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H G Britton

Publications and source records attributed to H G Britton.

At least 37 records · Page 2Linked to original sources
PubMed1978

Kinetics and mechanism of action of muscle pyruvate kinase.

1. The mechanism of rabbit muscle pyruvate kinase was investigated by measurements of fluxes, isotope trapping, steady-state velocity and binding of the substrates. All measurements were made at pH8.5 in Tris/HCl buffer and at 5mm-free Mg(2+). 2. Methods of preparing [(32)P]phosphoenolpyruvate from [(32)P]P(i) in high yield and determining [(32)P]-phosphoenolpyruvate and [8-(14)C]ADP are described. 3. The ratio Flux of ATP to ADP/Flux of ATP to phosphoenolpyruvate (measured at equilibrium) increased hyperbolically with ADP concentration from unity to about 2.1 at 2mm-ADP, but was unaffected by phosphoenolpyruvate concentration. Since the ratio is greater than unity, one pathway for the addition of substrates must involve phosphoenolpyruvate adding first to the enzyme in a rate-limiting step. However, the substrates must also add in the alternative order, because of the non-linear increase in the ratio with ADP concentration and because the rate of increase is very much less than that predicted from the steady-state velocity data for an ordered addition. The lack of influence of phosphoenolpyruvate on the ratio is consistent with the rapid addition of ADP in the alternative pathway. At low ADP concentrations the alternative pathway contributes less than 33% to the total reaction. 4. Isotope trapping was observed with [(32)P]phosphoenolpyruvate, confirming that when phosphoenolpyruvate adds first to the enzyme it is in a rate-limiting step. The release of phosphoenolpyruvate from the ternary complex must also be a slow step. Trapping was not observed with [8-(14)C]ADP, hence the addition of ADP to the free enzyme must be rapid unless its dissociation constant is very large (>20mm). 5. Binding studies showed that 4mol of [(32)P]phosphoenolpyruvate binds to 1mol of the enzyme, probably unligated to Mg(2+), with a dissociation constant appropriate to the mechanism indicated above. Binding of [8-(14)C]ADP could not be detected, and hence the binding of ADP occurs by a low-affinity step. The latter is also demanded by the steady-state velocity data. 6. The ratio Flux of phosphoenolpyruvate to ATP/Flux of phosphoenolpyruvate to pyruvate (determined from the incorporation of label into phosphoenolpyruvate from [3-(14)C]-pyruvate or [gamma-(32)P]ATP during the forward reaction) did not differ significantly from unity. Steady-state velocity data predicted grossly different flux ratios for ordered dissociations of the products, and the results indicate that the dissociation must be rapid and random. The data also exclude a Ping-Pong mechanism. 7. Permissible rate constants for the above mechanism are calculated. The results indicate a high degree of cooperativity in binding, whatever the order of addition of substrate.

Adenosine Diphosphate↗
PubMed1978

The supply of citrate to the sheep fetus.

In situ placental perfusion has been used to study the role of the sheep placenta in fetal citrate metabolism. The perfusate citrate concentrations rose steadily to values higher than in fetal plasma. This rise was not influenced by intravenous administration of citrate to the ewe. Concentration and electropotential gradients indicate that transplacental passage is unlikely and the rise is probably due to placental synthesis. However, the quantity of citrate supplied to the fetus was calculated to be small compared with the fetal metabolic rate. Comparison with the guinea pig shows close similarity in fetal plasma concentrations and the amount of citrate formed per unit weight of fetus.

Animals↗
PubMed1977

Calculation of steady-state rate equations and the fluxes between substrates and products in enzyme reactions.

