Energy metabolism of the anaerobic protozoon Giardia lamblia

Cells of the aerotolerant anaerobe Giardia lamblia respire in the presence of oxygen. Endogenous respiration is stimulated by glucose but not by other carbohydrates and Krebs cycle intermediates. Endogenous and glucose-stimulated respiration are insensitive to cyanide, malonate, and 2,4-dinitrophenol, but are inhibited by atabrin and iodoacetamide. G. lamblia produces ethanol, acetate and CO₂ both aerobically and anaerobically either from endogenous reserves or exogenous glucose. Molecular hydrogen is not produced. The following enzyme activities were detected in homogenates: hexokinase, fructose-biphosphate aldolase, pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malate dehydrogenase (decarboxylating), pyruvate synthase, acetyl-CoA synthetase, alcohol dehydrogenase (NADP⁺), NADH dehydrogenase, NADPH dehydrogenase, NADPH oxidoreductase and superoxide dismutase. The enzymes of energy and carbohydrate metabolism are nonsedimentable (109 000 × g for 30 min). Activities of lactate dehydrogenase, hydrogenase, phosphate acetyltransferase, acetate kinase, citrate synthase, succinate dehydrogenase, fumarate hydratase and catalase were below the limits of detection. The results suggest the occurrence of glycolysis, energy production by substrate level phosphorylation and a flavin, iron-sulfur protein mediated electron transport system as well as the absence of cytochrome mediated oxidative phosphorylation and a functional Krebs cycle.     

Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias.

Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm-1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.     

Histoenzymological study of selected dehydrogenase enzymes in Pneumocystis carinii.

The metabolic activity of Pneumocystis carinii cysts was studied histochemically by a tetrazolium dye technique to assess substrate-specific dehydrogenase activity. Lactate dehydrogenase, succinate dehydrogenase, and glutamate dehydrogenase produced moderate-to-strong reactions in the cysts, whereas glucose-6-phosphate dehydrogenase had little if any reactivity. These results suggest that pneumocystis cysts have some of the enzymes necessary for glycolysis, Krebs cycle activity, and intermediary protein metabolism. These studies provide a method of directly assessing metabolic pathways in P. carinii which circumvents the uncertainties of specificity inherent in previous investigations with partially purified suspensions.     

Histochemical research on metabolic pathways of glucose in some species of Mollusca Gastropoda

The metabolic pathways of glucose were studied by histochemical reactions in some species of gastropods living in different habitats. The glycolytic pathway is histochemically indicated by positive results for glucose-6-phosphate isomerase, fructose-1,6-biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and D-lactate dehydrogenase. The enzymes of the Krebs cycle gave different responses: isocitrate dehydrogenase and L-malate dehydrogenase were positive, whilst succinate dehydrogenase was constantly negative. Malate synthetase activity was also demonstrated. Despite L-glutamate dehydrogenase is undetectable, the presence of transaminase indicates the gluconeogenetic route. Phosphoglucomutase and glucose-6-phosphate phosphatase appear also positive. The metabolic meaning of our results were discussed.     

Histo- and cytochemical enzymatic characteristics of breast cancer

In most of the cases studied, histo- and cytoenzymochemical values of metabolism in cancer of the mammary gland correlate with the histological type, differentiation level and the degree of the tumor malignancy. As the histological degree of malignancy increases and the level of differentiation of the mammary cancer decreases, the activity of glycolysis and pentous shunt enzymes, lactate dehydrogenase and glucose-6-phosphate dehydrogenase, increases and that of the Krebs cycle enzymes, succinate and malate dehydrogenase, as well as the enzyme of glycerophosphate shuttle mechanism and oxidation of glycerol-L-glycerophosphate dehydrogenase decreases simultaneously. These data suggest that histo- and cytochemical methods of the enzyme activity determinations may be used for the specification of the prognosis and choosing of the auxiliary methods for the therapy of the mammary gland cancer based both on morphology and metabolism of the tumour.     

