Dissociation constants for alternate dirmcleotide substrates and competitive inhibitors suggest that the dinucleotide binding site of the Ascaris suum NAD-malic enzyme is hydrophobic in the vicinity of the nicotinamide ring. Changes in the divalent metal ion activator from Mg^2+ to Mn^2+ or Cd^2+ results in a decrease in the dinucleotide affinity and an increase in the affinity for malate. Primary deuterium and 13-C isotope effects obtained with the different metal ions suggest either a change in the transition state structure for the hydride transfer or decarboxylation steps or both. Deuterium isotope effects are finite whether reactants are maintained at saturating or limiting concentrations with all the metal ions and dinucleotide substrates used. With Cd^2+ as the divalent metal ion, inactivation of the enzyme occurs whether enzyme alone is present or is turning over. Upon inactivation only Cd^2+ ions are bound to the enzyme which becomes denatured. Modification of the enzyme to give an SCN-enzyme decreases the ability of Cd^2+ to cause inactivation. The modified enzyme generally exhibits increases in K_NAD and K_i_metai and decreases in V_max as the metal size increases from Mg^2+ to Mn^2+ or Cd^2+, indicative of crowding in the site. In all cases, affinity for malate greatly decreases, suggesting that malate does not bind optimally to the modified enzyme. For the native enzyme, primary deuterium isotope effects increase with a concomitant decrease in the 13-C effects when NAD is replaced by an alternate dinucleotide substrate different in redox potential. This suggests that when the alternate dinucleotides are used, a switch in the rate limitation of the chemical steps occurs with hydride transfer more rate limiting than decarboxylation. Deuteration of malate decreases the 13-C effect with NAD for the native enzyme, but an increase in 13-C effect is obtained with alternate dinucleotides. These suggest the presence of a …
The physiological significance of noradrenergic sympathohabenular ingrowth following medial septal lesions was investigated. Following septal lesions, sympathetic fibers originating in the superior cervical ganglia are known to sprout into the medial habenular nuclei, and into the hippocampal formation. Previous work involving sympathohippocampal ingrowth showed that firing rates in septal animals with no ingrowth showed that firing rates in septal animals with no ingrowth were higher than rates of septal animals with ingrowth and controls. Those results suggested that sympathetic ingrowth in the hippocampus had some functional capability in a modulatory manner. The primary aim of the present study was to determine if the peripheral sympathetic ingrowth into the medial habenular nuclei following a septal lesion is functionally significant. The results showed that firing rates of neurons of the medial habenulae in animals receiving septal lesions were significantly higher than rates of control animals and septal lesioned + ganglionectomized animals.
Glucose phosphate isomerase (GPI) occurs in different bovine tissues as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis and isoelectric focusing. GPI from bovine heart was purified to homogeneity and each of the isozymes was resolved. Four of the five isozymes were characterized with regard to their physical, chemical and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118 Kd) and subunit (59 Kd) molecular weights but had different apparent pi values of 7.2, 7.0, 6.8 and 6.6. Structural analyses showed that the amino terminus was blocked and the carboxyl terminal sequence was -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes (lower pi values) at pH extremes, at high ionic strength, in the presence of denaturants or upon exposure to proteases. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes. Identical amino acid composition and peptide mapping by chemical cleavage at methionine and cysteine residues of the isozymes suggest a postsynthetic modification rather then a genetic origin for the in vivo isozymes. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with electrophoretic properties identical to those found in vivo. The simultaneous release in ammonia along with the spontaneous shift to more acidic isozymes and changes in the specific cleavage of the Asn-Gly bonds by hydroxylamine of the acidic isozyme indicates deamidation as the probable molecular basis. In summary the isozymes appear to be the result of spontaneous, postsynthetic modifications involving the addition of an equal number of negative charges and are consistent with the deamidation process.
The isotope partitioning studies of the Ascaris suum NAD-malic enzyme reaction were examined with five transitory complexes including E:NAD, E:NAD:Mg, E:malate, E:Mg:malate, and E:NAD:malate. Three productive complexes, E:NAD, E:NAD:Mg, and E:Mg:malate, were obtained, suggesting a steady-state random mechanism. Data for trapping with E:14C-NAD indicate a rapid equilibrium addition of Mg2+ prior to the addition of malate. Trapping with 14C-malate could only be obtained from the E:Mg2+:14C-malate complex, while no trapping from E:14C-malate was obtained under feasible experimental conditions. Most likely, E:malate is non-productive, as has been suggested from the kinetic analysis. The experiment with E:NAD:malate could not be carried out due to the turnover of trace amounts of malate dehydrogenase in the pulse solution. The equations for the isotope partitioning studies varying two substrates in the chase solution in an ordered terreactant reaction were derived, allowing a determination of the relative rates of substrate dissociation to the catalytic reaction for each of the productive transitory complexes. NAD and malate are released from the central complex at an identical rate, equal to the catalytic rate.
The kinetic mechanism of the cAMP-dependent protein kinase has been determined to be random in the direction of MgADP phosphorylation by using initial velocity studies in the absence and presence of the product, phospho-Serpeptide (Leu-Arg-Arg-Ala-Ser[P]-Leu-Gly) , and dead-end inhibitors. In contrast to the kinetic parameters obtained in the direction of Serpeptide phosphorylation, the only kinetic parameters affected by Mg^2+ are the dissociation constants for E:phospho-Serpeptide and E:MgADP, which are decreased by about 4-fold. The dead-end analog MgAMPCP binds with an affinity equal to that of MgADP in contrast to MgAMPPCP, which binds weaker than MgATP. The ratio of the maximum velocities in the forward and reverse reactions is about 200, and the Haldane relationship gives a K-eq of (7.2 ± 2) x 10^2. The latter can be compared to the K-eq obtained by direct measurement of reactant concentrations (2.2 ± 0.4) x 10^3 and 31-P NMR (1 ± 0.5) x 10^3. Data for the pH dependence of kinetic parameters and inhibitor dissociation constants for the cAMP dependent protein kinase are consistent with a mechanism in which reactants selectively bind to an enzyme with the catalytic base unprotonated and an enzyme group required protonated for Ser-peptide binding. Preferentially MgATP binds fully ionized and requires an enzyme residue (probably lysine) to be protonated. The maximum velocity and V/K-MgATP are pH independent. The V/K for Serpeptide is bell-shaped with estimated pK values of 6.2 and 8.5. The dependence of 1/K-i for Leu-Arg-Arg-Ala-Ala-Leu-Gly is also bell-shaped, giving pK values identical with those obtained for V/K-Serpeptide, while the K-i for MgAMPPCP increases from a constant value of 650 μM above pH 8 to a constant value of 4 mM below pH 5.5. The K-i for uncomplexed Mg^2+ obtained from the Mg^2+ dependence of V and V/K-MgATP is apparently pH independent.
In these studies, a procedure which allowed the in vivo labeling and detection of poly(ADP-ribose) was combined with nuclear fractionation techniques to analyze the nuclear distribution of ADP-ribose polymers. The results from these studies suggest the occurrence of poly(ADP-ribose) metabolism in two compartments of chromatin; one that is nuclear matrix-associated and one that is not. The biological significance of this compartmentalization is conceptualization in a model. This model postulates that, under some physiological conditions, poly(ADP-ribose) metabolism accomplishes the reversible targeting of specific regions of chromatin to the nuclear matrix domain by modulating DNA-protein and or protein-protein interactions.
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