Respiration and glycolysis in the human diploid cell strain WI-38

Assessment of the respiratory and glycolytic capacity of non-growing WI-38 cells shows that, in the absence and presence of added glucose, the mean rates of oxygen consumption were 247 (QO₂ = 5.61) and 208 (QO₂ = 4.73) mμmoles/mg dry wt/hr., respectively. Mean glucose consumption was 225 mμmoles/mg dry wt/ hr. With uniformly labeled ¹⁴C glucose as substrate, 36 mμg atoms of carbon dioxide were produced, corresponding to 15–20% of the total cellular respiration. Mean values for lactate production in the presence and absence of glucose were 345 (Q_L^O2 = 7.85) and 196 (Q_L^O2 = 4.45) mμmoles/mg dry wt/hr., respectively. Human diploid cells in culture age, in the sense that their ability to proliferate decreases with time during serial subcultivation. Studies of their respiratory and glycolytic capacity as a function of the aging process showed that total respiration, glucose respiration and glycolytic capacity were relatively constant for cells in the middle and late passages and indicate that aging in this sense is not directly related to the respiratory and glycolytic capacity of the cell.     

Effects of L-thyroxine and L-triiodothyronine on protein and nucleic acid contents of liver of 6N-2-propylthiouracil treated hypothyroid singi fish, Heteropneustes fossilis bloch

Three consecutive injections of 12.5 X 10(-10) and 25 X 10(-10) moles/g of L-thyroxine (T4) or a single injection of L-triiodothyronine (T3) at 7.5 X 10(-10) moles/g to Singi fish caused an increase in liver protein and RNA contents, whereas similar injections of 50 X 10(-10) moles/g of T4 or 75 X 10(-10) moles/g of T3 caused a fall in these cellular constituents in liver. Treatments of Singi fish with thiourea (1 mg/ml) for 30 days caused a fall in the protein and RNA contents in liver which were restored to the euthyroid control level by a single injection of 7.5 X 10(-10) moles/g of T3 or three consecutive injections of T4 at 12.5 X 10(-10) moles/g dose. Administration of T4 (12.5 X 10(-10) moles/g, three consecutive injections) along with 6N-2-propylthiouracil (PTU) at 20 micrograms/g of b. w. in six consecutive injections to the thiourea treated (hypothyroid) fish failed to cause any change in hepatic protein and RNA contents in comparison to only PTU-treated hypothyroid fish, but a single injection of 7.5 X 10(-10) moles/g of T3 to the PTU-treated hypothyroid fish increased these cellular constituents of liver. A dose-dependent biphasic nature of thyroid hormone action, a higher potency of T3 than T4 and the probable 'prohormone' nature of T4 have been documented in case of Singi fish in the present experiments.     

5 Alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase activities in isolated canine prostatic epithelial cells

Kinetic parameters of two enzymes, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase, have been calculated for freshly isolated canine prostatic epithelial cells separated into secretory and non-secretory cells on the basis of their density in Percoll gradients. For 5 alpha-reductase, a Km value of 3 X 10(-6) M was obtained in both epithelial cell types, and similar Vmax values of 1.7 X 10(-12) and 1.9 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P greater than 0.50) were calculated in secretory and non-secretory cells, respectively. The Km of 3 alpha-hydroxysteroid dehydrogenase (dihydrotestosterone----3 alpha-androstanediol) varied between 2.2 and 2.8 X 10(-6) M (P greater than 0.50) with respective Vmax's of 9 and 24 X 10(-12) moles of 3 alpha-androstanediol formed/min/10(6) cells (P less than 0.005) in secretory and non-secretory cells. For the reverse reaction, that is the transformation of 3 alpha-androstanediol to dihydrotestosterone, the Km's obtained were 0.4 and 0.5 X 10(-6) M (P greater than 0.50) with Vmax's of 14 and 19 X 10(-12) moles of dihydrotestosterone formed/min/10(6) cells (P less than 0.50) in secretory and non-secretory cells, respectively. Vmax values for 3 alpha-hydroxysteroid dehydrogenase are 10-fold higher than the ones for 5 alpha-reductase. Moreover, Km values for the reaction 3 alpha-androstanediol----DHT are 5-fold lower than those calculated for the reverse reaction and for 5 alpha-reductase. This could explain why the major metabolic pathway in the canine prostate gland is the conversion of 3 alpha-androstanediol to dihydrotestosterone.     

