Ralph W. McKee

Interrelationships of metabolic pathways in the Ehrlich ascites carcinoma cells I. Glycolysis and respiration (Crabtree Effect)

Abstract 1. 1. Ehrlich ascites carcinoma cells grown for 7–8 days in C 57 /BL mice were used for in vitro chemical and metabolic time studies to evaluate the inhibition of oxygen consumption by the cell (Crabtree Effect). 2. 2. The time of onset of inhibited respiration was measured with an oxygen electrode. 3. 3. Determinations were made of the influence of monoiodoacetic acid and 2,4-dinitrophenol on the respiration of the tumor cells endogenously and in the presence of glucose. 4. 4. The rates of glucose and 2-desoxy- d -glucose metabolism and changes in certain metabolites (lactate, trioses, ketoses, pentose, ATP, ADP, AMP, DPN, TPN and total mono- and dinucleotides) were correlated with the onset, duration and termination of the Crabtree Effect. 5. 5. The influence of 2,4-dinitrophenol on glucose metabolism and changes in the same metabolites as indicated in point 4 were studied. 6. 6. The data obtained in these studies are evaluated in terms of the cell architecture, selective permeability of the mitochondrial membranes and the action of adenylate kinases. From these evaluations a tentative mechanism is suggested to explain the Crabtree Effect.

Lipid peroxidation and biosynthesis of l-ascorbic acid in rat liver microsomes☆

On incubating l-gulonolactone with rat liver microsomes, l-ascorbic acid is formed. l-Ascorbic acid, once formed, acts as a catalyst for the formation of a 2-thiobarbituric acid-reacting substance(s), tentatively lipid peroxide. The lipid peroxide, on the other hand, irreversibly inactivates the l-gulono dehydrogenase. Hence, in such a system, the amount of l-ascorbic acid formed does not give a true value of the enzymic activity. However, when a chelating agent or an antioxidant is added to the system, the formation of lipid peroxide is completely inhibited and the amount of l-ascorbic acid formed represents the true activity of the enzyme system. The use of a chelating agent has a further advantage. In the presence of 0.001 M KCN, it completely prevents the oxidation of l-ascorbic acid, so that the l-ascorbic acid synthesized can be estimated by simple titration with 2·6-dichlorophenol indophenol.

BIOSYNTHESIS OF L-ASCORBIC ACID IN RAT LIVER MICROSOMES: INFLUENCES OF AGE, SEX, DIETARY CHANGES, AND WHOLE-BODY X-IRRADIATION.

Abstract The activities of the liver microsomal enzymes in rat, namely, d -glucurono reductase, l -gulono dehydrogenase, and l -gulono oxidase, which convert d -glucuronolactone and l -gulonolactone into l -ascorbic acid, are very low in the embryonic stage. The activity increases after birth, attains a maximum on the 14th day, and then falls. Besides the effect of age, sex has also some influence on the biosynthetic capacity. Starvation or injection of l -ascorbic acid in rats results in a marked decrease in the enzymic activity of the liver microsomes. The biosynthetic capacity is also impaired after a lethal dose of whole-body X-irradiation, but this impairment again is caused by inanition rather than by a specific effect of X-irradiation. The results indicate that very probably the above-mentioned enzymes are adaptive enzymes.

Establishment of Resistance to Growth of Ehrlich Ascites Carcinoma in C57 Black Mice.

Summary It has been possible to immunize C57BL mice against Ehrlich carcinoma by giving 5 to 10 injections of Ehrlich carcinoma x-irradiated with 2000 to 4000 r. Of 70 mice so immunized 66 survived the first challenge with viable cells; and all 35 of the mice receiving 8 challenges survived. It appears that the carcinoma cells are altered by x-irradiation and thus stimulate production of resistance to both x-irradiated and completely viable Ehrlich carcinoma cells.

Effects of iodoacetate on glycolysis and respiration in Ehrlich-Lettré ascites carcinoma cells

Summary Iodoacetate added in optimum concentrations either to an Elirlich-Lettre hyperdiploid ascites carcinoma whole cell or to a mitochondria-dialyzed cell sap system from Ehrlich-Lettre cells stimulates oxygen consumption. This cell possesses within its cytoplasm an active α -glycerophosphate dehydrogenase enzyme system but an even more active lactate dehydrogenase (EC 1.1.1.27). The latter system is so active that it allows very little NADH to be utilized by the α -glycerophosphate dehydrogenase (EC 1.1.1.8) enzyme. However, with partial iodoacetate blockage of the glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) enzyme system it appears that more substrate and NADH are available for the α -glycerophosphate dehydrogenase to produce α -glycerophosphate. Since the mitochondria contain a flavin-mediated α -glycerophosphate dehydrogenase more oxidation results via that pathway. Partial blockage of α -glycerophosphate dehydrogenase decreases the amount of lactate formed but still produces adequate pyruvate to allow maximum mitochondrial oxidation of this substrate. Thus with pyruvate oxidation at or near a maximum, plus an enhanced NADH oxidation via α -glycerophosphate inside the mitochondria, there is a marked increase of the total oxygen consumption rate with optimum partial blockage of α -glycerophosphate dehydrogenase with iodoacetate. The precise reasons for this shift and increase of oxygen utilization are not known, but probably lie in the kinetics of the lactate dehydrogenase and α -glycerophosphate dehydrogenase enzyme systems. Further investigations are being conducted to determine details of the mechanism.

