Low Concentrations Of Fructose Research Articles

Glucokinase regulatory protein may interact with glucokinase in the hepatocyte nucleus.

Glucokinase (GK) plays a central role in the sensing of glucose in pancreatic beta-cells and parenchymal cells of the liver. Glucokinase regulatory protein is a physiological inhibitor of GK in the liver. To understand the role of the interaction of these two proteins in glucose sensing, we carried out a series of experiments to localize the protein in the liver cell. The regulatory protein was found to be present mainly in the nucleus of the cell under a variety of conditions that mimicked the glucose status of the fed and fasted state. GK was localized in the nucleus when the cells were exposed to low glucose concentrations. At higher glucose concentrations or in the presence of low concentrations of fructose, GK translocated to the cytoplasm. The effect of fructose was more robust and rapid than the effect of high glucose concentrations. Furthermore, the effect of fructose and high glucose on the translocation of GK from the nucleus could be partially reversed by glucagon. This unusual localization and behavior suggests a role for GK and its regulatory protein in hepatic energy metabolism that may be broader than glucose phosphorylation.

Improvement on the Efficiency of Somatic Embryogenesis from Spinach Root Tissues by Applying Various Sugars.

Spinach (Spinacia oleracea L.) root segments were cultured on a medium containing a half strength of inorganic salts in Murashige and Skoog's medium, 10 μM 1-naphthaleneacetic acid (NAA), 0.1 μM gibberellic acid (GA3) and various kinds and concentrations of carbohydrates. For callus induction and somatic embryogenesis, spinach root segments effectively utilized glucose, fructose, sucrose, maltose, or raffinose as a carbon source. Frequency of embryo formation increased inversely to sugar concentration. Fructose or raffinose increased the number of embryos per callus. Levels of sugars remained were measured in a raffinose-containing liquid culture medium in which root segments were cultured continuously. The fact indicates that fructose was preferentially utilized by the tissues of spinach. These results show that a low concentration of fructose in the culture medium promotes a high frequency of embryogenesis in spinach tissue culture.

Fructose metabolism and cell survival in freshly isolated rat hepatocytes incubated under hypoxic conditions: Proposals for potential clinical use

The protective effect of fructose with regard to hypoxia-induced cell injury was investigated. The addition of fructose (2 to 20 mmol/L) protected hepatocytes against hypoxia-mediated cell lysis in a concentration-dependent way. The intracellular ATP content was initially decreased as a result of fructose-1-phosphate formation, but it remained constant during the hypoxic incubation. Conversely, high initial ATP values observed at low fructose concentrations progressively declined. Cellular protection was observed only when fructose was added before (and not after) the start of hypoxia. In addition, a sufficient amount of fructose-1-phosphate rapidly accumulated before the induction of hypoxia, and the linear production of lactate, during hypoxic incubation, indicated that cells synthesized ATP continuously. The lack of cell protection by fructose added after the onset of the hypoxia may be explained by a lesser fructose-1-phosphate formation and a subsequently low accumulation leading to insufficient glycolytic ATP production. Under aerobic conditions, both glycolysis (lactate formation) and gluconeogenesis (glucose formation) were carried out in fructose-1-phosphate-loaded cells with the same initial rates, whereas under hypoxic conditions glycolysis was the main metabolic event. The fact that protein synthesis activity recovered faster during reoxygenation of previously hypoxic fructose-treated cells than in glucose-treated cells led us to hypothesize that in situ perfusion of liver with fructose, before its removal, would improve its metabolic capacity during the hypoxic cold preservation and subsequent transplantation.