Energy Metabolism in Capnocytophaga ochracea

Among the microflora of the gingival sulcus are members of the genus Capnocytophaga which have been implicated as possible etiological agents of juvenile periodontitis and systemic infectious diseases. In this study, the pathway used by C. ochracea strain 25 for generating energy from glucose was investigated. When grown in a complex medium supplemented with glucose and NaHCO₃, the major end products formed were acetate (4.6 mmol), succinate (11.0 mmol), pyruvate (4.3 mmol), and oxalacetate (3.6 mmol), and the molar growth yield was 58. Addition of yeast extract to the growth medium caused (i) an increase in acetate (9.2 mmol) and succinate (14.3 mmol), (ii) a decrease in pyruvate (0 mmol) and oxalacetate (1.1 mmol), and (iii) the molar growth yield increased to 75. Glucose was transported by a phosphoenolpyruvate:phosphotransferase system and then catabolized to phosphoenolpyruvate by enzymes of the Embden-Meyerhof-Parnas pathway. No activities were detected for the key enzymes of the Warburg-Dickens, Entner-Douderoff, or hexose phosphoketolase pathways. During growth in the yeast extract-supplemented medium, approximately 37% of the phosphoenolpyruvate carbon was converted to acetate by pyruvate kinase, a pyruvate-decarboxylating enzyme activity, and acetate kinase; the remaining 63% was converted to succinate via phosphoenolpyruvate carboxykinase, malate dehydrogenase, fumarate hydratase, and fumarate reductase.     

Age-related changes in glucose metabolizing enzymes in spleen, thymus, and pulmonary lavage cells from F344 rats

Healthy male Fischer 344 rats were sampled at 6, 12, 18, and 24 months of age. There was no gross pathological evidence or deviations in body weight, hematology, or clinical chemistry that were indicative of disease. Mixed populations of thymus, spleen, and pulmonary cells were obtained for enzymatic analyses. Key enzymes from the hexose monophosphate shunt, glycolysis and the tricarboxylic acid cycle were evaluated to determine if there were tissue-specific or pathway-specific changes that occurred during aging. The enzyme responses among the tissues were not consistent during the aging process. Generally the activities of the glucose metabolizing enzymes in thymus and pulmonary lavage cells decreased with age whereas they increased in the spleen cells. Between 18 and 24 months enzymes representative of all three glucose metabolic pathways decreased in pulmonary lavage cells, whereas the decreases in thymic cells were mainly restricted to glycolytic enzymes. By contrast there were two- to ten-fold increases during aging in all of the splenic enzymes measured except malate dehydrogenase. The alterations in tissue enzyme activities probably reflected the changing cellular populations during aging, and in the thymic and pulmonary lavage cellular environment resulted in a loss of energy production by glucose oxidation, compared to the vigorous activity maintained in spleen.     

A quantitative cytochemical analysis of resident and exudate macrophages

Quantitative cytochemical investigations have detected individual variations between murine peritoneal macrophages and have shown distinct difference between resident and exudate populations. The latter generally contain greater amounts of protein, RNA, acid phosphatase, succinate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and NADH dehydrogenase. On te other hand, no differences were detected in the cellular content of DNA, not-specific esterase, and NADPH dehydrogenase. In many instances they reflect the biochemical findings of other investigators including the stimulation of glycolysis, tricarboxylic acid cycle and hexose monophosphate shunt pathways, which can occur in elicited or activated macrophages. Although cytochemical differences between the two populations exist, it cannot be stated whether they represent distinct cell lines or different functional states of the same cell population.     

Energy metabolism in cancer cells: How to explain the Warburg and Crabtree effects?

