Arrizabalaga ◽  
F. J. Andr�s-Ordax ◽  
M. Y. Fern�ndez-Ar�nguiz ◽  
R. Peche
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Kinetic Studies ◽  
Carbon Chain

scholarly journals Molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II of Acinetobacter calcoaceticus

1998 ◽  
Vol 330 (3) ◽  
pp. 1375-1381 ◽  
Author(s):  
J. David GILLOOLY ◽  
G. S. Alan ROBERTSON ◽  
A. Charles FEWSON
Keyword(s):  
Amino Acids ◽  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Catalytic Mechanism ◽  
Acinetobacter Calcoaceticus ◽  
Higher Plant ◽  
Ph Optimum ◽  
Alcohol Dehydrogenases ◽  
Kinetic Coefficients ◽  
Benzaldehyde Dehydrogenase

The nucleotide sequences of xylB and xylC from Acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II, were determined. The complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. Benzaldehyde dehydrogenase II is a 51654 Da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenases. Benzyl alcohol dehydrogenase has a subunit Mr of 38923 consisting of 370 amino acids, it stereospecifically transfers the proR hydride of NADH, and it is a member of the family of zinc-dependent long-chain alcohol dehydrogenases. The enzyme appears to be more similar to animal and higher-plant alcohol dehydrogenases than it is to most other microbial alcohol dehydrogenases. Residue His-51 of zinc-dependent alcohol dehydrogenases is thought to be necessary as a general base for catalysis in this category of alcohol dehydrogenases. However, this residue was found to be replaced in benzyl alcohol dehydrogenase from A. calcoaceticus by an isoleucine, and the introduction of a histidine residue in this position did not alter the kinetic coefficients, pH optimum or substrate specificity of the enzyme. Other workers have shown that His-51 is also absent from the TOL-plasmid-encoded benzyl alcohol dehydrogenase of Pseudomonas putida and so these two closely related enzymes presumably have a catalytic mechanism that differs from that of the archetypal zinc-dependent alcohol dehydrogenases.

Purification and characterization of tonin

1976 ◽  
Vol 54 (9) ◽  
pp. 788-795 ◽  
Author(s):  
S. Demassieux ◽  
R. Boucher ◽  
C. Grisé ◽  
J. Genest
Keyword(s):  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Potassium Phosphate ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Sedimentation Equilibrium ◽  
Angiotensin I ◽  
Ph Optimum ◽  
Ammonium Sulphate Precipitation

Tonin was purified from rat submaxillary glands by differential centrifugation, ammonium sulphate precipitation, gel filtration on Sephadex G150, and by ion-exchange chromatography on DEAE-cellulose, phospho-cellulose, SP-Sephadex C25, and SP-Sephadex C50. Purified tonin was shown to be homogeneous by analytical electrophoresis and by analytical ultracentrifugation analysis. Purified tonin was very stable when stored in buffers of low pH values or when incubated at high temperatures in neutral solutions. The molecular weight estimated by sedimentation equilibrium was 28 700. The pH optimum was near 6.8 in a 0.1 M potassium phosphate buffer. The Michaelis–Menten constant for tonin using angiotensin I as substrate was about 4 × 10−5 M. Tonin activity was strongly inhibited by plasma. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by plasma was of the non-competitive type.

Methionine transport by mycelia of Fusarium oxysporum f. sp. lycopersici

1981 ◽  
Vol 27 (8) ◽  
pp. 743-747 ◽  
Author(s):  
Leslie R. Barran
Keyword(s):  
Amino Acids ◽  
Fusarium Oxysporum ◽  
Dry Weight ◽  
Carbon Chain ◽  
Temperature Optimum ◽  
Ph Optimum ◽  
Methionine Uptake ◽  
Energy Dependent ◽  
Methionine Transport ◽  
Unidentified Metabolite

Mycelia of Fusarium oxysporum f. sp. lycopersici accumulated L-methionine by an energy-dependent process, and the energy required for uptake may be derived from either respiration or glycolysis. The pH optimum for transport was 4 and the temperature optimum was 35 °C. The apparent Km for methionine uptake was 2.5–3.3 μM and the Vmax was 0.24–0.30 nmol∙min−1∙mg dry weight−1. S-adenosylhomocysteine (Ado-Hcys) was the major metabolic product of methionine although S-adenosylmethionine (Ado-Met), homocysteine (Hcys), and an unidentified metabolite (compound X) were also detected. The failure to demonstrate efflux of accumulated methionine in the presence of the uncoupler 2,4-dinitrophenol or excess unlabeled methionine was probably due to the fact that methionine was rapidly metabolized within the cells.Acidic and basic amino acids, and those amino acids having less than a four-carbon chain, did not inhibit methionine uptake. The rate of uptake of methionine, which was greatest in log phase mycelia, decreased substantially as the cells entered the stationary phase.