Coccidian parasites (Apicomplexa: Eimeriidae) from insectivores. VII. Six new species from the hairy-tailed mole, Parascalops breweri

Sixteen hairy-tailed moles, Parascalops breweri, collected from the northeastern U.S.A. were examined for coccidian oocysts; all were infected with multiple species of coccidia and 3 genera were represented. Two cyclosporans, 2 eimerians, and 2 isosporans are described as new species. Sporulated oocysts of Cyclospora ashtabulensis n. sp. are subspheroid to ellipsoid, 18 X 14 (14-23 X 11-19) microns, and sporocysts are ovoid, 12 X 7 (8-14 X 5-9) microns; C. ashtabulensis was found in 7 of 16 (44%) moles. Sporulated oocysts of Cyclospora parascalopi n. sp. are spheroid, 17 X 14 (13-20 X 11-20) microns, and sporocysts are ovoid, 11 X 7 (8-14 X 5-8) microns; C. parascalopi was found in 8 of 16 (50%) moles. Sporulated oocysts of Eimeria aethiospora n. sp. are subspheroid to ellipsoid, 19 X 13 (15-24 X 10-16) microns, and sporocysts are ovoid, 11 X 6 (8-13 X 4-7) microns; E. aethiospora was found in 4 of 16 (25%) moles. Sporulated oocysts of Eimeria titthus n. sp. are subspheroid, 16 X 14 (13-19 X 11-17) microns, and sporocysts are ellipsoid, 11 X 6 (9-13 X 4-7) microns; E. titthus was found in 4 of 16 (25%) moles. Sporulated oocysts of Isospora ashtabulensis n. sp. are ellipsoid, 20 X 14 (16-24 X 10-18) microns, and sporocysts are ovoid, 10 X 7 (7-14 X 5-10) microns; I. ashtabulensis was found in 5 of 16 (31%) moles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of four aromatic organic pollutants on microbial glucose metabolism and thymidine incorporation in marine sediments.

The metabolism of D-[U-14C]glucose and the incorporation of [methyl-3H]thymidine by aerobic and anaerobic marine sediment microbes exposed to 1 to 1,000 ppm anthracene, naphthalene, p,p'-dichlorodiphenyltrichloroethane, and pentachlorophenol were examined. Cell-specific rates of [14C]glucose metabolism averaged 1.7 X 10(-21) and 0.5 X 10(-21) mol/min per cell for aerobic and anaerobic sediment slurries, respectively; [3H]thymidine incorporation rates averaged 43 X 10(-24) and 9 X 10(-24) mol/min per cell for aerobic and anaerobic slurries, respectively. Aerobic sediments exposed to three of the organic pollutants for 2 to 7 days showed recovery of both activities. Anaerobic sediments showed little recovery after 2 days of pre-exposure to the pollutants. We conclude that (i) anaerobic sediments are more sensitive than aerobic sediments to pollutant additions; (ii) [3H]thymidine incorporation is more sensitive to pollutant additions than is [14C]glucose metabolism; and (iii) the toxicity of the pollutants increased in the following order: anthracene, p,p'-dichlorodiphenyltrichloroethane, naphthalene, and pentachlorophenol.     

Effects of four aromatic organic pollutants on microbial glucose metabolism and thymidine incorporation in marine sediments

The metabolism of D-[U-14C]glucose and the incorporation of [methyl-3H]thymidine by aerobic and anaerobic marine sediment microbes exposed to 1 to 1,000 ppm anthracene, naphthalene, p,p'-dichlorodiphenyltrichloroethane, and pentachlorophenol were examined. Cell-specific rates of [14C]glucose metabolism averaged 1.7 X 10(-21) and 0.5 X 10(-21) mol/min per cell for aerobic and anaerobic sediment slurries, respectively; [3H]thymidine incorporation rates averaged 43 X 10(-24) and 9 X 10(-24) mol/min per cell for aerobic and anaerobic slurries, respectively. Aerobic sediments exposed to three of the organic pollutants for 2 to 7 days showed recovery of both activities. Anaerobic sediments showed little recovery after 2 days of pre-exposure to the pollutants. We conclude that (i) anaerobic sediments are more sensitive than aerobic sediments to pollutant additions; (ii) [3H]thymidine incorporation is more sensitive to pollutant additions than is [14C]glucose metabolism; and (iii) the toxicity of the pollutants increased in the following order: anthracene, p,p'-dichlorodiphenyltrichloroethane, naphthalene, and pentachlorophenol.     