Cancer cells have a greater need for energy and a ready supply of the building blocks necessary for the synthesis of macromolecules (nucleotides, protein, lipids) in order to duplicate genome and biomass. The hypothesis can be postulated that those precursors for synthetic processes, which can only be furnished by glycolysis, cannot be sufficiently recruited from external sources (the blood stream) and that glycolysis is necessarily markedly activated. It can also be hypothesized that the Krebs cycle, which also furnishes precursors for macromolecule synthesis to meet the requirements of proliferating cells, is depleted of intermediates. In view of its cyclic nature requiring not only pyruvate but also oxalacetate as the "last" metabolite of the reaction sequence for its sustenance, the Krebs cycle may be partially inactivated. While anaplerotic reactions and other sources (amino acids and fatty acids) could supply the cycle with intermediates, those pathways constitute futile cycles for amino and fatty acids as they would be partially degraded in the cycle and the intermediates thus obtained would be exported into the cytoplasm for synthetic processes with no advantage for the cell. It is also hypothesized that glutamine, an important fuel for cancer cells and playing a critical role in anaplerosis, may not contribute to reinforce the cycle; malate and α-ketoglutarate, two products of glutamine metabolism, might be exported from the mitochondria as precursors of biosynthetic pathways. It is possible then that malate, used for NADPH production required in the biosynthetic pathways, and glycerol-phosphate, too used for biosynthetic purposes (lipid biosynthesis), are unable to sustain the mitochondrial redox shuttles reducing the respiratory capacity of the mitochondria. Low shuttle capacity implies that NADH generated by glycolysis needs to be continuously re-oxidized in the cytoplasm via lactate dehydrogenase to maintain glycolysis fully activated, causing the abnormal lactate production observed in cancer. The paper goes onto discuss the essential role of glucose in cancer cell proliferation also in inducing the Crabtree effect. It is finally hypothesized that respiration inhibition after cancer cells have been supplied with glucose is due to reactivation in a suited medium of biosynthetic pathways with the consequences described above.     

Enzymatic activities of cell-free extracts of Rickettsia typhi.

Cell-free extracts of Rickettsia typhi were tested for activities of enzymes of the tricarboxylic acid cycle, of glutamate catabolism, and of glycolysis. The organisms were grown in the yolk sacs of chicken embryos, harvested shortly before the time of embryo death, purified by Renografin density gradient centrifugation, and ruptured in a French pressure cell. The following enzymatic activities were demonstrated: high levels of malate dehydrogenase (MDH), moderate levels of glutamate-oxaloacetate transaminase, glutamate, succinate, and isocitrate dehydrogenases, and citrate synthase, and low levels of glutamate-pyruvate transaminase. The specific activities of some of these enzymes were higher when the rickettsiae were harvested at a time of active proliferation, 3 to 4 days prior to embryo death. Rickettsial MDH was differentiated from host MDH by its migration pattern on polyacrylamide gel electrophoresis. The activities of MDH and two other dehydrogenases, demonstrable after the cells had been disrupted, were absent from purified, intact rickettsial preparations. No activity was detected for glucose-6-phosphate, 6-phosphogluconate, glyceraldehyde-3-phosphate, lactate dehydrogenases, phosphoglucose isomerase, fructoaldolase, or pyruvate kinase. Our results suggest that extracts of R. typhi that contain demonstrable enzymes involved in the catabolism of glutamate and tricarboxylic acid cycle intermediates, unlike Coxiella burnetti, lack detectable glycolytic activity.     

Arterial enzymes and their relation to atherosclerosis in pigeons.