1. Two methods are described for deriving the steady-state velocity of an enzyme reaction from a consideration of fluxes between enzyme intermediates. The equivalent-reaction technique, in which enzyme intermediates are systematically eliminated and replaced by equivalent reactions, appears the most generally useful. The methods are applicable to all enzyme mechanisms, including three-substrate and random Bi Bi Ping Pong mechanisms. Solutions are obtained in algebraic form and these are presented for the common random Bi Bi mechanisms. The steady-state quantities of the enzyme intermediates may also be calculated. Additional steps may be introduced into enzyme mechanisms for which the steady-state velocity equation is already known. 2. The calculation of fluxes between substrates and products in three-substrate and random Bi Bi Ping Pong mechanisms is described. 3. It is concluded that the new methods may offer advantages in ease of calculation and in the analysis of the effects of individual steps on the overall reaction. The methods are used to show that an ordered addition of two substrates to an enzyme which is activated by another ligand will not necessarily give hyperbolic steady-state-velocity kinetics or the flux ratios characteristic of an ordered addition, if the dissociation of the ligand from the enzyme is random.

Enzymes↗
PubMed1977

Mechanism of "L"-type pyruvate kinase from rabbit liver. Evidence against phosphoenzyme formation.

The "L"-type pyruvate kinase from rabbit liver does not catalyse exchange between phosphoenol[1-14C]pyruvate and pyruvate at either pH 8.5 or 6.2. Spectrophotometric experiments at pH 8.5 and 6;2 and gel-filtration experiments with [32P]phosphoenolpyruvate at pH 8,5 also fail to demonstrate phosphoenzyme formation. It is concluded that it is very unlikely that the enzyme has a phosphoenzyme mechanism.

Animals↗
PubMed1976

A study of the permeability of the guinea pig placenta to citrate using recirculating placental perfusion technique.

The role of the guinea pig placenta in fetal citrate metabolism has been studied by perfusing the placenta in situ. The initial citrate concentrations in the perfusate plasma were lower than in the maternal plasma but rose progressively until, in some cases, they exceeded those in the mother. The rise was uninfluenced by intravenous administration of citrate to the mother. Citrate therefore does not enter the perfusate by simple diffusion from the maternal compartment. It may be synthesised within the placenta. From the rate of accumulation of citrate in the perfusate, the supply to the fetus was calculated to be small compared to the fetal metabolic rate. Citrate added to the perfusate became distributed in a space similar to the extracellular space and no change occurred in maternal citrate concentrations. Thus, the placenta does not appear to metabolise citrate or allow transport to the mother.

Animals↗
PubMed1974

The reaction of diethyl pyrocarbonate with pyruvate kinase.

Diethyl pyrocarbonate inactivates muscle pyruvate kinase with the substitution of 3-4 histidine residues per subunit. Phosphoenolpyruvate, ATP and ADP to a lesser extent, and Mg(2+) and pyruvate to a small extent, protect against inactivation.

Adenosine Diphosphate↗
PubMed1974

The mechanism of phosphoglucomutase from Micrococcus lysodeikticus.

The mechanism of the phosphoglucomutase from Micrococcus lysodeikticus was investigated. Induced-transport tests at low substrate concentrations (0.15mm) showed co-transport of the (32)P label but no induced transport of the (14)C label, which is in quantitative agreement with a phosphoenzyme mechanism with a rapid isomerization of the phosphoenzyme. The results excluded an intramolecular transfer of phosphate and could only have been compatible with a sequential mechanism if the K(m) for glucose 1-phosphate had been over 20 times smaller than the measured value. The results of induced-transport tests at intermediate concentrations (1mm) with both labels agreed quantitatively with a phosphoenzyme mechanism, and induced-transport tests with (14)C-labelled substrates at high concentrations (26mm) indicated that the rate constants for isomerization of the phosphoenzyme must be greater than about 3x10(6)s(-1). Consistent with these findings is the fact that (14)C label exchanged between the substrates twice as rapidly as the (32)P label at chemical equilibrium. Further, since the (14)C label exchanged between the substrates about ten times more rapidly than between the substrates and glucose 1,6-diphosphate, glucose 1,6-diphosphate is not an obligatory intermediate in the interconversion of the substrates. It is concluded that, contrary to previous evidence, the mechanism of the enzyme from M. lysodeikticus is essentially that of the rabbit muscle enzyme. To account for the rapid isomerization of the phosphoenzyme in both cases a mechanism is proposed in which there is no formal isomerization of the phosphoenzyme.

Animals↗
PubMed1974

Preparation and properties of a phosphoenzyme from the phosphoglucomutase of Micrococcus lysodeikticus.

