David N. Dietzler

Osmotic stress drastically inhibits active transport of carbohydrates by Escherichiacoli

In intact Escherichia coli cells, severe osmotic stress almost totally inhibited active transport of carbohydrate by all of the systems known to transport carbohydrates in E. coli: group translocation (glucose), binding-protein mediated transport (maltose), proton symport (lactose), and sodium cotransport (melibiose). Detailed study of glucose transport showed that this inhibition of transport was not secondary to the inhibition of growth by osmotic stress, but rather that the inhibition of transport of a source of carbon and energy was sufficient to cause the complete inhibition of growth observed during severe osmotic upshock. Transport and growth inhibition did not result from cell death; upshocked cells were viable and metabolically active.

Restoration of cell volume and the reversal of carbohydrate transport and growth inhibition of osmotically upshocked Escherichiacoli

Resumption of growth in osmotically upshocked Escherichia coli was effected only by an external stimulus (betaine treatment) in severe upshock, but was spontaneous in less severe upshock. In either case, growth resumption was preceded by a reversal of glucose transport inhibition, and that reversal was preceded by a recovery of cell volume. We hypothesize that deformation of the membrane by osmotic stress results in conversion of a membrane component of the transport system to a less functional conformation, which results in the inhibition of transport and the consequent inhibition of growth. Relief of the deformation would then allow recovery to a more functional conformation, reversal of transport inhibition, and then resumption of growth.

Simultaneous increases of the adenylate energy charge and the rate of glycogen synthesis in nitrogen-starved Escherichia coli W4597(K).

When exogenous nitrogen is exhausted in cultures of E. coli W4597 (K) containing excess glucose, the rate of glycogen synthesis increases (3.33-fold), adenylate energy charge increases from 0.74 to 0.87 and FDP decreases (77%). This is the first observation of parallel changes in vivo in the adenylate energy charge and the rate of glycogen synthesis, and of an increase in the adenylate energy charge in vivo with maintenance of the adenine nucleotide pool size and adenylate kinase mass action ratio when growth is limited. This report is also the first direct experimental evidence of the major elements of the Preiss group hypothesis concerning in vivo regulation of bacterial glycogen synthesis by FDP, NADPH, and PLP, the primary activators of ADPG pyrophosphorylase, the rate-limiting enzyme in this synthetic pathway. By taking into account in vitro activities of the enzymes of glycogen metabolism in E. coli W4597(K) and in E. coli B we have concluded that the decrease in FDP is offset by the increase in energy charge and that FDP contributes to the increased rate of glycogen synthesis we observe, while activation by NADPH is improbable. Additional activation by PLP remains a possibility and is discussed.

Rates of glycogen synthesis and the cellular levels of ATP and FDP during exponential growth and the nitrogen-limited stationary phase of Escherichia coli W4597 (K)☆

Abstract The cessation of growth in a culture of E. coli W4597(K) which occurs when the nitrogen source ( NH 4 + ) is exhausted in the presence of excess glucose is accompanied by a 4.17-fold increase in the rate of glycogen synthesis and the cellular ATP level increases 50% while the cellular FDP level decreases 76%. These data provide the first experimental evaluation concerning certain elements of the hypotheses of previous investigators to account for the increased accumulation of glycogen which occurs when bacterial growth is limited in the presence of an excess of a carbon and energy source.

Maintenance of the energy charge in the presence of large decreases in the total adenylate pool of Escherichia coli and concurrent changes in glucose-6-P, fructose-P2 and glycogen synthesis

Abstract Lowered aeration and lowered aeration in the presence of chloramphenicol cause a respective 40% and 60% decrease in the total adenylate concentration of nitrogen-starved E. coli W4597 (K). These decreases are accompanied by maintenance of the adenylate energy charge and adenylate kinase mass action ratio, increases in fructose-1,6-P2 and glucose-6-P, and decreases in the net rates of glycogen synthesis. The important new observation is that a drastic decrease in the total adenylate pool is accompanied by complete maintenance of the energy charge. These decreases in the adenylate pool are correlated with the accompanying metabolic changes.

