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Biomedical subjects

N E Tolbert

Publications and source records attributed to N E Tolbert.

At least 91 records · Page 5Linked to original sources
PubMed1978

NADH-Nitrate Reductase Inhibitor from Soybean Leaves.

A NADH-nitrate reductase inhibitor has been isolated from young soybean (Glycine max L. Merr. Var. Amsoy) leaves that had been in the dark for 54 hours. The presence of the inhibitor was first suggested by the absence of nitrate reductase activity in the homogenate until the inhibitor was removed by diethylaminoethyl (DEAE)-cellulose chromatography. The inhibitor inactivated the enzyme in homogenates of leaves harvested in the light. Nitrate reductases in single whole cells isolated through a sucrose gradient were equally active from leaves grown in light or darkness, but were inhibited by addition of the active inhibitor.The NADH-nitrate reductase inhibitor was purified 2,500-fold to an electrophoretic homogeneous protein by a procedure involving DEAE- cellulose chromatography, Sephadex G-100 filtration, and ammonium sulfate precipitation followed by dialysis. The assay was based on nitrate reductase inhibition. A rapid partial isolation procedure was also developed to separate nitrate reductase from the inhibitor by DEAE-cellulose chromatography and elution with KNO(3). The inhibitor was a heat-labile protein of about 31,000 molecular weight with two identical subunits. After electrophoresis on polyacrylamide gel two adjacent bands of protein were present; an active form and an inactive form that developed on standing. The active factor inhibited leaf NADH-nitrate reductase but not NADPH-nitrate reductase, the bacterial nitrate reductase or other enzymes tested. The site of inhibition was probably at the reduced flavin adenine dinucleotide-NR reaction, since it did not block the partial reaction of NADH-cytochrome c reductase. The inhibitor did not appear to be a protease. Some form of association of the active inhibitor with nitrate reductase was indicated by a change of inhibitor mobility through Sephadex G-75 in the presence of the enzyme. The inhibition of nitrate reductase was noncompetitive with nitrate but caused a decrease in V(max).The isolated inhibitor was inactivated in the light, but after 24 hours in the dark full inhibitory activity returned. Equal amounts of inhibitor were present in leaves harvested from light or darkness, except that the inhibitor was at first inactive when rapidly isolated from leaves in light. Photoinactivation of yellow impure inhibitor required no additional components, but inactivation of the purified colorless inhibitor required the addition of flavin.Preliminary evidence and a procedure are given for partial isolation of a component by DEAE-cellulose chromatography that stimulated nitrate reductase. The data suggest that light-dark changes in nitrate reductase activity are regulated by specific protein inhibitors and stimulators.

Journal Article↗
PubMed1977

Inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase by xylulose 1,5-bisphosphate.

Xylulose 1,5-bisphosphate is a potent inhibitor of ribulose-1,5-bisphosphate carboxylase/oxygenase. The enzyme was inhibited 50% at a xylulose 1,5-bisphosphate concentration of 0.56 micrometer and an enzyme concentration of 0.14 micrometer. When both ribulose 1,5-bisphosphate and xylulose 1,5-bisphosphate were added simultaneously to the enzyme, this inhibition appeared to be competitive. A preincubation of 20 to 30 min was needed for maximum inhibition. The inhibitory effect of this compound is probably exerted through its binding at the ribulose 1,5-bisphosphate active site, since both the carboxylase and oxygenase activities were inhibited similarly.

Carboxy-Lyases↗
PubMed1976

Glyoxylate aminotransferase in peroxisomes from rat liver and kidney.