1. Phosphoglucomutase from Micrococcus lysodeikticus was incubated with (14)C- and (32)P-labelled glucose 1,6-diphosphate and separated from the cofactor on a Sephadex column. (32)P-labelled phosphate (0.7mol/mol of enzyme) was associated with the enzyme, but no (14)C label was. 2. The (32)P-labelled enzyme exchanged its label with the substrates. When the labelled enzyme was incubated in Tris buffer, pH8.3, at 30 degrees C the proportion of exchangeable label slowly fell indicating a half-life of the phosphoenzyme of about 50h. 3. When HClO(4) was added to the labelled phosphoenzyme all of the label was precipitated with the protein and none was released as P(i). On alkaline hydrolysis P(i) was released at a rate comparable with the rate of hydrolysis of the phosphoenzyme from rabbit muscle. 4. We conclude that the phosphoenzyme from Micrococcus lysodeikticus yields a relatively stable, catalytically active phosphoenzyme when treated with cofactor, and that there is no evidence for the formation of an enzyme-glucose 1,6-diphosphate complex. The properties of the phosphoenzyme, which resemble those of rabbit muscle phosphoglucomutase, suggest that the phosphate may be bound to serine.

Animals↗
PubMed1974

The equilibrium constant of the phosphoglyceromutase reaction.

The equilibrium constant of the phosphoglyceromutase reaction was determined over a range of pH (5.4-7.9), in solutions of different ionic strength (0.06-0.3) and in the presence of Mg(2+), at 30 degrees C and at 20 degrees C. The values obtained (8.65-11.65) differ substantially from previously published values. The third acid dissociation constants were redetermined for 2- and 3-phosphoglycerate, and in contrast with previous reports the pK values (7.03 and 6.97 respectively at zero ionic strength) were closely similar. The Mg(2+)-binding constants were measured spectrophotometrically and the values, 286mm(-1) and 255mm(-1) for 2- and 3-phosphoglycerate at pH7 and ionic strength 0.02, were also very similar. From the relative lack of effect of temperature, pH and ionic strength it is concluded that the equilibrium constant differs from unity largely because of entropic factors. At low ionic strength, in the neutral region, the pH-dependence can be attributed to the small difference in the acid dissociation constants, but the difference in dissociation constants does not explain the pH-dependence in the acid region or at high ionic strength. Within physiological ranges of pH, Mg(2+) concentration and ionic strength there will be little variation in equilibrium constant.

Acid-Base Equilibrium↗
PubMed1973

Methods of determining rate constants in single-substrate-single-product enzyme reactions. Use of induced transport: limitations of product inhibition.

1. The calculation of the rate constants from steady-state kinetics of a single-substrate-single-product enzyme reaction in which there is an isomerization of the enzyme is described. 2. It is shown that even with the use of isotopically labelled substrates a set of solutions for the constants is obtained rather than a unique solution. However, limits are derived within which they must lie. 3. The most appropriate observations to determine the rate constants are measurements of V(max.) and K(m) for both substrate and product, and measurement of the degree of countertransport in an induced-transport test. 4. Experimental procedures for induced-transport tests and the quantitative interpretation of the results obtained are discussed. 5. Product inhibition is shown to be an ambiguous and imprecise means of determining the rate constants. Further, the absence of a [substrate]x[product] term in the denominator of the steady-state rate equation does not necessarily mean that the isomerization of the enzyme is rapid, since the term also disappears when the isomerization is very slow. 6. Similar considerations apply to carrier mechanisms.

Binding Sites↗
PubMed1973

The reactive serine residue in phosphoglucomutase of Micrococcus lysodeikiticus.

1. Phosphoglucomutase of Micrococcus lysodeikticus was labelled at the active site by exchange with (32)P-labelled substrates of high specific radioactivity. 2. Partial acid hydrolysis gave rise to radioactive peptides; serine phosphate was identified as one of the derivatives. 3. Comparison of the other (32)P-labelled peptides with the peptides obtained from the (32)P-labelled rabbit muscle phosphoglucomutase indicates that the sequence around the reactive serine residue is identical in both enzymes.

Autoradiography↗