Evidence for the Degradation of Nicotinamide Adenine Dinucleotide Phosphate-Dependent Glutamate Dehydrogenase of Candida utilis During Rapid Enzyme Inactivation

The nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase (NADP-GDH) from the food yeast Candida utilis was found to be rapidly inactivated when cultures were starved of a carbon source. The addition of glutamate or alanine to the starvation medium stimulated the rate of inactivation. Loss of enzyme activity was irreversible since the reappearance of enzyme activity, following the addition of glucose to carbon-starved cultures, was blocked by cycloheximide. A specific rabbit antibody was prepared against the NADP-GDH from C. utilis and used to quantitate the enzyme during inactivation promoted by carbon starvation. The amount of precipitable antigenic material paralleled the rapid decrease of enzyme activity observed after transition of cells from NH(4) (+)-glucose to glutamate medium. No additional small-molecular-weight protein was precipitated by the antibody as a result of the inactivation, suggesting that the enzyme is considerably altered during the primary steps of the inactivation process. Analysis by immunoprecipitation of the reappearance of enzyme activity after enzyme inactivation showed that increase of NADP-GDH activity was almost totally due to de novo synthesis, ruling out the possibility that enzyme activity modulation is achieved by reversible covalent modification. Enzyme degradation was also measured during steady-state growth and other changes in nitrogen and carbon status of the culture media. In all instances so far estimated, the enzyme was found to be very stable and not normally subject to high rates of degradation. Therefore, the possibility that inactivation was caused by a change in the ratio of synthesis to degradation can be excluded.     

The functional significance of variants of fructose 1,6-diphosphatase in the gill and hypodermis of a marine crustacean. A kinetic study

1. In the hypodermis and gill of the Crustacea fructose 1,6-diphosphatase (EC 3.1.3.11) functions at a primary branch point between glycogen and chitin synthesis. In these tissues of the Arctic king-crab, Paralithodes camtchatica, fructose diphosphatase occurs in two electrophoretically distinguishable forms. 2. Fructose diphosphatase I (pI7.2-7.5) accounts for 70 and 10% of total fructose diphosphatase activity in the hypodermis and gill respectively, whereas fructose diphosphatase II (pI5.3) accounts for 30 and 90% of the total activity in the two tissues. Both forms display a neutral pH optimum, have an absolute requirement for a bivalent cation, and are potently inhibited by high concentrations of AMP and substrate. 3. Fructose 1,6-diphosphate saturation follows Michaelis-Menten kinetics for both fructose diphosphatases; the K(m) (fructose diphosphate) for fructose diphosphatase I is somewhat higher than for fructose diphosphatase II. In the presence of 50-200mm-K(+), the K(m) (fructose diphosphate) increases and at high concentrations of K(+) fructose diphosphate saturation follows sigmoidal kinetics. 4. UDP-N-acetylglucosamine and UDP-glucose at high concentrations specifically and potently inhibit fructose diphosphatase II, but do not significantly affect fructose diphosphatase I activity. 5. Low concentrations of UDP-N-acetylglucosamine activate fructose diphosphatase II by a decrease in the apparent K(m) (fructose diphosphate), but fructose diphosphatase I is again refractory to UDP-N-acetylglucosamine under these conditions. 6. In the presence of K(+) and UDP-N-acetylglucosamine, fructose diphosphatase II is able to compete for limiting fructose diphosphate about three times more effectively than is fructose diphosphatase I. 7. AMP inhibition of both forms of the enzyme is subject to three independent variables: (a) alkaline pH increases the K(i) (AMP), (b) K(+) decreases the K(i), increases the sigmoidicity of inhibition kinetics, increases the maximum inhibition attained, and abolishes the effect of pH on AMP inhibition, and (c) Mg(2+) strongly de-inhibits AMP-inhibited fructose diphosphatase. 8. It is postulated that the presence of two forms of fructose diphosphatase aids controlled channelling of carbon through the fructose diphosphatase ;bottleneck' either towards glycogen synthesis or chitin synthesis, but not towards both simultaneously.     

