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Biomedical subjects

N E Tolbert

Publications and source records attributed to N E Tolbert.

At least 73 records · Page 4Linked to original sources
PubMed1980

Interaction of ribulosebisphosphate carboxylase/oxygenase with transition-state analogues.

2-C-Carboxy-D-ribitol 1,5-bisphosphate and 2-C-carboxy-D-arabinitol 1,5-bisphosphate have been synthesized, purified, and characterized. In the presence of Mg2+, 2-C-carboxy-D-arabinitol 1,5-bisphosphate binds to ribulose-1,5-bisphosphate carboxylase/oxygenase by a two-step mechanism. The first, rapid step is similar to the binding of ribulose 1,5-bisphosphate or its structural analogues. The second step is a slower process (k = 0.04 s-1) and accounts for the tighter binding of 2-C-carboxy-D-arabinitol 1,5-bisphosphate (Kd less than or approximately to 10(-11) M) than of 2-C-carboxy-D-ribitol 1,5-bisphosphate (Kd = 1.5 X 10(6) M). Both carboxypentitol bisphosphates exhibit competitive inhibition with respect to ribulose 1,5-bisphosphate. 2-C-(Hydroxymethyl)-D-ribitol 1,5-bisphosphate and 2-C-(hydroxymethyl)-D-arabinitol 1,5-bisphosphate were also synthesized; both are competitive inhibitors with respect to ribulose 1,5-bisphosphate with Ki = 8.0 X 10(-5) M and Ki = 5.0 X 10(-6) M, respectively. Thus, the carboxyl group of 2-C-carboxy-D-arabinitol 1,5-bisphosphate is necessary for maximal interaction with the enzyme. Additionally, Mg2+ is essential for the tight binding of 2-C-carboxy-D-arabinitol 1,5-bisophsphate. A model for catalysis of ribulose 1,5-bisphosphate carboxylation is discussed which includes a functional role for Mg2+ in the stabilization of the intermediate 2-C-carboxy-3-keto-D-arabinitol 1,5-bisphosphate. Mechanistic implications that arise from the stereochemistry of this intermediate are also discussed.

Carboxy-Lyases↗
PubMed1980

Mass spectrometric analysis of the reactions of ribulosebisphosphate carboxylase/oxygenase.

The products of the reaction of D-[2-13C]ribulose 1,5-bisphosphate with molecular oxygen in the presence of D-ribulose 1,5-bisphosphate carboxylase/oxygenase were analyzed, after dephosphorylation, as the trimethylsilyl derivatives of glycolate and glycerate by mass spectrometry. The extent of isotopic incorporation into [1-13C]glycolate from [1-13C]glycolate 2-phosphate produced in the oxidation reaction demonstrates that at least 95% of the glycolate 2-phosphate produced arises from carbon atoms 1 and 2 of D-ribulose 1,5-bisphosphate. When D-[2-18O]ribulose 1,5-bisphosphate was used, a significant amount of [1-18O]glycolate 2-phosphate was formed, indicating that O-2 of D-ribulose 1,5-bisphosphate is retained in the carboxyl oxygens of glycolate 2-phosphate. In addition, analyses of the products of the reaction between D-[2-13C]ribulose 1,5-bisphosphate and [13C]O2 confirm and extend the conclusions of an earlier report (Müllhofer, G., and Rose, I. A. (1965) J. Biol. Chem. 240, 1341-1346) that cleavage of D-ribulose 1,5-bisphosphate occurs between carbon atoms 2 and 3 during its enzymatic carboxylation. The results eliminate possible mechanisms involving the obligatory loss of O-2 of D-ribulose 1,5-bisphosphate during its enzymatic oxidation and confirm the specificity of carbon--carbon bond cleavage in both the oxygenase and carboxylase reactions.

