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Regulation of the oxidative phase of the pentose phosphate cycle in mussels.

ABSTRACT: 1. The mechanisms that control the oxidative phase of the pentose phosphate cycle in mussel hepatopancreas were investigated. 2. The effects of GSSG (oxidized glutathione) on the inhibition of glucose 6-phosphate dehydrogenase by NADPH [Eggleston & Krebs (1974) Biochem. J. 138, 425-435] extend to 6-phosphogluconate dehydrogenase. 3. The effect of GSSG on both enzymes increases as the [NADP+1]/[NADPH] ratio decreases; greater percentage deinhibition always was obtained for 6-phosphogluconate dehydrogenase. 4. Increasing concentration of GSSG increased the percentage deinhibition. This effect is more pronounced with 6-phosphogluconate dehydrogenase. 5. We confirmed the apparent imbalance between the activities of the two enzymes [sapag-Hagar, Lagunas & Sols (1973) Biochem. Biophys. Res. Commun, 50, 179-185] in the presence of 10mM-Mg2+. 6. The imbalance practically disappears when the substrate concentrations are less than saturating and Mg2+ approaches physiological concentrations. 7. The addition of GSSG at physiological concentrations allows the activities of both enzymes to be measured at high [NADPH]/[NADP+] ratios ratios and the co-operative action of GSSG and Mg2+ on the imbalance between the two enzymes to be verified. 8. The control of the activity of the two enzymes of the pentose cycle could be carried out by deinhibition of the two dehydrogenases and by the intracellular concentrations of substrates and inorganic ions.

SUBMITTER: Rodriguez-Segade S

PROVIDER: S-EPMC1183934 | biostudies-other | 1978 Mar

REPOSITORIES: biostudies-other

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Project description:1. A search was made for mechanisms which may exert a ;fine' control of the glucose 6-phosphate dehydrogenase reaction in rat liver, the rate-limiting step of the oxidative pentose phosphate cycle. 2. The glucose 6-phosphate dehydrogenase reaction is expected to go virtually to completion because the primary product (6-phosphogluconate lactone) is rapidly hydrolysed and the equilibrium of the joint dehydrogenase and lactonase reactions is in favour of virtually complete formation of phosphogluconate. However, the reaction does not go to completion, because glucose 6-phosphate dehydrogenase is inhibited by NADPH (Neglein & Haas, 1935). 3. Measurements of the inhibition (which is competitive with NADP(+)) show that at physiological concentrations of free NADP(+) and free NADPH the enzyme is almost completely inhibited. This indicates that the regulation of the enzyme activity is a matter of de-inhibition. 4. Among over 100 cell constituents tested only GSSG and AMP counteracted the inhibition by NADPH; only GSSG was highly effective at concentrations that may be taken to occur physiologically. 5. The effect of GSSG was not due to the GSSG reductase activity of liver extracts, because under the test conditions the activity of this enzyme was very weak, and complete inhibition of the reductase by Zn(2+) did not abolish the GSSG effect. 6. Preincubation of the enzyme preparation with GSSG in the presence of Mg(2+) and NADP(+) before the addition of glucose 6-phosphate and NADPH much increased the GSSG effect. 7. Dialysis of liver extracts and purification of glucose 6-phosphate dehydrogenase abolished the GSSG effect, indicating the participation of a cofactor in the action of GSSG. 8. The cofactor removed by dialysis or purification is very unstable. The cofactor could be separated from glucose 6-phosphate dehydrogenase by ultrafiltration of liver homogenates. Some properties of the cofactor are described. 9. The hypothesis that GSSG exerts a fine control of the pentose phosphate cycle by counteracting the NADPH inhibition of glucose 6-phosphate dehydrogenase is discussed.

Project description:The maintenance of advanced malignancies relies on continued activity of driver oncogenes, although their rate-limiting role is highly context-dependent with respect to tumor types and associated genetic alterations. Oncogenic Kras mutation is the signature event in human pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible KrasG12D-driven p53 mutant PDAC mouse model establishes that advanced PDAC remains strictly dependent on continued KrasG12D expression and that KrasG12D serves a vital role in the control of tumor metabolism, through stimulation of glucose uptake and channeling of glucose intermediates through the hexosamine biosynthesis pathway (HBP) and the pentose phosphate pathway (PPP). Notably, these studies reveal that oncogenic Kras regulates ribose biogenesis. Unlike canonical models of PPP-mediated ribose biogenesis, we demonstrate that oncogenic Kras drives intermediates from enhanced glycolytic flux into the non-oxidative arm of the PPP, thereby decoupling ribose biogenesis from NADPNADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in Kras-driven PDAC. Primary pancreatic tumor lines were established from p48Cre tetO_LKrasG12D ROSA_rtTAL+ p53L+ mice. Five independent tumor lines (iKras1-5) were used for pancreatic injection into nude mice to generate orthotopic tumors. The mice were kept on doxycycline for 2 weeks until obvious tumor formation. Half of the animals were pulled off doxycycline for 24 hours. Tumors with over 75% cellularity were collected for total RNA prepartion. For in vitro expression profiles, the same five tumor lines were cultured in the presence or absence of doxycycline for 24 hours and total cellular RNA was prepared. For control samples, two independent tumor lines from LSL-KrasG12D p53L+ tumors were cultured in the presence or absence of doxycycline for 24 hours and total cellular RNA was prepared.

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Regulation of the oxidative phase of the pentose phosphate cycle in mussels.

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