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Biomedical subjects

D D Randall

Publications and source records attributed to D D Randall.

At least 37 records · Page 2Linked to original sources
PubMed1992

Autophosphorylation of the pea mitochondrial heat-shock protein homolog.

Highly purified mitochondria isolated from 14-day-old pea (Pisum sativum L., cv Little Marvel) seedlings contain a homolog of the 70,000 molecular weight heat-shock protein. The amount of this heat-shock cognate (Hsc70) was not reduced by limited proteolysis of intact mitochondria or by preparation of mitoplasts, indicating that the protein is located within the matrix compartment. Pea mitochondrial Hsc70 binds to immobilized ATP and reacts on western blots with anti-tomato Hsc70 antiserum. When a mitochondrial matrix fraction was incubated with [gamma-(32)P]ATP, there was phosphorylation of Hsc70. The extent of phosphorylation was increased by including calcium chloride in the reactions. Phospho amino acid analysis of purified mitochondrial Hsc70, phosphorylated in the calcium-stimulated reaction, revealed only phosphothreonine. Pea mitochondrial Hsc70, purified by a combination of ATP-agarose affinity chromatography and gel permeation chromatography, was labeled when incubated with ATP plus calcium, suggesting autophosphorylation rather than phosphorylation by an associated kinase. In analogy to mammalian cells and yeast, it is likely that mitochondrial Hsc70 acts as a molecular chaperone, and it is possible that phosphorylation plays a role in chaperone function.

Journal Article↗
PubMed1992

Asparagine and boric Acid cause allantoate accumulation in soybean leaves by inhibiting manganese-dependent allantoate amidohydrolase.

Our previous work demonstrated substantial accumulation of allantoate in leaf tissue of nodulated soybeans (Glycine max L. Merr., cv Williams) in response to nitrogen fertilization. Research was continued to determine the effect of nitrate and asparagine on ureide assimilation in soybean leaves. Stem infusion of asparagine into ureide-transporting soybeans resulted in a significant increase in allantoate concentration in leaf tissue. Accumulation of allantoate was also observed when asparagine was supplied in the presence of allopurinol, an inhibitor of xanthine dehydrogenase in the pathway of ureide biosynthesis. In vitro, asparagine was found to have an inhibitory effect on the activity of allantoate amidohydrolase, a Mn(2+)-dependent enzyme catalyzing allantoate breakdown in soybean leaves. The inhibition was partially overcome by supplemental Mn(2+) in enzyme assays. Another inhibitor of allantoate amidohydrolase, boric acid, applied foliarly on field-grown nodulated soybeans, caused up to a 10-fold increase in allantoate content of leaf tissue. Accumulation of allantoate in response to boric acid was either eliminated or greatly reduced in plants presprayed with Mn(2+). We conclude that elevated levels of allantoate in leaves of ureide-transporting soybeans fertilized with ammonium nitrate result from inhibition of allantoate degradation by asparagine and that Mn(2+) is a critical factor in this inhibition. Furthermore, our studies with asparagine and boric acid indicate that availability of Mn(2+) has a direct effect on ureide catabolism in soybean.

Journal Article↗
PubMed1991

Oxalate as a potent and selective inhibitor of spinach (Spinacia oleracea) leaf NADPH-dependent hydroxypyruvate reductase.

Purified spinach (Spinacia oleracea) NADPH-preferring hydroxypyruvate reductase (HPR-2) was potently and selectively inhibited by oxalate, an end product of metabolism in plants. Both hydroxypyruvate- and glyoxylate-dependent rates of the HPR-2 enzyme were affected. Oxalate acted as an uncompetitive inhibitor of the enzyme, with Ki values of 7 and 36 microM for the NADPH/hydroxypyruvate and NADPH/glyoxylate pairs of reactants respectively. Oxalate, at millimolar levels, caused less than 10% inhibition of purified spinach NADH-preferring HPR (HPR-1) and had no effect on purified spinach NADPH-preferring glyoxylate-specific reductase (GR-1). The inhibition of spinach HPR-2 by oxalate is by far the strongest for any known inhibitor of leaf HPR and GR activities. In photosynthetic tissues, oxalate could potentially act as a primary regulator of extraperoxisomal metabolism of hydroxypyruvate and glyoxylate.