Carbon Isotopes↗
PubMed1980

Ribulose-1,5-bisphosphate Carboxylase/Oxygenase and Polyphenol Oxidase in the Tobacco Mutant Su/su and Three Green Revertant Plants.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) was crystallized from a heterozygous tobacco (Nicotiana tabacum L.) aurea mutant (Su/su), its wild-type sibling (su/su), and green revertant plants regenerated from green spots found on leaves of haploid Su plants. No differences were found in the specific activity or kinetic parameters of this enzyme, when comparing Su/su and su/su plants of the same age, which had been grown under identical conditions. The enzyme crystallized from revertant plants was also identical to the enzyme from wild-type plants with the exception of one clone, designated R2. R2 has a chromosome number approximately double that of the wild-type (87.0 +/- 11.1 versus 48). The enzyme from R2 had a lower V(max) for CO(2), although the K(m) values were identical to those for the enzyme from the wild-type plant. The enzyme from all mutant plants had identical isoelectric points, identical molecular weight as demonstrated by migration on native and sodium dodecyl sulfate (SDS)-polyacrylamide gels, and the same ratio of large to small subunits as the enzyme from the wild-type. The large subunit of the enzyme from tobacco leaves exhibited a different electrophoretic pattern than did the large subunit from spinach; there were two to three bands on SDS-polyacrylamide gels for the tobacco enzyme whereas the enzyme from spinach had only one species of large subunit.Total polyphenol oxidase activity was the same in leaves from the heterozygous mutant (Su/su) and wild-type (su/su) plants when correlated with developmental age as represented by morphology rather than by the chronological age of the plants. There was a marked increase in the soluble activity of this enzyme with increasing age of both plant types and also as a result of varying environmental conditions. Ribulose-1,5-bisphosphate carboxylase/oxygenase activity correlated inversely with increases in the soluble activity of polyphenol oxidase in crude homogenates from which the carboxylase/oxygenase was crystallized over a generation of Su/su and su/su plants. Criteria are outlined for determining if differences in activity of ribulose-1,5-bisphosphate carboxylase/oxygenase are caused by an effect of polyphenol oxidase activity and/or by some other extrinsic parameter.

Journal Article↗
PubMed1980

Properties of a Membrane-bound Phosphatase from the Thylakoids of Spinach Chloroplasts.

A 3-phosphoglycerate phosphatase activity of about 2 micromoles per minute per milligram chlorophyll is associated with the thylakoid membranes of spinach chloroplasts. The K(m) for 3-phosphoglycerate is 3 millimolar. The enzyme can be solubilized from thylakoid membranes by treatment with 0.33 molar MgCl(2) or sodium deoxycholate. The activity is not stimulated by sulfhydryl reagents or the addition of 10 millimolar MgCl(2). The enzymic activity is insensitive to ethylenediaminetetraacetate. The pH optimum is broad, between 5.5 to 7.5. Although the substrate specificity is broad, 3-phosphoglycerate is the best substrate of those tested at neutral pH. However, p-nitrophenyl phosphate was a more effective substrate at pH 5.5. The enzyme exhibits the general characteristics of an acid phosphatase.

Journal Article↗
PubMed1979

Postnatal development of peroxisomal and mitochondrial enzymes in rat liver.

Subcellular organellles from livers of rats three days prenatal to 50 weeks postnatal were separated on sucrose gradients. The peroxisomes had a constant density of 1.243 g/ml throughout the life of the animal. The density of the mitochondria changed from about 1.236 g/ml at birth to a constant value of 1.200 g/ml after two weeks. The peroxisomal and mitochondrial fatty acid beta-oxidation and the peroxisomal and supernatant activities of catalase and glycerol-3-phosphate dehydrogenase were measured at each age, as well as the peroxisomal core enzyme, urate oxidase, and the mitochondrial matrix enzyme, glutamate dehydrogenase. All of these activities were very low or undetectable before birth. Mitochondrial glutamate dehydrogenase and peroxisomal urate oxidase reached maximal activities per g of liver at two and five weeks of age, respectively. Fatty acid beta-oxidation in both peroxisomes and mitochondria and peroxisomal glycerol-3-phosphate dehydrogenase exhibited maximum activities per g of liver between one and two weeks of age before weaning and then decreased to steady state levels in the adult. Peroxisomal beta-oxidation accounted for at least 10% of the total beta-oxidation activity in the young rat liver, but became 30% of the total in the liver of the adult female and 20% in the adult male due to a decrease in mitochondrial beta-oxidation after two weeks of age. The greatest change in beta-oxidation was in the mitochondrial fraction rather than in the peroxisomes. At two weeks of age, four times as much beta-oxidation activity was in the mitochondria as in the peroxisomal fraction. Peroxisomal glycerol-3-phosphate dehydrogenase activity accounted for 5% to 7% of the total activity in animals younger than one week, but only 1% to 2% in animals older than one week. Up to three weeks of age, 85% to 90% of the liver catalase was recovered in the peroxisomes. The activity of peroxisomal catalase per g of rat liver remained constant after three weeks of age, but the total activity of catalase further increased 2.5- to 3-fold, and all of the increased activity was in the supernatant fraction.

