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Biomedical subjects

B G Harris

Publications and source records attributed to B G Harris.

At least 55 records · Page 3Linked to original sources
PubMed1987

Ascaris suum phosphofructokinase. Phosphorylation by protein kinase and sequence of the phosphopeptide.

Phosphorylation of the ascarid phosphofructokinase with the catalytic subunit of beef heart cyclic AMP-dependent protein kinase results in the incorporation of 1 mol of P/mol of subunit. Accompanying the phosphorylation there is a 3-4-fold increase in catalytic activity when measured at pH 6.8 with inhibitory levels of ATP. Studies on the effect of phosphorylation on the ATP saturation curve demonstrated that phosphorylation decreased the inhibitory action of ATP. The apparent Km of the catalytic subunit for the phosphofructokinase was 11.2 microM. Chymotryptic or subtilisin digestion of the labeled enzyme released distinct but overlapping phosphopeptides that were purified by high pressure liquid chromatography and sequenced by gas phase peptide sequencing. The sequence of the chymotryptic peptide was Ala-Lys-Gly-Arg-Ser-Asp-Ser(P)-Ile-Val-Pro-Thr. Based on these results and earlier observations, it is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of energy metabolism in the parasitic helminth.

Adenosine Triphosphate↗
PubMed1986

Reversible activation and inactivation of phosphofructokinase from Ascaris suum by the action of tissue-homologous protein phosphorylating and dephosphorylating enzymes.

In the presence of ATP-Mg2+, purified phosphofructokinase from Ascaris suum muscle was effectively phosphorylated and activated in vitro by a protein kinase purified from the same tissue. Both effects were reversed by the action of a purified protein phosphatase from the same tissue. The findings suggest the presence of a highly potent interconversion mechanism for phosphofructokinase in the muscle of the parasitic nematode.

Ascaris↗
PubMed1986

pH dependence of kinetic parameters for oxalacetate decarboxylation and pyruvate reduction reactions catalyzed by malic enzyme.

Both chicken liver NADP-malic enzyme and Ascaris suum NAD-malic enzyme catalyze the metal-dependent decarboxylation of oxalacetate. Both enzymes catalyze the reaction either in the presence or in the absence of dinucleotide. The presence of dinucleotide increases the affinity of oxalacetate for the chicken liver NADP-malic enzyme, but this information could not be obtained in the case of A. suum NAD-malic enzyme because of the low affinity of free enzyme for NAD. The kinetic mechanism for oxalacetate decarboxylation by the chicken liver NADP-malic enzyme is equilibrium ordered at pH values below 5.0 with NADP adding to enzyme first. The Ki for NADP increases by a factor of 10 per pH unit below pH 5.0. An enzyme residue is required protonated for oxalacetate decarboxylation (by both enzymes) and pyruvate reduction (by the NAD-malic enzyme), but the beta-carboxyl of oxalacetate must be unprotonated for reaction (by both enzymes). The pK of the enzyme residue of the chicken liver NADP-malic enzyme decreases from a value of 6.4 in the absence of NADP to about 5.5 with Mg2+ and 4.8 with Mn2+ in the presence of NADP. The pK value of the enzyme residue required protonated for either oxalacetate decarboxylation or pyruvate reduction for the A. suum NAD-malic enzyme is about 5.5-6.0. Although oxalacetate binds equally well to protonated and unprotonated forms of the NADP-enzyme, the NAD-enzyme requires that oxalacetate or pyruvate selectively bind to the protonated form of the enzyme. Both enzymes prefer Mn2+ over Mg2+ for oxalacetate decarboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Protonation mechanism and location of rate-determining steps for the Ascaris suum nicotinamide adenine dinucleotide-malic enzyme reaction from isotope effects and pH studies.

