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J S Easterby

Publications and source records attributed to J S Easterby.

At least 19 recordsLinked to original sources
PubMed1996

The fusion of control analysis and temporal analysis of metabolic systems.

Metabolic control analysis and the study of the transient response of metabolic systems had coincident births in 1973. They developed along parallel lines until in 1989/90 their complete fusion occurred. It was evident that the control of the transient response of metabolism could be described in terms of general control properties, such as the flux and concentration control coefficients and elasticities. Consequently, it is possible to define temporal control coefficients which relate to the lifetimes of individual metabolite pools or to the total system temporal response. These control coefficients are readily expressed in terms of the flux and concentration control coefficients. Therefore, to analyse the control of metabolism is also to analyse its temporal response.

Animals↗
PubMed1994

Isolation of multiple dimeric forms of phosphoribulokinase from an alga and a higher plant.

Dimeric phosphoribulokinase from either spinach (Spinacia oleracea) leaf or from the green alga, Scenedesmus obliquus can be separated into three distinct forms by hydrophobic interaction chromatography. Variation of the redox conditions prior to and during chromatography resulted in specific forms of phosphoribulokinase being eluted. It is suggested that three dimeric forms of phosphoribulokinase differ in the extent of disulfide bond formation between Cys-16 and Cys-55 in each of the two subunits. Phosphoribulokinase-3, isolated under the most oxidising conditions and exhibiting unusual kinetics, has properties consistent with those expected of an oxidised form of the enzyme in which Cys-16 and Cys-55 are completely oxidised to form a disulfide bond in each subunit. Phosphoribulokinase-1 is the completely reduced form predominating following incubation of extracts with dithiothreitol. Phosphoribulokinase-2, the intermediate species in which only one subunit possesses the disulfide, predominates only when extracts, previously reduced by high concentrations of 2-mercaptoethanol, are allowed to stand overnight in the presence of air prior to chromatography.

Chlorophyta↗
PubMed1994

Metabolic channeling versus free diffusion: transition-time analysis.

Metabolic channeling is the term used to describe the restricted flow of substrates and products in multienzyme systems. It has been argued for some time that free diffusion is sufficiently rapid to obviate the need for channeling and, furthermore, that it is also fast enough to prevent competing side reactions from interfering with the metabolic flow. In this article we argue that a thorough consideration of the temporal behavior of metabolite pools suggests that channeling is important in many cases.

Biological Transport↗
PubMed1993

Temporal analysis of metabolic systems and its application to metabolite channelling.

When a metabolic system undergoes a transition between steady states, the lag or transition time of the system is determined by the aggregated lifetimes of the metabolite pools. This allows the transition time, and hence the temporal responsiveness of the system, to be estimated from a knowledge of the starting and finishing steady states and obviates the need for dynamic measurements. The analysis of temporal response in metabolic systems may be integrated with the general field of metabolic control analysis by the definition of a temporal control coefficient (Cei tau) in terms of flux and concentration control coefficients. The temporal control coefficient exhibits summation and other properties analogous to the flux and concentration control coefficients. For systems in which static metabolite channels exist, the major kinetic advantage of channelling is a reduction in pool sizes and, as a result, a more rapid system response reflected in a reduced transition time. The extent of the channelling advantage may therefore be assessed from a knowledge of the system transition time. This reveals that no channelling advantage is achieved at high enzyme concentrations (i.e., comparable to Km) or, in the case of 'leaky' channels, where rapid equilibrium kinetic mechanisms obtain. In the case of a perfect channel with no leakage and direct transfer of metabolite between adjacent enzyme active sites, the transition time is minimized and equal to the lifetime of the enzyme-substrate complex.

Binding Sites↗
PubMed1992

Turnover of glycogen phosphorylase in the pectoralis muscle of broiler and layer chickens.

Glycogen phosphorylase is a major sarcoplasmic protein in chicken pectoralis muscle, constituting approx. 4% of the total protein complement. In slow-growing layer chicks phosphorylase accumulated in parallel with muscle accretion, but in fast-growing broiler chicks the concentration of phosphorylase in the muscle increased (from 5 to 8 mg/g wet wt.) with time. In a 5-week period, the total amount of phosphorylase in the pectoralis muscles increased 18-fold in broiler chicks (from approx. 75 to 1400 mg total), but only 3-fold (from approx. 100 to 270 mg total) in layers. Pyridoxal phosphate, the cofactor of the enzyme glycogen phosphorylase, was used as a specific label to measure the rate of degradation of the enzyme in the pectoralis muscle of growing broiler and layer chickens in vivo. In young animals, the fractional rate of phosphorylase synthesis was similar in broiler and layer chickens (approx. 15%/day), but the rate of degradation in layers (5%/day) was 5-fold higher than in broilers (1%/day). As the animals aged, the rate of synthesis decreased, but more so in layers than in broilers. The rate of degradation of phosphorylase also decreased in layers, but in broilers it remained at the low level seen in young animals. The dramatically higher rate of phosphorylase accretion in the pectoralis muscles of the broilers is therefore achieved by an initial lower rate of degradation combined with a sustained difference between rates of synthesis and degradation.