Regulation of ADP-glucose synthetase, the rate-limiting enzyme of bacterial glycogen synthesis, by the pleiotropic nucleotides ppGpp and pppGpp

Summary Stimulated physiological concentrations of guanosine 5′-diphosphate 3′-diphosphate (ppGpp) and guanosine 5′-triphosphate 3′-diphosphate (pppGpp) cause a marked inhibition of ADP-glucose synthetase activity in crude extracts of Escherichia coli W4597(K) but basal cellular concentrations only weakly affect the activity. Inhibitory concentrations decrease the maximal velocity and increase the amount of fructose-P 2 and glucose-1-P needed for half-maximal activity. ADP-glucose synthetase is apparently the rate-limiting enzyme in bacterial glycogen synthesis. Thus, the data presented here provide the first evidence that the highly phosphorylated guanine nucleotides may play a role in the regulation of bacterial glycogen synthesis.

Evidence for the regulation of bacterial glycogen synthesis by cyclic AMP.

Summary Exogenous 3′,5′-cyclic AMP causes a 1.5-fold increase in the rate of glycogen synthesis in E. coli W4597(K) using glucose. This increase is effected without increases in known factors (i.e., the cellular level of the energy charge, fructose-P 2 or glucose-1-P) which increase the velocity of the rate-limiting enzyme of bacterial glycogen synthesis, ADP-glucose synthetase and without an increase in the level of this enzyme. The inhibition by glucose of the rate in cultures using succinate and the antagonism of this inhibition by cyclic AMP cannot be explained by changes in the known factors or the enzyme level. These results and others presented here provide the first evidence that cyclic AMP plays a role in regulating bacterial glycogen synthesis.

Evidence for the allosteric regulation of bacterial glycogen synthesis in vivo

Abstract In stationary-phase cultures of either Escherichia coli W4597(K) or G34 in various nutrient conditions there is a 10-fold range of steady-state rates of glycogen synthesis with an essentially constant steady-state level of ATP, presumably reflecting an essentially constant energy charge. The steady-state level of fructose-1,6-diphosphate in these cultures varies from experiment to experiment as a function of the observed rate of glycogen synthesis. These data were fitted to the Hill equation using an assumed Hill coefficient of 2: a plot of [Fru- P 2 ] 2 / rate of glycogen synthesis versus [Fru- P 2 ] 2 is linear with a correlation coefficient greater than 0.999, indicating a causal relationship between the concentration of Fru- P 2 and the rate of glycogen synthesis. These data provide further evidence that allosteric effects observed in vitro function in vivo .

Purification of five creatine kinase-MM variants from human heart and skeletal muscle

Variants of creatine kinase-MM (variant of ATP:creatine N-phosphotransferase, EC 2.7.3.2), present in human heart and skeletal muscle, have been purified to homogeneity using DEAE-Sepharose column chromatography and column chromatofocusing techniques. Creatine kinase-MM I-IV were present in both heart and skeletal muscle, while MM-V was found only in heart. The number, ratio and elution profile of the variants during chromatofocusing remained identical even when they were purified in the presence of proteinase inhibitors. MM-I-V, on chromatofocusing, were eluted at pH 8.3, 7.9, 7.6, 7.2 and 6.8, respectively. Isoelectric focusing revealed the pI of MM-I-V to be 7.2, 6.9, 6.7, 6.4 and 6.2. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a doublet pattern for creatine kinase-MM variants III-V. However, polyacrylamide gel electrophoresis without SDS indicated homogeneity because each variant showed a single band. The doublet pattern observed in the presence of SDS may reflect the presence of two subunits of slightly different mass.

Adaptation of the EMIT theophylline assay to kinetic analyzers: the relationship of reaction kinetics to calculation procedures.

We investigated the kinetic characteristics of an enzyme immunoassay system (EMIT) for the determination of theophylline. Less than 10% of the glucose-6-phosphate and NAD+ are consumed and the glucose-6-P-dehydrogenase-theophylline complex is essentially saturated with these substrates during the course of the reaction. However, apparently as the result of antibody heterogeneity, the rate does change during the course of the reaction. As a result, the values of the kinetic parameters for the theophylline saturation curve vary with the timing interval chosen for measurement. We show that the values of these parameters govern the application of the available methods of calculating EMIT data, the graphical procedure suggested by the manufacturer, logit-log, log-log, and curve fitting. These studies (1) explain the basis of the graphical procedure and why it does not always provide proper calibration, (2) show that to appropriately understand the application of any of the calculation procedures one should determine the values of the kinetic constants of the theophylline saturation curve in the particular assay conditions, and (3) illustrate simple, practical procedures for determining the values of these constants for any instrument-EMIT assay system. Specific illustrations are shown for the EMIT theophylline system with two kinetic analyzers, the Abbott ABA-100 and Gilford 3500, with which markedly different reaction conditions are used.