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D W Yates

Publications and source records attributed to D W Yates.

86 records · Page 5Linked to original sources
PubMed1974

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates.

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.

Acid-Base Equilibrium↗
PubMed1972

Protein fluorescence of nicotinamide nucleotide-dependent dehydrogenases.

1. The decrease in the protein fluorescence (F) of Neurospora crassa glutamate dehydrogenase is linearly related to the increase in the fraction of the coenzyme sites occupied by NADPH (alpha) at pH6.35. Under these conditions NADPH causes this enzyme to dissociate to monomers. 2. There is a non-linear relationship of F to alpha for NADH binding to give the alcohol dehydrogenase-NADH-isobutyramide complex, the l-glycerol 3-phosphate dehydrogenase-NADH complex and the bovine glutamate dehydrogenase-NADH-glutamate complex. The non-linearity is accurately represented by F=[1-alpha(1-x)](n) where n is the number of NADH-binding sites per protein molecule. 3. The co-operative binding of GTP to bovine glutamate dehydrogenase in the presence of NADH gives a linear relationship between F and alpha. 4. The prediction from the equation F=[1-alpha(1-x)](n) that initial tangents to non-linear protein-fluorescence-quenching curves will intercept the fluorescence when alpha=1 at a value of total ligand concentration less than the sum of the concentration of binding sites in the solution plus the dissociation constant of ligand is quantitatively fulfilled. 5. Non-linear protein-fluorescence titrations may be used to obtain information about the distribution of ligand among the protein molecules in solution.

Alcohol Oxidoreductases↗
PubMed1971

Dihydroxyacetone phosphate. Its structure and reactivity with -glycerophosphate dehydrogenase, aldolase and triose phosphate isomerase and some possible metabolic implications.

1. Dihydroxyacetone phosphate exists in neutral aqueous solution at 20 degrees C as a mixture of keto, gem-diol and enolic forms in the ratio 55:44:1. 2. The three forms are freely interconvertible and rate constants for these reactions have been determined. 3. Keto-dihydroxyacetone phosphate is the primary reactive species in the reactions catalysed by alpha-glycerophosphate dehydrogenase, aldolase and triose phosphate isomerase. 4. The proportion of keto form to gem-diol forms of dihydroxyacetone phosphate is temperature-dependent. At 37 degrees C, 83% is keto-dihydroxyacetone phosphate. 5. The enzymological and metabolic consequences of these results are discussed.

Acetone↗
PubMed1970

Carnitine palmitoyltransferase activities (EC 2.3.1.-) of rat liver mitochondria.

1. A continuously recording and sensitive fluorimetric assay is described for carnitine palmitoyltransferase. This assay has been applied to whole or disintegrated mitochondria and to soluble protein fractions. 2. When rat liver mitochondria had been disintegrated by ultrasound, the specific activity of carnitine palmitoyltransferase was 15-20m-units/mg of protein. Only one-fifth of this activity was assayable (with added substrates) before mitochondrial disintegration. 3. It is concluded that there are two carnitine palmitoyltransferase activities in rat liver mitochondria, of which one (type I) is relatively superficial in location and catalyses an acyl-group transfer between added CoA and carnitine, whereas the other (type II) is less superficial and catalyses an acyl-group transfer in unbroken mitochondria between added carnitine and intramitochondrial CoA. The existence of two distinct carnitine palmitoyltransferases was predicted by Fritz & Yue (1963). 4. In unbroken mitochondria, type I transferase is accessible to the inhibitor 2-bromostearoyl-CoA whereas the type II transferase is inaccessible. 5. A major part of the total carnitine palmitoyltransferase activity of rat liver mitochondria is membrane-bound and of type II. 6. These observations, when considered in conjunction with the penetration of mitochondria by CoASH or carnitine, indicate that the type II transferase is attached to the inner mitochondrial membrane.

Acyltransferases↗
PubMed1970

Spectrophotometric studies of acyl-coenzyme A synthetases of rat liver mitochondria.