Alcohol Oxidoreductases↗
PubMed1991

Acetyl-coenzyme a can regulate activity of the mitochondrial pyruvate dehydrogenase complex in situ.

In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon K(m) and K(i) relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [1987] Plant Physiol 83:306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or l-carnitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.

Journal Article↗
PubMed1991

Spinach leaf acetyl-coenzyme a synthetase: purification and characterization.

Acetyl-coenzyme A (CoA) synthetase was purified 364-fold from leaves of spinach (Spinacia oleracea L.) using ammonium sulfate fractionation followed by ion exchange, dye-ligand, and gel permeation chromatography. The final specific activity was 2.77 units per milligram protein. The average M(r) value of the native enzyme was about 73,000. The Michaelis constants determined for Mg-ATP, acetate, and coenzyme A were 150, 57, and 5 micromolar, respectively. The purified enzyme was sensitive to substrate inhibition by CoA with an apparent K(i) for CoA of 700 micromolar. The enzyme was specific for acetate; other short and long chain fatty acids were ineffective as substrates. Several intermediates and end products of fatty acid synthesis were examined as potential inhibitors of acetyl-CoA synthetase activity, but none of the compounds tested significantly inhibited acetyl-CoA synthetase activity in vitro. The properties of the purified enzyme support the postulated role of acetyl-CoA synthetase as a primary source of chloroplast acetyl-CoA.

Journal Article↗
PubMed1990

Mechanism of pyruvate inhibition of plant pyruvate dehydrogenase kinase and synergism with ADP.

The effects of various metabolites on pyruvate dehydrogenase (PDH) kinase-catalyzed inactivation of the pyruvate dehydrogenase complex (PDC) were studied in extracts of mitochondria purified from green leaf tissue of Pisum sativum L. Pyruvate was an uncompetitive inhibitor of PDH kinase with respect to ATP whereas ADP was a competitive inhibitor. In the absence of pyruvate a fivefold excess of ADP over ATP was required to inhibit PDH kinase, however, in the presence of pyruvate much lower ADP concentrations were required. Inhibition of PDH kinase by pyruvate and ADP was synergistic and the addition of ADP changed pyruvate from an uncompetitive inhibitor to a noncompetitive inhibitor. This result indicates that pyruvate acts as a "dead-end" inhibitor, binding to the PDH kinase-ADP reaction intermediate. Evidence is also presented that inhibition by pyruvate in the presence of thiamine pyrophosphate is due to the formation of hydroxyethyl thiamine pyrophosphate. The results are discussed in terms of the regulation of PDC activity by pyruvate and ADP during periods of increased demand for carbon skeleton biosynthesis by way of the tricarboxylic acid (TCA) cycle despite constraints imposed on TCA cycle flux by a high ATP/ADP ratio.

Adenosine Diphosphate↗
PubMed1990

Pea leaf mitochondrial pyruvate dehydrogenase complex is inactivated in vivo in a light-dependent manner.

We examined the effect of light on the activity of the mitochondrial pyruvate dehydrogenase complex (mt-PDC) by using intact green pea (Pisum sativum) seedlings. Upon illumination there is an initial drop in mtPDC activity followed by oscillations that dampen during the initial period of photosynthesis to a steady-state level of one-fourth or less of the mtPDC activity measured in the dark. The initial light-dependent decrease in mtPDC activity is inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (an inhibitor of photosystem II of photosynthesis) and does not occur in etiolated seedlings. Therefore, the effect of light is indirect and most likely associated with photosynthesis and/or photorespiration. Conditions that would be unfavorable for photorespiration also inhibited the light-dependent decrease in mtPDC activity.

Journal Article↗
PubMed1990

Identification and Characterization of Mitochondrial Acetyl-Coenzyme A Hydrolase from Pisum sativum L. Seedlings.