Glucose-glucose 6-phosphate cycling in hepatocytes determined by incorporation of 3HOH and D2O. Effect of glycosyns and fructose

The phosphorylation of glucose and recycling between glucose and glucose-6-P was determined in hepatocytes from fasted rats by a novel method. The cells were incubated with [U-14C]glucose as sole substrate in media containing 3HOH and D2O. Recycling was calculated from the yield of protons in glucose and glycogen. Results with 3HOH and D2O were identical. Phosphorylation was obtained as the sum of recycling plus the U-14C yields in products. At 10 mM glucose, more than 4 out of 5 molecules of glucose-6-P were recycled. About 1.2 mumol of glucose min/g of liver was phosphorylated, 1 mumol was recycled, and 0.2 mumol was glycolyzed. The effect of two phenacylimidazolium compounds (designated as glycosyns) and low concentrations of fructose (0.05-0.2 mM) on phosphorylation and recycling were examined. The glycosyns doubled glucose uptake, mainly as glycogen, nearly abolished glycolysis, and decreased recycling from 80 to 50-60%. There was little change in phosphorylation. Fructose doubled the yield of tritium from [2-3H]glucose in short term incubations (20-30 min), confirming the results of Van Schaftingen ((1993) Diabetologia 36, 582-588). The effect was transient, and cells became refractory to fructose. There was no glycogen synthesis and little effect on recycling. A new phenacylimidazolium compound stimulated glycogen synthesis and suppressed glycolysis and recycling, like the compound designated as proglycosyn by Yamanuchi et al. ((1992) Arch. Biochem. Biophys. 294, 609-615). This new compound (glycosyn-2) was fully active at lower concentrations (maximal effect at 0.02 mM).

Respective roles of glucose, fructose, and insulin in the regulation of the liver-specific pyruvate kinase gene promoter.

The L-type pyruvate kinase (L-PK) is a key enzyme of the glycolytic pathway mainly expressed in the liver. Rat liver contains a regulatory protein that inhibits glucokinase (GK) activity. The effect of this protein is greatly reinforced by the fructose 6-phosphate and antagonized by the fructose 1-phosphate (Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179-184). In hepatocytes, fructose in low concentrations is phosphorylated into fructose 1-phosphate, and therefore is able to active GK in the absence of insulin via the regulatory protein in the liver. In primary culture of rat hepatocytes, 0.2 mM fructose in the presence of 20 or 40 mM glucose stimulated the activity of the L-PK gene promoter fused with the chloramphenicol acetyltransferase reporter gene, regardless of the addition of insulin, through the glucose/insulin response element. A constitutive GK expression vector co-transfected with the L-PK/chloramphenicol acetyltransferase construct is also able to confer an insulin-independent glucose responsiveness in hepatocytes. Thus, the insulin effect on glucose-dependent activation of the L-PK promoter is, under these experimental conditions, to permit glucose phosphorylation through the stimulation of the GK synthesis. In the presence of glucose, the L-PK promoter can also be activated by a post-translational GK activation, mediated by a low concentration of fructose acting via the regulatory protein of glucokinase.

Conversion of fructose to glucose in the rabbit small intestine. A reappraisal of the direct pathway.

Gopher et al [Gopher, A., Vaisman, N., Mandel, H. & Lapidot, A. (1990) Proc. Natl Acad. Sci. USA 87, 5449-5453] recently reported that about 50% of the glucose formed from [U-13C]fructose infused nasogastrically in children contained 13C3 adjacent to 13C4. Assuming a high isotopic dilution of the triosephosphate pool, the authors concluded that about 50% of the fructose converted to glucose in liver and intestine bypassed the classical aldolase pathway, utilizing a hypothetical direct pathway that would involve the phosphorylation of fructose 1-phosphate to fructose 1,6-bisphosphate. The present work was undertaken in order to establish to what extent the conversion of fructose to glucose in the intestine could account for this unexpected isotopic distribution. The technique of everted sleeves was used to define the rate of conversion of [U-14C]glucose and [U-14C]fructose in the small intestine of 24-h-fasted rabbits. It appeared that, at the low concentration of fructose used by Gopher et al., almost as much fructose was converted to glucose as remained unmodified in the tissue. Fructose uptake was not inhibited by glucose, and the presence of all the necessary enzymes in the tissue indicated that the fructose to glucose conversion occurred by the aldolase pathway. Remarkably, this conversion operated with an isotopic dilution not exceeding 25%, due to the low rate of glucose metabolism and the near absence of gluconeogenesis from lactate. It can, therefore, be postulated that, in the presence of pure [U13C]fructose, the triosephosphate pool is highly enriched in 13C with little dilution by 12C, essentially giving rise to [U-13C]glucose, as reported by Gopher et al. There is, therefore, no need to postulate the participation of a direct pathway.