Sulphate uptake and metabolism in the chrysomonad, monochrysis lutheri.

The intracellular concentration of inorganic 35SO4 in Monochrysis lutheri cells exposed to 0.513 mM Na235SO4 for up to 6-hr remained constant at about 0.038 mM. The exchange rate of this 35SO4 with the external unlabelled sulphate was negligible compared to the rate of influx across the plasmalemma (0.032 mu moles/g cells/hr). The flux of free 35SO4 to organic 35S was 0.029 mu moles/g cells/hr. Assuming an internal electrical potential in the cells of -70 mV, this intracellular concentration of inorganic 35SO4 was well in excess of that obtainable by passive diffusion as calculated from the Nernst equation. These results indicate that sulphate is accumulated by an active mechanism rather than by facilitated diffusion. Sulphate uptake appears to occur via a carrier-mediated membrane transport system which conforms to Michaelis-Menten type saturation kinetics with a Km of 3.2 X 10(-5) M and Vmax of 7.9 X 10(-5) mu moles sulphate/hr/10(5) cells. Uptake was dependent on a source of energy since the metabolic inhibitor CCCP almost completely inhibited uptake under both light and dark conditions and DCMU caused a 50% decrease in uptake under light conditions. Under dark conditions, uptake remained at about 80% of that observed under light conditions and was little affected by DCMU, indicating that the energy for uptake could be supplied by either photosynthesis or respiration. A charge and size recognition site in the cell is implied by the finding that sulphate uptake was inhibited by chromate and selenate but not by tungstate, molybdate, nitrate or phosphate. Chromate did not inhibit photosynthesis. Cysteine and methionine added to the culture medium were apparently capable of exerting inhibition of sulphate uptake in both unstarved and sulphate-starved cells. Cycloheximide slightly inhibited sulphate uptake over an 8-hr period indicating, either a slow rate of entry of the inhibitor into the cells or a slow turnover of the protein(s) associated with sulphate transport.     

Presence of nuclear triiodothyronine (T3) receptors in mouse fibroblasts]

The present preliminary data obtained from intact fibroblasts of adult mice (polyploid stem L 929) suggest that this cell system possesses high-affinity and saturable nuclear binding sites for triiodothyronine. As estimated by the Scatchard analysis, the equilibrium dissociation constant is approximately 2 X 10(-10) moles, the maximal binding capacity is about 2 000 sites for T3 per cell nucleus.     

Iodinated fibroblast beta-glucuronidase as a ligand for receptor-mediated endocytosis.

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 X 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 X 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.     

Facilitated Transport of Glucose from Blood into Peripheral Nerve

D-Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood-nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D-[14C]glucose, L-[14C]glucose, and [14C]3-O-methyl-D-glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D-glucose. The permeability-surface area product (PA) for D-[14C]glucose and [14C]3-O-methyl-D-glucose decreased, whereas the PA for L-[14C]glucose remained constant, as the perfusate concentration of D-glucose was increased. In the presence of no added unlabelled D-glucose in the perfusate, the PA for L-[14C]glucose equaled one-fifth the PA for D-[14C]glucose. These results demonstrate self-saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D-monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half-saturation constant and maximal rate of transport for the saturable component of D-glucose influx equaled 23 +/- 11 mumol X ml-1 and 6.6 +/- 3.2 X 10(-3) mumol X s-1 X g-1, respectively. The constant of nonsaturable glucose influx equaled 0.5 +/- 0.1 X 10(-4) s-1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D-glucose influx into nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of alloxan by microsomal electron transport proteins

Alloxan behaves as a substrate for NADH:ferricytochrome b5 oxidoreductase (EC 1.6.2.2). The apparent Km for alloxan was 10 mM in liver microsomes and 20 mM with the enzyme prepared by lysosomal digestion. The apparent Km for NADH was the same with microsomes and the isolated enzyme (30 microM). The maximum turnover rate was calculated as 426 moles electrons/min X mole enzyme. Cytochrome b5 was shown to reduce alloxan nonenzymatically.     