The pH dependence of the kinetic parameters and the primary deuterium isotope effects with nicotinamide adenine dinucleotide (NAD) and also thionicotinamide adenine dinucleotide (thio-NAD) as the nucleotide substrates were determined in order to obtain information about the chemical mechanism and location of rate-determining steps for the Ascaris suum NAD-malic enzyme reaction. The maximum velocity with thio-NAD as the nucleotide is pH-independent from pH 4.2 to 9.6, while with NAD, V decreases below a pK of 4.8. V/K for both nucleotides decreases below a pK of 5.6 and above a pK of 8.9. Both the tartronate pKi and V/Kmalate decrease below a pK of 4.8 and above a pK of 8.9. Oxalate is competitive vs. malate above pH 7 and noncompetitive below pH 7 with NAD as the nucleotide. The oxalate Kis increases from a constant value above a pK of 4.9 to another constant value above a pK of 6.7. The oxalate Kii also increases above a pK of 4.9, and this inhibition is enhanced by NADH. In the presence of thio-NAD the inhibition by oxalate is competitive vs. malate below pH 7. For thio-NAD, both DV and D(V/K) are pH-independent and equal to 1.7. With NAD as the nucleotide, DV decreases to 1.0 below a pK of 4.9, while D(V/KNAD) and D(V/Kmalate) are pH-independent. Above pH 7 the isotope effects on V and the V/K values for NAD and malate are equal to 1.45, the pH-independent value of DV above pH 7. From the above data, the following conclusions can be made concerning the mechanism for this enzyme. Substrates bind to only the correctly protonated form of the enzyme. Two enzyme groups are necessary for binding of substrates and catalysis. Both NAD and malate are released from the Michaelis complex at equal rates which are equal to the rate of NADH release from E-NADH above pH 7. Below pH 7 NADH release becomes more rate-determining as the pH decreases until at pH 4.0 it completely limits the overall rate of the reaction.

Animals↗
PubMed1986

Accelerated autoimmune disease and lymphoreticular neoplasms in F1 hybrid PN/NZB and NZB/PN mice.

This report describes the first studies of inheritance of autoimmunity in inbred Palmerston North (PN) mice, a model of systemic lupus erythematosus (SLE). Mating of PN mice with the nonautoimmune DBA/2 strain produced evidence that PN disease had a recessive mode of inheritance. When PN mice were crossed with autoimmune NZB mice, female offspring from both crosses developed anti-DNA antibodies and died prematurely with vasculitis, renal disease, and lymphomas. In contrast, reciprocal hybrid males had different patterns of mortality; PN/NZB males from the PN female X NZB male mating had moderately prolonged life spans, whereas NZB/PN males from the opposite cross (NZB female X PN male) had prolonged survival to the mean age of 104 weeks. To determine if testicular hormones were solely responsible for increased longevity in hybrid males, PN/NZB and NZB/PN mice were castrated at 2 weeks of age and compared to sham-operated littermate controls. Prepubertal castration did not influence longevity in PN/NZB males, but loss of gonadal hormones significantly reduced life spans in reciprocal NZB/PN males. Female hybrids were not affected by oophorectomy. Because castration changed disease expression only in male hybrids from the NZB female X PN male cross, it was concluded that parentage influenced sensitivity to the protective effects of male hormones. Although surgical sterilization had disparate effects on males, castrated PN/NZB and NZB/PN males consistently outlived oophorectomized females. The lack of clear-cut reversal of disease in males subjected to early castration suggested that nonhormonal, possibly genetic, factors contributed to longevity in both groups of male hybrids.

Animals↗
PubMed1985

Diethylpyrocarbonate inactivation of NAD-malic enzyme from Ascaris suum.

Treatment with diethylpyrocarbonate results in a first-order loss of the malate oxidative decarboxylase activity of NAD-malic enzyme. First-order plots are biphasic, with about 40-50% activity loss in the first phase. The inactivation process is not saturable, and the second-order rate constant is 4.7 M-1 S-1. Malate (250 mM) provides complete protection against inactivation (as measured by a decrease in the inactivation rate), and less malate is required with Mg2+ present. Partial protection (50%) is afforded by either NAD+ (1 mM) or Mg2+ (50 mM). Treatment of modified (inactive) enzyme with hydroxylamine restores activity to 100% of the control when corrected for the effect of hydroxylamine on unmodified enzyme. A total of 10-13 histidine residues/subunit are acylated concomitant with loss of activity while 1-2 tyrosines are modified prior to any activity loss. The presence of Mg2+ and malate at saturating concentrations protect 1-2 histidine residues/subunit. The intrinsic fluorescence of the enzyme decreases with time after addition of diethylpyrocarbonate, but the rate constant for this process is at least 10-fold too low to account for the biphasicity observed in the first order plots. The histidine modified which is responsible for loss of activity has a pK of 8.3 as determined from the pH dependence of the rate of inactivation. The histidine titrated is still modified under conditions where the residue is completely protonated but at a rate 1/100 the rate of the unprotonated histidine. The results suggest that 1-2 histidines are in or near the malate binding site and are required for malate oxidative decarboxylation.