Mitochondria from Pisum sativum seedlings purified free of peroxisomal and chlorophyll contamination were examined for acetyl-coenzyme A (CoA) hydrolase activity. Acetyl-CoA hydrolase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of the mitochondria. The products, acetate and CoA, were quantified by two independent methods and verified that the observed activity was an acetyl-CoA hydrolase. The pea mitochondrial acetyl-CoA hydrolase showed a K(m) for acetyl-CoA of 74 micromolar and a V(max) of 6.1 nanomoles per minute per milligram protein. CoA was a linear competitive inhibitor of the enzyme with a K(is) of 16 micromolar. The sensitivity of the enzyme to changes in mole fraction of acetyl-CoA suggested that the changes in the intramitochondrial acetyl-CoA/CoA ratio may be an effective mechanism of control. The widespread distribution of mitochondrial acetyl-CoA hydrolase activity among different plant species indicated that this may be a general mechanism in plants for synthesizing acetate.

Journal Article↗
PubMed1990

Light as a signal influencing the phosphorylation status of plant proteins.

The phosphorylation-status of a number of plant enzymes has been shown to be altered in response to light. Phosphoenolpyruvate carboxylase is phosphorylated (more active) in C(4) plants in the light but CAM phosphoenolpyruvate carboxylase is phosphorylated (more active) in the dark. C(4) plant pyruvate, Pi dikinase is dephosphorylated (activated) in the light and sucrose phosphate synthase is less phosphorylated (more active) in the light. The mitochondrial pyruvate dehydrogenase is inactivated (phosphorylated) in the light. The reversal of these events occurs in the dark or when photosynthesis is inhibited. Phytochrome and blue light receptors also alter the phosphorylation-status of proteins. The evidence is rapidly increasing in support of signal transduction networks in plants that involve light reception.

Journal Article↗
PubMed1989

A synthetic peptide-directed antibody as a probe of the phosphorylation site of pyruvate dehydrogenase.

A synthetic peptide, corresponding to the 14-amino acid tryptic fragment containing phosphorylation sites one and two of bovine mitochondrial pyruvate dehydrogenase, was coupled to Limulus polyphemus hemocyanin and used to produce rabbit polyclonal antibodies. A positive signal was observed when Western blots of bovine, porcine, or yeast mitochondrial pyruvate dehydrogenase complexes were probed with the antibodies but not with blots of bacterial, cellular slime mold, plant mitochondrial, or plant plastid pyruvate dehydrogenase complexes. The antibodies also gave a positive signal when used to probe blots of the bovine kidney branched chain 2-oxo acid dehydrogenase complex. The ATP-dependent phosphorylation/inactivation of rat liver mitochondrial pyruvate dehydrogenase complex could be inhibited by prior incubation with the anti-peptide antibodies.

Amino Acid Sequence↗
PubMed1989

Regulation of pea mitochondrial pyruvate dehydrogenase complex : does photorespiratory ammonium influence mitochondrial carbon metabolism?

Inactivation of the pyruvate dehydrogenase complex catalyzed by pyruvate dehydrogenase kinase was studied using intact mitochondria purified from green leaf tissue of pea (Pisum sativum L.) and dialyzed mitochondrial extracts. Thiamine pyrophosphate was inhibitory in dialyzed extracts but not in intact mitochondria, except in the presence of high concentrations of Na(+). NH(4) (+), at concentrations as low as 20 micromolar, markedly stimulated inactivation in dialyzed extracts. K(+) in the range 1 to 10 millimolar also enhanced inactivation. In contrast, Na(+) was without affect at lower concentrations but was inhibitory at 10 to 100 millimolar levels. The effect of NH(4) (+) is discussed in relation to a possible regulatory interaction between photorespiratory NH(4) (+) production and the entry of carbon into the tricarboxylic acid cycle by way of the pyruvate dehydrogenase complex.

Journal Article↗
PubMed1989

Whole Leaf Carbon Exchange Characteristics of Phosphate Deficient Soybeans (Glycine max L.).

