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D L Purich

Publications and source records attributed to D L Purich.

At least 91 records · Page 5Linked to original sources
PubMed1979

Nucleotide release from tubulin and nucleoside-5'-diphosphate kinase action in microtubule assembly.

ATP and UTP support microtubule assembly through the action of brain nucleoside-5'-diphosphate kinase on GDP. Penningroth and Kirschner (1977) J. Mol. Biol. 115, 643-673) have proposed that microtubule assembly may occur by either of two mechanisms: indirectly, through nucleoside-5'-diphosphate kinase-catalyzed phosphorylation of uncomplexed GDP and directly by nucleoside-5'-diphosphate kinase-mediated transphosphorylation of tubulin-bound GDP at low tubulin concentrations. We find the rates of GDP and GTP release (0.68 and 0.32 min-1, respectively) are sufficiently fast relative to assembly to permit GDP release, phosphorylation, and GTP binding as the sole mechanism of nucleoside-5'-diphosphate kinase action in microtubule assembly. Computer simulation studies accord with the conclusion that GDP release is rapid relative to microtubule assembly. The specific activity of the nucleoside-5'-diphosphate kinase is 1.7 nmol/min/mg of microtubular protein under the conditions studied. Pulse-chase experiments with tubulin . [14C]GDP complex and the rapidity of GDP phosphorylation by the kinase are in agreement with this scheme. Finally, it was observed that the extent and rate of microtubule assembly depends upon the [ATP]/[ADP] ratio.

Adenosine Diphosphate↗
PubMed1979

Participation of guanine nucleotides in nucleation and elongation steps of microtubule assembly.

Critical concentrations for formation of microtubules from subunits with GTP and its beta, gamma-imido and beta, gamma-methylene analogs are similar when adequate time is given for equilibration. Dilution of microtubules into GTP and GDP yielded values of 0.1 and 0.19 mg/ml for the critical concentration, results similar to those reported by Carlier and Pantaloni [Carlier, M. & Pantaloni, D. (1978) Biochemistry 17, 1908-1915]. GDP is capable of supporting elongation of preformed microtubules, but it efficiently poisons the nucleation events. Reported experiments also demonstrate that the critical tubulin concentration of the tubulin-GDP complex can be accurately measured in both the assembly and disassembly directions. Evidence is presented that GTP is involved in early nucleation events but that microtubules are stabilized in the presence of either GTP or GDP. These results are discussed in terms of a condensation-equilibrium model in which tubulin subunits equilibrate rapidly with microtubule ends, and their affinity for the ends is governed by the nucleotide ligand at the exchangeable site.

Animals↗
PubMed1978

Stoichiometry and role of GTP hydrolysis in bovine neurotubule assembly.

A method is given for preparing tubulin with 1 mol of exchangeably bound [gamma-32P]GTP/mol of 6 S dimer. Bovine tubulin is shown to hydrolyze 1 mol of GTP/mol of 6 S dimer added to assembling microtubules at 37 degrees. Hydrolysis and assembly occur at the same rate and to the same extent. When microtubule-associated proteins (MAPs) are removed, both hydrolysis and assembly fail to occur. Readdition of the MAPs restores both activities. Tubulin with exchangeable GDP will co-assemble with GTP.tubulin even at equimolar levels. Exchangeability is demonstrated by pulse-chase experiments with GDP or GTP. GDP is also a potent inhibitor of assembly under these conditions, and the rate of assembly is reduced by 50% at 10 micron GDP. One mole of inorganic phosphate is released to the solvent per mole of exchangeable GTP hydrolyzed. An assembly mechanism is proposed in which exchangeable GTP is hydrolyzed without intermediate transphosphorylation of nonexchangeable GDP.

Animals↗
PubMed1977

Autophosphorylation of cardiac 3',5'-cyclic AMP-stimulated protein kinase. Kinetic evidence for the regulatory subunit directly acting at the active site in the R2C2 complex.

The mechanism of the autophosphorylation reaction of bovine heart cyclic AMP-dependent protein kinase (ATP: protein phosphotranferase, EC 2.7.1.31) has been further examined using a kinetic approach. The reaction is first order in both ATP and protein kinase when both are present at comparable concentrations. Dilution has no effect on the fraction of regulatory subunit phosphorylated over a given interval of time, and this finding is in accord with the autophosphorylation proceeding via an intramolecular (or, more appropriately in this case, by an intracomplex) reaction. The possibility of regulatory subunit phosphorylation by uncomplex catalytic subunit or another R2C2 complex (the protein kinase complex of two catalytic subunits and the regulatory dimer) was clearly eliminated. These results are compatible with a subunit geometry permitting the regulatory subunit to bind at the protein substrate region of the kinase's active site and to undergo subsequent phosphorylation.

Animals↗
PubMed1977

Steady state and equilibrium exchange kinetic studies of the sheep brain glutamine synthetase reaction.

The kinetic mechanism of the sheep brain glutamine synthetase has been examined by both initial rate kinetics using the glutamate analog beta-glutamate and by isotope exchange measurements at equilibrium. Results of the initial rate studies were compatible with a number of sequential mechanisms but not with a partially or fully ordered rapid equilibrium or a ping-pong mechanism. Kinetic parameters at 37 degrees and pH 7.2 were K beta-Glu = 16 mM, KATP = 0.28 mM, and KNH2OH = 1.4 mM. For all equilibrium exchanges studied (ATP in equilibrium ADP, ATP in equilibrium Pi, and Glu in equilibrium Gln), the rate of exchange rose smoothly to a maximum as all substrates and products were simultaneously raised in a constant ratio. This result is in accord with a random order of substrate addition. A brief treatment of equilibrium exchange rates in cases where all substrate/product pairs are varied together is also presented.

Adenosine Diphosphate↗
PubMed1977

On the role of the tubulin nonexchangeable GTP site in bovine neurotubule assembly.

A procedure for radiolabeling the terminal phosphoryl group of the tubulin nonexchangeable GTP site using bacterial acetate kinase and acetyl-32P is described. Warming such samples to 37 degrees results in microtubule assembly and hydrolysis of the nonexchangeable site GTP in a parallel fashion. Removal of the microtubule-associated protein fraction from lebeled tubulin prevents hydrolysis and assembly, and recombination of these components restores both processes again in a parallel fashion. These and other experiments indicate that the nonexchangeable site GTP hydrolysis and assembly are intimately linked. The experiments also demonstrate that GRP is not required at the exchangeable nucleotide site for assembly to occur.

Animals↗
PubMed1975

Use of the sodium borohydride reduction technique to identify a gamma-glutamyl phosphate intermediary in the Escherichia coli glutamine synthetase reaction.

Incubation of unadenylylated Escherichia coli glutamine synthetase with ATP, L-[14C]glutamate and metal ion results in the formation of gamma-glutamyl-P which can under appropriate conditions be reduced by sodium borohydride. The acyl-P compound is formed catalytically as judged by the quantity of radioactive alpha-amino-delta-hydroxyvalerate produced compared to the concentration of enzyme subunits. Formation of the glutamyl-P compound occurs in the presence of magnesium or manganous ions, and the relation of this apparent lack of metal ion specificity with regard to the highly specific Mg2+-supported biosynthetic activity of the unadenylylated form is discussed.

Adenosine Triphosphate↗