Fructose 1‐phosphate and the regulation of glucokinase activity in isolated hepatocytes

Fructose 1-phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1-phosphate and fructose. The concentration of fructose 1-phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate. Addition of fructose (0.05-1 mM) caused a concentration-dependent and transient increase in the fructose 1-phosphate content. Glucagon (1 microM) and ethanol (10 mM) caused a severalfold decrease in the concentration of fructose 1-phosphate in cells incubated with fructose, whereas the addition of 0.1 microM vasopressin or 10 mM glycerone, or raising the concentration of glucose from 5 mM to 20 mM had the opposite effect. All these agents caused changes in the concentration of triose phosphates that almost paralleled those of the fructose 1-phosphate concentration. Sorbitol had a similar effect to fructose in causing the formation of fructose 1-phosphate. D-Glyceraldehyde was much less potent in this respect than the ketose and its effect disappeared earlier. The effect of D-glyceraldehyde was reinforced by an increase in the glucose concentration and decreased by glucagon. Both fructose and D-glyceraldehyde stimulated the phosphorylation of glucose as estimated by the release of 3H2O from [2-3H]glucose, but the triose was less potent in this respect than fructose and its effect disappeared earlier. Glucagon and ethanol antagonised the effect of low concentrations of fructose or D-glyceraldehyde on the detritiation of glucose. These results support the proposal that fructose 1-phosphate mediates the effects of fructose, D-glyceraldehyde and sorbitol by relieving the inhibition exerted on glucokinase by a regulatory protein.

Induction and metabolite regulation of levanase synthesis in Bacillus subtilis

Levanase expression in Bacillus subtilis was studied by using transcriptional and translational fusions. It was shown that the degradative products of levan or inulin and low concentrations of fructose were able to induce levanase expression. In the wild-type strain and in a constitutive overproducing sacL mutant, levanase synthesis was repressed by glucose or fructose. This catabolite repression was partially abolished in the derepressed alpha-amylase gra-26 mutant. The levanase gene (sacC) appears to be the distal gene of an operon transcribed from a fructose-inducible promoter. Deletion mapping experiments and primer extension analysis revealed a transcriptional start point located 2.7 kilobases upstream from the sacC gene. Two constitutive sacL mutations were shown to be closely linked by transformation to the sacC gene. The sacL6 and sacL8 mutations were mapped in the promoter-proximal region of the operon.

Gluconeogenesis from fructose predominates in periportal regions of the liver lobule.

Gluconeogenesis from fructose was studied in periportal and pericentral regions of the liver lobule in perfused livers from fasted, phenobarbital-treated rats. When fructose was infused in increasing concentrations from 0.25 to 4 mM, corresponding stepwise increases in glucose formation by the perfused liver were observed as expected. Rates of glucose and lactate production from 4 mM fructose were around 100 and 75 mumol/g/h, respectively. Rates of fructose uptake were around 190 mumol/g/h when 4 mM fructose was infused. 3-Mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, decreased glucose formation from fructose maximally by 20% suggesting that a fraction of the lactate formed from fructose is used for glucose synthesis. A good correlation (r = 0.92) between extra oxygen consumed and glucose produced from fructose was observed. At low fructose concentrations (less than 0.5 mM), the extra oxygen uptake was much greater than could be accounted for by glucose synthesis possibly reflecting fructose 1-phosphate accumulation. Furthermore, fructose diminished ATP/ADP ratios from about 4.0 to 2.0 in periportal and pericentral regions of the liver lobule indicating that the initial phosphorylation of fructose via fructokinase occurs in both regions of the liver lobule. Basal rates of oxygen uptake measured with miniature oxygen electrodes were 2- to 3-fold higher in periportal than in pericentral regions of the liver lobule during perfusions in the anterograde direction. Infusion of fructose increased oxygen uptake by 65 mumol/g/h in periportal areas but had no effect in pericentral regions of the liver lobule indicating higher local rates of gluconeogenesis in hepatocytes located around the portal vein. When perfusion was in the retrograde direction, however, glucose was synthesized nearly exclusively from fructose in upstream, pericentral regions. Thus, gluconeogenesis from fructose is confined to oxygen-rich upstream regions of the liver lobule in the perfused liver.

