Effectors of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver.

Summary The lower Vmax of 6PGDH with respect to G6PDH and its higher sensitivity to inhibition by NADPH, suggest the existence of an imbalance between the two dehydrogenases of the pentose phosphate pathway in rat liver. Possible modulators of these activities, particularly in relation with the inhibition by NADPH in physiological conditions, have been investigated. The results suggest that in both cases the inhibition by NADPH is strictly isosteric and that the relative affinities for the reduced and oxidized forms of the pyridine nucleotide are unaffected by glutathion, the intermediates of the pentose phosphate shunt or some divalent ions.Abbreviations G6PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - 6PGDH 6-phosphogluconate dehydrogenase (EC 1.1.1.44) On leave from the Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.     

A two-step purification procedure for sheep liver 6-phosphogluconate dehydrogenase

A two-step procedure for the purification of 6-phosphogluconate dehydrogenase (EC 1.1.1.44; 6-PGDH) from sheep liver is described. The enzyme is directly bound to cellulose phosphate by batch extraction and eluted with a linear salt gradient. Purification is completed by affinity chromatography using NADP(+)-agarose. The result is 6-PGDH of high purity, greatly increased yield, and the highest specific activity yet achieved, with a significant reduction in the purification time.     

Importance in catalysis of the 6-phosphate-binding site of 6-phosphogluconate in sheep liver 6-phosphogluconate dehydrogenase

The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K(6PG) that ranges from 5- to 800-fold. There is also a less pronounced increase in K(NADP), ranging from 3- to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E(t) (600- and 300-fold, respectively) as well as in V/K(6PG)E(t) (10(5) - and 10(4)-fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E(t) was observed for the Y191A mutant enzyme, whereas 20- and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K(6PG)E(t) range from 3- to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13C-isotope effect -1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.     

Kinetic studies of 6-phosphogluconate dehydrogenase from sheep liver

The steady-state kinetics of the oxidative decarboxylation of 6-phosphogluconate catalysed by 6-phosphogluconate dehydrogenase from sheep liver in triethanolamine and phosphate buffers (pH 7.0) have been reinvestigated. In triethanolamine buffer the enzyme is inhibited by high NADP+ concentrations in the presence of low fixed concentrations of 6-phosphogluconate. Data are consistent with an asymmetric sequential mechanism in which NADP+ and 6-phosphogluconate bind randomly and product release is ordered. The pathway through the enzyme--6-phosphogluconate complex appears to be preferred in triethanolamine buffer. Pre-steady-state studies of the oxidative decarboxylation reaction at pH 6.0-8.0 show that hydride transfer is greater than 900 s-1. After the fast formation of NADPH in amounts equivalent to about half of the enzyme-active-centre concentration, the rate of NADPH formation is equal to the steady-state rate. Two possible interpretations are considered. Rapid fluorescence measurements of the displacement of NADPH from its complex with the enzyme at pH 6.0 and 7.0 indicate that the dissociation of NADPH is fast (greater than 800 s-1) and cannot be the rate-limiting step in oxidative decarboxylation. Coenzyme binding studies at equilibrium have been extended to include the determination of the dissociation constants for the binary complexes of enzyme with NADPH and NADP+ at pH 6.0-8.0 and the dissociation constant for NADPH in the ternary enzyme--6-phosphogluconate--NADPH complex in triethanolamine buffer, pH 7.0.     

Purification and properties of 6-phosphogluconate dehydrogenase from sheep liver

A method is described for the isolation of 6-phosphogluconate dehydrogenase from sheep liver. The product appears to be homogeneous in polyacrylamide-gel electrophoresis and in sedimentation-velocity and sedimentation-equilibrium studies in the ultracentrifuge. The molecular weight is estimated as 129000 from equilibrium sedimentation.     

Activation of phosphofructokinase from rat liver by 6-phosphogluconate

GuHCl induced unfolding-refolding of T₄ lysozyme has been studied at equilibrium and by kinetic analysis using fluorescence emission and circular dichroïsm at 223 nm. Both approaches have indicated the occurrence of intermediates in the folding pathway. A plausible model has been proposed.     

