Age-dependent metabolic adaptation to starvation-refeeding in rats

Age-dependent metabolic adaptation to starvation-refeeding in rats

CLONING AND EXPRESSION IN ESCHERICHIA COLI OF A RAT HEPATOMA CELL cDNA CODING FOR 6-PHOSPHOFRUCBTOSE-2,6-BISPHOSPHATASE. K.M. Cre in M.I. Darville, L...

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CLONING AND EXPRESSION IN ESCHERICHIA COLI OF A RAT HEPATOMA CELL cDNA CODING FOR 6-PHOSPHOFRUCBTOSE-2,6-BISPHOSPHATASE. K.M. Cre in M.I. Darville, L. Hue and G.G. Rousseau. Hormone and Metabolic Research Unit, u-J-mversity of Louvain Medical School, UCLICP 7529, Avenue Hippocrate 75, B- 1200 Brussels, Belgium. In liver, the 470-residue bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6bisphosphatase (PFK-2/FBPase-2) catalyzes the synthesis and degradation of fructose 2,6_bisphosphate, a potent stimulator of glycolysis. In rat hepatoma HTC cells, this ey has kinetic, antigenic, and regulatory properties such as insensitivity to cyc ic AMP-dependent protein kmase, that differ from those in liver. To compare the sequence of the HTC enzyme to that of the liver enzyme, we have cloned the corresponding fully-coding cDNA from HTC cells. This cDNA predicts a protein of 448 residues in which the first 32 residues of liver PFK-2/FBPase-2 including the cyclic AMP target sequence have been replaced by a unique N-terminal decapeptide. The rest of the protein is identical to the liver enzyme. A N-terminally truncated recombinant peptide of 380 residues containing the PFK-2 and FBPase-2 domains was expressed in E. coli as a fi-galactosidase fusion protein. It was recognized by anti-PFK-2 antIIbut its PFK-2 and FBPase-2 activities were barely detectable. In contrast, a cDNA fully-coding for the HTC enzyme could be expressed in E. coli as a P-galactosidase-free peptide that exhibited both PFK-2 and FBPase-2 Z’flVXZs. This peptide had the same PFK-2 kinetic properties as the HTC enzyme, again different from those of the liver enzyme. Moreover, the FBPase-2 activity was inhibited by fructose 6-phosphate with a Ki different from that of the liver enzyme. Besides this novel type of PFK-2/FBPase-2 mRNA, HTC cells also contained low concentrations of the liver-type mRNA. Unlike in liver, neither mRNA was induced by glucocorticoids in these cells. These data provide a molecular basis for some of the properties of the PFK-2/FBPase2 isozymes in liver and HTC cells.

HUMAN RECOMBINANT ADENOVIRUS USED TO CORRECT A MOUSE ENZYME DEFICIENCY. J.F. Chasse", M. Levrero+, M. Minet*, P. Kamoun*, P. Briand*, M. ferric-, Laboratoire de Biochimie Genetique, Hopital Necker, 149 rue de SBvres, 15743 PARIS, FRANCE. l Laboratoire de Genetique des virus oncogenes, Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 VILLEJUIF, FRANCE. Human ornithine trancarbamylase deficiency is one of the most inherited frequent hereditary hyperammoneamia. This X-linked disease leads to death of 15% of the affected males, whatever the treatement. Thus, although several types of mutations leading to various levels of residual activity have been described, the evolution is in most cases very dramatic. We have used the spfash animals exhibiting an OTC deficiency characterized by an equal reduction in the amounts of OTC activity, OTC protein and specific OTC mRNA (but without any modification of the system leading to the mature enzyme in mitochondria), to attempt a correction of the enzymatic defect by somatic gene transfer. A human recombinant adenovirus which harbors the OTC gene under the control of the viral major late promotor, has been constructed. Following the injection of the recombinant virus into the liver of mutant mice, an elevated enzymatic activity was found in several animals and this, at different times after the last injection. Concomitantly to the expression of the introduced OTC gene, a corrected phenotype could be observed in some animals.