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 Characterization of vitamin K-dependent carboxylase from the livers from the adult ox and dicoumarol-treated calf.

1982 ◽  
Vol 201 (2) ◽  
pp. 249-258 ◽  
Author(s):  
L Uotila ◽  
J W Suttie
Keyword(s):  
Rat Liver ◽  
Vitamin K ◽  
Pyridoxal Phosphate ◽  
Liver Microsomes ◽  
Rat Liver Microsomes ◽  
Ph Optimum ◽  
Carboxylase Activity ◽  
Triton X ◽  
Assay Conditions

The properties of the microsomal vitamin K-dependent carboxylase from the livers of the adult ox and dicoumarol-treated calf were investigated. The enzymes from both sources utilized glutamic residues of synthetic peptides as substrates and could be solubilized with Triton X-100 similarly to the enzyme from vitamin K-deficient rat liver. Under the optimal assay conditions, the microsomes from calf liver had peptide carboxylase activity comparable with that of the rat liver microsomes and 6.5-fold that of adult ox liver microsomes. The apparent Km for reduced vitamin K and the ionic strength optima of the calf and adult ox enzyme clearly differ from those of the rat enzyme. Pyridoxal phosphate activated the adult ox carboxylase only slightly, whereas the calf enzyme was activated by pyridoxal phosphate as effectively as was the enzyme from the vitamin K-deficient rat. Mn2+ activated the adult ox enzyme 9-fold and calf enzyme 22-fold under optimal conditions (no KCl). Three other divalent metal cations (Ca2+, Ba2+, and Mg2+) activated the adult ox and calf enzymes to about half the extent caused by Mn2+, KCl inhibited this activation. The vitamin K-dependent carboxylase from the dicoumarol-treated calf is apparently more tightly bound to the microsomal membrane than is the adult ox enzyme. In many other respects (pH optimum), temperature optimum, Km values for peptide substrate, substrate specificity, inhibitor effects), the properties of the adult ox and calf enzymes resemble closely those of the rat enzyme.

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.

scholarly journals Characterization of mouse liver α-l-fucosidase. Demonstration of unusual basic isoelectric forms of the enzyme that appear to be developmentally regulated

1985 ◽  
Vol 230 (1) ◽  
pp. 75-82 ◽  
Author(s):  
L D Laury-Kleintop ◽  
I Damjanov ◽  
J A Alhadeff
Keyword(s):  
Postnatal Development ◽  
Liver Enzyme ◽  
Human Liver ◽  
Mouse Liver ◽  
Kinetic Studies ◽  
Total Activity ◽  
Cross Reactivity ◽  
Ph Optimum ◽  
Neuraminidase Treatment ◽  
Mouse Tissues

Mouse tissues contain unusual basic isoelectric forms of α-L-fucosidase (with approximate isoelectric points of 8.3 and 9.0) in addition to the usual acidic and neutral forms previously described in tissues of other species. These unusual forms are very prominent in placenta and foetal tissues and comprise approx, 50-80% of total activity up to 11 days of postnatal development. By 15 days of postnatal development, the basic forms are diminished in amount and comprise not more than 25% of total activity. Neuraminidase treatment of adult mouse liver α-L-fucosidase led to significantly decreased amounts of acidic forms and increased amounts of the basic forms, suggesting that these forms are chemically related at least in part by sialic acid residues. Comparative kinetic studies on mouse liver, human liver and mouse placental α-L-fucosidases indicated that they have the same Km (0.05-0.06 mM) for 4-methylumbelliferyl α-L-fucopyranoside but different pH optima and thermostability properties. Mouse liver α-L-fucosidase has one pH optimum (5.5) and an acidic shoulder (centred around pH 4.0) compared with two distinct optima (4.3 and 6.8) for the human liver enzyme. Mouse placental α-L-fucosidase has a pH-activity curve comparable with that of the mouse liver enzyme except that the acidic shoulder is absent. Mouse liver α-L-fucosidase is considerably more thermolabile after preincubation at 50 degrees C than are the human liver and mouse placental enzymes, which gave similar thermodenaturation curves. Immunochemical studies indicated that mouse and human α-L-fucosidases are dissimilar antigenically but exhibit some cross-reactivity. The IgG fraction of antibody prepared in goat against human liver α-L-fucosidase was ineffective by itself in immunoprecipitating mouse liver α-L-fucosidase, but 63% and 72% of the mouse liver and placental enzymes respectively could be immunoprecipitated in the double-antibody experiments under conditions that immunoprecipitated 92% of the human liver enzyme.