Properties of locust muscle 6-phosphofructokinase and their importance in the regulation of glycolytic flux during prolonged flight

6-Phosphofructokinase (PFK, EC 2.7.1.11) from the flight muscle of the locust (Locusta migratoria) was purified to a specific activity of 80 μmol min−1 (mg protein)−1 (at 25°C). 1. The enzyme is made up from subunits ofMr-81600, and the smallest catalytically active form is likely to be a tetramer. 2. PFK activity is markedly affected by the pH of the assay; the optimum pH was at about 8. 3. Physiological concentrations of ATP strongly inhibit locust PFK by shifting the S0.5 for fructose 6-phosphate (concentration required for 50% of maximum activity) out of the physiological concentration range. At pH 7.4 and about physiological concentrations of ATP, the curve of PFK activity against the concentration of fructose 6-phosphate is highly sigmoidal with S0.5 several hundred-fold higher than the concentration of fructose 6-phosphate in vivo. 4. The sigmoidicity and the S0.5 values for fructose 6-phosphate are decreased by addition of activators such as NH 4 + , inorganic phosphate, AMP and fructose 2,6-bisphosphate. The sensitivity of the response to these activators is increased by synergistic effects; marked synergistic effects were observed between inorganic phosphate and NH 4 + , fructose 2,6-bisphosphate or AMP as well as between AMP and fructose 2,6-bisphosphate. 5. Fructose 1,6-bisphosphate and fructose 2,6-bisphosphate are both activators of locust muscle PFK but the latter is much more potent than the former. Either of these compounds modifies the effect of the other; when phosphofructokinase is activated by fructose 2,6-bisphosphate, addition of fructose 1,6-bisphosphate results in inhibition of the enzyme activity; and when fructose 1,6-bisphosphate is present in the assay, higher concentrations of fructose 2,6-bisphosphate are required to increase the activity of the enzyme. 6. At low AMP concentrations, fructose 2,6-bisphosphate is not very effective in activating PFK, but AMP is effective at low concentrations of fructose 2,6-bisphosphate. 7. The properties of locust PFK suggest that the marked decrease in the concentration of fructose 2,6-bisphosphate that occurs in the locust flight muscle in the early stages of flight is, in part, responsible for the decrease in PFK activity and thus conservation of carbohydrate during prolonged flight, when lipid is the major fuel. At any stage of flight, however, an increase in energy demand could bring about an increase in PFK activity via increased concentrations of AMP and phosphate.

The reactive cysteine residue of pig kidney fructose 1,6-bisphosphatase is related to a fructose 2,6-bisphosphate allosteric site

Modification of a highly reactive cysteine residue of pig kidney fructose 1,6-bisphosphatase with N-ethylmaleimide results in the loss of activation of the enzyme by monovalent cations. Low concentrations of fructose 2,6-bisphosphate or high (inhibitory) levels of fructose 1,6-bisphosphate protect the enzyme against the loss of monovalent cation activation, while non-inhibitory concentrations of the substrate gave partial protection. The allosteric inhibitor AMP markedly increases the reactivity of the cysteine residue. The results indicate that fructose 2,6-bisphosphate can protect the enzyme against the loss of potassium activation by binding to an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit the enzyme by binding to this allosteric site.

Parasite population biology and host nutrition: dietary fructose and Moniliformis (Acanthocephala).