The activities of fructose diphosphatase in flight muscles from the bumble-bee and role of this enzyme in heat generation

1. The maximum catalytic activities of fructose diphosphatase from flight muscles of bumble-bees (Bombus spp.) are at least 30-fold those reported for the enzyme from other tissues. The maximum activity of fructose diphosphatase in the flight muscle of any particular bee is similar to that of phosphofructokinase in the same muscle, and the activity of hexokinase is similar to or greater than the activity of phosphofructokinase. There is no detectable activity of glucose 6-phosphatase and only a very low activity of glucose 6-phosphate dehydrogenase in these muscles. The activities of both fructose diphosphatase and phosphofructokinase vary inversely with the body weight of the bee, whereas that of hexokinase is relatively constant. 2. There is no significant hydrolysis of fructose 1-phosphate, fructose 6-phosphate, glucose 1,6-diphosphate and glycerol 3-phosphate by extracts of bumble-bee flight muscle. 3. Fructose 1,6-diphosphatase from bumble-bee flight muscle and from other muscles is inhibited by Mn(2+) and univalent cations; the potency of inhibition by the latter varies in the order Li(+)>Na(+)>K(+). However, the fructose diphosphatase from bumble-bee flight muscle is different from the enzyme from other tissues in that it is not inhibited by AMP. 4. The contents of ATP, hexose monophosphates, fructose diphosphate and triose phosphates in bumble-bee flight muscle showed no significant changes between rest and flight. 5. It is proposed that both fructose diphosphatase and phosphofructokinase are simultaneously active and catalyse a cycle between fructose 6-phosphate and fructose diphosphate in resting bumble-bee flight muscle. Such a cycle would produce continuous hydrolysis of ATP, with the release of energy as heat, which would help to maintain the thoracic temperature during rest periods at a level adequate for flight.     

Temperature and the regulation of enzyme activity in poikilotherms. Regulatory properties of fructose diphosphatase from muscle of the Alaskan king-crab

1. The properties of fructose diphosphatase from skeletal muscle of the Alaskan king-crab (Paralithodes camtschatica) were examined over the physiological temperature range of the animal. 2. King-crab muscle fructose diphosphatase is first activated by Na(+) and NH(4) (+) and is then partially inhibited by these cations at concentrations higher than 10mm at 0 degrees , 8 degrees and 15 degrees C. Enzyme activity is stimulated by K(+) at 0 degrees C, but is curtailed at 8 degrees C and 15 degrees C, an effect that could render rate independent of temperature. 3. Affinity for substrate increases with decreasing temperature; below the temperature of acclimatization, K(m) for fructose 1,6-diphosphate increases, resulting in a complex U-shaped temperature-K(m) curve. 4. King-crab muscle fructose diphosphatase is inhibited by low concentrations of AMP. As with enzymes of other poikilotherms, inhibition by AMP is sensitive to temperature; the enzyme is least sensitive to inhibition by AMP near the temperature of acclimatization. 5. The affinity of fructose diphosphatase for fructose 1,6-diphosphate is enhanced by phosphoenolpyruvate, and this activation is temperature-sensitive; 0.5mm-phosphoenolpyruvate causes a sevenfold decrease in K(m) for fructose 1,6-diphosphate at 15 degrees C but a 25-fold decrease at 0 degrees C. 6. Phosphoenolpyruvate appears to decrease the affinity of king-crab muscle fructose diphosphatase for AMP at low temperature, whereas at the higher temperature it appears to enhance inhibition by AMP. Phosphoenolpyruvate was not observed to cause a reversal of inhibition of fructose diphosphatase activity by AMP. The identification of phosphoenolpyruvate as an activator of a rate-limiting step in gluconeogenesis permits the suggestion of a coupling of the controlling mechanisms of several steps in the glycolytic and gluconeogenic chains. 7. These findings suggest mechanisms for the maintenance and regulation of control of fructose diphosphatase activity in king-crab skeletal muscle at low temperature and under conditions that favour concomitant activity of phosphofructokinase.     

Induction of rat kidney gluconeogenesis during acute liver intoxication by carbon tetrachloride.