Isolation and properties of lactate dehydrogenase isozyme X from swiss mice

Abstract Lactate dehydrogenase X, a sixth LDH isozyme in testes and mature sperm, has been purified to homogeneity from adult mouse testes. The isozyme has a molecular weight of 139,000 ± 6000. Dissociation of the molecule with 6 m guanidine produced a homogeneous material with mol wt 37,700 ± 800. Pyruvate at 37 ° was reduced optimally at about pH 7.25 and a concentration of 0.25 m m . Alpha-ketobutyrate has an optimum pH of 7.75 and concentration of 0.5 m m for reduction with a rate about 50% greater than that of pyruvate. Alpha-ketoglutarate has an optimum pH of about 5.5 and is reduced at only about one-fourth the rate of pyruvate. Pyruvate reduction is considerably inhibited by 2.5 m m lactate. Alpha-hydroxybutyrate and α-hydroxyvalerate are oxidized at about the same rate and under similar optimum conditions as lactate. Alpha-hydroxyglutarate is oxidized at only about one-twentieth the rate of lactate.

Determination of nicotinamide and metabolic products in urine by high-performance liquid chromatography.

A high-performance liquid chromatographic procedure has been developed for the quantitation of nicotinamide, nicotinic acid, nicotinuric acid, 1-methylnicotinamide and 1-methyl-2-pyridone-5-carboxamide in rat and human urines. The procedure utilizes a Varian Model 5020 liquid chromatograph with a UV detector, and an Altex 15 cm X 4.6 mm Ultrasphere-ODS column, employing a linear ion-pair mobile phase gradient. Solvent A contains 10 mM concentrations of pentanesulfonic acid (PSA), tetramethylammonium chloride (TMA) and KH2PO4, and solvent B contains PSA, TMA and acetonitrile. Different pH values for solvent A vary the retention times and thus the separation of the five compounds. Temperature of the system is critical. The conditions found most satisfactory were pH 3.30 and 24.5 degrees C.

Influence of lodoacetate on glycolytic intermediates and on respiration in ehrlich-lettre ascites carcinoma cells

Abstract Glycolytic intermediates have been determined in whole tumor and tumor cells, before and after washing, at intervals of time after glucose and glucose plus iodoacetate (IAA) addition to washed tumor cells. The influences of IAA on glycolysis and respiration have been determined on washed mitochondria plus cell sap. The effects of added α-glycerophosphate (α-GP), pyruvate, and both on glycolytic intermediates and respiration of washed whole cells and of mitochondria plus cell sap also have been determined, and the oxidation of α-GP and appearance of dihydroxyacetone phosphate (DHAP) were measured in five-times washed mitochondria. The findings definitely show that the addition of an optimum amount of iodoacetate to either whole cells or mitochondria plus cell sap causes an increased accumulation of DHAP and α-GP, as well as pyruvate, which in turn is responsible for a stimulation of respiration.

RESPIRATION AND GLYCOLYSIS IN X-IRRADIATED EHRLICH MOUSE ASCITES CARCINOMA CELLS.

>Ehrlich ascites tumor cells exposed in vitro at a concentration of 80 mg dry-weight equivalent per milliliter to 500 to 4000 r showed no significant alterations in glycolytic or oxidative metabolism relative to unirradiated controls. At 8000 r, however, there was some indication of damage to glycolysis. Cells irradiated with 500 to 8000 r all showed an increase in ATP concentration, the increase being greater at the higher levels of radiation. These observations are discussed with relation to the fact that cells irradiated with 2000 to 4000 r no longer kill their hosts, but instead grow briefly and then regress, producing an immunity toward the nonirradiated ascites cell. (auth)

Effects of inanition, protein depletion and repletion on serum lactic acid dehydrogenase levels in rats.

Summary The effects of inanition, protein depletion and repletion upon serum lactic acid dehydrogenase (SLD), total serum protein and hematocrit values have been determined in adult, male Sprague-Dawley rats. Significant increases in concentration of SLD concomitant with decreases in serum protein levels followed inanition and protein depletion. Upon repletion, subnormal SLD values occurred in groups fed a diet containing 17% or more of protein. Serum protein values for the groups were significantly increased. The results are discussed with respect to possible causes for observed changes.