Aging↗
PubMed1991

Properties of two high-molecular-mass forms of glyceraldehyde-3-phosphate dehydrogenase from spinach leaf, one of which also possesses latent phosphoribulokinase activity.

Two high-Mr forms of chloroplast glyceraldehyde-3-phosphate dehydrogenase from spinach leaf can be separated by DEAE-cellulose chromatography. One form, the high-Mr glyceraldehyde-3-phosphate dehydrogenase, resembles an enzyme previously described [Yonuschot, G.R., Ortwerth, B.J. & Koeppe, O.J. (1970) J. Biol. Chem. 245, 4193-4198]. The other, a glyceraldehyde-3-phosphate dehydrogenase/phosphoribulokinase complex, is characterised by possession of latent phosphoribulokinase activity, only expressed following incubation with dithiothreitol. This complex is composed not only of subunits A (39.5 kDa) and B (41.5 kDa) characteristic of the high-Mr glyceraldehyde-3-phosphate dehydrogenase, but also of a third subunit, R (40.5 kDa) comigrating with that from the active phosphoribulokinase of spinach. Incubation of the complex with dithiothreitol markedly stimulated both its phosphoribulokinase and NADPH-dependent dehydrogenase activities. This dithiothreitol-induced activation was accompanied by depolymerisation to give two predominantly NADPH-linked tetrameric glyceraldehyde-3-phosphate dehydrogenases (the homotetramer, A4, and the heterotetramer, A2B2) as well as the active dimeric phosphoribulokinase. Incubation of the high-Mr glyceraldehyde-3-phosphate dehydrogenase with dithiothreitol promoted complete depolymerisation yielding only the heterotetramer (A2B2). Possible structures suggested for the glyceraldehyde-3-phosphate dehydrogenase/phosphoribulokinase complex are (A2B2)2A4R2 or (A2B2)(A4)2R2.

Blotting, Western↗
PubMed1990

Integration of temporal analysis and control analysis of metabolic systems.

A theory is developed that integrates approaches to the analysis of pathway transient response and metabolic control analysis. A Temporal Control Coefficient is defined that is a measure of the system's transient response to modulation of enzyme activity or concentration. The approach allows for the analysis of the establishment of a steady state from rest, of the system's 'agility' of response to minor perturbations of a pre-existing steady state and of the macroscopic transition between steady states. In the last-mentioned case it is shown that, like the transient time itself, the control of transient response retains the property of independence from the mechanism of the transition. In consequence, the Temporal Control Coefficient can be defined in terms of the control properties of the initial and final states alone without reference to the mechanism of transition. A summation property is shown to apply to the Temporal Control Coefficients in each case. Connectivity relationships between elasticities and Temporal Control Coefficients are also established.

Enzymes↗
PubMed1989

The analysis of metabolite channelling in multienzyme complexes and multifunctional proteins.

Multienzyme complexes and multifunctional proteins may confer a kinetic advantage by channelling reaction intermediates between consecutive enzymes and reducing the transient time for the establishment of steady states. A general means for quantitatively assessing the contribution of channelling to the reduction of pool size and transient time is presented. Restrictions to the kinetic advantage are identified, and it is shown that no channelling advantage is obtained at high enzyme concentration or for enzymes which exhibit rapid-equilibrium kinetic behaviour.

Kinetics↗
PubMed1987

Properties of a multimeric protein complex from chloroplasts possessing potential activities of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase.