THE INVERTASE OF THE CORN RADICLE AND ITS ACTIVITY IN SUCCESSIVE STAGES OF GROWTH

1962 ◽  
Vol 40 (1) ◽  
pp. 113-126 ◽  
Author(s):  
J. A. Hellebust ◽  
D. F. Forward
Keyword(s):  
Reducing Sugars ◽  
Sugar Content ◽  
Kinetic Studies ◽  
Invertase Activity ◽  
Cell Number ◽  
Permeability Barrier ◽  
Ph Optimum ◽  
Intact Cells ◽  
Stages Of Growth ◽  
Growing Cell

Segments of the first 10 millimeters of corn radicle tips have been analyzed in terms of invertase activity, cell number, fresh and dry weights, and sugar content. Invertase activity per cell increased 40-fold as the meristematic cell advanced to the stage of most rapid elongation, and again subsided as the cell ceased to elongate and entered the stage of maturation. In the growing cell, the concentration of sucrose remained low while that of reducing sugars increased fivefold.The corn radicle invertase was found to be a β-fructofuranosidase with a Km of 0.006 M and a pH optimum of 4.6. Kinetic studies indicate that there is no change in the nature of the corn radicle invertase during cell growth. Equivalent activities of intact cells or segments and homogenates is consistent with the assumption that the enzyme is located outside the permeability barrier of the cells.

STUDIES ON A LIPOXIDASE SYSTEM FROM SUNFLOWER SEEDS AND SEEDLINGS

1958 ◽  
Vol 36 (11) ◽  
pp. 1149-1157 ◽  
Author(s):  
Oluf L. Gamborg ◽  
Saul Zalik
Keyword(s):  
Kinetic Studies ◽  
Maximum Activity ◽  
Sunflower Seeds ◽  
Reaction Products ◽  
Total Oxidation ◽  
Ph Optimum ◽  
Potassium Oleate ◽  
Enzyme Preparations ◽  
Cytoplasmic Proteins ◽  
Total Fat

Lipoxidase activity was obtained in enzyme preparations from sunflower seeds and seedlings. A partly purified preparation from seedlings was used for enzyme kinetic studies. The pH optimum was 6.8 and 100% oxygen was required for maximum activity. The Michaelis constant, with potassium linoleate as substrate, was 1.64 × 10−3 M. The reaction products were conjugated dienes. Enzyme activity was not affected by various metal and sulphydryl inhibitors nor by α-tocopherol, but catechol, α-naphthol, ethanol, and potassium oleate were inhibitory. Oil from flax, rape, and sunflower seeds reduced total oxidation of linoleate by the enzyme. Copper sulphate increased the rate and total oxidation of the linoleate–lipoxidase system, but iron, manganese, magnesium, and calcium were without effect. Lipoxidase activity was associated with mitochondrial (15,000 × g), intermediate (25,000 × g), and microsomal (100,000 × g) fractions, as well as with the soluble cytoplasmic proteins. Lipoxidase activity in seedlings increased during initial stages of germination, then decreased. The most rapid depletion of total fat in the seedlings coincided with maximum lipoxidase activity.

Isolation and Purification to Apparent Homogeneity of 4,5-Dioxovalerate Aminotransferase from Scenedesmus obliquus Mutant C-2 A′

1988 ◽  
Vol 43 (7-8) ◽  
pp. 563-571 ◽  
Author(s):  
A. Kah ◽  
D. Dörnemann ◽  
H. Senger
Keyword(s):  
Molecular Weight ◽  
Green Alga ◽  
Isoelectric Point ◽  
Pyridoxal Phosphate ◽  
Scenedesmus Obliquus ◽  
Ph Optimum ◽  
Isolation And Purification ◽  
Apparent Homogeneity ◽  
Unicellular Green Alga ◽  
Glyoxylate Aminotransferase

In the present paper the purification of a specific 4,5-dioxovalerate transaminase from pigment mutant C-2 A′ of the unicellular green alga Scenedesmus obliquus to apparent homogeneity is described. The newly isolated enzyme ʟ-glutamate: 4,5-dioxovalerate aminotransferase is not identical with ʟ-alanine: 4,5-dioxovalerate aminotransferase (EC 2.6.1.43) and ʟ-alanine: glyoxylate aminotransferase (EC 2.6.1.44). A procedure for the purification is described and the resulting homogeneous protein is characterized by its Kᴍ-values for oxo-substrates and amino donors, its pyridoxal phosphate requirement, reversability of the catalysis, pH-optimum, isoelectric point and its molecular weight.

scholarly journals Substrate-specificities of acid and alkaline ceramidases in fibroblasts from patients with Farber disease and controls

1982 ◽  
Vol 205 (2) ◽  
pp. 419-425 ◽  
Author(s):  
Toru Momoi ◽  
Yoav Ben-Yoseph ◽  
Henry L. Nadler
Keyword(s):  
Fatty Acids ◽  
Unsaturated Fatty Acids ◽  
Specific Activity ◽  
Kinetic Studies ◽  
Significant Activity ◽  
Acid Ceramidase ◽  
Ph Optimum ◽  
Farber Disease ◽  
Normal Controls