1. Glucose production from L-lactate was completely inhibited 24h after carbon tetrachloride treatment in liver from 48h-starved rats. The activities of phosphoenolpyruvate carboxykinase, fructose diphosphatase and glucose 6-phosphatase were decreased by this treatment in fed and starved rats, whereas lactate dehydrogenase activity was only decreased in fed animals. 2. The production of glucose by renal cortical slices from fed rats previously treated with carbon tetrachloride was enhanced when L-lactate, pyruvate and glutamine but not fructose were used as glucose precursors. Renal phosphoenolpyruvate carboxykinase activity was increased in this condition. 3. This increase was counteracted by cycloheximide or actinomycin D, suggesting that the effect was due to the synthesis de novo of the enzyme. 4. The pattern of hepatic gluconeogenic metabolites in treated animals was characterized by an increase in lactate, pyruvate, malate and citrate as well as a decrease in glucose 6-phosphate, suggesting an impairment of liver gluconeogenesis in vivo. 5. In contrast, the profile of renal metabolites suggested that gluconeogenesis was operative in the treated rats, as indicated by the marked increase in the content of phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate and glucose 6-phosphate. 6. It is postulated that renal gluconeogenesis could contribute to the maintenance of glycaemia in carbon tetrachloride-treated rats.     

The glucose-dependent transport of l-malate in Zygosaccharomyces bailii

Zygosaccharomyces bailii possesses a constitutive malic enzyme, but only small amounts of malate are decomposed when the cells ferment fructose. Cells growing anaerobically on glucose (glucose cells) decompose malate, whereas fructose cells do not. Only glucose cells show an increase in the intracellular concentration of malate when suspended in a malate-containing solution. The transport system for malate is induced by glucose, but it is repressed by fructose. The synthesis of this transport system is inhibited by cycloheximide. Of the two enantiomers l-malate is transported preferentially. The transport of malate by induced cells is not only inhibited by addition of fructose but also inactivated. This inactivation is independent of the presence of cycloheximide. The transport of malate is inhibited by uranyl ions; various other inhibitors of transport and phosphorylation were of little influence. It is assumed that the inducible protein carrier for malate operates by facilitated diffusion. Fructose cells of Z. bailii and cells of Saccharomyces cerevisiae do not contain a transport system for malate.This research was supported in part by a grant from the Forschungsring des Deutschen Weinbaus.     

Purification and Regulatory Properties of Fructose 1,6-Diphosphatase from Hydrogenomonas eutropha

Fructose diphosphatase of Hydrogenomonas eutropha H 16, produced during autotrophic growth, was purified 247-fold from extracts of cells. The molecular weight of the enzyme was estimated to be 170,000. The enzyme showed a pH optimum of 8.5 in both crude extracts and purified preparation. The shape of the pH curve was not changed in the presence of ethylenediaminetetraacetic acid. The enzyme required Mg²⁺ for activity. The MgCl₂ saturation curve was sigmoidal and the degree of positive cooperativity increased at lower fructose diphosphate concentrations. Mn²⁺ can replace Mg²⁺, but maximal activity was lower than that observed with Mg²⁺ and the optimal concentration range was narrow. The fructose diphosphate curve was also sigmoidal. The purified enzyme also hydrolyzed sedoheptulose diphosphate but at a much lower rate than fructose diphosphate. The enzyme was not inhibited by adenosine 5′-monophosphate but was inhibited by ribulose 5-phosphate and adenosine 5′-triphosphate. Adenosine 5′-triphosphate did not affect the degree of cooperativity among the sites for fructose diphosphate. The inhibition by adenosine 5′-triphosphate was mixed and by ribulose 5-phosphate was noncompetitive. An attempt was made to correlate the properties of fructose diphosphatase from H. eutropha with its physiological role during autotrophic growth.     

Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts.

Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinites for fructose diphosphate and Mg-2+ and were similarly activated by EDTA, dithiothreitol and cystamine. Mg-2+, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.     

Properties and regulation of C-1-fructose-1,6-diphosphatase from spinach chloroplasts

Chloroplast fructose diphosphatase (EC 3.1.3.11) was purified according to the procedures of Racker and Schroeder [1] and Buchanan et al. [2] and the properties compared. Neither preparation contained fructose diphosphatase from the cytoplasm. The preparations had similar molecular weights, pH optima, affinities for fructose diphosphate and Mg²⁺ and were similarly activated by EDTA, dithiothreitol and cystamine.Mg²⁺, fructose diphosphate and dithiothreitol all activate chloroplast fructose diphosphatase more so at suboptimal pH values. The combined effects of these substances under estimated physiological conditions in the chloroplast stroma in the light and in darkness were consistent with almost full activity of the enzyme during illumination but no activity in the dark.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cell. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ‘Crabtree effect’, was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate hydrogenase was subject to glucose inactivation.     

