Lillian Garfinkel

Magnesium in cardiac energy metabolism

Free intracellular magnesium ion, which influences many metabolic processes, is the subject of ongoing research. Its concentration has been difficult to measure because the available methods, including dye injection, microelectrodes, and nuclear magnetic resonance measurements, are invasive or indirect. Concentrations ranging from 0.1 mM in frog muscle to 6 mM in barnacle muscle have been reported. We describe recent experimental evidence regarding the concentration of free intracellular magnesium and consider the limitations of these methods. A substantial body of evidence, including our models of cardiac energy metabolism and its magnesium-related processes, indicates that intracellular concentrations of free magnesium are low (ca. 0.4 mM) and vary with time and conditions.

Simulation of the detailed regulation of glycolysis in a heart supernatant preparation

Abstract A study of the regulation of the glycolytic pathway has been carried out by simulating in detail the behavior of three glycolyzing beef heart supernatant preparations. Enzyme activity profiles as well as kinetic data on glycolytic intermediates and adenine nucleotides were obtained in the laboratory using glucose-6-phosphate as a substrate. Phosphoglycerate mutase appears to be the limiting enzyme, while triosephosphate isomerase, the most active enzyme when measured separately, is relatively inactive in the complete system. A model consisting of 74 simultaneous differential equations representing 107 chemical reactions were fitted to these results. Incorporated into this model were detailed models of 13 enzymes and crude models of the pentose shunt and ATPase. For most data points, the fit appears fairly good. Most of the enzyme models show strong inhibition as glycolysis proceeds. Individual enzymes were modeled with data from the literature obtained from isolated enzymes. The action of only one enzyme could be satisfactorily simulated without some modification in the form of additional activations, inhibitions, and enzyme-enzyme interactions. This model suggests that the activity of many enzymes is a function of several glycolytic intermediates or products arranged to slow the rate of glycolysis as glycolytic products pile up.

Computer modeling of muscle phosphofructokinase kinetics.

The kinetics of the phosphofructokinase reaction were studied by computer modeling. A general random order, two-state allosteric model, of which the Monod--Wyman--Changeux model is a limiting case, was found to most accurately reproduce the experimental observations of Pettigrew & Frieden (1979 a,b). A simplified model with Hill coefficients was found to fit almost as well. In these models substrates bind preferentially to and stabilize the enzyme in the R state, and ATPH3-, the inhibitory species, binds preferentially to and stabilizes the enzyme in the T state. Enzymatic activity is regulated by conversion from the R to the T state, which is effected by protonation, especially of the uncomplexed enzyme, but the experimental data are inadequate for accurate estimation of the pKa of the enzyme. Random order binding of substrates is an important cause of sigmoidal kinetics. Additional experiments that would aid in the discrimination among rival models are described.

A computer model of pancreatic islet glycolysis

We have modeled an experiment with perifused pancreatic islet cells using our BIOSSIM language. The experiment and the resulting model are concerned with glucose uptake and glycolysis by the beta-cells of pancreatic islets. Although glycolysis appears to be involved in insulin release, we do not have enough information to represent insulin release in detail. The rapid entry of glucose into the beta-cell is promoted by a carrier having a very high tissue capacity. Phosphorylation of glucose by the low affinity enzyme glucokinase appears to be limiting for glycolysis. The effects of several hexose diphosphate activators of phosphofructokinase are modeled. Model behavior is described. The kinetic parameters of the enzyme submodels are given. Because of the difficulties of preparing large amounts of experimental material, information on pancreatic islet metabolism is limited. This model is a plausible explanation of the experimental results. Recent work on the genetically engineered glucose transporter and glucokinase is discussed.

Computer Simulation as a Means of Physiological Integration of Biochemical Systems

Traditionally biochemistry has been concerned with analyzing the components of living systems, and has paid less attention to how they interact. While the information available about individual entities such as proteins and nucleic acids is now immense and still growing rapidly, the task of determining how they interact to form a living system is now only beginning, especially with respect to quantitative behavior. In reviewing a recent book, Dr. J. S. King (1975), the editor of Clinical Chemistry has written “biochemistry was mainly a reductionist science; only lately has it become possible to fit together facts and observations into what is beginning to be a coherent -- and thus more interesting -- whole”.

Computer technology for the realistic calculation of properties of enzyme systems

Traditional pencil-and-paper analyses of enzyme kinetic experiments assume so much simplification that the results have limited biological significance. Althouqh performing initial velocity experiments with negligibly low enzyme concentrations in the presence of single inhibitors facilitates interpretation, there are a number of enzymes for which this method fails completely. Software has been developed for the economical simulation of enzyme behavior under realistic conditions. Enzyme activity is computed as a function of either time or concentration by solution of either differential or algebraic equations with any desired ratio of enzyme/substrate concentrations. Simulation of experiments with hexo-kinase from mouse ascites cells has permitted resolution of some apparently contradictory results and indicated guidelines for assuring reliability of data. Applications of lab. analyzers to improve interpretation of experiments and perhaps actually perform them are discussed.