Nuclear precursors for ceruloplasmin-coding messenger RNA from rat liver

The distribution of the sequences coding for ceruloplasmin (CP) in rat liver heterogeneous nuclear RNA (hnRNA) was studied using highly specific CP cDNA as a hybridization probe. The content of CP-coding sequences in poly(A)-containing and poly(A)-free subfractions of hnRNA was shown to be respectively 1 and 27 equivalents of CP mRNA molecule per one hepatocyte. The gel electrophoresis of hnRNA under strongly denaturing conditions with the subsequent transfer of RNA to diazobenzyloxymethyl paper and hybridization with [32P]-cDNA probe showed that CP mRNA sequences were of multiple molecular weight distribution. In particular, 9.0, 6.6, 2.4 and 1.6 megadalton fractions of non-polyadenylate hnRNA carried CP-coding sequences while the only hand that hybridized to CP cDNA was detected in polyadenylated hnRNA. This band was of a molecular weight 1.1-1.2 megadaltons corresponding to that of cytoplasmic CP mRNA. The hybridization of high molecular weight hnRNA with full-length CP cDNA followed by the determination of the size of cDNA fragments protected against SI nuclease demonstrated that coding sequences of CP pre-mRNA are interrupted by intervening sequences.     

Isolation of rat liver albumin messenger RNA.

Rat liver albumin messenger RNA has been purified to apparent homogeneity by means of polysome immunoprecipitation and poly(U)-Sepharose affinity chromatography. Specific polysomes synthesizing albumin were separated from total liver polysomes through a double antibody technique which allowed isolation of a specific immunoprecipitate. The albumin-polysome immunoprecipitate was dissolved in detergent and the polysomal RNA was separated from protein by sucrose gradient centrifugation. Albumin mRNA was then separated from ribosomal RNA by affinity chromatography through the binding of poly(U)-Sepharose to the polyadenylate 3' terminus of the mRNA. Pure albumin mRNA migrated as an 18 S peak on 85% formamide-containing linear sucrose gradients and as a 22 S peak on 2.5% polyacrylamide gels in sodium dodecyl sulfate. It coded for the translation of authentic liver albumin when added to a heterologous protein-synthesizing cell-free system derived from either rabbit reticulocyte lysates or wheat germ extracts. Translation analysis in reticulocyte lysates indicated that albumin polysomes were purified approximately 9-fold from total liver polysomes, and that albumin mRNA was purified approximately 74-fold from albumin polysomal RNA. The total translation product in the mRNA-dependent wheat germ system, upon addition of the pure mRNA, was identified as authentic albumin by means of gel electrophoresis and tryptic peptide chromatography.     

Growth-rate-dependent alteration of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase levels in Escherichia coli K-12.

The levels of 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase are subject to metabolic regulation; they increased three- to fivefold with increasing growth rate.     

Highly purified ceruloplasmin messenger RNA from rat liver

Summary Highly purified ceruloplasmin mRNA was isolated from rat liver polyribosomes. The molecular weight of ceruloplasmin mRNA is in a range from 1.05 to 1.25 · 10⁶ daltons which is large enough to code for a putative precursor of ceruloplasmin (∼700 amino acids). Ceruloplasmin mRNA contains 3′-terminal poly(A) the length of which varies from 38 to 165 nucleotides. The 5′-end of ceruloplasmin mRNA is blocked with confronting m⁷G residue which is a component of cap I (m⁷G^5′ppp^5′X^mpAp). The addition of ceruloplasmin mRNA to wheat-germ cell-free system programmed the synthesis of a product that was largely precipitated by anti-ceruloplasmin immunoglobulins. The translation product was homogeneous in polyacrylamide gel-sodium dodecylsulfate electrophoresis. Cell-free translation of ceruloplasmin mRNA was sensitive to inhibition by cap analogue.     

Purification of messenger RNA coding for glycine methyltransferase from rat liver

Rat liver messenger RNA coding for glycine methyltransferase was associated preferentially with free polysomes. The mRNA was purified about 1000-fold over the total poly(A)-containing RNA by specific immunoadsorption of polysomes to protein A-Sepharose followed by oligo(dT)-cellulose column chromatography. Sodium dodecyl sulfate-gel electrophoresis of the in vitro translation products in a rabbit reticulocyte lysate system revealed only one major band which migrated to the position of the purified glycine methyltransferase subunit. The result shows that the mRNA isolated is nearly homogeneous and suggests that no precursor form of the enzyme existed. The mRNA sedimented at the position slightly smaller than 18 S rRNA in a sucrose density-gradient centrifugation and was shown to contain about 1,300 nucleotides by the Northern blot hybridization analysis with a cDNA probe.