An aminotransferase was isolated from peroxisomes that had been separated by isopycnic centrifugation of homogenates from rat liver or kidney. The enzyme was located only in the peroxisomes and in the soluble fraction, presumably from broken peroxisomes. Within the peroxisomes, this aminotransferase was in the soluble matrix. This specific aminotransferase was not found in spinach leaves. The enzyme was relatively specific for glyoxylate as the amino group acceptor. L-Leucine and L-phenylalanine were the preferred amino donors; other amino acids were less efficiently utilized. Rates were 181 nmol X min-1 of peroxisomal protein with leucine, and 134 with phenylalanine. The rate with serine was only 28% as fast and there was no reaction with glutamate. The reactions were essentially irreversible. Treatment of peroxisomes with 0.04% Triton X-100 increased enzyme activity 80%. The enzyme in the peroxisomes was stable at 50 degrees. The enzyme was purified 100-fold. Activities with leucine, phenylalanine, and histidine could not be separated by gel filtration and DEAE-cellulose chromatography. Its molecular weight was estimated to be 72,000. Reaction kinetics were ping-pong. The Km (glyoxylate) was 0.5 mM with leucine and 0.67 mM with phenylalanine. Km (leucine) was 2.5 mM and Km (phenylalanine) was 2.8 mM. Substrate inhibition occurred at over 4 mM glyoxylate but did not occur with the amino donors. pH optima were 8.5 for leucine and phenylalanine and 6.2 for histidine. There was no requirement for exogenous pyridoxal phosphate, but activity was inhibited by phenylhydrazine and isonicotinic acid hydrazide. The glyoxylate aminotransferase developed postnatally and increased with age until rats were 40 days old. There was more activity in female than male rats. About 50% of the activity disappeared if rats were starved overnight. Clofibrate treatment of male rats increased this enzyme activity in isolated peroxisomes. Rats on a high casein diet had slightly higher enzyme activity.

Animals↗
PubMed1976

Variables Affecting the CO(2) Compensation Point.

Some factors influencing dark respiration, photorespiration, and photosynthesis were examined for their effect on the CO(2) compensation point (70 mul/l) of detached soybean (Glycine max) leaf discs. A higher compensation point in young leaves decreased to the constant value after leaf expansion and maturation, but increased again during senescence. The compensation point was 40 to 50% higher in plants grown in the summer than in the winter. The compensation point and dark respiration increased with temperatures above 17 C. Below 17 C dark respiration continued to decrease, but the compensation point did not decrease further. Increasing light intensities did not affect the compensation point.The effect of selected chemicals on the compensation point were surveyed. Some buffer components did not greatly alter the compensation point but organic solvents lowered it. Potassium phosphate and pyrophosphate greatly increased it. Inhibitors of photosynthesis increased the compensation point. Hydroxypyridinemethanesulfonate and sodium bisulfite severely inhibited photosynthesis in soybean leaves, stimulated dark respiration, and increased the compensation point.

Journal Article↗
PubMed1976

Photosynthetic Carbon Metabolism in the Palisade Parenchyma and Spongy Parenchyma of Vicia faba L.

Palisade parenchyma cells and spongy parenchyma cells were isolated separately from Vicia faba L. leaflets. Extracts of the cell isolates were assayed for several enzymes involved in CO(2) fixation and photorespiration. When compared on a chlorophyll basis, the levels of enzyme activities either were equal in the different cell types or were greater in the spongy parenchyma; this difference is a reflection, perhaps, of the higher protein-chlorophyll ratio in the latter tissue. The distribution of radioactivity in the products of photosynthesis by each cell type was the same at various times after exposure to NaH(14)CO(3), and the kinetics of (14)C incorporation into these compounds was similar. However, a larger percentage of radioactivity was incorporated by the cell isolates into the 80% ethanol-insoluble fraction and correspondingly less into the neutral fraction as compared to whole leaf. It was concluded that photosynthetic CO(2) fixation is similar in the different mesophyll tissues from which these cells were derived.

Journal Article↗
PubMed1975

Ribulose diphosphate carboxylase/oxygenase. IV. Regulation by phosphate esters.