Comparison of metabolic processes between the atherosclerosis-resistant Show Racer (SR) and susceptible White Carneau (WC) pigeon strains offers an opportunity to study factors which may predispose to atherosclerosis. The activity of several glycolytic enzymes, Krebs cycle enzymes, enzymes of the ATP cycle and of the glycerol phosphate shuttle was studied in SR and WC arteries.In 4–6 yr old pigeons the activity of lipoamide dehydrogenase and malate dehydrogenase is significantly lower in WC than SR arteries. The differences are not the result of aging or atherosclerosis, because they were also detected in arteries of very young 5–8 wk old pigeons. Furthermore, the arteries of the young pigeons revealed a significantly higher activity of two glycolytic enzymes, namely phosphofructokinase and aldolase, in the WC arteries as compared with SR arteries.The results in the young birds indicate that the differences between the two pigeon strains are of an inherited (genetic?) nature. It is suggested that low activity of lipoamide and malate dehydrogenases slows down the Krebs cycle and leads to low citrate and ATP production. The latter factor is an essential part of the feedback control adjustments that regulate the efficiency of glycolysis via phosphofructokinase. Increased dependence of the WC arteries on glycolysis appears to facilitate the development of atherosclerosis in these birds, and the mechanism may be similar to the mechanism by which tissue hypoxia induces lipid accumulation and connective tissue alterations in the arterial wall.An additional finding in these studies is the higher activity in female than male arteries of phosphofructokinase, aldolase, isocitrate dehydrogenase, glycerokinase, ATPase and creatine phosphokinase.     

Carbon dioxide metabolism by Actinomyces viscosus: pathways for succinate and aspartate production

14C-labeled bicarbonate was incorporated into trichloroacetic acid-insoluble material by cell suspensions of A. viscosus strain M100 and also into the four-carbon fermentation product, succinate, but not into the three-carbon fermentation product, lactate. The initial step in the conversion of 14C-labeled bicarbonate into both trichloroacetic acid-insoluble material and succinate was catalyzed by the enzyme phosphoenolypyruvate carboxylase, which served to convert the glycolytic intermediate, phosphoenolpyruvate, and bicarbonate to the four-carbon compound, oxalacetate. The metabolic fate of oxalacetate was its conversion to either trichloroacetic acid-insoluble material or succinate. One pathway by which oxalacetate may be metabolized into acid-insoluble material is via its conversion to the biosynthetic precursor aspartate by the action of glutamate aspartate aminotransferase. One source of the alpha-amino group of aspartate was the ammonium ion, which could be incorporated into glutamate, the substrate of the glutamate aspartate aminotransferase reaction, by the action of a reduced nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase whose reducing equivalents could be derived from the nicotinamide adenine dinucleotide phosphate-dependent oxidative reactions of the hexose monophosphate pathway catalyzed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Alternatively, oxalacetate was converted to the fermentation product, succinate, through the sequential action of malate dehydrogenase, fumarase, and succinic dehydrogenase. The resolution and partial purification of phosphoenolpyruvate carboxylase, glutamate aspartate aminotransferase, glutamate dehydrogenase, malate dehydrogenase, fumarase, and succinic dehydrogenase are also reported.     

Circadian organization of thirteen liver and six brain enzymes of the mouse

The activities of 13 liver and 6 brain enzymes were studied in 7–12 week old CD₂F₁ male mice that had been fed ad libitum and standardized either to 12 hours of light (0600–1800) alternating with 12 hours of darkness (1800–0600) (LD 12:12), or to a reversed light-dark cycle (darkness 0600–1800; light 1800–0600) (DL 12:12). Three separate studies were performed on two different days; in each experiment, subgroups of 14 animals were sacrificed at 3-hour intervals. Livers were assayed for: isocitrate dehydrogenase, glutamate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, glutathione reductase, glyoxylate reductase, L-alanine aminotransferase, glutamate oxalacetate transaminase, pyruvate decarboxylase, fructose-1-phosphate aldolase, fructose diphosphate aldolase, fructose 1,6-diphosphatase, and fatty acid synthetase. Brains were assayed for phosphoglucose isomerase, adenosine triphosphatase, creatine phosphokinase, pyruvate kinase, adenylate kinase, and malate dehydrogenase. All 19 enzymes demonstrated a prominent circadian rhythm in at least one experiment. Moreover, each rhythmic variable showed a statistically significant fit to a 24-hour cosine(sine) curve by the method of least squares. In general, peak activities of the liver enzymes analyzed were associated with the beginning of the dark cycle and initiation of the animal's activity, while the group of brain enzymes had peak activities which occurred at the beginning of the animals' rest span and were near the beginning of the light cycle. The phasing of each of the rhythms could be reversed within a two-week span after reversing the environmental light-dark cycle 180°.     

