S. Pontremoli

Rabbit liver fructose 1,6-diphosphatase. Properties of the native enzyme and their modification by subtilisin☆

Abstract A simple two-step procedure is described for the purification of fructose 1, 6-diphosphatase from rabbit liver. The enzyme shows activity at neutral pH, in contrast to the previously described “alkaline” fructose diphosphatase. The neutral fructose diphosphatase has a higher molecular weight, 140,000, as compared with 130,000 for the alkaline enzyme and a somewhat different amino acid composition. Most significant is the presence of one residue of tryptophan per subunit; this amino acid is not present in the alkaline fructose diphosphatase. The subunit molecular weight of the neutral enzyme is 36,000, and it contains 4 moles of COOH-terminal alanine. Digestion with subtilisin converts the neutral enzyme to one with properties resembling those of the alkaline form. The pH optimum is shifted to pH 9 and the sensitivity toward inhibition by AMP is decreased. The molecular weight of the modified enzyme is 120,000. The results suggest that the changes in catalytic properties are associated with the removal of a tryptophan-containing peptide or peptides with molecular weights totaling approximately 6000 per subunit, and that the alkaline enzyme previously studied in this and other laboratories is a modified form of the native neutral fructose diphosphatase.

A specific interaction of pyridoxal 5′-phosphate and 6-phosphogluconic dehydrogenase

Abstract The enzymic activity of crystalline 6-phosphogluconic dehydrogenase from Candida utilis is inhibited by incubation at neutral pH with very low concentrations of pyridoxal 5′-phosphate. No inactivation is observed when the enzyme is incubated with pyridoxal itself, or with pyridoxine or pyridoxamine phosphate. The inhibition is reversed by addition of 6-phosphogluconate, by dilution or dialysis, or upon addition of amino acids. The effect of pyridoxal phosphate is competitive with 6-phosphogluconate. The inhibition of the enzymic activity is due to the formation of a Schiff base between the aldehydric group of the inhibitor and the ϵ-amino group of a single lysine residue at the active center of the enzyme. This was established by reduction with sodium borohydride and the isolation of N 6 -pyridoxyllysine from acid hydrolyzates. Pyridoxal also forms a Schiff base derivative with the enzyme, but in this case both the Schiff base and the reduced complex are enzymically active. The presence of a phosphate group appears to be important in directing the inhibitor to the particular sensitive lysine residue at the active center of the enzyme.

Fructose 1, 6-diphosphatase from liver: Isolation of the native form with optimal activity at neutral pH

A new procedure has been developed for the purification of rabbit liver fructose 1,6-diphosphatase which yields homogeneous enzyme preparations with optimum activity at neutral pH. The properties of this protein differ from those of previous preparations, which show alkaline pH optima. The neutral fructose diphosphatase has a higher molecular weight and yields only one species of subunit in SDS disc-gel electrophoresis. The Km values for substrate and divalent cations were not significantly different from those of the alkaline fructose diphosphatase. It is, however, more sensitive to AMP inhibition, and there are also differences in the reactivity of some sulfhydryl groups with p-mercuribenzoate. It appears to represent a native form of fructose diphosphatase.

Pyridoxal 5'-phosphate as a specific photosensitizer for histidine residue at the active site of 6-phosphogluconate dehydrogenase.

Abstract Photooxidation of 6-phosphogluconate dehydrogenase in the presence of pyridoxal 5′-phosphate (which binds reversibly to a lysine residue at the active center) leads to an irreversible loss of the enzymatic activity. Loss of activity follows first-order kinetics. Photoinactivation was correlated with the specific destruction of only two histidine residues. Binding to the active center lysine seems to be a requisite for the efficiency of the pyridoxal phosphate as a photoinactivating agent. Pyridoxal, which does not react with this lysine residue, is ineffective as photosensitizer. The substrate 6-phosphogluconate prevents the binding of the pyridoxal phosphate and protects against photoinactivation. These findings suggest that pyridoxal phosphate may be useful as a specific photosensitizer for those enzymes which are inhibited by specific binding with this compound.

