Mary P. Leckie

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.

d‐β‐HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D‐β‐hydroxybutyrate (β‐OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3‐14C]β‐OHB or [2‐14C]glucose was injected subcu‐taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3‐14C]β‐OHB rapidly labelled brain amino acids at all ages whereas [2‐14C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3‐14C]β‐OHB from birth‐15 days of age whereas brain alanine was more heavily labelled by [2‐14C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β‐OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [14C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2‐,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3‐14C]β‐OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [14C]label from [2‐14C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [14C]label into the brain amino acids of the 15‐day‐old [3‐14C]β‐OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?‐OHB at 6 days was 19 8 min and at 15 days was 12‐2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3‐14C]β‐OHB injected rats compared to the [2‐14C]glucose injected rats despite a heavier labelling of brain glutamate in the [3‐14C]β‐OHB injected group. We interpreted these data to mean that β‐OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.

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.

Subcellular distribution of ketone body metabolizing enzymes in the rat brain.

RECENT interest in the utilization of ketone bodies by brain tissue has directed attention towards the three responsible enzymes: D(-)-P-hydroxybutyrate dehydrogenase (EC 1.1.1.30), 3-oxoacid CoA transferase (EC 2.8.3.5) and acetoacetyl CoA thiolase (EC 2.3.1.9). Both the dehydrogenase and transferase enzymes are located within the crude mitochondria] fraction (KLEE & SOKOLOFF, 1967: TILDON ei al., 1971; WILLIAMSON et al., 1971), whereas the total thiolase activity reflects the activities of two isozymes, one cytoplasmic and the other mitochondria1 (MIDDLETON, 1973). Changes in the activities of thesc three enzymes during cerebral development of the rat has been investigated in several laboratories (KLEE & SOKOLOFF, 1967; TILDON et al., 1971; PAGE et a!., 1971; DIERKS-VENTLING & CONE, 1971 ; MIDDLETON, 1973). These studies have shown that the three bound enzymes involved in the cerebral oxidation of ketone bodies are induced during the suckling period and then drop after weaning. The cytoplasmic thiolase activity is not induced during the suckling period. Rather, it is maximal at birth and gradually falls throughout development to a lower activity characteristic of the mature brain. There is no information concerning the subcellular distribution of the cerebral enzymes involved in ketonebody metabolism in the immature and mature rat brain, despite these several reports describing their developmental profile. This report concerns the subcellular distribution of these three ketone body metabolizing enzymes in the immature and mature brain of the rat to further our understanding of the role played by ketone bodies in energy metabolism during cerebral development (DEVIVO et al., 1975). A preliminary report of these subcellular fractionation studies has been presented (DEVIVO et a/., 1975a).

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 .