Fed-batch fermentation studies with Bacillus thuringiensis H-14 synthesising endotoxin.

Cell yield and toxicity of B. thuringiensis H-14 was improved markedly by adopting a simple fed-batch fermentation technique based on controlling glucose concentration. Maintenance of steady glucose concentration (0.3-0.5%) in the culture medium was achieved by the continuous addition of concentrated glucose solution. Addition of glucose at 3 g/hr/l of culture starting from 3rd hr till 16th hr of fermentation was found to yield cell densities of 80 g/l (wet weight) which represented a nearly 3-fold increase over the batch mode. A fivefold increase in toxicity was obtained by fed-batch fermentation. Cultivation of B. thuringiensis H-14 to high cell densities had no negative effect on sporulation and toxin synthesis. The rate of pH drop and dissolved oxygen level were within manageable limits.     

Use of fluorescence-labelled cerebroside as a substrate for galactosylceramidase of human skin fibroblasts

Possible use of the fluorescently labelled cerebroside, 1-O-(beta-D-galactosyl)-2-N-[6-(2-antroyl)hexanoyl]-4-sphingeni n (Gal-A-sphingenin) as a substrate for galactosylceramidase (GC) from human skin fibroblasts was investigated. Studies involving TLC and fluorimetric methods revealed enzymatic splitting of Gal-A-sphingenin whose degree correlated with the amount of the enzyme and incubation time. Some kinetic parameters of GC were determined, using Gal-A-sphingenin as substrate. It was shown that the values of specific activity of GC (1.6 nmol/mg protein/hr) and Km (0.025 mM) for Gal-A-sphingenin agree well with the corresponding values obtained with the use of the galactocerebroside as a natural substrate. In experiments with mixtures of Gal-A-sphingening and galactocerebroside used as substrates in different molar ratios, it was shown that the enzyme splits each of the substrates with equal velocity. The results of experiments with the enzyme samples from skin fibroblasts of healthy individuals and of patients with GM1-gangliosidosis (GM1-beta-D-galactosidase deficiency) suggest that Gal-A-sphingenin is a specific substrate for GC.     

Replication of X chromosomes in complete moles

Summary DNA replication patterns of X chromosomes in complete hydatidiform moles were studied using cultured fibroblasts from three 46,XX moles resulting from duplication of a haploid sperm, and from a 46,XY mole originating from dispermy. Control cultures included skin fibroblasts from an adult woman and a female fetus as well as PB lymphocytes from an adult woman. Cultures were treated with 5-bromodeoxyuridine for the last 2–4h of the S phase, and the chromosome slides prepared were stained by the Hoechst 33258-Giemsa procedure. Each of the three XX moles studied revealed one early-replicating and one late-replicating X chromosomes, while the XY mole revealed one early-replicating X chromosome. DNA replication patterns of molar X chromosomes were similar to those of adult and fetal fibroblasts, but different from those in adult lymphocytes. These findings indicate that DNA replication kinetics of molar fibroblasts are tissue-specific rather than origin- or developmental-stage specific.     

AN ISOTOPIC MICROMETHOD FOR THE MEASUREMENT OF CHOLINESTERASE ACTIVITY IN INDIVIDUAL CELLS

—A microisotopic method for measuring acetylcholinesterase activity in isolated cells is described. The assay employs [¹⁴C]acetylcholine and can measure 7 × 10^-12 moles of acetylcholine hydrolysed/hr in 50-150 samples per experiment. The method described has been applied to the measurement of cholinesterase activity in individual sympathetic ganglion cells of the cat. It has been shown that under standard conditions the substrate has complete access to the enzymatic site.     

Gaucher disease. III. Substrate specificity of glucocerebrosidase and the use of nonlabeled natural substrates for the investigation of patients.

