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Relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives by Pseudomonas aeruginosa.

The relationship between catabolism of glycerol and metabolism of hexosephosphate derivatives in Pseudomonas aeruginosa was studied by comparing the growth on glycerol and enzymatic constitution of strain PAO with these characteristics of glucose-catabolic mutants and revertants. Growth of strain PAO on glycerol induced a catabolic oxidized nicotinamide adenine dinucleotide-linked glyceraldehyde-phosphate dehydrogenase and seven glucose-catabolic enzymes. The results indicated that these enzymes were induced by a six-carbon metabolite of glucose. All strains possessed a constitutive anabolic Embden-Meyerhof-Parnas pathway allowing limited conversion of glycerol-derived triosephosphate to hexosephosphate derivatives, which was consistent with induction of these enzymes by glycerol. Phosphogluconate dehydratase-deficient mutants grew on glycerol. However, mutants lacking both phosphogluconate dehydrogenase and phosphogluconate dehydratase were unable to grow on glycerol, although these strains possessed all of the enzymes needed for degradation of glycerol. These mutants apparently were inhibited by hexosephosphate derivatives, which originated from glycerol-derived triosephosphate and could not be dissimilated. This conclusion was supported by the fact that revertants regaining only a limited capacity to degrade 6-phosphogluconate were glycerol positive but remained glucose negative.

Gluconates

[Experience in using hexosephosphate in the surgical clinic].

The authors' experience with the clinical use of an antihypoxic substnace--hexosophosphate representing the complex of tetraphosphoric glucose and fructose esters is reported. The substance was studied experimentally on 20 dogs and clinically in 126 patients with diseases of vessels, abdominal organs and the kidneys. A positive effect was gained in peroral and intravenous use of the substance both with theaim of preparative management of patients to be subjected to major surgery and postoperatively. HP, being a readily absorbed energetic substance, renders a normalizing effect on the hemodynamics, the bowel activity, improves general patients'state, that is of special value in patients whose state is aggravated by concomitant lesions.

Adult

Physiological effects of seven different blocks in glycolysis in Saccharomyces cerevisiae.

Saccharomyces cerevisiae mutants unable to grow and ferment glucose have been isolated. Of 45 clones isolated, 25 had single enzyme defects of one of the following activities: phosphoglucose isomerase (pgi), phosphofructokinase (pfk), triosephosphate isomerase (tpi), phosphoglycerate kinase (pgk), phosphoglyceromutase (pgm), and pyruvate kinase (pyk). Phosphofructokinase activities in crude extracts of the pfk mutant were only 2% of the wild-type level. However, normal growth on glucose medium and normal fermentation of glucose suggested either that the mutant enzyme was considerably more active in vivo or, alternatively, that 2% residual activity was sufficient for normal glycolysis. All other mutants were moderately to strongly inhibited by glucose. Unusually high concentrations of glycolytic metabolites were observed before the reaction catalyzed by the enzyme which was absent in a given mutant strain when incubated on glucose. This confirmed at the cellular level the location of the defect as determined by enzyme assays. With adh (lacks all three alcohol dehydrogenase isozymes) and pgk mutants, accumulation of the typical levels of hexosephosphates was prevented when respiration was blocked with antimycin A. A typical feature of all glycolytic mutants described here was the rapid depletion of the intracellular adenosine 5'-triphosphate pool after transfer to glucose medium. No correlation of low or high levels of fructose-1,6-bisphosphate with the degree of catabolite repression and inactivation could be found. This observation does not support the concept that hexose metabolites are directly involved in these regulatory mechanisms in yeast.

Adenosine Triphosphate

[Alternativity of methane assimilation pathways in obligate methylotrophs].

The activity of key enzymes involved in the primary pathways of methane assimilation and enzymes of the citrate cycle was determined in various obligate methylotrophs: mesophilic, thermotolerant, and thermophilic. The bacteria are characterized by the membrane ultrastructure of the I type, high activity of hexosephosphate synthase, NAD- and NADP-specific isocitrate dehydrogenase, and the absence of alpha-ketoglutarate dehydrogenase. The bacteria also displayed the activity of key enzymes of the serine cycle, hydroxypyruvate reductase, and serineglyoxylate aminotransferase. Therefore, both the ribulose monophosphate and serine pathways are involved in methane assimilation, and the division of methanotrophs into two groups, according to their metabolism, is tentative.

Aldehyde-Lyases

Metabolism of carbohydrate derivatives by Pseudomonas acidovorans.

