Elmon L. Coe

Activities of glycolytic enzymes during the early stages of differentiation in the cellular slime mold Dictyostelium discoideum

Abstract Activities of fructose diphosphatase (EC 3.1.3.11) and the enzymes in the Embden-Meyerhof glycolytic sequence from hexokinase (EC 2.7.1.1) through lactate dehydrogenase (EC 1.1.1.27) were estimated in extracts of starved myxamoebae and cells at the preculmination stage of Dictyostelium discoideum. The activities, in terms of μmoles of substrate or product/min per 100 mg soluble protein at 22°, ranged from 0.1 or less and 0.4 for fructose diphosphatase and hexokinase, resp., to 35 to 49 for glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and phosphoglycerate kinase (EC 2.7.2.3), resp., in starved myxamoebae. Activities tended to increase slightly on progression to the preculmination stage, but in no case did the increase exceed a factor of 3, and in some instances (aldolase (EC 4.1.2.7) for example) a slight decline occured. Allowing the possible exception of pyruvate kinase (EC 2.7.1.40) the least active enzyme in the direction of gluconeogenesis was fructose diphosphatase, and there was no detectable increase in its activity during differentiation. It is concluded that any great acceleration in the rate of gluconeogenesis via glycolysis during culmination is brought about by changing substrate levels or by intracellular relocalization rather than by an increase in total enzyme activity. Comparison of the profile of glycolytic enzyme activities in the slime mold with those of liver, brain, and ascites tumor (literature values) revealed that the slime mold profile resembled that of liver most closely, but differed from all in its low lactate dehydrogenase activity.

ANALYSIS OF APPROACHES USED IN STUDYING DIFFERENTIATION OF THE CELLULAR SLIME MOLD

AbstractTwo approaches currently utilized in studying intracellular regulation of differentiation in D. discoideum have been analyzed. One tends to implicate transcription and translation as the most critical types of control during differentiation. The other approach assumes a priori that no single cellular event or component has inherent causal significance, but rather attempts to assess various factors limiting the rate of differentiation through a kinetic analysis of the relationships between enzymes, metabolites, and in vivo reaction rates. We have attempted to justify our prejudice for the latter approach, and have presented a kinetic analysis of the metabolism involved in the synthesis of carbohydrate end products. In addition, data pertaining to the control of energy metabolism are discussed in considering directions for expansion of the kinetic model.

Correlations between adenine nucleotide levels and the velocities of rate-determining steps in the glycolysis and respiration of intact Ehrlich ascites carcinoma cells.

Summary Changes were measured in the rates of respiration and in the levels of glycolytic intermediates during the first 4 min after addition of 0.77 mM glucose to respiring suspensions of Ehrlich ascites carcinoma cells in 54 mM phosphate buffer (pH 7.3) at 30°. The measured glycolytic products fully accounted for the glucose utilized. The respiratory rate was proportional to the ADP concentration. After 10 sec, the rate of the terminal segment of glycolysis was proportional to the ADP:ATP ratio, whereas the rate of the initial segment was proportional to the square of the ADP ATP ratio. A rapid nonglycolytic oxidation of glycolytically generated NADH occurred during the first 15 sec but not thereafter. Discrepancies in the lactate dehydrogenase (EC 1.1.1.27) and α -glycerophosphate dehydrogenase (EC 1.1.1.8) substrate ratios suggested an activation of a-glycerophosphate oxidase (EC 1.1.2.1) during glycolysis. At a period when glucose utilization had momentarily ceased, the theoretical rates of glycolytic and respiratory ATP synthesis were both substantial and nearly equal, indicating that mitochondrial trapping of ATP was not responsible for this phenomenon. Control of the initial steps of glycolysis is attributed to ADP activation of phosphofructokinase (EC 2.7.1.11) and limitation of hexokinase (EC 2.7.1.1) by product inhibition.

Inhibition of glycolysis in ascites tumor cells preincubated with 2-deoxy-2-fluoro-d-glucose

Abstract 2-Deoxy-2-fluoro- d -glucose is phosphorylated by hexokinase (EC 2.7.1.1) and is consumed by Ehrlich ascites carcinoma cells. In comparison with cells preincubated in buffer alone, cells preincubated with 0.5 mM 2-deoxy-2-F-Glc exhibited the following alterations in glycolysis between 5 and 25 sec after addition of 1 mM glucose: broaded maxima and slower declines in the glucose-6-P and fructose-6-P accumulation curves; and a much lower rate of fructose-1,6-P2 accumulation. Accumulations of lactate and other glycolytic intermediates in 2-deoxy-2-F-Glc-treated cells were either equal to or slightly higher than those in control cells. Preincubation with 2-deoxy-2-F-Glc also caused a 30% inhibition of 2- d -deoxyglucose consumption at both low and high concentrations of 2-deoxyglucose. Calculation of the velocities of glycolytic enzymes from accumulation rates between 5 and 15 sec after glucose addition revealed a 20–40% inhibition of hexokinase, phosphoglucose isomerase (EC 5.3.1.9), and fructose-6-P kinase (EC 2.7.1.11) and a slight stimulation of the remainder of glycolysis. These results are interpreted to mean that 2-deoxy-2-F-Glc and/or its derivatives specifically inhibit the hexokinase and fructose-6-P kinase steps and that the hexokinase inhibition is noncompetitive. The exact identity of the inhibiting species is not known.