Experiments were carried out to investigate the possible influence of host-dietary fructose concentration on the relationship between worm burden and the fecundity of Moniliformis dubius. Worm length was found to be dependent both on the fructose concentration of the host's diet and on worm burden. The number of free ovaries/female worm was found to increase linearly with increasing dietary fructose concentration but was independent of worm burden. The length of the ovaries increased with increasing dietary fructose concentration and decreased in response to increases in worm density. The magnitude of the density-dependent reduction in the volume of ovarian tissue/female was decreased in worms from rats fed on diets containing low concentrations of fructose. The numbers of eggs present in the body cavities of the female worms was found to be positively correlated with dietary fructose concentration. At 5 weeks post-infection, mature eggs (containing fully developed acanthor larvae) were found in the body cavities of female worms from rats whose diets contained 3 and 12% fructose, but not in those from rats whose diets contained 1 and 2% fructose (w/w). The experimental results are discussed in the wider context of the influence of host nutrition on parasite population biology.

The interaction of fructose 2,6-bisphosphate with an allosteric site of rat liver fructose 1,6-bisphosphatase

Rat liver fructose 1,6-bisphosphatase can be protected against partial inactivation by N-ethylmaleimide by low concentrations of fructose 2,6-bisphosphate or high concentrations of fructose 1,6-bisphosphate. The partially inactivated enzyme has a much reduced sensitivity to high substrate inhibition and has lost the sigmoid component of the inhibition by fructose 2,6-bisphosphate; this compound is a simple linear competitive inhibitor of the modified enzyme. The results suggest that fructose 2,6-bisphosphate can bind to the enzyme at two distinct sites, the catalytic site and an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit by binding to the allosteric site.

Activation of the plastid isozyme of phosphofructokinase from developing endosperm of [formula omitted] by fructose 2,6-bisphosphate

The plastid isozyme of phosphofructokinase from developing castor oil seeds is stimulated by low concentrations of fructose 2,6-bisphosphate when assayed at pH 7.0. The stimulation involves a shift in fructose 6-phosphate kinetics from sigmoidal to near hyperbolic. The plastid isozyme is unaffected by fructose 2,6-bisphosphate when assayed at pH 8.0, and the cytosolic isozyme is unaffected at either pH 7.0 or 8.0. There is no interaction between fructose 2,6-bisphosphate and the other regulators of the Ricinus phosphofructokinases; phosphoenolpyruvate, 2-phosphoglycerate, 3-phosphoglycerate and inorganic phosphate.

Effects of vasopressin on fructose and glycogen metabolism in hepatocytes from fed and fasted rats

Hepatocytes were isolated from fed and 24-hr-fasted rats and incubated with lactate plus pyruvate in the presence and absence of 30 m m glucose. The effects of fructose and/ or vasopressin (ADH) on the activities of glycogen synthase and phosphorylase were examined and correlated with their effects on glycogen content. Effects of glucose and vasopressin on the metabolism of [U- 14C]fructose were also studied. ADH-stimulated net glycogenolysis in hepatocytes from fed rats was inhibited by each fructose concentration studied. In the presence of glucose, lactate, and pyruvate, low concentrations of fructose (up to 5 m m) markedly increased hepatocyte glycogen content, but high concentrations (20–40 m m) prevented net glycogen synthesis. In the presence or absence of glucose or of ADH, incubation of hepatocytes with 2 m m fructose generally caused either no change or a slight activation of both glycogen synthase and phosphorylase. About 80% of the radioactivity initially present as [U- 14C]fructose was incorporated after 80 min into glucose and about 1% into glycogen in hepatocytes from both fed and fasted rats. Addition of glucose reduced the amount of radioactivity incorporated into glucose to 65%, and increased that into glycogen about fivefold. ADH had no effect on the percentage of radioactivity incorporated into glucose, CO 2, or anionic metabolites, but it decreased the percentage of radioactivity incorporated into glycogen by about fivefold. Thus the only observed effect of ADH on [U- 14C]fructose metabolism in hepatocytes from fed or fasted rats in the presence or absence of 30 m m glucose was to decrease deposition of radioactive glycogen, an effect consistent with the observed ADH-mediated inactivation of glycogen synthase and activation of phosphorylase.