Age Factors↗
PubMed1979

Changes in Activity of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Three Peroxisomal Enzymes during Tomato Fruit Development and Ripening.

Ribulose-1,5-bisphosphate carboxylase/oxygenase, catalase, glycolate oxidase, and hydroxypyruvate reductase activities on a protein and fresh weight basis were measured over seven stages of tomato fruit development and ripening. Ribulose-1,5-bisphosphate carboxylase decreased steadily during fruit development from 23 +/- 8 nmoles per minute per milligram protein at the mature green stage to 13.4 +/- 2 at the table ripe stage. There was no change in partially purified preparations of the enzyme in the ratio of carboxylase to oxygenase activity, which was about 10. Catalase activity reached a maximum during the climacteric, simultaneously with increased ethylene and CO(2) formation. Glycolate oxidase activity decreased during early stages of development and was barely detectable at the climacteric. Hydroxypyruvate reductase, associated with serine formation by the glycerate pathway, increased in specific activity during early stages of tomato fruit ripening. In the fruit of the rin tomato mutant, which does not ripen normally, none of these changes in enzyme activity occurred.

Journal Article↗
PubMed1978

Active site studies of ribulose-1,5-bisphosphate carboxylase/oxygenase with pyridoxal 5'-phosphate.

There are 16 epsilon-amino groups of lysyl residues which are essential for the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These lysyl residues formed a Schiff base with pyridoxal 5'-phosphate which was stabilized by NaBH4 reduction. The stoichiometry of covalently bound pyridoxal 5'-phosphate after NaBH4 reduction was determined spectrophotometrically with a derived molar extinction coefficient of 4800 M-1 cm-1. The incorporation of pyridoxal 5'-phosphate into the protein was accompanied by loss of the carboxylase and oxygenase activities, but the ratio of their activities remained constant. Vmax, but not Km, values were changed by this modification of the amino acid groups. Half of the epsilon-amino groups of lysine appeared to be at the 8 catalytic sites and half at the 8 activator sites for CO2, as indicated by kinetics of reactivation of the enzyme activity during dissociation of the Schiff base between pyridoxal 5'-phosphate and the protein. Reduction with NaB3H4 revealed that all 16 of the lysyl residues were on the large subunit. Ribulose-1,5-bis-phosphate alone protected 16 amino groups from Schiff base formation with pyridoxal 5'-phosphate, and the enzyme activity was fully conserved. NaHCO3 increased and MgCl2 lowered slightly the protective effect of ribulose-1,5-bisphosphate. Modification of sulfhydryl groups by p-chloromercuribenzoic acid inhibited the enzyme and excluded binding of 8 equivalents of pyridoxal 5'-phosphate. Upon removal of the mercuribenzoate groups with excess dithiothreitol, the loss of enzyme activity was exponentially correlated with the binding of 8 mol of pyridoxal 5'-phosphate/mol of enzyme. In contrast to p-chloromercuribenzoic acid, iodoacetamide, which inhibited the catalysis, had no influence on the binding of the 16 pyridoxal 5'-phosphate/mol of enzyme. It is suggested that the CO2 activator site with one epsilon-amino group for binding of CO2 and the catalytic site with one epsilon-amino group of a yet unknown function are located closely together on the large subunits of the enzyme. The results are consistent with a sulfhydryl group as the proton acceptor opposite carbon 3 of ribulose-1,5-bisphosphate, and when this sulfhydryl group is blocked by the bulky mercuribenzoate group, but not by the small carboxyamidomethyl group, pyridoxal 5'-phosphate binding is modified.