Low phosphate nutrition results in increased chlorophyll fluorescence, reduced photosynthetic rate, accumulation of starch and sucrose in leaves, and low crop yields. This study investigated physiological responses of soybean (Glycine max [L.] Merr.) leaves to low inorganic phosphate (Pi) conditions. Responses of photosynthesis to light and CO(2) were examined for leaves of soybean grown at high (0.50 millimolar) or low (0.05 millimolar) Pi. Leaves of low Pi plants exhibited paraheliotropic orientation on bright sunny days rather than the normal diaheliotropic orientation exhibited by leaves of high Pi soybeans. Leaves of plants grown at high Pi had significantly higher light saturation points (1000 versus 630 micromole photons [400-700 nanometers] per square meter per second) and higher apparent quantum efficiency (0.062 versus 0.044 mole CO(2) per mole photons) at ambient (34 pascals) CO(2) than did low Pi leaves, yet stomatal conductances were similar. High Pi leaves also had significantly higher carboxylation efficiency (2.90 versus 0.49 micromole CO(2) per square meter per second per pascal), a lower CO(2) compensation point (6.9 versus 11.9 pascals), and a higher photosynthetic rate at 34 pascals CO(2) (19.5 versus 6.7 micromoles CO(2) per square meter per second) than did low Pi leaves. Soluble protein (0.94 versus 0.73 milligram per square centimeter), ribulose-1,5-bisphosphate carboxylase/oxygenase content (0.33 versus 0.25 milligram per square centimeter), and ribulose-1,5-bisphosphate carboxylase/oxygenase specific activity (25.0 versus 16.7 micromoles per square meter per second) were significantly greater in leaves of plants in the high Pi treatment. The data indicate that Pi stress alters the plant's CO(2) reduction characteristics, which may in turn affect the plant's capacity to accommodate normal radiation loads.

Journal Article↗
PubMed1988

Purification and characterization of a novel NADPH(NADH)-dependent hydroxypyruvate reductase from spinach leaves. Comparison of immunological properties of leaf hydroxypyruvate reductases.

A novel hydroxypyruvate reductase preferring NADPH to NADH as a cofactor was purified over 1500-fold from spinach leaf extracts. The enzyme was an oligomer of about 70 kDa, composed of two subunits of 38 kDa each. The Km for hydroxypyruvate (with NADPH) was about 0.8 mM in the pH range 5.5-6.5, and 0.3 mM at pH 8.2. The Vmax. was highest in the pH range 5.5-6.5 and decreased by about 65% at pH 8.2. Above pH 6.0, the enzyme was prone to a strong substrate inhibition by hydroxypyruvate. The reductase could use glyoxylate as an alternative substrate, with rates up to one-quarter of those with hydroxypyruvate. This glyoxylate-dependent activity preferred NADPH to NADH as a cofactor. Rabbit antibodies prepared against NADPH(NADH)-hydroxypyruvate reductase were highly specific for this enzyme and did not cross-react with peroxisomal NADH(NADPH)-dependent hydroxypyruvate reductase, as found by Western immunoblots of proteins from leaf extracts of spinach, pea and wheat. Antibodies raised against purified NADH(NADPH)-hydroxypyruvate reductase were also highly specific, recognizing only their own antigen. To our knowledge, this is the first report in the literature of the occurrence of NADPH(NADH)-hydroxypyruvate reductase in leaves, and the first to provide immunological comparison of leaf hydroxypyruvate reductases. Because of the relatively high rates of the novel reductase in leaf extracts (at least 20 mumol/h per mg of chlorophyll), this enzyme might be an important side-component of the glycollate pathway (photorespiration), possibly utilizing hydroxypyruvate 'leaked' from peroxisomes, and thus contributing to the glycerate pool derived from glycollate. Because of the glyoxylate-dependent activity, the enzyme may also contribute to glycollate formation in leaves.

Alcohol Oxidoreductases↗
PubMed1988

Ureide Catabolism in Soybeans : III. Ureidoglycolate Amidohydrolase and Allantoate Amidohydrolase Are Activities of an Allantoate Degrading Enzyme Complex.