Dependence of the motor function of the rabbit uterus on activity of the enzymes of glucose metabolism

The motor function of the uterus of intact and pregnant rabbits was investigated in vitro during inhibition of the key enzymes of the pentose phosphate cycle (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) and of the Embden-Meierhof-Krebs cycle (glyceraldehyde-3-phosphate dehydrogenase, enolase, and succinate dehydrogenase). Under these conditions regular changes took place in the contractile function of the uterine cornua. During pregnancy activation of the enzymes responsible for direct oxidation of glucose in the pentose phosphate cycle was observed in the smooth-muscle cells of the myometrium, and this could be a factor maintaining pregnancy.Experimental Division, Kiev Research Institute of Pediatrics, Obstetrics, and Gynecology. (Presented by Academician of the Academy of Medical Sciences of the USSR, N. N. Sirotinin.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 79, No. 1, pp. 11–14, January, 1975.     

Fructose-6-phosphate substrate cycling and glucose and insulin regulation of gluconeogenesis in vivo.

The question whether glucose or insulin regulates gluconeogenesis by effecting changes in the fructose-6-phosphate (F-6-P) substrate cycle (phosphofructokinase (PFK), fructose-1,6-diphosphatase (FDPase)) was investigated in vivo in fasted normal rats using [3-3H,U-14C]- or [3-3H,6-14C]glucose. The plasma glucose 3H/14C ratio was used as an index of substrate cycling because 3H loss from the liver hexose phosphate pool is limited by the activities of PFK and FDPase during gluconeogenesis and glycolysis, respectively. The 3H/14C ratio was corrected where necessary for glucose or insulin-induced changes in reincorporation of 14C from C-6 to C-1-3 of plasma glucose. A glucose infusion producing hyperglycemia and insulinemia was accompanied by decreased hepatic glucose production and diminished F-6-P substrate cycling, i.e., decreased FDPase activity. When insulin was infused along with glucose to produce high plasma insulin levels and avoid hypo- or hyperglycemia, the 3H/14C decay rate did not change, suggesting that the hormone does not influence basal rates of gluconeogenesis or PFK or FDPase activities. These in vivo results suggest that increased blood glucose levels inhibit gluconeogenesis and depress F-6-P substrate cycling. Whether these cycle changes constitute primary regulatory actions of glucose or occur secondarily to other metabolic events resulting from excess hexose (e.g., increased glycogen synthetase activity) cannot now be concluded.     

Inhibition of indoleamine 2,3-dioxygenase in mixed lymphocyte reaction affects glucose influx and enzymes involved in aerobic glycolysis and glutaminolysis in alloreactive T-cells

Indoleamine 2,3-dioxygenase (IDO) suppresses adaptive immunity. T-cell proliferation and differentiation to effector cells require increased glucose consumption, aerobic glycolysis and glutaminolysis. The effect of IDO on the above metabolic pathways was evaluated in alloreactive T-cells. Mixed lymphocyte reaction (MLR) in the presence or not of the IDO inhibitor, 1-methyl-dl-tryptophan (1-MT), was used. In MLRs, 1-MT decreased tryptophan consumption, increased cell proliferation, glucose influx and lactate production, whereas it decreased tricarboxylic acid cycle activity. In T-cells, from the two pathways that could sense tryptophan depletion, i.e. general control nonrepressed 2 (GCN2) kinase and mammalian target of rapamycin complex 1, 1-MT reduced only the activity of the GCN2 kinase. Additionally 1-MT treatment of MLRs altered the expression and/or the phosphorylation state of glucose transporter-1 and of key enzymes involved in glucose metabolism and glutaminolysis in alloreactive T-cells in a way that favors glucose influx, aerobic glycolysis and glutaminolysis. Thus in alloreactive T-cells, IDO through activation of the GCN2 kinase, decreases glucose influx and alters key enzymes involved in metabolism, decreasing aerobic glycolysis and glutaminolysis. Acting in such a way, IDO could be considered as a constraining factor for alloreactive T-cell proliferation and differentiation to effector T-cell subtypes.     