Animals↗
PubMed1984

Kinetic mechanism in the direction of oxidative decarboxylation for NAD-malic enzyme from Ascaris suum.

Measurement of the initial rate of the malic enzyme reaction varying the concentration of NAD at several different fixed levels of Mg2+ (0.25-1.0 mM) and a single malate concentration gave a pattern which intersects to the left of the ordinate. Repetition of this initial velocity pattern at several additional malate concentrations and treatment in terms of a terreactant mechanism suggests an ordered mechanism in which NAD adds prior to Mg2+ which must add prior to malate. On the other hand, when a broader concentration range of Mg2+ (0.25-50 mM) is used, data are consistent with a random mechanism in which Mg2+ must add prior to malate. By use of product inhibition studies, pyruvate is competitive vs. malate and noncompetitive vs. NAD, while NADH is competitive vs. NAD and noncompetitive vs. malate. These results are consistent with the random addition of substrates and further suggest rapid equilibrium random release of products. Tartronate, a dead-end analogue of malate, is competitive vs. malate and noncompetitive vs. NAD. Thio-NAD is a slow substrate which is used at 2.4% the maximum rate of NAD. When used as a dead-end analogue of NAD, thio-NAD is competitive vs. NAD and gives a complex inhibition pattern vs. malate in which competitive inhibition is apparent at low concentrations of malate (less than 12.5 mM), and this changes to uncompetitive inhibition at high concentrations of malate (greater than 12.5 mM). These data are consistent with a steady-state random mechanism in the direction of oxidative decarboxylation in which Mg2+ adds in rapid equilibrium prior to malate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Determination of dissociation constants for enzyme-reactant complexes for NAD-malic enzyme by modulation of the thiol inactivation rate.

Incubation of NAD-malic enzyme from Ascaris suum with the sulfhydryl reagents N-ethylmaleimide (NEM), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), or 4,4'-dithiodipyridine (4-PDS) results in rapid and complete loss of malate oxidative decarboxylase and pyruvate reductive carboxylase activities. With DTNB, this loss of activity occurs concomitantly with the modification of about 1 thiol group per subunit. The majority of the activity is lost when 0.5 thiol per subunit is modified, indicative of possible half-site reactivity with DTNB. Complete restoration of activity follows addition of dithiothreitol to enzyme inactivated by DTNB and 4-PDS but not with NEM. With the DTNB-inactivated enzyme, replacement of the thionitrobenzoate moiety with cyanide restores activity. The presence of a divalent metal ion (Mg2+ or Mn2+) results in enhancement of the inactivation rate with all sulfhydryl reagents. However, malate alone or competitors of malate provide protection which is more effective in the presence of Mg2+, while NAD provides only about 25% protection. Thus, the Ascaris suum NAD-malic enzyme has a thiol group probably located in or near the malate binding site, which is not essential for enzyme activity. The changes in the rate of inactivation in the presence of reactants were used to determine the dissociation constants for enzyme-reactant complexes. These data suggest that all three possible binary and all three possible ternary complexes form. The binding of malate to free enzyme exhibits negative cooperativity, which is eliminated by the presence of either NAD or Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1983

Purification and characterization of phosphorylase B from Ascaris suum.