Composition of rabbit semen and the origin of several constituents.

Semen was collected twice a day every other day (2X/48 h) for 36 days from 12 Dutch-belted rabbits. Sex drive, ejaculate volume, microscopic appearance of the semen and sperm counts were recorded. Seminal plasma was analyzed for itcontent of fructose, citric acid, glycerylphos- phorylcholine (GPC) and the minerals Na, K, P, Mg, Ca and Zn. Six males were exsanguinated 24 h after the last semen collection and 6 were exsanguinated 5 weeks later. The organs of the reproductive tract were weighed and analyzed for their contents of spermatozoa, fructose, citric acid and GPC. Rabbits differed in libido and several characteristics of semen. Second ejaculates had lower concentrations of fructose, citric acid, K and Zn than did the first ejaculates. Fructose was found chiefly in the different portions of the prostate gland and major concentrations of citric acid were found in the ampulla of the ductus deferens and in the vesicular gland. GPC concentrations were highest in the caput and cauda epididymides. Correlations among different constituents were calculated and their significance is discussed.

Escherichia coli phosphoenolpyruvate carboxylase: Studies on the mechanism of multiple allosteric interactions

Some kinetic studies of the interactions between Escherichia coli phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylase (phosphorylating) EC 4.1.1.31) acetyl coenzyme A, fructose 1,6-bisphosphate, and aspartate were performed. Activation of the enzyme by fructose 1,6-bisphosphate is anomalous by comparison with acetyl coenzyme A in that it confers hysteretic properties on the enzyme. In the presence of both activators and aspartate, hysteresis is observed also, but the approach to optimum catalytic activity can be fit to an equation for a second-order reaction with respect to enzyme concentration. Since, however, hysteresis is not a result of any apparent association-dissociation reaction, the apparent fit to a second-order kinetic equation is probably not real but is the result of a multistep activation mechanism. Hysteresis is not eliminated by preincubation of the enzyme with fructose 1,6-bisphosphate, acetyl coenzyme A, or phosphoenolpyruvate singly or in any pair of combinations. Hysteresis is associated, therefore, with the slow conformation change from the inactive species to the active species under the influence of all three of those reactants. The enzyme complex resulting from the binding of each activator, including phosphoenolpyruvate, has an increased affinity for the other activators. A kinetic method for estimating the relative changes in affinity of these complexes for some of the other reactants is presented. At concentrations of the activators below their K a , synergistic effects are evident, particularly in their ability to relieve aspartate inhibition. Aspartate inhibition is competitive with acetyl coenzyme A both in the absence and in the presence of low concentrations of fructose 1,6-bisphosphate. Increasing the concentrations of fructose 1,6-bisphosphate results in an increase in the apparent K l for aspartate, suggesting that synergistic activation by fructose 1,6-bisphosphate is a result of the increased affinity of the fructose 1,6-bisphosphate-enzyme complex for acetyl coenzyme A, and a shift in the concentration of enzyme species away from the one(s) to which aspartate can bind most easily. In the presence of fructose 1,6-bisphosphate alone optimal activation can be achieved, but the concentrations required in vitro are high and suggest that fructose 1,6-bisphosphate alone does not function in that capacity physiologically, but primes the enzyme for more effective activation by acetyl coenzyme A and/or phosphoenolpyruvate.

Size-dependent allosteric effects of monovalent cations on rabbit liver fructose-1,6-bisphosphatase.