Binding Sites↗
PubMed1978

Phosphoglycolate phosphatase. Purification and properties.

Phosphoglycolate phosphatase (EC 3.1.3.18) was purified 1500-fold from field-grown tobacco leaves by acetone fractionation, DEAE-cellulose and molecular sieve chromatography, and preparative polyacrylamide gel electrophoresis. Preparations were judged 90 to 95% homogeneous by chromatography on DEAE-cellulose, polyacrylamide gel electrophoresis, and by isoelectric focusing. The highest specific activity obtained was 468 mumol of phosphate released/min/mg of protein. The native protein has a molecular weight of 80,500 by Ferguson plot analysis and 86,300 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 20,700, indicating the P-glycolate phosphatase is a tetramer with identical or near identical subunits. The enzyme, freshly purified or in crude homogenates, had a pI of 3.8 to 3.9 pH units by isoelectric focusing. Phosphosphoglycolate phosphatase from spinach leaves has a molecular weight of 93,000 and, unlike the enzyme from tobacco leaves, it is extremely unstable after DEAE-cellulose chromatography and is inactivated by lipase (EC 3.1.1.3). The phosphatase from both plants was stabilized by the addition of citrate or isocitrate in the buffers. Ribose 5-phosphate is a competitive inhibitor of phosphoglycolate phosphatase at physiological concentration, while other phosphate esters of the photosynthetic carbon cycle were without effect.

Glycolates↗
PubMed1978

Mechanism of phosphoglycolate phosphatase. Studies of hydrolysis and transphosphorylation, substrate analogs, and sulfhydryl inhibition.

Enzymatic hydrolysis of phosphoglycolate proceeds through O-P bond cleavage as determined by reaction in H218O and analysis of the trimethylsilyl derivatives of the reaction products by mass spectrometry. No phosphate, hydroxyl, or carboxyl exchange occurred. End product inhibition was consistent with an ordered release of products, first the alcoholic product, glycolate, then phosphate. Analysis of the data indicated that the phosphate.enzyme complex dissociated very rapidly, and this was confirmed by use of alternative phosphomonoester substrates. Maximum velocity with these alternate substrates was found to be proportional to the pKa of of the corresponding alcoholic product, indicating the rate-limiting step in the reaction was protonation of the bridge oxygen. The use of substrate analogs further suggested that enzymatic specificity residues in exacting steric requirements for binding, and that large alkyl groups were excluded on this basis. Phosphoglycolate phosphatase catalyzed transphorylation to a wide range of acceptors and was inhibited at the active site by diisopropyl-fluorophosphate. The data suggest that the reaction sequence proceeds via a phosphoenzyme intermediate. N-Ethylmaleimide slowly inactivated the enzyme, the rate being greatly increased by P-glycolate, but not by magnesium or phosphate ions. The data suggest a conformational change is necessary to induce the transition state complex and phosphoenzyme formation. This may account for the phosphate acceptor specificity and is consistent with the failure to observe an enzyme-mediated H2O-phosphate oxygen exchange.

Glycolates↗
PubMed1978

Phosphoglycolate phosphatase. Effect of cation and pH on activity.

P-glycolate phosphatase requires divalent cations for activity. Activity-pH curves identified 2 active site residues with pK values at pH 5.7 and pH 9.1 in the presence of magnesium and at pH 5.7 and pH 7.5 in the presence of manganese or cobalt. Saturation velocity kinetics enabled the identification of two distinct divalent cation binding sites. The first, nonspecific site has a K0.5 of 2 to 7 x 10(-5) M, depending on the cation and the pH. The second site, which is specific for magnesium, binds this cation in a negatively cooperative fashion. The affinity at pH 8.1 varies approximately 100-fold from the first magnesium bound to the fourth. The negative cooperativity is greatest at high pH. Because the pH range of activity is very broad, both the phosphate monoanion and dianion of P-glycolate must be bound as the substrate. The concentration of these two species at the apparent Km is independent of magnesium concentration. The P-glycolate.magnesium complex is kinetically inactive.

Cobalt↗