A hypothesis relating oil : Water partition coefficients and vapor pressures of nonelectrolytes to their penetration rates through biological membranes

The energy barrier model of a cellular membrane proposed by Danielli 1952 and modified by Zwolinski, Eyring & Reese 1949 is employed in conjunction with the equations derived from absolute reaction rate theory by the latter investigators. It is assumed, in agreement with Danielli, that the major barrier to entry of a slowly penetrating nonelectrolyte molecule into a lipid membrane is at the membrane-water interface rather than in the membrane itself. It is further assumed that the molecule passing from water into a lipid membrane goes through a partially vaporized or “exposed” state where a statistical fraction of the penetrating molecules is associated with neither water nor lipid. The peak free energy of activation for entry into the membrane, ΔFM∗, may be equated to (1 − γ)ΔFL + γΔFV + ΔFB∗, where γ is related to the fraction of the molecule exposed during passage, ΔFL and ΔFV are the Gibbs free energy changes on going from an aqueous phase to a lipid phase and from an aqueous phase to a vapor phase, respectively, and ΔFB∗ is an additional activation energy barrier which is assumed constant for similar molecules. For a molecule passing completely into a dissociated or a vapor state, γ = 1·0; for a molecule completely unexposed or entirely in contact with either one or both phases, γ = 0. The free energy values are evaluated as follows: ΔFM∗ in terms of the permeability constant at 20°C, Φ; ΔFL in terms of the olive oil : water partition coefficient, C; and ΔFV in terms of the vapor pressure of the pure solute at 20°C, P. The following equation is derived for determination of γ: log (ΦC) = γ log (PC) + constant. Graphs of log (ΦC) versus log (PC) illustrate that most oxygen containing nonelectrolytes, ranging from the slowly penetrating erythritol to the rapidly penetrating propanol, fall close to a straight line. The slope of the line, γ, is near 0·6 for animal cells (ox and rabbit erythrocytes, sipunculid worm hemolymph cells, sea urchin eggs and rat lymphocytes) and near 0·4 for an algal cell (Chara).

Conversion of asparatic acid to glucose during culmination in Dictyostelium discoideum

Abstract Incorporation of [ 4 C]aspartate into alkali-insoluble carbohydrtae of the cellular slime mold Dictyostelium discoideum during culmination was used to evaluated the contribution of gluconeogenesis to polysaccharide synthesis. Bacterial and chemical degradation of glucose released by cellulase digestion of the alkali-insoluble carbohydrate indicated that 84% of the 14 C incorporated from [I- 14 C] aspartate was localized in the C-3 and C-4 positions of glucose and implicated glycolysis as the major pathway in gluconeogenesis. Greater randomization of label from [I- 14 C]aspartate was evident in glucose from trehalose, 73% being in the C-3 and C-4 positions of glucose. Slime mold undergoing culmination in the presence of a concentration of iodoacetate sufficient to decrease incorporation of [ 14 C]aspartate into polyasccharide by 50% exhibited no inteference with morphogenesis and only a slight delay in the rate of development. Approximations of teh rate of incorporation of intracellular [ 14 C]aspartate into alkali-insoluble carbohydrate gave values of 3 nmoles/min per ml cells or less, equivalent to less than 5% of the total rate of glucose conversion to polysaccharide during culmination. An indirect calculation of the minimal specific activity of the intracellular asparate pool from the specific activities of protein and nucleic acid indicated that less than 2% of the alkali-insoluble carbohydrate in the sorocarp was derived from aspartate. It is concluded that some gluconeogenesis from aspartate does occur but that it is not an essential source of the glucose utilized for stalk-building.

Bromoperoxidase from the marine snail, Murex trunculus

Abstract 1. 1. Extracts of a 25,000 g sediment of Murex trunculus hypobranchial gland homogenate contain an enzyme which brominates monochlorodimedon in the presence of bromide and H 2 O 2 . 2. 2. Dependence of activity on H 2 O 2 concentration exhibits a sharp optimum which increases from 20 μM at pH 7.4 to 400 μM at pH 4.0. 3. 3. Dependence on Br − concentration follows a simple saturation curve with K m values of 7 mM at pH 7.4 and 30 mM at pH 5.0. 4. 4. The pH optima depend on H 2 O 2 concentration, ranging from pH 6.6 at 10 μM to pH 4.8 at 200 μM. 5. 5. The enzyme is totally inactive with Cl − and F − , although both these ions inhibit bromination.