Glycogen phosphorylase b (EC 2.4.1.1) has been purified from the muscle of the roundworm, Ascaris suum. The 223-fold purified enzyme was shown to be homogenous by high performance liquid chromatography (HPLC), gel filtration column chromatography and sodium dodecyl sulfate (SDS) gel electrophoresis. The apparent native molecular weight of the enzyme determined by size exclusion chromatography by HPLC and gel filtration corresponded to 200 000 and 199 000, respectively. The subunit molecular weight of the enzyme was determined to be 100 000 by electrophoresis in the presence of SDS. Therefore, the enzyme appears to be a dimer with identical or near identical subunits. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol subunit and exhibited an absorbance index E1% 280 of 13.8. The apparent isoelectric point of the enzyme is 5.53. The enzyme, inactive in the absence of AMP, can be converted to the active form by rabbit muscle phosphorylase kinase and MgATP. The molecular weight of the activated form of the enzyme is 200 000. Kinetic studies showed apparent Km values of 0.17% for glycogen, 36 mM for Pi and 52 mM for glucose-1-P. The apparent Ka for AMP was 0.22 mM.

Animals↗
PubMed1983

The role of cyclic AMP-mediated regulation of glycogen metabolism in levamisole-perfused Ascaris suum muscle.

The effects of levamisole on muscle contraction and glycogen metabolism have been examined in isolated muscle-cuticle sections of the roundworm Ascaris suum. Muscle contraction occurred when various levels of levamisole were perfused through the preparation. At a levamisole concentration of 0.42 mM, the period of contraction lasted only about 6 min and was followed by a period of relaxation. During this relaxation period, there was an activation of glycogen synthase (EC 2.4.1.11), as evidenced by a decrease in the Ka values of glucose 6-phosphate for glycogen synthase to 0.26 mM from control values of 0.50 mM. The glycogen phosphorylase (EC 2.4.1.1) activity ratio decreased from 0.85 to 0.65, which indicated an inactivation of this enzyme. Concomitant with this activation of glycogen synthase and inactivation of phosphorylase there was an increased synthesis of glycogen. In addition, the presence of levamisole prevented both the serotonin-induced cyclic AMP accumulation and the activation of the cyclic AMP-dependent protein kinase (EC 2.7.1.37). However, levamisole did not significantly affect the changes in glycogen synthase and phosphorylase brought about by perfusion with the neurostimulator acetylcholine. Collectively, the data indicated that levamisole caused a transient muscle contraction followed by muscle relaxation, and the muscle relaxation effect appeared to be the result of a levamisole-inhibited cyclic AMP-mediated pathway of glycogen utilization.

Acetylcholine↗
PubMed1982

Phosphofructokinase from Ascaris suum. Purification and properties.

A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.

Amino Acids↗
PubMed1982

Correlation of muscle activity with glycogen metabolism in muscle of Ascaris suum.

Isolated muscle segments from the parasitic roundworm Ascaris suum were shown to contract when perfused with acetylcholine (ACh). The muscle responded to ACh concentrations of 1 microM and was maximally contracted at 50 microM ACh. In fed muscle segments perfused with saturating levels of ACh the glycogen synthase Ka values for glucose 6-phosphate increased from 0.5 to 0.95 mM. In starved segments stimulated by ACh, the muscle utilized glycogen at a rate that was 1.41 micrograms.min-1.g tissue-1 greater than the saline-perfused controls. The cyclic AMP (cAMP) levels remained relatively constant at 0.34 +/- 0.08 nmol/g muscle during perfusion with ACh. Contraction in the muscle could be inhibited in a dose-dependent manner by gamma-aminobutyric acid (GABA). The presence of GABA in starved muscle prevented the decrease in Ka values and phosphorylase activity ratios brought about by glucose. Perfusion of GABA did not change cAMP levels in the muscle. Starved muscle perfused with GABA utilized glycogen at a rate that was 0.41 microgram.min-1.g-1 greater than saline-perfused controls. The results indicated that muscle contraction could be elicited by ACh, and that the energy for this process was derived from endogenous glycogen stores, which were depleted during contraction. Muscle contraction was also correlated with inactivation of glycogen synthase and activation of phosphorylase. These processes appeared to function via a cAMP-independent mechanisms.

Acetylcholine↗