Wild-type Pseudomonas acidovorans strain A1 was unable to grow on glycerol or glucose as sole source of carbon and energy although it grew well on gluconate. Spontaneous glycerol-positive mutants, which apparently had become permeable to glycerol, were readily isolated, but glucose-positive mutants did not occur. P. acidovorans lacked glucose dehydrogenase and glucokinase, which were sufficient to account for its inability to grow on glucose. Gluconate was degraded exclusively via a noncoordinately induced Entner-Doudoroff pathway. Phosphogluconate dehydrogenase was undetectable. In contrast to P. aeruginosa, P. acidovorans possessed a single glyceraldehyde-phosphate dehydrogenase activity, which was NAD+ specific and constitutive, and an inducible pyruvate kinase. Moreover, growth of glycerol-positive strain K2 on glycerol did not induce any of the enzymes related to metabolism of hexosephosphate derivatives as occurs in fluorescent pseudomonads.

Gluconates

Further studies on creatine kinase activity in human skeletal muscle.

Creatine kinase (CK) was measured in muscle samples from 9 female and 9 male adult subjects using comparatively two procedures. The improved method, using N-acetylcysteine as activator gives results about 1.5 times higher than that with reduced glutathione. The significant correlation existing between activities of CK and hexosephosphate isomerase was confirmed using the improved method. There also exists a correlation between CK and enolase activity. In this respect, there are no differences between muscle samples from male and female subjects.

Adult

The effect of enzymes upon metabolism, storage, and release of carbohydrates in normal and abnormal endometria.

This paper presents preliminary data concerning the relationship of various components of glandular epithelium and effect of enzymes on metabolism, storage, and release of certain substances in normal and abnormal endometria. Activity of these endometrial enzymes has been compared between two groups: 252 patients with normal menstrual histories and 156 patients, all over the age of 40, with abnormal uterine bleeding. Material was obtained by endometrial biopsy or curettage. In the pathologic classification of the group of 156, 30 patients had secretory endometria, 88 patients had endometria classified as proliferative, 24 were classified as endometrial hyperplasia, and 14 were classified as adenocarcinoma. All tissue was studied by histologic, histochemical, and biochemical methods. Glycogen synthetase activity caused synthesis of glucose to glycogen, increasing in amount until midcycle, when glycogen phosphorylase activity caused the breakdown to glucose during the regressive stage of endometrial activity. This normal cyclic activity did not occur in the abnormal endometria, where activity of both enzymes continued at low constant tempo. Only the I form of glycogen synthetase increased as the tissue became more hyperplastic. With the constant glycogen content and the increased activity of both the TPN isocitric dehydrogenase and glucose-6-phosphate dehydrogenase in the hyperplastic and cancerous endometria, tissue energy was created, resulting in abnormal cell proliferation. These altered biochemical and cellular activities may be the basis for malignant cell growth.

Adenocarcinoma

Effect of chemotherapeutic agents on metabolic and bactericidal activity of polymorphonuclear leukocytes.

Blood was obtained on 36 occasions from 12 healthy adult volunteers and the polymorphonuclear leukocytes (PMNL) were separated. PMNL hexose monophosphate shunt activity of whole blood and ability of separated cells to phagocytize and kill E. coli were evaluated when the PMNL were incubated with normal pooled sera and sera containing therapeutic concentrations of either 15 cancer chemotherapeutic drugs singly and in combination or 9 antibiotics. Resting and stimulated HMPS activity was significantly (p less than 0.025 to p less than 0.001) decreased by cyclophosphamide, carmustine (BCNU), high dose prednisone (pred), vinblastine (vinbl) and vincristine (vinc) and significantly (p less than 0.025 to p less than 0.01) increased by combinations of vinc-pred, vinc-predasparaginase, 6-mercaptopurine (6MP)-methotrexate (Mtx) and 6MP-Mtx-pred when compared to controls. No significant differences in HMPS activity of PMNL were found when exposed to various antimicrobial agents singly or in combination. The killing of E. coli by PMNL was significantly (p less than 0.001) decreased when exposed to BCNU, high concentration pred or combinations of 6MP-Mtx-pred, 6MP-Mtx and vinc-vinbl-pred but not when exposed to other chemotherapeutic agents. This study shows a disparity in results obtained when evaluating PMNL function by HMPS activity and bactericidal assay. In addition, a functional impairment in PMNL exposed to various antimetabolites occurred at a time when they exhibited normal morphology.

Anti-Bacterial Agents

Metabolic factors and the utilization of phosphorus by plants.

The overall process of entry and transport of phosphate by plants has been separated into its component parts. Rapid esterification is involved but a small proportion of the total transport may occur by a non-metabolic route. Mannose alters the metabolism of phosphate in roots of cereals and thereby reduces the transport to the shoot by as much as 99% whereas dicotyledonous species are much less sensitive. The sequestration of phosphate as mannose 6-phosphate is reversible in some species depending on the extent of its conversion into fructose 6-phosphate and it is possible that in vivo controls of this type operate in whole plants. The factors involved in the control of distribution of inorganic phosphate between cellular organelles and throughout the plant are discussed. Enzymic hydrolysis of organic forms by roots and associated microorganisms may also be important in soils where available orthophosphate is limiting. Other inorganic nutrients, particularly boron, play an important role during the transport of phosphate across membranes. Major differences have been found in the capacities of different species to transport absorbed phosphate to the shoot. Peas and field beans absorb efficiently in the early weeks of growth but the rate of transport is about one thirtieth that of oats, barley, mung bean or sunflower. The relevance of the experimental findings to the wider problem of efficiency of fertilizer use and the possible selection of genotypes with high capacities for absorption and transport are discussed.