1. Deca-2,4,6,8-tetraenoic acid is a substrate for both ATP-specific (EC 6.2.1.2 or 3) and GTP-specific (EC 6.2.1.-) acyl-CoA synthetases of rat liver mitochondria. The enzymic synthesis of decatetraenoyl-CoA results in new spectral characteristics. The difference spectrum for the acyl-CoA minus free acid has a maximum at 376nm with epsilon(mM) 34. Isosbestic points are at 345nm and 440nm. 2. The acylation of CoA by decatetraenoate in mitochondrial suspensions can be continuously measured with a dual-wavelength spectrophotometer. 3. By using this technique, three distinct types of acyl-CoA synthetase activity were demonstrated in rat liver mitochondria. One of these utilized added CoA and ATP, required added Mg(2+) and corresponded to a previously described ;external' acyl-CoA synthetase. The other two acyl-CoA synthetase activities utilized intramitochondrial CoA and did not require added Mg(2+). Of these two ;internal' acyl-CoA synthetases, one was insensitive to uncoupling agents, was inhibited by phosphate or arsenate, and corresponded to the GTP-specific enzyme. The other corresponded to the ATP-specific enzyme. 4. Atractylate inhibited the activity of the two internal acyl-CoA synthetases only when the energy source was added ATP. 5. The amount of intramitochondrial CoA acylated by decatetraenoate was independent of whether the internal ATP-specific or GTP-specific acyl-CoA synthetase was active. It is concluded that these two internal acyl-CoA synthetases have access to the same intramitochondrial pool of CoA. 6. The amount of intramitochondrial CoA that could be acylated with decatetraenoate was decreased by the addition of palmitoyl-dl-carnitine, 2-oxoglutarate, or pyruvate. These observations indicated that pyruvate dehydrogenase (EC 1.2.4.1), oxoglutarate dehydrogenase (EC 1.2.4.2), carnitine palmitoyltransferase (EC 2.3.1.-), citrate synthase (EC 4.1.3.7), and succinyl-CoA synthetase (EC 6.2.1.4) all have access to the same intramitochondrial pool of CoA as do the two internal acyl-CoA synthetases.

Acyltransferases↗
PubMed1970

The localization of some coenzyme A-dependent enzymes in rat liver mitochondria.

1. CoA, acetyl-CoA, l-carnitine and acetyl-l-carnitine when added to rat liver mitochondria equilibrate with approximately two-thirds of the total intramitochondrial water. The mitochondrial space calculated to be freely permeable to these solutes was identical with that obtained for sucrose. 2. Acetyl-CoA is rapidly deacylated by rat liver mitochondria at 0 degrees C, and special precautions are required to measure its mitochondrial permeation. 3. Rat liver mitochondria were separated into fractions that correspond to the inner membrane, the outer membrane, and the soluble proteins of the matrix and intermembrane compartment. Soluble enzymes considered to be located in the matrix were citrate synthase (EC 4.1.3.7), palmitoyl-CoA dehydrogenase (EC 1.3.2.2), electron-transferring flavoprotein, medium-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.2), l-3-hydroxybutyryl-CoA dehydrogenase (EC 1.1.1.35) and 3-keto-acyl-CoA thiolase (EC 2.3.1.16). Carnitine palmitoyltransferase (EC 2.3.1.-) is largely associated with the inner-membrane fraction. A long-chain-length ATP-specific fatty acyl-CoA synthetase (EC 6.2.1.3) is associated with the outer-membrane fraction.

Acyltransferases↗
PubMed1965

Steady-state concentrations of coenzyme A, acetyl-coenzyme A and long-chain fatty acyl-coenzyme A in rat-liver mitochondria oxidizing palmitate.

1. Fluorimetric assays are described for CoASH, acetyl-CoA and long-chain fatty acyl-CoA, and are sensitive to at least 50mumumoles of each. 2. Application of these assays to rat-liver mitochondria oxidizing palmitate in the absence and presence of carnitine indicated two pools of intramitochondrial CoA. One pool could be acylated by palmitate and ATP, and the other pool acylated by palmitate with ATP and carnitine, or by palmitoylcarnitine alone. 3. The intramitochondrial content of acetyl-CoA is increased by the oxidation of palmitate both in the absence and presence of l-malate. 4. The conversion of palmitoyl-CoA into acetyl-CoA by beta-oxidation takes place without detectable accumulation of acyl-CoA intermediates.

Journal Article↗
PubMedDate not supplied

Trends in head injury outcome from 1989 to 2003 and the effect of neurosurgical care: an observational study.

BACKGROUND: Case fatality rates after all types of blunt injury have not improved since 1994 in England and Wales, possibly because not all patients with severe head injury are treated in a neurosurgical centre. Our aims were to investigate the case fatality trends in major trauma patients with and without head injury, and to establish the effect of neurosurgical care on mortality after severe head injury. METHODS: We analysed prospectively collected data from the Trauma Audit and Research Network database for patients presenting between 1989 and 2003. Mortality and odds of death adjusted for case mix were compared for patients with and without head injury, and for those treated in a neurosurgical versus a non-neurosurgical centre. FINDINGS: Patients with head injury (n=22,216) had a ten-fold higher mortality and showed less improvement in the adjusted odds of death since 1989 than did patients without head injury (n=154,231). 2305 (33%) of patients with severe head injury (presenting between 1996 and 2003) were treated only in non-neurosurgical centres; such treatment was associated with a 26% increase in mortality and a 2.15-fold increase (95% CI 1.77-2.60) in the odds of death adjusted for case mix compared with patients treated at a neurosurgical centre. INTERPRETATION: Since 1989 trauma system changes in England and Wales have delivered greater benefit to patients without head injury. Our data lend support to current guidelines, suggesting that treatment in a neurosurgical centre represents an important strategy in the management of severe head injury.

Adolescent↗