The stimulation or inhibition of ribulose diphosphate oxygenase by a variety of compounds is compared with the reported effects on these compounds on the ribulose diphosphate carboxylase activity. A possible transition state analog of ribulose diphosphate, 2-carboxyribitol 1, 5-diphosphate, at a molar ratio of inhibitor to enzyme of 10 to 1, irreversibly inactivates the oxygenase and carboxylase activities. This is consistent with the hypothesis that there may be a single active site for both the carboxylase and oxygenase activities. Several compounds of the reductive pentose photosynthetic carbon cycle act as effectors of the ribulose diphosphate oxygenase in a manner complementary to their reported effect upon the carboxylase. Ribose 5-phosphate inhibits the oxygenase with an apparent Ki of 1.8 mM, but it is reported to activate the carboxylase; fructose 6-phosphate and glucose 6-phosphate act similarly but are less effective than ribose 5-phosphate. Fructose 1. 6-diphosphate stimulates the oxygenase at low magnesium ion concentrations. The stimulatory effect of 6-phosphogluconate on the oxygenase is associated with a 3-fold reduction of the Km (Mg2+). ATP inhibits the oxygenase but has been reported to stimulate the carboxylase; pyrophosphate acts in an opposite manner. From these results it appears that the ratio of carboxylase to oxygenase activity may be a variable factor with predictable subsequent alteration in the ratio between photosynthetic CO2 fixation and photorespiration.

Adenosine Triphosphate↗
PubMed1975

Ribulose diphosphate carboxylase/oxygenase. III. Isolation and properties.

Similarities in properties of ribulose diphosphate carboxylase and oxygenase activities further substantiate the hypothesis that the same protein catalyzes both reactions. The Km (ribulose diphosphate) is 0.33 mM for the ribulose diphosphate oxygenase, when assayed in air with an oxygen electrode. Maximum activity is obtained with 10 to 35 mM MgCl2. Higher MgCl2 concentrations are inhibitory, but they shift the pH optimum from 9.3 or 9.4 to 8.7 or 9.0. MnCl2 is an effective cofactor of the oxygenase and some activity is obtained with CoCl2. Both the ribulose diphosphate carboxylase and oxygenase activity of the purified protein from spinach leaves are slowly inactivated by storage at 0 degrees and reactivated in 10 min at 50 degrees, provided both 25 mM MgCl2 and 1 mM dithiothreitol are present. The sulfhydryl groups of the enzyme which react rapidly with 5,5'-dithiobis(2-nitrobenzoic acid) are approximately 4 at pH 7.8 and 11 at pH 9.4. At both pH values ribulose diphosphate prevents two of these sulfhydryl groups from reacting with this reagent. About 50% inhibition of the oxygenase activity at pH 9.0 occurs with 50 mM bicarbonate in the presence of 3 mM ribulose diphosphate, and from variations in these parameters the inhibition is attributed to the CO2 species. The purified enzyme of acrylamide gels prevented the reduction of nitroblue tetrazolium in the presence of the superoxide radical, but the enzyme in solution did not react as a superoxide dismutase.

Bicarbonates↗
PubMed1974

Enzymes related to lactate metabolism in green algae and lower land plants.

Cell-free extracts of Chlorella pyrenoidosa contained two enzymes capable of oxidizing d-lactate; these were glycolate dehydrogenase and NAD(+)-dependent d-lactate dehydrogenase. The two enzymes could be distinguished by differential centrifugation, glycolate dehydrogenase being largely particulate and NAD(+)-d-lactate dehydrogenase being soluble. The reduction of pyruvate by NADH proceeded more rapidly than the reverse reaction, and the apparent Michaelis constants for pyruvate and NADH were lower than for d-lactate and NAD(+). These data indicated that under physiological conditions, the NAD(+)-linked d-lactate dehydrogenase probably functions to produce d-lactate from pyruvate.Lactate dehydrogenase activity dependent on NAD(+) was found in a number of other green algae and in the green tissues of a few lower land plants. When present in species which contain glycolate oxidase rather than glycolate dehydrogenase, the enzyme was specific for l-lactate rather than d-lactate. A cyclic system revolving around the production and utilization of d-lactate in some species and l-lactate in certain others is proposed.

Journal Article↗