Clinical consequences of enzyme deficiencies in the erythrocyte

The anucleate mature erythrocyte also lacks ribosomes and mitochondria and thus cannot synthesize enzymes or derive energy from the Krebs citric acid cycle. Nevertheless, the red blood cell is metabolically active and contains numerous residual enzymes and their products which are essential for its survival and normal functioning. Enzyme deficiencies in the Embden-Myerhoff glycolytic pathway can result in nonspherocytic hemolytic anemia (NSHA), and some are also associated with neuromuscular or neurologic disorders. Glucose-6-phosphate dehydrogenase deficiency in the hexose monophosphate shunt also results in hemolytic anemia, especially following exposure to various drugs. Defects in glutathione synthesis and pyrimidine 5'-nucleotidase deficiency also cause NSHA, as does increased adenosine deaminase activity. Gluthathione synthetase deficiency which is not limited to the red cell also presents as oxoprolinuria with neurologic signs. All red cell enzyme defects appear as single gene errors, in most cases recessive in inheritance, either autosomal of X-linked.     

Setaria cervi: Enzymes of glycolysis and PEP-succinate pathway

Summary Setaria cervi, the filarial parasite inhabiting the Indian water buffalo (Bubalus bubalis Linn.) contained almost all the enzymes involved in glycogen degradation. Significant activities of glycogen phosphorylase, glucokinase, phosphoglucomutase, phosphoglucose isomerase, phosphofructokinase, FDP-aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate hydratase, pyruvate kinase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were detected in cell-free extracts of whole worms.The presence of PEP-carboxykinase, malate dehydrogenase, fumarase and fumarate reductase revealed the functioning of the PEP-succinate pathway in addition to phosphorylating glycolysis and pentose phosphate pathway in the parasite. Excepting fumarate reductase all other enzymes were localized in the particulate-free cytosol fraction, although small amounts of glycogen phosphorylase, aldolase and lactate dehydrogenase were also detected in the mitochondrial fraction.Communication No. 2112 from the Central Drug Research Institute, Lucknow—226001, India. Part of the work was presented at the Annual Meeting of the Society of Biological Chemists, Ludhiana, India (November 1974).     

Fermentation and the properties of some enzymes of carbohydrate metabolism in the trematode Calicophoron ijimai

Glycogen content, glucose consumption and the production of metabolic end products by Calicophoron ijimai were determined under aerobic and anaerobic conditions. The major end products of fermentation were identified as lactic, acetic, propionic, isobutyric and α-methylbutyric acids, propionic acid predominating. The activities and properties of some of the enzymes of carbohydrate metabolism were determined. The worms showed high phosphoenolpyruvate carboxykinase, malate dehydrogenase and malate dehydrogenase (decarboxylating) but relatively low pyruvate kinase and very low lactate dehydrogenase activities. The pH optima, coenzyme, cofactor and ionic requirements of the enzymes were similar to those of other helminths. Malate dehydrogenase had an 8-fold greater affinity for oxaloacetate than malate, and was about 14 times more active for oxaloacetate reduction than malate oxidation. Phosphoenolpyruvate carboxykinase was 2.4 times more active and had a 2-fold greater affinity for phosphoenolpyruvate and dinucleotide than pyruvate kinase. The low activities of lactate dehydrogenase and pyruvate kinase but high activities of malate dehydrogenase and phosphoenolpyruvate carboxykinase suggest that anaerobic carbohydrate catabolism follows the fumarate reductase pathway.