Effects of monovalent cations on the neutral rabbit liver fructose-1,6-bisphosphatase are multifunctional and dependent on their nonhydrated ionic size. (a) The maximal velocity is increased by addition of monovalent cations with the optimum stimulation occurring with a nonhydrated ionic radius of 1.2 A in the presence of a chelating agent such as EDTA. (B) Activation curves are sigmoidal with n values varying from 1.5 to 2.3 as ionic radius of monovalent cation increases. The apparent Ka values from 16.0 to 180 mM, obtained for various monovalent cations, have a linear relationship to ionic radii of cations. (c) At lower concentrations of fructose 1,6-bisphosphate monovalent cations show the inhibitory effect and the apparent Km for fructose 1,6-bisphosphate is increased as the concentration of monovalent cation is increased. A linear relationship is obtained between the slopes of increase in the Km and the reciprocals of ionic volume of monovalent cations. (d) The apparent Ka for Mg2+ is also increased as the concentration of monovalent cation is increased, and a linear relationship is obtained again between the increases in Ka and the reciprocals of ionic volume of monovalent cations. The cooperative nature for Mg2+ saturation is decreased as the Ka increases. (e) The apparent Ki for AMP is also linearly altered as the concentration of monovalent cation is varied. However, the alteration of the Ki is unusual, that is, the smaller cations than K+ increase the Ki (Li+ greater than Na+ greater than NH4+), whereas the larger cations decrease the value ((CH2CH2OH)3N+ greater than Cs+ greater than Rb+). The effect of K+ is insignificant. Alterations in the Ki are also linearly related to the reciprocals of ionic volume of monovalent cations. The cooperative nature for AMP inhibition is decreased or increased as the Ki increased or decreased. (f) In the absence of the chelating agent, the curves for Mg2+ saturation and AMP inhibition were hyperbolic without monovalent cations. By addition of monovalent cation the Ka for Mg+2+ or Ki for AMP is increased and cooperative natures for binding of both ligands are induced. For nonspherical monovalent cations, the application of "functional ionic radius" is proposed. Functional ionic radii of NH4+, (CH2OH)3CNH3+, and (CH2CH2OH)3N+ are estimated to be 1.17, 2.55, and 2.87 A, respectively. The presence of two distinct sites for the actions of monovalent cations is suggested.

Location of L-iditol: NAD oxidoreductase (D-sorbital tetrazolium reductase or ketose-reductase) in the genital tract of the bull.

SUMMARY The histochemical technique for the demonstration of the d-sorbitol dehydrogenase activity gave intensely positive results in the epithelium of the seminal vesicles and the glands of the ampulla of the ductus deferens of the bull. The enzymic activity was lower in the prostatic epithelium of the ductus deferens and the epididymis. The reaction was negative in the epithelium of the bulbo-urethral glands of Cowper. The enzymic activity detected is connected with the synthesis of fructose, but in organs with a low concentration of fructose, such as the prostate gland and the ductus epididymidis the presence of the enzyme may be connected with the synthesis of sorbitol, also present in the semen. In the seminal vesicles d-sorbitol dehydrogenase activity was found in the epithelium of the glandular tubules and in the material contained in those tubules. Using this technique, we have observed in the epithelium different types of cells which may correspond to different stages of a secretory cycle. The cells with greater deposits of diformazan appear to be related to the secretion of a product with enzymic activity.

The Dissociation of d-Fructose 1,6-Diphosphatase by Adenosine 5'-Phosphate

Abstract Adenosine 5'-monophosphate, an inhibitor of the d-fructose 1,6-diphosphatase isolated from Candida utilis, induces a specific dissociation of this enzyme at pH 9.2. On the basis of sucrose density gradient centrifugation and gel filtration on Sephadex G-100, it may be concluded that the molecule has dissociated into subunits having one-half the molecular weight of the native enzyme. The dissociation occurs only at alkaline pH and in the cold. It is prevented by low concentrations of fructose 1,6-diphosphate (0.01 mm). d-Fructose 1,6-diphosphatase which has been desensitized to the inhibitory action of 5'-AMP is not dissociated. The results suggest that the dissociation induced by 5'-AMP may be an extreme manifestation of more subtle alterations in the quaternary structure of the enzyme which occur in the presence of substrate at physiological pH.