Edwin M. Chance

Comparison of the kinetic properties of the lipid- and protein-kinase activities of the p110alpha and p110beta catalytic subunits of class-Ia phosphoinositide 3-kinases.

Growth factors regulate a wide range of cellular processes via activation of the class-Ia phosphoinositide 3-kinases (PI 3-kinases). We directly compared kinetic properties of lipid- and protein-kinase activities of the widely expressed p110alpha and p110beta isoforms. The lipid-kinase activity did not display Michaelis-Menten kinetics but modelling the kinetic data demonstrated that p110alpha has a higher V(max) and a 25-fold higher K(m) for PtdIns than p110beta. A similar situation occurs with PtdIns(4,5)P(2), because at low concentration of PtdIns(4,5)P(2) p110beta is a better PtdIns(4,5)P(2) kinase than p110alpha, although this is reversed at high concentrations. These differences suggest different functional roles and we hypothesize that p110beta functions better in areas of membranes containing low levels of substrate whereas p110alpha would work best in areas of high substrate density such as membrane lipid rafts. We also compared protein-kinase activities. We found that p110beta phosphorylated p85 to a lower degree than did p110alpha. We used a novel peptide-based assay to compare the kinetics of the protein-kinase activities of p110alpha and p110beta. These studies revealed that, like the lipid-kinase activity, the protein-kinase activity of p110alpha has a higher K(m) (550 microM) than p110beta (K(m) 8 microgM). Similarly, the relative V(max) towards peptide substrate of p110alpha was three times higher than that of p110beta. This implies differences in the rates of regulatory autophosphorylation in vivo, which are likely to mean differential regulation of the lipid-kinase activities of p110alpha and p110beta in vivo.

Metabolism of alternative substrates and the bioenergetic status of EMT6 tumor cell spheroids

In order to evaluate the ability of EMT6/Ro multicellular spheroids to utilize various pathways of energy production, 13C and 31P MRS have been employed to monitor the metabolism of glucose, glutamine, acetate and propionate. EMT6/Ro spheroids perfused with culture medium containing 5.5 mM glucose maintain stable levels of nucleotide triphosphates (NTP) and phosphocreatine (PCr) for up to 48 h, even in the absence of glutamine. The metabolism of 1‐13C‐glucose was almost entirely to 3‐13C‐lactate (88 ± 12%, n = 7), even though the perfusion medium was equilibrated with 95% O2. Labeling was also observed in other glycolytic metabolites, primarily alanine and α‐glycerolphosphate. A low level of 13C labeling in glutamate, indicative of mitochondrial oxidative metabolism (TCA cycle), was consistently detected when spheroids were perfused with 1‐13C‐glucose, almost exclusively in the C4 position of glutamate. Labeling of glutamate C2 and C3 was always less than 20% of the labeling in C4 and was usually undetectable. No evidence of adjacent carbon labeling in individual glutamate molecules (indicative of multiple cycles of label incorporation) was found, even in high‐resolution 13C NMR spectra of extracts from cells or spheroids. Despite the predominantly glycolytic metabolism of glucose, the mitochondrial substrate glutamine (2 mM, in the presence of ≤0.5 mM glucose from fetal bovine serum), supported stable levels of NTP and PCr in the tumor cells for up to 12 h. In the presence of 2.5 mM acetate, the bioenergetic status of cells in EMT6 spheroids declined slowly but measurably, and no incorporation of label from 2‐13C‐acetate into other metabolites was detected either in intact perfused spheroids or in high‐resolution spectra of extracts. In contrast, when the anaplerotic TCA cycle substrate 3‐13C‐propionate replaced acetate, the high‐energy phosphate levels in EMT6/Ro spheroids were somewhat reduced, but stabilized at a new lower level. Incubation of spheroids with 3‐13C‐propionate (with natural abundance glucose and glutamine) resulted in label detectable in the C2 and C3 of glutamate, but the primary labeled compound was methylmalonate, an intermediate in propionate metabolism. Addition of vitamin B12, a cofactor for methylmalonyl CoA reductase, to the growth medium 24 h prior to perfusion with propionate resulted in the elimination of the methylmalonate resonance. A variety of 2‐ and 3‐labeled metabolites were detected, including succinate, malate and glutamate. Labeling of C2 and C3 of lactate implicated cytoplasmic malic enzyme activity. Copyright

Automatic techniques in enzymes simulation

Abstract This paper describes a digital computer program written in Fortran IV which will translate a system of chemical reactions into simultaneous differential equations based on mass action kinetics. This theoretical representation of the complex chemical system is required to take overall account of the behavior of such a system when only a small number of experimental variables can be observed at any one time.