The specific activity of acid ceramidase (N-acylsphingosine deacylase, EC 3.5.1.23) was measured at pH4.5 in normal fibroblasts and in fibroblasts from patients with Farber disease and obligate heterozygotes. Greater activity was found when the synthetically made ceramide substrates contained shorter-chain fatty acids or higher content of double bonds. Acid ceramidase activities towards N-lauroyl- (C12:0), N-myristoyl- (C14:0) and N-palmitoyl- (C16:0) sphingosine (C18:1) were respectively about 38, 26 and 6 times higher than the activity towards the N-stearoyl (C18:0) substrate. The activity towards N-linolenoylsphingosine (C18:3/C18:1), N-linoleoylsphingosine (C18:2/C18:1) and N-oleoylsphingosine (C18:1/C18:1) were respectively about 5, 4 and 3 times higher than the activity towards N-stearoylsphingosine (C18:0/C18:1). The activity towards N-stearoyldihydrosphingosine (C18:0/C18:0) was about 40% of that towards N-stearoylsphingosine. Fibroblast alkaline ceramidase possessed significant activity only towards ceramides of unsaturated fatty acids, with a pH optimum of about 9.0. Deficiency of acid ceramidase activity in fibroblasts from patients with Farber disease and intermediate activities in obligate heterozygotes were demonstrated with all ceramides examined except for N-hexanoylsphingosine (C6:0/C18:1), whereas alkaline ceramidase activity was unaffected. Comparative kinetic studies of acid ceramidase activity with N-lauroylsphingosine and N-oleoylsphingosine demonstrated about 5 (2–12)-fold and 7 (4–17)-fold higher Km values in fibroblasts from patients with Farber disease as compared with normal controls. N-Lauroylsphingosine, towards which acid ceramidase activity in control fibroblasts was about 10 times higher than that towards N-oleoylsphingosine, may serve as a better substrate for enzymic diagnosis of Farber disease as well as for further characterization of the catalytically defective acid ceramidase.

Extracellular alkaline phosphatase from alveolar secretions of patients with pulmonary alveolar proteinosis

1981 ◽  
Vol 59 (4) ◽  
pp. 290-300 ◽  
Author(s):  
Denis Nadeau ◽  
Mark J. Reasor ◽  
Gary E. R. Hook
Keyword(s):  
Molecular Weight ◽  
Alkaline Phosphatase ◽  
Pulmonary Alveolar Proteinosis ◽  
Extracellular Enzyme ◽  
Soluble Form ◽  
Kinetic Properties ◽  
Ph Optimum ◽  
High Molecular Weight Complex ◽  
Heat Stable ◽  
Alveolar Proteinosis

Alkaline phosphatases in alveolar secretions from the lungs of patients with pulmonary alveolar proteinosis have been studied. A soluble form of alkaline phosphatase was isolated from the secretions and characterized. The extracellular enzyme had a pH optimum at 9.95; was stimulated by Mg2+, Ni2+, and Mn2+; was inhibited by Zn2+, Be2+, Cu2+, and low concentrations (8 mM) of L-homoarginine and imidazole; and was heat-stable at 55 °C. The soluble phosphatase existed primarily as a high molecular weight complex (excluded from Sepharose 4B) and could be dispersed into low molecular weight forms (205 000 – 285 000) by treatment with n-butanol. Following butanol treatment, the thermostability of the enzyme was markedly decreased but the kinetic properties such as the Km values, activation energies, and responses to various inhibitors were unchanged.The alkaline phosphatase may originate from unusual type 2 cells present in the alveoli of patients with pulmonary alveolar proteinosis.

THE ENZYMICALLY CATALYZED OXIDATION OF INDOLEACETIC ACID

1956 ◽  
Vol 34 (6) ◽  
pp. 905-926 ◽  
Author(s):  
E. R. Waygood ◽  
Ann Oaks ◽  
G. A. Maclachlan
Keyword(s):  
Indoleacetic Acid ◽  
Maleic Hydrazide ◽  
Organic Peroxide ◽  
Horse Radish Peroxidase ◽  
Reaction Sequence ◽  
Leaf Extracts ◽  
Naturally Occurring ◽  
Wheat Leaf ◽  
Wheat Leaves ◽  
Catalyzed Oxidation

Dialyzed wheat leaf extracts, catalase, and horse-radish peroxidase catalyze the decarboxylation and oxidation of indoleacetic acid at pH 5.0–6.0 in the presence of critical concentrations of manganese and monohydric phenols or resorcinol. The equivalent of 1 mole of carbon dioxide is liberated and 1 mole of oxygen consumed per mole of substrate. Manganic ions formed by a phenol–peroxidase–peroxide system initiate the decarboxylation and oxidation. A naturally occurring ether soluble factor from wheat leaves, and maleic hydrazide, can substitute for the active phenols. Catechol, hydroquinone, pyrogallol, seopoletin, and riboflavin, etc. competitively inhibit the oxidation. The nature of the active peroxide is discussed and a reaction sequence involving an organic peroxide or radical rather than hydrogen peroxide is submitted as being a possibility.

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