Theoretical phosphorylation rates after addition of a small amount of glucose to intact ascites tumor cells

Abstract Changes were measured in glycolytic and respiratory rates during the entire period of glycolysis and respiratory inhibition after addition of 0.08 or 0.15 mM glucose to Ehrlich ascites carcinoma cells in 54 mM phosphate buffer (pH 7.3) at 37°. Glycolytic products fully accounted for the glucose utilized. Theoretical rates of glycolytic ATP synthesis were calculated from the rates of accumulation of glycolytic products, and rates of oxidative phosphorylation were calculated from respiratory rates, assuming a P:O ratio of 3.0. The maximum in the glycolytic phosphorylation rate curve preceded the minimum in the respiratory phosphorylation rate curve. As a consequence, the total phosphorylation rate curve was biphasic, first rising above, then falling below, and finally returning to the initial, pre-glucose rate. The area under the early rise approximately equalled the area above the later dip and corresponded to between 1 and 2 μmoles of ATP/ml cells. The low rate of change in the ATP content of the cells indicated that most of the change in phosphorylation rate represented changes in both ATP synthesis and ATP utilization. It is hypothesized that ATP synthesized by glycolysis is more readily available to the ATP-utilizing systems. On addition of glucose, ATP is shifted from a respiratory to a glycolytic reservoir and a period of more rapid ATP utilization associated with a decrease in the level of endogenous substrates involved in the ATP-utilizing reactions ensues; after cessation of glycolysis, the process is reversed, and ATP utilization is slowed for a period while the endogenous substrates increase again.

Fatty acid compositions of lipid fractions from vegetative cells and mature sorocarps of the cellular slime moldDictyostelium discoideum

A wild-type strain ofDictyostelium discoideum was grown uponAerobacter aerogenes. Fatty acid compositions of lipid fractions and of total lipids obtained from vegetative amoebae and mature sorocarps were compared. Fatty acids isolated from vegetative cells were found to include large quantities of 17- and 19-carbon cyclopropane fatty acids while straight-chain, saturated fatty acids represented only 10% (w/w) of total fatty acids. These cyclopropane fatty acids appear to be derived from ingested bacteria and are preferentially incorporated into neutral lipids of the slime mold. Development of amoebae to mature sorocarps is accompanied by a substantial decrease in cyclopropane fatty acid content and a concomitant increase in unsaturated fatty acids, mostly as octadeca-5,11-dienoic acid. The †-22 stigmastenyl ester fraction is the richest source of this acid. Fully 65% of the fatty acids in this fraction are the octadecadienoate.

Possible regulatory interactions between compartmentalized glycolytic systems during initiation of glycolylsis in ascites tumor cells

Abstract Changes were measured in the rates of respiration and in the levels of glycolytic intermediates during the first 5 min after addition of 1.6 mM glucose to a suspension (5%, v/v) of respiring Ehrlich ascites carcinoma cells incubated in an isotonic 50 mM tris(hydroxymethyl)methylglycine buffer (pH 7.4) at 38 °C. The rates of accumulation of lactate and glycolytic intermediates were used to calculate the in vitro velocities of glycolytic enzymes. The initial velocities of hexokinase (EC 2.7.1.1), fructose-6-phosphate kinase (EC 2.7.1.11) and lactate dehydrogenase (EC 1.1.1.27) in μmoles glucose equivalents/ ml cells per min were 14, 11 and 4, respectively. The velocities of the two kinases fell sharply to less than 5 between 5 and 10 s, while the velocity of the dehydrogenase declined gradually over the first minute. The initial burst of activity in the kinases, which lasted for about 8 s, was associated with a rapid accumulation of phosphate ester and a negative net ATP generation by glycolysis. The accumulation of phosphate ester is almost exactly matched by the generation of ATP by the “tail end” of glycolysis (triose- P to lactate) in this period. After this time (10–25 s) the rate of oxidative phosphorylation calculated as six times the rate of O 2 consumption, is nearly identical to the combined rate of ATP utilization by hexokinase and fructose-6-phosphate kinase. As observed previously, oxamate (42 mM) blocked lactate dehydrogenase but did not depress the rate of phosphate ester accumulation. These various observations and correlations can be interpreted in terms of a dual glycolytic system. The accumulation of phosphate ester during the first 8 s is attributed to the operation of a partial glycolytic system, System B, which includes only the first three or four enzymes of glycolysis, and which draws upon an ATP pool (Pool I) previously employed in assorted cytoplasmic phosphorylations. The ADP generated by System B is rephosphorylated by and regulates the rate of a complete glycolytic system A, which converts glucose to lactate with little intermediate accumulation. The tail end of System A generates a new pool of ATP (Pool II) and controls the rate of glucose input through its head end, which is supplied by ATP being produced by oxidative phosphorylation. This scheme of interlocking controls is transient and alters after 8 s, when System B slows to a stop.