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P C Poat

Publications and source records attributed to P C Poat.

At least 19 recordsLinked to original sources
PubMed1982

Residual analysis in determining the error structure in enzyme kinetic data. Simulation experiments and observations on Carcinus maenas phosphofructokinase.

The nature of the experimental error in the initial velocities of an enzyme-catalysed reaction is required if meaningful least-squares regression is to be applied. When a rate equation more complex that that of Michaelis and Menten is to be solved least-squares techniques are the method of choice and so determination of the error structure becomes mandatory. The use of residual analysis and Tukey's T statistics to determine the weights to use are described. This method has the advantage of requiring no additional experimentation over that required for the primary investigation. Using data obtained for Carcinus maenas phosphofructokinase the variance was found to increase with velocity and was approximated by either an empirical power function, var (vi) alpha vi1.8 or by the function, var (vi) alpha 0.007 + vi2. The latter function is preferred and suggests that the data contains both a constant absolute error and a constant percentage error component.

Animals↗
PubMed1980

An investigation into the apparent inhibition by arginine phosphate of the activity of Carcinus maenas type-M pyruvate kinase.

An enzymic synthesis utilising arginine kinase for preparing arginine phosphate in a high state of purity is described. The dissociation constant of magnesium arginine phosphate, determined by gel filtration, was 30.0 +/- 0.9 mM. That for potassium arginine phosphate was calculated to be 63.0 +/- 4.0 mM measured by the effect of potassium on the apparent magnesium dissociation constant. The effect of KCl on the reaction catalysed by the type-M pyruvate kinase from Carcinus maenas (the common shore crab) pincer and leg muscle was investigated. No effect was seen on the C. maenas pyruvate kinase activity, apart from that due to alteration of the K+ concentration, on adding up to 70 mM potassium arginine phosphate to the reaction medium. The less pure form of arginine phosphate was found to give an apparent noncompetitive inhibition of the enzyme when phosphoenolpyruvate was the varied substrate. This apparent inhibition can be accounted for by the removal of ADP from the assay medium in a side reaction involving arginine kinase and arginine phosphate. These results are discussed in terms of the possible physiological control of the type-M pyruvate kinase from C. maenas.

Adenosine Diphosphate↗
PubMed1980

A steady-state kinetic analysis of the fructose 1,6-bisphosphate-activated pyruvate kinase from Carcinus maenas hepatopancreas.

1. An investigation of the reaction mechanism of the fructose 1,6-bisphosphate-activated pyruvate kinase isolated from the hepatopancreas of the crab Carcinus maenas was conducted. The enzyme was assayed in the presence of 500 microns-fructose 1,6-bisphosphate, 75 mM-KCl and 8 mM-Mg2+free at 25 degrees C. The results are consistent with a rapid-equilibrium random mechanism. 2. Evidence is presented that suggests the formation of two mixed-substrate-product dead-end complexes, enzyme-ADP-pyruvate and enzyme-ADP-ATP. 3. Competitive substrate inhibition was observed for both substrates, ADP and phosphoenolpyruvate, suggesting the formation of the complexes enzyme-ADP-ADP and enzyme-phosphoenolpyruvate-phosphoenolpyruvate in the suggested mechanism. 4. Data from the ATP product-inhibition studies indicate the formation of the complex enzyme-ATP-ATP. This suggests that in the reverse reaction ATP also will show substrate inhibition. 5. The presence of a saturating concentration of fructose 1,6-bisphosphate does not cause full activation of the purified preparations of the enzyme. 6. Pyruvate kinase activity in the supernatant of a hepatopancreas homogenate was completely activated by fructose 1,6-bisphosphate, suggesting that the binding of this ligand to the purified pyruvate kinase was impaired.

Adenosine Diphosphate↗
PubMed1977

An investigation of the interactions of the allosteric modifiers of pyruvate kinase with the enzyme from Carcinus maenas hepatopancreas.