A reproducible and convenient method for assaying glucocerebrosidase activity using the natural substrates has been developed. From the insoluble pellet fraction of cultured skin fibroblast homogenates, released glucose was measured enzymically using hexokinase coupled with the glucose-6-phosphate dehydrogenase (G6PD) and nicotinamide adenine dinucleotide phosphate (NADP) system. Optimal enzyme assay conditions required both Triton X-100 and sodium taurocholate, pH 4.8. Glucocerebrosidase activities from three patients with type 1 Gaucher disease were 17.5%, 15.8%, and 11.2% of normal (normal = 198 +/- 14 nmol/hr per mg protein, n = 3). The first patient had normal beta-glucosidase activity with the artificial fluorogenic umbelliferone substrate. Interference with the accuracy of the glucose-dependent assay system by either glycolytic or gluconeogenic enzyme activites was not detected under these experimental conditions, and when substrates with long fatty-acid chain lengths (C = 22) were used, markedly decreased glucocerebrosidase activity occurred in both normal individuals and patients. The apparent Km's for the natural substrates were 0.56 +/- 0.05 mM for controls and 2.2-3.3 mM for Gaucher fibroblasts. These data further support the hypothesis that a structurally altered and catalytically deficient enzyme is synthesized in patients with type 1 Gaucher disease and illustrate the value of the natural substrate in investigating patients.     

S-adenosylmethionine: protein-arginine methyltransferase. Purification and mechanism of the enzyme.

Protein methylase I (S-adenosylmethionine: protein-arginine methyltransferase, EC 2.1.1.23) has been purified from calf brain approximately 120-fold with a 14% yield. The final preparation is completely free of any other protein-specific methyltransferases and endogenous substrate protein. The enzyme has an optimum pH of 7.2 and pI value of 5.1. The Km values for S-adenosyl-L-methionine, histone H4, and an ancephalitogenic basic protein are 7.6 X 10(-6), 2.5 X 10(-5), and 7.1 X 10(-5) M, respectively, and the Ki value for S-adenosyl-L-homocysteine is 2.62 X 10(-6) M. The enzyme is highly specific for the arginine residues of protein, and the end products after hydrolysis of the methylated protein are NG,NG-di(asymmetric), NG,N'G-di(symmetric), and NG-monomethylarginine. The ratio of [14C]methyl incorporation into these derivatives by enzyme preparation at varying stages of purification remains unchanged at 40:5:55, strongly indicating that a single enzyme is involved in the synthesis of the three arginine derivatives. The kinetic mechanism of the protein methylase I reaction was studied with the purified enzyme. Initial velocity patterns converging at a point on the extended axis of abscissas were obtained with either histone H4 or S-adenosyl-L-methionine as the varied substrate. Product inhibition by S-adenosyl-L-homocysteine with S-adenosyl-L-methionine as the varied substrate was competitive regardless of whether or not the enzyme was saturated with histone H4. On the other hand, when histone H4 is the variable substrate, noncompetitive inhibition was obtained with S-adenosyl-L-homocysteine under conditions where the enzyme is not saturated with the other substrate, S-adenosyl-L-methionine. These results suggest that the mechanism of the protein methylase I reaction is a Sequential Ordered Bi Bi mechanism with S-adenosyl-L-methionine as the first substrate, histone H4 as the second substrate, methylated histone H4 as the first product, and S-adenosyl-L-homocysteine as the second product released.     

Rat uterine microbiochemistry: metabolic enzyme activities stimulated by 17-beta-estradiol are localized in epithelial cells

Lactate dehydrogenase (LDH; EC 1.1.1.27), citrate synthase (CS; EC 4.1.3.7), and beta-hydroxyacyl-CoA-dehydrogenase (beta-OH-acyl-CoA-DH; EC 1.1.1.35) activities were determined in each of the three major cell types of rat uterus, i.e., epithelial, stromal, and smooth muscle, using quantitative microanalytical techniques. Adult ovariectomized rats were treated with 17-beta-estradiol to determine the time course and dose response (0.025-50 micrograms/300-g rat) effect of estrogen on enzyme activity of each type of uterine cell. The use of "oil well" and enzyme-cycling microtechniques to determine the time course and the dose responses of enzyme activity changes required microassays involving 1595 microdissected single cell specimens. Estradiol treatment increased epithelial LDH, CS and beta-OH-acyl-CoA-DH activity but had no effect on these enzymes in the stroma or in smooth muscle cells. The estradiol-stimulated peak enzyme activities on Day 4 in the intervention group are compared with those in the ovariectomized rat controls as follows: LDH, 44.5 +/- 3.5 vs 22.3 +/- 3.9; CS, 3.5 +/- 0.2 vs 1.5 +/- 0.6; beta-OH-acyl-CoA-H, 3.5 +/- 0.32 vs 2.2 +/- 0.2 (mean +/- standard deviation; mol/kg/hr). Stromal cell activities (LDH, 7.4 +/- 1.0; CS, 1.2 +/- 0.2; beta-OH-acyl-CoA-DH, 0.9 +/- 0.1) were significantly lower than epithelial cell levels and were similar to smooth muscle levels. Therefore, even in the ovariectomized animal epithelial cells have markedly higher metabolic activity compared with adjacent cells. The enzyme activities are expressed as moles of substrate reacting per kilogram of dry weight per hour. All three enzymes exhibited a 17-beta-estradiol-induced dose response between 0.025-0.15 micrograms/300-g rat. The three enzymes studied all had similar response patterns to estrogen. The effect of estradiol was restricted to epithelial cells, with enzyme activities increasing to maximal levels after approximately 96 hr of hormone treatment. This study therefore not only confirms the specific and differential metabolic responses of uterine cells to estradiol treatment, but clearly demonstrates that marked metabolic differences exist between epithelial cells and stromal or smooth muscle uterine cells.     