Temperature and the regulation of enzyme activity in poikilotherms. Properties of lungfish fructose diphosphatase

1. The properties of fructose diphosphatase from liver of South American lungfish (Lepidosiren paradoxa) were examined. 2. Saturation curves for substrate (fructose diphosphate) and both cofactors (Mn(2+) and Mg(2+)) are sigmoidal and Hill plots of these results suggest about 2 interacting substrate and cofactor sites/molecule of enzyme. 3. Mn(2+) is an efficient positive modulator of the enzyme and K(a) for Mn(2+) is about 20-30-fold lower than the K(a) for Mg(2+). 4. Lungfish fructose diphosphatase is inhibited by low concentrations of AMP, and the affinity of the enzyme for AMP is insensitive to temperature. 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be dependent on temperature, whereas affinity for Mg(2+) is temperature-independent. 6. The pH optimum of the enzyme depends on the presence of the particular cofactor. As pH increases, the K(a) values of both cations are lowered, maximum velocities are increased and the saturation curves for cofactor become hyperbolic. 7. The possible roles of these ions, pH and substrate in the modulation of fructose diphosphatase and gluconeogenic activity in the lungfish are discussed in relation to aestivation and temperature adaptation.     

Regulation of enzymes and isoenzymes of carbohydrate metabolism in the yeast Saccharomyces cerevisiae

An electrophoretic method has been devised to investigate the changes in the enzymes and isoenzymes of carbohydrate metabolism, upon adding glucose to derepressed yeast cells. (i) Of the glycolytic enzymes tested, enolase II, pyruvate kinase and pyruvate decarboxylase were markedly increased. This increase was accompanied by an overall increase in glycolytic activity and was prevented by cycloheximide, an inhibitor of protein synthesis. (ii) In contrast, respiratory activity decreased after adding glucose. This decrease was clearly shown to be the result of repression of respiratory enzymes. A rapid decrease within a few minutes of adding glucose, by analogy with the so-called ' Crabtree effect', was not observed in yeast. (iii) The gluconeogenic enzymes, fructose-1,6-bisphosphatase and malate dehydrogenase, which are inactivated after adding glucose, showed no significant changes in electrophoretic mobilities. Hence, there was no evidence of enzyme modifications, which were postulated as initiating degradation. However, it was possible to investigate cytoplasmic and mitochondrial malate dehydrogenase isoenzymes separately. Synthesis of the mitochondrial isoenzyme was repressed, whereas only cytoplasmic malate dehydrogenase was subject to glucose inactivation.     

The activities of fructose 1,6-diphosphatase, phosphofructokinase and phosphoenolpyruvate carboxykinase in white muscle and red muscle

1. The activities of fructose 1,6-diphosphatase were measured in extracts of muscles of various physiological function, and compared with the activities of other enzymes including phosphofructokinase, phosphoenolpyruvate carboxykinase and the lactate-dehydrogenase isoenzymes. 2. The activity of phosphofructokinase greatly exceeded that of fructose diphosphatase in all muscles tested, and it is concluded that fructose diphosphatase could not play any significant role in the regulation of fructose 6-phosphate phosphorylation in muscle. 3. Fructose-diphosphatase activity was highest in white muscle and low in red muscle. No activity was detected in heart or a deep-red skeletal muscle, rabbit semitendinosus. 4. The lactate-dehydrogenase isoenzyme ratio (activities at high and low substrate concentration) was measured in various muscles because a low ratio is characteristic of muscles that are more dependent on glycolysis for their energy production. As the ratio decreased the activity of fructose diphosphatase increased, which suggests that highest fructose-diphosphatase activity is found in muscles that depend most on glycolysis. 5. There was a good correlation between the activities of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle, where the activities of these enzymes were similar to those of liver and kidney cortex. However, the activities of pyruvate carboxylase and glucose 6-phosphatase were very low in white muscle, thereby excluding the possibility of gluconeogenesis from pyruvate and lactate. 6. It is suggested that the presence of fructose diphosphatase and phosphoenolpyruvate carboxykinase in white muscle may be related to operation of the alpha-glycerophosphate-dihydroxyacetone phosphate and malate-oxaloacetate cycles in this tissue.     