We demonstrate that allantoate is catabolized in soybean seedcoat extracts by an enzyme complex that has allantoate amidohydrolase and ureidoglycolate amidohydrolase activities. Soybean seedcoat extracts released (14)CO(2) from [ureido-(14)C]ureidoglycolate under conditions in which urease is not detectable. CO(2) and glyoxylate are enzymically released in a one to one ratio indicating that ureidoglycolate amidohydrolase is the responsible activity. Ureidoglycolate amidohydrolase has a K(m) of 85 micromolar for ureidoglycolate. Glyoxylate and CO(2) are enzymically released from allantoate at linear rates in a one to 2.3 ratio from 5 to 30 min. This ratio is consistent with the degradation of allantoate to two CO(2) and one glyoxylate with approximately 23% of the allantoate degraded reacting with 2-mercaptoethanol to yield 2-hydroxyethylthio, 2'-ureido, acetate (RG Winkler, JC Polacco, DG Blevins, DD Randall 1985 Plant Physiol 79: 787-793). That [(14)C]urea production from [2,7-(14)C]allantoate is not detectable indicates that allantoate-dependent glyoxylate production is enzymic and not a result of nonenzymic hydrolysis of a ureido intermediate (nonenzymic hydrolysis releases urea). These results and those from intact tissue studies (RG Winkler DG Blevins, JC Polacco, DD Randall 1987 Plant Physiol 83: 585-591) suggest that soybeans have a second amidohydrolase reaction (ureidoglycolate amidohydrolase) that follows allantoate amidohydrolase in allantoate catabolism. The rate of (14)CO(2) release from [2,7-(14)C]allantoate is not reduced when the volume of the reaction mixture is increased, suggesting that the release of (14)CO(2) is not dependent on the accumulation of free intermediates. That [2,7-(14)C]allantoate dependent (14)CO(2) release is not proportionally diluted by unlabeled ureidoglycolate indicates that the reaction is carried out by an enzyme complex. This is the first report of ureidoglycolate amidohydrolase activity in any organism and the first in vitro demonstration in plants that the ureido-carbons of allantoate can be completely degraded to CO(2) without a urea intermediate.

Journal Article↗
PubMed1988

Regulation of steady state pyruvate dehydrogenase complex activity in plant mitochondria : reactivation constraints.

The requirements for reactivation (dephosphorylation) of the pea (Pisum sativum L.) leaf mitochondrial pyruvate dehydrogenase complex (PDC) were studied in terms of magnesium and ATP effects with intact and permeabilized mitochondria. The requirement for high concentrations of magnesium for reactivation previously reported with partially purified PDC is shown to affect inactivation rather than reactivation. The observed rate of inactivation catalyzed by pyruvate dehydrogenase (PDH) kinase is always greater than the reactivation rate catalyzed by PDH-P phosphatase. Thus, reactivation would only occur if ATP becomes limiting. However, pyruvate which is a potent inhibitor of inactivation in the presence of thiamine pyrophosphate, results in increased PDC activity. Analysis of the dynamics of the phosphorylation-dephosphorylation cycle indicated that the covalent modification was under steady state control. The steady state activity of PDC was increased by addition of pyruvate. PDH kinase activity increased threefold during storage of mitochondria suggesting that there may be an unknown level of regulation exerted on the enzyme complex.

Journal Article↗
PubMed1988

Regulation of the phosphorylation of mitochondrial pyruvate dehydrogenase complex in situ: effects of respiratory substrates and calcium.

The activity of the pyruvate dehydrogenase complex (PDC), as controlled by reversible phosphorylation, was studied in situ with mitochondria oxidizing dfifferent substrates. PDCs from both plant and animal tissues were inactivated when pyruvate became limiting. The PDC did not inactivate in the presence of saturating levels of pyruvate. Calcium stimulated reactivation of PDC in chicken heart but not pea (Pisum sativum L.) leaf mitochondria. With pea leaf mitochondria oxidizing malate, inactivation of PDC was pH dependent corresponding to the production of pyruvate via malic enzyme. When pea leaf mitochondria oxidized succinate or glycine, PDC was inactivated. This inactivation was reversed by the addition of pyruvate. Reactivation by pyruvate was enhanced by the addition of thiamine pyrophosphate, as previously observed with nonrespiring mitochondria. These results indicate a major role for pyruvate in regulating the covalent modification of the PDC.

Journal Article↗