Modeling of Oxygen Diffusion and Metabolism from Capillary to Muscle

In the bioengineering and sports training field, there are interests in obtaining information on oxygen transport and metabolism during muscle exercise. The goal of our study was to develop a time-dependent model which can simulate the changes in oxygen diffusion and metabolism in muscle tissue, especially from capillary to mitochondria. We postulate that oxygen diffuses into the tissue at a rate proportional to the oxygen concentration in plasma when applied to a steady state condition. As we know, any build up of oxygen in the tissue is directly related to oxygen leaving the capillary into the tissue and the consumption of oxygen by the mitochondria, or the metabolic demand. We designed a model which consisted of four compartments: (1) capillary; (2) interstitial space between capillary and myofibril tissue cell; (3) parenchymal cell through myofibril to mitochondria in muscle tissue; (4) at mitochondria. Oxygen comes from arteriole to capillary, then diffuses to mitochondria through interstitial space and myofibril. Oxygen is reduced to water in mitochondria, and ATP is generated through proton transport. Our model simulates the measured oxygen uptake during rest and muscle exercise as the input oxygen uptake represented as H2O formation.

CO BINDING TO MITOCHONDRIAL MIXED VALENCE STATE CYTOCHROME OXIDASE AT LOW TEMPERATURES

The kinetics and thermodynamics of the reaction of mixed valence state membrane-bound cytochrome oxidase with CO over the 178-203 K range has been studied by multichannel optical spectroscopy at three wavelength pairs (444-463 nm in the Soret region, and 590-630 and 608-630 nm in the alpha region) and analysed by non-linear optimization techniques. As in the case of the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore, G.M. and Chance, E.M. (1978) Biochem J. 175, 709-725), the normalized progress curves at the three wavelength pairs are significantly different indicating, on the basis of Beers law, the presence of a minimum of three optically distinct species. The only model that satisfies the triple statistical requirement of a standard deviation within the standard error of the data, a random distribution of residuals and good determination of the optimized parameters, is a two species sequential mechanism: flash photolysis of the mixed valence state cytochrome oxidase-CO complex (species IIMC) yields unliganded mixed valence state cytochrome oxidase (species EM) and free CO which then recombine to form species IMC; species IMC is then converted into species IIMC. All the thermodynamic parameters describing the model are calculated and compared to those obtained for the fully reduced membrane-bound cytochrome oxidase-CO reaction (Clore and Chance (1978) Biochem. J. 175, 709-725). Although there are some qualitative similarities in the kinetics and thermodynamics of the reactions of mixed valence state (alpha 23+Cu+B.ALPHA 3+Cu2+A) and fully reduced (a3 2+Cu B + . a2+Cu A+) cytochrome oxidase with CO, there are large and significant quantitative differences in zero-point activation energies and frequency factors; over the temperature range studied, the mixed valence state cytochrome oxidase-CO reaction is found to proceed at a significantly slower rate than the fully reduced cytochrome oxidase-CO reaction. These differences indicate that changing the valence states of cytochrome a and CuA has a significant effect on the CO binding properties of cytochrome a 3 and possibly CuB.

Oxygen Delivery to Tissue: Calculation of Oxygen Gradients in the Cardiac Cell

In this study, a two dimensional mathematical model of a cross section of a single heart muscle cell 30.0 u in diameter, was constructed from which one would be able to calculate the rate of oxygen delivery from the capillary blood supply to the mitochondria by passive oxygen diffusion, myoglobin facilitation of oxygen delivery, and subsequent utilization of oxygen by cytochrome oxidase. From such calculations, the conditions under which the two hemoprotein indicators, myoglobin and cytochrome oxidase, behave in a coherent manner, as observed by Tamura and his colleagues (1978), could be established and the loci of oxygen gradients identified.

A computer model of allosteric kinetics involving binding of a single substrate molecule.

Rabin* has presented a possible alternative to the theory of Monod, Wyman and Changeux2 to account for cooperative activation of enzymic reaction velocity by the enzyme’s substrate in terms of a monomeric mechanism. Kinetics of atlosteric enzymes, in which binding studies are not yet possible, can be accounted for by the Monod theory using the Hill plot as an indirect indication of the number of substrate binding sites available or as a measure of cooperativity. Furthermore, sigmoidal progress curves have been observed in certain cases by Hess.” in this paper, the Rabin mechanism has been simulated by computer to yield a solution of a general case, which gives both cooperative saturation and sigmoidal progress curves. This mechanism postulates the binding of one substrate molecule to the enzyme and in all cases in which a cooperative saturation curve was obtained, Hill exponents greater than 1 .O were observed.