Non-oxidative synthesis of pentose 5-phosphate from hexose 6-phosphate and triose phosphate by the L-type pentose pathway

1. Ribose 5-phosphate was non-oxidatively synthesized from glucose 6-phosphate and triose phosphate by an enzyme extract prepared from rat liver (RLEP). Analysis of the intermediates by GLC, ion-exchange chromatography and specific enzymatic analysis, revealed the presence of the following intermediates of the L-type pentose pathway: altro-heptulose 1,7-bisphosphate, arabinose 5-phosphate and D-glycero D-ido octulose 8-phosphate. 2. With either [1-14C] or [2-14C]glucose 6-phosphate as diagnostic substrates, the distribution of 14C in ribose 5-phosphate was determined. At early time intervals (0.5-8 hr), [1-14C]glucose 6-phosphate introduced 14C into C-1, C-3 and C-5 of ribose 5-phosphate, at 17 hr 14C was confined to C-1. With [2-14C]glucose 6-phosphate as substrate, 14C was confined to C-2, C-3 and C-5 of ribose 5-phosphate during early times (0.5-8 hr), while at 17 hr 14C was located in C-2. 3. The transketolase exchange reaction, [14C]ribose 5-phosphate + altro-heptulose 7-phosphate in equilibrium ribose 5-phosphate + [14C]altro-heptulose 7-phosphate, was demonstrated for the first time using purified transketolase, its activity was measured and it is proposed to play a major role in the relocation of 14C into C-3 and C-5 or ribose 5-phosphate during the prediction labelling experiments. 4. The coupled transketolase-transaldolase reactions, 2 fructose 6-phosphate in equilibrium altro-heptulose 7-phosphate + xylulose 5-phosphate and 2 altro-heptulose 7-phosphate in equilibrium fructose 6-phosphate + D-glycero D-altro octulose 8-phosphate were demonstrated with purified enzymes, but are concluded to play a minor role in the non-oxidative synthesis of pentose 5-phosphate and octulose phosphate by (RLEP). 5. The formation of gem diol and dimers of erythrose 4-phosphate is proposed to account in part for the failure to detect monomeric erythrose 4-phosphate in the carbon balance studies. 6. The equilibrium value for the pentose pathway acting by the reverse mode in vitro was measured and contrasted with the value for the pathway acting in the forward direction. The initial specific rates of the pentose pathway reactions in vitro for the reverse and forward directions are measured. 7. The study which includes carbon balance, time course changes and 14C prediction labelling experiments reports a comprehensive investigation of the mechanism of the pentose pathway acting reversibly.     

Studies on gluconeogenesis and protein synthesis in isolated hepatocytes.

Glucose production was studied in isolated hepatocytes using various substrates and with increasing substrate concentrations (0-10 mM). Fructose was the best gluconeogenic substrate while other substrates studied stimulated net glucose production in the following decreasing order: lactate, pyruvate, glycerol, galactose, alanine, and succinate. Studies on oxygen consumption showed that endogenous respiration was linear for 60 min and was not altered by extracellular calcium. Studies on the incorporation of 14C-leucine into protein was linear for only 3-4 hr in cells containing low glycogen. However, cells containing high glycogen incorporated 14C-leucine into protein linearly for 8-10 hr. About 3 mg of protein per g per hr was synthesized by isolated cells when incubated for 4 hr with amino acids mixture, glucose, lactate, and insulin.