The inhibition of skeletal-muscle fructose 1,6-diphosphatase by adenosine monophosphate

1. The present study extends the finding of Krebs & Woodford (1965) that muscle fructose diphosphatase is more sensitive to AMP inhibition than liver fructose diphosphatase. 2. Hen breast fructose diphosphatase has a K(i) for AMP of 0.1mum; the plot of percentage inhibition is non-sigmoid and the reciprocal plot of activity against AMP concentration is sometimes linear. 3. Percentage inhibition plots for other muscle fructose diphosphatases are sigmoid curves which exhibit different threshold responses to the AMP concentration. 4. The intracellular content of AMP in all muscles tested exceeds the inhibition concentration range of AMP. 5. The sensitivity of muscle fructose diphosphatase to AMP inhibition is decreased by the presence of Mg(2+) or Mn(2+) ions; in the presence of Mn(2+) the inhibition curve for hen breast fructose diphosphatase becomes sigmoid. 6. From the formation constants for the Mg(2+) and Mn(2+) chelates, the effect of these ions in chelation of AMP can be calculated. Although chelation of AMP can explain the Mg(2+) effect, it cannot explain the marked relief of AMP inhibition by Mn(2+). 7. It is suggested that Mn(2+) has a specific effect on this enzyme which reduces the sensitivity to AMP inhibition.     

Regulation of sugar transport in Neurospora crassa

Sugar uptake systems in Neurospora crassa are catabolically repressed by glucose. Synthesis of a low K(m) glucose uptake system (system II) in Neurospora is derepressed during starvation for an externally supplied source of carbon and energy. Fasting also results in the derepression of uptake systems for fructose, galactose, and lactose. In contrast to the repression observed when cells were grown on glucose, sucrose, or fructose, system II was not repressed by growth on tryptone and casein hydrolysate. System II was inactivated in the presence of 0.1 m glucose and glucose plus cycloheximide but not by cycloheximide alone. Inactivation followed first-order kinetics with a half-time of 40 min. The addition of glycerol to the uptake medium had no significant effect on the kinetics of 3-0-methyl glucose uptake, suggesting that the system was not feedback inhibitable by catabolites of glycerol metabolism.     

Temperature and the regulation of enzyme activity in poikilotherms. Properties of rainbow-trout fructose diphosphatase

1. The properties of fructose diphosphatase from the liver of rainbow trout (Salmo gairdnerii) were examined over the physiological temperature range of the organism. 2. Saturation curves for substrate (fructose 1,6-diphosphate) and a cofactor (Mg(2+)) are sigmoidal, and Hill plots of the results suggest a minimum of two interacting fructose 1,6-diphosphate sites and two interacting Mg(2+) sites per molecule of enzyme. 3. Mn(2+)-saturation curves are hyperbolic, and the K(a) for Mn(2+), which inhibits the enzyme at high concentrations, is 50-100-fold lower than the K(a) for Mg(2+). 4. Fructose diphosphatase is inhibited by low concentrations of AMP; this inhibition appears to be decreased and reversed by increasing the concentrations of Mg(2+) and Mn(2+). Higher concentrations of AMP are required to inhibit the trout fructose diphosphatase in the presence of Mn(2+). 5. The affinities of fructose diphosphatase for fructose diphosphate and Mn(2+) appear to be temperature-independent, whereas the affinities for Mg(2+) and AMP are highly temperature-dependent. 6. The pH optimum of the enzyme depends on the concentrations of Mg(2+) and Mn(2+). In addition, pH determines the K(a) for Mg(2+); at high pH, K(a) for Mg(2+) is lowered. 7. The enzyme is inhibited by Ca(2+) and Zn(2+), and the inhibition is competitive with respect to both cations. 8. The possible roles of these ions and AMP in the modulation of fructose diphosphatase and gluconeogenic activity are discussed in relation to temperature adaptation.     