1. Pyruvate kinase purified from the hepatopancrease of Carcinus maenas exhibited sigmoidal saturation kinetics with respect to the substrate phosphoenolpyruvate in the absence of the allosteric activator fructose 1,6-bisphosphate, but normal hyperbolic saturation was seen in the presence of this activator. The activation appears to be the result of a decrease in the s0.5 (phosphoenolpyruvate) and not to a change in Vmax. 2. In the presence of ADP and ATP at a constant nucleotide-pool size the results indicate that phosphoenolpyruvate co-operativity is lost on increasing the [ATP]/[ADP] ratio. 3. Paralleling this change is the observation that the fructose 1,6-bisphosphate activation became less at the [ATP]/[ATP] ratio was increased. This was due to the enzyme exhibiting a near-maximal activity in the absence of activator. 4. L-Alanine inhibited the enzyme, but homotropic co-operative interactions were only seen with a cruder (1000000g supernatant) enzyme preparation. The inhibition by alanine could be overcome by increasing the concentration of either phosphoenolpyruvate or fructose 1,6-bisphosphate, although increasing the L-alanine concentration did not appear to be able to reverse the activation by fructose 1,6-bisphosphate. 5. In the presence of a low concentration of phosphoenolpyruvate, increasing the concentration of the product, ATP, caused an initial increase in enzyme activity, followed by an inhibitory phase. In the presence of either fructose 1,6-bisphosphate or L-alanine only inhibition was seen. 6. The inhibition by ATP could not be completely reversed by fructose 1,6-bisphosphate.

Adenosine Triphosphate↗
PubMed1976

The kinetics of rabbit muscle pyruvate kinase. Initial-velocity, substrate- and product-inhibition and isotopic-exchange studies of the reverse reaction.

1. An assay, based on the transfer of label from [gamma-32P]ATP to [32P]phosphoenolpyruvate, suitable for a steady-state kinetic analysis of pyruvate kinase in the reverse direction (i.e. phosphoenolpyruvate synthesis), is described. 2. This assay was used in a kinetic investigation of the rabbit muscle enzyme including initial-rate and product-inhibition experiments, at a pH of 7.4 and constant concentrations of total K+ and free Mg2+. 3. These studies indicate that there is a random release of ADP and phosphoenolpyruvate from the enzyme and that there is a competitive substrate inhibition by ATP. Some of the results were suggestive that the rapid-equilibrium assumption, generally used for this enzyme was not valid. 4. Techniques were developed to measure the rate of isotopic exchange between all the substrate-product pairs. 5. By using these techniques the rates of isotopic exchange at chemical equilibrium were measured. The results indicate that this enzyme does not catalyse a truly rapid-equilibrium random mechanism, although in the forward reaction all initial-rate data obtained to date are consistent with this assumption.

Adenosine Diphosphate↗
PubMed1976

Purification and properties of pyruvate kinase from the hepatopancreas of Carcinus maenas.

Pyruvatekinase from the hepatopancreas of the common shore crab, Carcinus maenas, was purified to a specific activity of 240 units/mg of protein in the assay conditions described. 2. In one method of purification the enzymic activity could be resolved into two fractions after chromatography on DEAE-cellulose. Fructose 1, 6-diphosphate was able to effect the conversion of one form (peak 1) into the second (peak 2). 3. In the presence of a saturating concentration of fructose 1, 6-diphosphate both forms of the enzyme were kinetically similar. 4. Polyacrylamide-gel electrophoresis of the enzyme 1 day after preparation showed a single protein band. On storage at least three protein bands became visible, all of which were associated with pyruvate kinase activity. 5. Chromatography of the enzyme on Sephadex G-200 indicated a mol.wt. of 247000, but in the presence of fructose 1, 6-diphosphate the elution volume of the enzyme increased corresponding to a mol.wt. of 193000. 6 Dissociation of the enzyme in sodium dodecyl sulphate and 2-mercaptoethanol followed by polyacrylamide-gel electrophoresis produced one major protein band with a mol.wt. of 55000.

Animals↗