Absorption

Abnormal erythrocyte metabolism in hepatic disease: effect of NADP repletion.

Erythrocytes from ten patients with severe liver disease displayed low methylene blue-stimulated hexose monophosphate (HMP) shunt activity and glucose recycling despite elevated total glucose consumption when compared to controls. Heinz body formation was increased and reduced glutathione concentration significantly decreased. After hemolysis, no differences in methylene-blue estimulated HMP shunt activity or glucose recycling could be demonstrated between patients and controls. The addition of 2- and 4-mM NADP to the hemolysates produced significantly greater HMP shunt activity and glucose recycling in the patients' hemolysates. The addition of NADPH to the incubation mixture produced no significant stimulation of either HMP shunt activity or glucose recycling, unless methylene blue was also added. Omission of NAD or phosphate from the incubation mixture produced no change in shunt metabolism. The absence of supplemental ATP resulted in extremely low shunt metabolism and refractoriness to NADP stimulation in both patients and controls. In the absence of additional magnesium, a reduction of shunt metabolism was noted. These data suggest that the defect in stimulated shunt metabolism in the intact erythrocytes of patients with hepatic disease does not result from an absolute enzyme deficiency, but rather from an unavailability of NADP or other cofactor.

Adult

Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells. I. Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line.

In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway. These enzymes are galactokinase, alpha-D-galactose-1-phosphate: UDP glucose uridyl transferase and UDP galactose 4-epimerase. A cell strain from a patient with galactosemia had no detectable activity for the transferase. The substitution of galactose for glucose in the medium of these cells (which has been shown to cause the cells to accumulate galactose-1-phosphate) also failed to affect cellular activity for the three enzymes. Similarly, the three activities failed to respond to the substitution of galactose for glucose in cultures of a rat hepatoma line. Cells of this line have been shown by others to perform a number of the tissue-specific functions of liver. The failure of galactose to stimulate increasd cellular activity for the three enzymes represents a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.

Bucladesine

Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells. II. A search for quantitative interrelationships between the three enzyme activities.

Studies on a normal human diploid cell strain revealed that the specific activity of the cell protein, for each of the three enzymes of the Leloir pathway, changed significantly as the cells grew. The kinetics of change in specific activity varied according to the enzyme being studied, and the kinetics for each enzyme varied from experiment to experiment. Within each experiment, there was no consistent correlation between specific activity for any one enzyme and specific activity for the other two. The ratios between the specific activities did not tend to remain constant as the absolute levels of specific activity changed. Hence, the activities did not behave coordinately. The kinetics of change in these ratios varied from experiment to experiment. The failure of galactose to stimulate increased cellular activity for the three enzymes (shown in the preceding paper), and the absence of a coordinate relationship between the activities, represent a striking difference between the behavior of these enzymes in human diploid cell strains and their behavior in E. coli.

Carbohydrate Epimerases

Comparative studies of glucose-fed and glucose-starved hamster cell cultures: responses in galactose metabolism.

The metabolic flow of trace amounts of D-[14C]-galactose was followed in cultures of transformed and untransformed hamster cells over a period ranging from five minutes to two hours. The results of chromatographic and enzymatic analyses of the soluble pools are described. Non-glycolytic cells(previously deprived of sugar periods of up to 24 hours) convert D-galactose to galactose-1-phosphate and uridine diphosphoglucuronic acid in 10 to 20 minutes. In the same short assay time, glycolytic cells which have been maintained for 24 hours in media containing glucose or galactose convert D-galactose to uridine diphsphogalactose and uridine diphosphoglucose (ratio 1.4:1). Long term diprivation of sugar also results in 3- to 4-fold increases in the uptake of galactose. In addition, the incorporation of galactose label into chloroformethanol soluble material appears to be influenced by the culture conditions of the untransformed cells while incorporation in the transformed cells appears unaffected. When cycloheximide is included in the maintenance medium for extended periods, the non-glycolytic cells also show increases in galactose uptake rates but the glucose-fed, glycolytic cells llose uptake ability. UDPhexose is the main galactose metabolic peak in the soluble pools of the cycloheximide-treated, glycolytic and the cycloheximide-treated, non-glycolytic cells. The results of these experiments suggests that uptake of galactose and its subsequent metabolism are under separate control.

Animals