Induction of lipogenic enzymes in primary cultures of rat hepatocytes. Relationship between lipogenesis and carbohydrate metabolism

Using primary cultures of adult rat hepatocytes, the regulation of the following lipogenic enzymes was studied: glucose-6-phosphate dehydrogenase, malic enzyme, ATP-citrate lyase, acetyl-CoA carboxylase, fatty acid synthetase, and stearoyl-CoA desaturase. The addition to the culture medium of either insulin or triiodothyronine produced a 2-3-fold increase in each of the individual enzyme activities whereas glucagon slightly decreased enzyme activities. The addition to the medium of 8-bromoguanosine 3,'5'-monophosphate had no effect on any of the enzyme activities unless glucose was also added to the culture medium. Glucose addition alone to the culture medium was without any effect; however, glucose enhanced the stimulation of enzyme activity due to insulin. The addition of fructose or glycerol, even in the absence of insulin, increased the activities of each of the enzymes studied 2-3-fold. The increases in enzyme activity brought about by insulin or fructose were apparently the result of de novo enzyme synthesis, as indicated by the observation that the increases were not noted in the presence of cordycepin or cycloheximide. Immunoprecipitation of ATP-citrate lyase from hepatocytes pulse-labeled with [3H]leucine indicated that the induction of this enzyme in response to the addition of fructose or glycerol to the culture medium was the result of an increase in the rate of synthesis of the enzyme. These results indicate that the activity and synthesis of individual enzymes involved in lipogenesis are increased in response to the metabolism of carbohydrate independently in part from hormonal effects.     

Increase in nucleoside diphosphatase in rat brain striatum lesioned with kainic acid

The activity of ammoniagenesis from guanine nucleotides was found to increase significantly in rat brain after infusion of kainic acid into the striatum. Among the enzymes involved in degrading guanine nucleotides, nucleoside diphosphatase was markedly increased in the lesioned striatum. The enzyme activity began to increase 2 days after the infusion, and reached the maximum on the 13th day, the level being 4 times as high as that of the intact contralateral region. The increased activity was due to Type L enzyme, judging from its substrate specificity. Puromycin and cycloheximide inhibited this increase, indicating that the increased activity resulted from an increase in the net synthesis of the enzyme. These findings suggest that Type L NDPase might play some important roles in gliosis after neuronal lesion.     

Hormonal regulation of the system a amino acid transport adaptive response mechanism in a kidney epithelial cell line (MDCK)

When mamalian cells are starved for amino acids, the activity of the A amino acid transport system increases, a phenomenon called adaptive regulation. We have examined the effects of those factors which support Madin-Darby canine kidney (MDCK) cell growth in a defined medium on the derepression of System A activity. Of the five factors which supported MDCK cell growth, insulin was found to be an absolute requirement for derepression. In contrast, PGE₁ was a negative controlling factor for the transport system. Growth of MDCK cells in the absence of PGE₁ resulted in elevated System A activity which derepressed poorly upon amino acid starvation. Kinetic analysis of α-(methylamino) isobutyric acid (mAIB) uptake as a function of substrate concentration showed that the elevated A activity observed when cells were grown in the absence of PGE₁ was kinetically similar to the activity induced by starvation for amino acids. Transport of mAIB by amino-acid-fed cells grown in the presence of PGE₁ was characterized by a linear Eadie-Hofstee graph and by a relatively low Vmax. Transport by cells starved for amino acids or by cells grown in the absence of PGE₁ was characterized by biphasic kinetics for mAIB transport and by elevated Vmax values. An influence of growth factors on the inactivation of derepressed A activity was also observed. In the presence of cycloheximide the rate of loss of A activity in amino-acid-starved cells was 1/4–1/2 that of amino-acid-fed cells. Insulin slowed inactivation in the absence of most amino acids in a protein-synthesis-independent manner, but insulin did not influence the more rapid inactivation observed in amino-acid-fed cells. These results indicate that the level of System A activity observed in response to regulation by amino acids represents a balance between carrier synthesis and inactivation, which can be positively or negatively influenced by growth factors.     

P-nitrophenyl phosphate as substrate for rabbit liver fructose diphosphatase

Purified rabbit liver fructose diphosphatase has been found to catalyze the hydrolysis of p-nitrophenyl phosphate, PNPP. It has been established that the hydrolysis of p-nitrophenyl phosphate is due to fructose diphosphatase through studies of the chromatographic properties of the enzyme, its temperature sensitivity, dependence on divalent cations and its inhibition by fructose diphosphate. The K_m for PNPP is 6 × 10⁻³M at pH 9.2, 5 × 10⁻⁴M at pH 7.5. This substrate should facilitate studies of the kinetics and mechanism of action of fructose diphosphatase and the comparison of this enzyme with other alkaline phosphatases.