Kinetic Behaviour of Amylase According to pH: A New Perspective for Starch Hydrolysis Process

2020 ◽  
Vol 16 (2) ◽  
pp. 135-144
Author(s):  
Ravneet K. Grewal ◽  
Baldeep Kaur ◽  
Gagandeep Kaur
Keyword(s):  
Metal Ions ◽  
Competitive Inhibition ◽  
Deae Cellulose ◽  
Kinetic Studies ◽  
Cost Effective ◽  
Starch Hydrolysis ◽  
Divalent Metal Ions ◽  
Activity Data ◽  
Time Saving ◽  
Alkaline Conditions

Background: Amylases are the most widely used biocatalysts in starch saccharification and detergent industries. However, commercially available amylases have few limitations viz. limited activity at low or high pH and Ca2+ dependency. Objective: The quest for exploiting amylase for diverse applications to improve the industrial processes in terms of efficiency and feasibility led us to investigate the kinetics of amylase in the presence of metal ions as a function of pH. Methods: The crude extract from soil fungal isolate cultures is subjected to salt precipitation, dialysis and DEAE cellulose chromatography followed by amylase extraction and is incubated with divalent metal ions (i.e., Ca2+, Fe2+, Cu2+, and Hg2+); Michaelis-Menton constant (Km), and maximum reaction velocity (Vmax) are calculated by plotting the activity data obtained in the absence and presence of ions, as a function of substrate concentration in Lineweaver-Burk Plot. Results: Kinetic studies reveal that amylase is inhibited un-competitively at 5mM Cu2+ at pH 4.5 and 7.5, but non-competitively at pH 9.5. Non-competitive inhibition of amylase catalyzed starch hydrolysis is observed with 5mM Hg2+ at pH 9.5, which changes to mixed inhibition at pH 4.5 and 7.5. At pH 4.5, Ca2+ induces K- and V-type activation of amylase catalyzed starch hydrolysis; however, the enzyme has V-type activation at 7mM Ca2+ under alkaline conditions. Also, K- and V-type of activation of amylase is observed in the presence of 7mM Fe2+ at pH 4.5 and 9.5. Conclusion: These findings suggest that divalent ions modulation of amylase is pH dependent. Furthermore, a time-saving and cost-effective solution is proposed to overcome the challenges of the existing methodology of starch hydrolysis in starch and detergent industries.

scholarly journals Synthesis of Organic Matter in Aqueous Environments Simulating Small Bodies in the Solar System and the Effects of Minerals on Amino Acid Formation

Life ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 32
Author(s):  
Walaa Elmasry ◽  
Yoko Kebukawa ◽  
Kensei Kobayashi
Keyword(s):  
Amino Acids ◽  
Organic Matter ◽  
Amino Acid ◽  
Solar System ◽  
Acid Formation ◽  
Gel Chromatography ◽  
Small Bodies ◽  
Enhanced Production ◽  
Aqueous Environments ◽  
Heating Duration

The extraterrestrial delivery of organics to primitive Earth has been supported by many laboratory and space experiments. Minerals played an important role in the evolution of meteoritic organic matter. In this study, we simulated aqueous alteration in small bodies by using a solution mixture of H2CO and NH3 in the presence of water at 150 °C under different heating durations, which produced amino acids after acid hydrolysis. Moreover, minerals were added to the previous mixture to examine their catalyzing/inhibiting impact on amino acid formation. Without minerals, glycine was the dominant amino acid obtained at 1 d of the heating experiment, while alanine and β-alanine increased significantly and became dominant after 3 to 7 d. Minerals enhanced the yield of amino acids at short heating duration (1 d); however, they induced their decomposition at longer heating duration (7 d). Additionally, montmorillonite enhanced amino acid production at 1 d, while olivine and serpentine enhanced production at 3 d. Molecular weight distribution in the whole of the products obtained by gel chromatography showed that minerals enhanced both decomposition and combination of molecules. Our results indicate that minerals affected the formation of amino acids in aqueous environments in small Solar System bodies and that the amino acids could have different response behaviors according to different minerals.

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