A homogeneous multimeric protein isolated from the green alga, Scenedesmus obliquus, has both latent phosphoribulokinase activity and glyceraldehyde-3-phosphate dehydrogenase activity. The glyceraldehyde-3-phosphate dehydrogenase was active with both NADPH and NADH, but predominantly with NADH. Incubation with 20 mM dithiothreitol and 1 mM NADPH promoted the coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase, accompanied by a decrease in the glyceraldehyde-3-phosphate dehydrogenase activity linked to NADH. The multimeric enzyme had a Mr of 560,000 and was of apparent subunit composition 8G6R. R represents a subunit of Mr 42,000 conferring phosphoribulokinase activity and G a subunit of 39,000 responsible for the glyceraldehyde-3-phosphate dehydrogenase activity. On SDS-PAGE the Mr-42,000 subunit comigrates with the subunit of the active form of phosphoribulokinase whereas that of Mr-39,000 corresponds to that of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. The multimeric enzyme had a S20,W of 14.2 S. Following activation with dithiothreitol and NADPH, sedimenting boundaries of 7.4 S and 4.4 S were formed due to the depolymerization of the multimeric protein to NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (4G) and active phosphoribulokinase (2R). It has been possible to isolate these two enzymes from the activated preparation by DEAE-cellulose chromatography. Prolonged activation of the multimeric protein by dithiothreitol in the absence of nucleotide produced a single sedimenting boundary of 4.6 S, representing a mixture of the active form of phosphoribulokinase and an inactive dimeric form of glyceraldehyde-3-phosphate dehydrogenase. Algal thioredoxin, in the presence of 1 mM dithiothreitol and 1 mM NADPH, stimulated the depolymerization of the multimeric protein with resulting coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. Light-induced depolymerization of the multimeric protein, mediated by reduced thioredoxin, is postulated as the mechanism of light activation in vivo. Consistent with such a postulate is the presence of high concentrations of the active forms of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase in extracts from photoheterotrophically grown algae. By contrast, in extracts from the dark-grown algae the multimeric enzyme predominates.

Chlorophyta↗
PubMed1986

The effect of feedback on pathway transient response.

The effect of variation of the rate of input of material on the transient behaviour of metabolic pathways is examined. This reveals the existence of three transient times which make up the overall pathway transient. Two of these have been described previously and represent the times required for the accumulation of the free intermediate pool and the pool of enzyme-bound intermediate. They are state functions and as such are independent of the way in which the steady state was reached. The third is attributable to the variation in the rate of input of material to the pathway. It is dependent on three further factors. These are (a) the time required for the initial enzyme to reach its own steady state, (b) substrate depletion and (c) feedback. The description of the transient is: (Formula: see text) where V0 represents the rate of input and Vss represents the steady-state flux. The transient time associated with the transition between steady-states is shown to be a simple function of the transients for the establishment of each steady state from rest and may be expressed as: tau = tau b-Va/Vb . tau a where Va and Vb refer to the fluxes in the two steady states and tau a and tau b represent the transient times for the establishment of each of the steady-states from rest. The total pathway transient may now be completely defined as: (formula: see text) where summation over all intermediates, I, is implied. The significance of this to the analysis of pathway behaviour is discussed with more general examples of pathway transient analysis.

Enzymes↗
PubMed1984

The purification of yeast glucose 6-phosphate dehydrogenase by dye-ligand chromatography.

Glucose 6-phosphate dehydrogenase (EC 1.1.1.39) has been purified to homogeneity from baker's yeast by a simple procedure involving affinity elution from a column of red triazine dye, H-8BN, immobilized to Sepharose 6B. Eight milligrams of homogeneous protein is obtained in 53% yield from 200 g of dried yeast. This represents the first published purification of the enzyme from Saccharomyces Cerevisiae.

Chromatography↗
PubMed1984

The kinetics of consecutive enzyme reactions. The design of coupled assays and the temporal response of pathways.

A regime is proposed for the design of coupled enzyme assays in which auxiliary enzymes are added at concentrations proportional to their Km values. Under these conditions it is possible to calculate the complete time course of the assay including the time required for the system to approach its steady state. The consequence of increasing the number of coupling enzymes is shown to be a considerable decrease in time required to reach the steady state provided that the overall transient time remains the same. The method is extended to the general consideration of pathways and shows that pathways of the same length exhibit identical temporal responses provided that the units of concentration and time used are based on the steady-state concentration of intermediates and the transient time respectively. An unexpected finding is that increasing the number of intermediates in a pathway can decrease the time required to enter a steady state.

Enzymes↗
PubMed1981

A generalized theory of the transition time for sequential enzyme reactions.

In a sequence of coupled enzyme reactions the steady-state production of product is preceded by a lag period or transition time during which the intermediates of the sequence are accumulating. Provided that a steady state is eventually reached, the magnitude of this lag may be calculated, even when the differentiation equations describing the process have no analytical solution. The calculation may be made for simple systems in which the enzymes obey Michaelis-Menten kinetics or for more complex pathways in which intermediates act as modifiers of the enzymes. The transition time associated with each intermediate in the sequence is given by the ratio of the appropriate steady-state intermediate concentration to the steady-state flux. The theory is also applicable to the transition between steady states produced by flux changes. Application of the theory to coupled enzyme assays allows a definition of the minimum requirements for successful operation of the assay. The theory can be extended to deal with sequences in which the enzyme concentration exceeds substrate concentration.

Enzymes↗