Fructose diphosphatase from rabbit muscle. II. Amino acid composition and activation by sulfhydryl reagents.

Abstract The properties of rabbit muscle and rabbit liver fructose 1,6-diphosphatases are compared. It may be concluded that the two enzymes are different proteins with a number of common properties. They differ significantly in primary structure, as indicated by amino acid analysis, although there may be extensive homology. They are similarly activated by treatment with dinitrofluorobenzene or p -mercuribenzoate, or by disulfide exchange with 5,5′-dithio bis(2-nitrobenzoic) acid (DTNB). However, the muscle enzyme is not activated by cystamine, the only known natural activator of liver fructose diphosphatase. The enzyme activated with fluorodinitrobenzene retains normal sensitivity to the allosteric inhibitor AMP; after activation with DTNB the enzyme is somewhat less sensitive.

The Correlation between Red‐Cell Survival and Excess of α‐Globin Synthesis in β‐Thalassaemia

In 13 subjects affected by β‐thalassaemia major, in three subjects affected by β‐thalassaemia minor and in five normal healthy persons haemoglobin synthesis and the survival of red cells transfused into normal, group compatible, healthy recipients has been studied. The existence of an excess of newly synthesized α‐chains and of a negative correlation between the excess α‐chain and the red‐cell survival has been demonstrated. The harmful role of the α‐chain excess on the erythrocyte and the implications of this finding are discussed.

Rabbit liver and rabbit kidney fructose diphosphatases: Catalytic properties of enzymes activated by coenzyme A and acyl carrier protein

Abstract The catalytic properties of rabbit liver and rabbit kidney fructose diphosphatases are altered when these enzymes are treated with CoA or acyl carrier protein from Escherichia coli . The activity in the neutral pH range is increased several fold, and the pH optima are shifted from pH 8.8 to pH 7.5 in the presence of MgCl 2 , and from pH 9.1 to pH 8.2 when MnCl 2 is the cofactor. Maximum activity requires the presence of a chelating agent such as EDTA, histidine, or glycine. The untreated enzymes are inhibited by excess fructose-1,6- P 2 , whereas the activated enzymes are not, although the K m for this substrate is increased by approximately 10-fold. The modified enzymes are also more sensitive to inhibition by AMP. The reactions with CoA or acyl carrier protein are prevented by the addition of high concentrations of substrate, but not by AMP. In the activated enzyme approximately two sulfhydryl groups appear to be blocked, and the changes in catalytic properties are reversed by treatment with sulhydryl compounds such as cysteine or glutathione. This preliminary evidence indicates that activation involves the formation of disulfide linkages between CoA or acyl carrier protein and the enzyme. Activation by CoA or an ACP-like protein may represent a physiological mechanism for the reciprocal control of gluconeogenesis and fatty acid synthesis.

Absence of β-globin synthesis and excess of α-globin synthesis in homozygous β-thalassaemic subjects from the Ferrara region.

β-THALASSAEMIA is a genetically determined anaemia of man, characterized by a decrease in production1–6 or the absence7 of β-globin, and by an excess of α-globin synthesis4,6–8, which results in the α/(β + γ + δ) globin synthesis ratio largely exceeding the normal value of 1.0.

[26] 6-Phosphogluconate dehydrogenase—Crystalline

Publisher Summary This chapter discusses the determination of crystalline 6-phosphogluconate dehydrogenase. The method of determination of enzymatic activity is based on the spectrophotometric measurement of reduced triphosphopyridine nucleotide (TPNH) + H + formed during oxidation and decarboxylation of D-gluconate 6-phosphate to D-ribulose 5-phosphate and CO 2 . In the course of purification procedure, the dried yeast preparation (50 g) is suspended in 150 ml of 0.1 N NaHCO 3 . After 5 hours at 37° the autolyzed suspension is centrifuged for 20 minutes at 20,000 g . The extract (90 ml) is kept frozen overnight without loss of activity and is diluted with 117 ml of cold water for the subsequent procedures. The crystalline enzyme catalyzes the reversible decarboxylation of D-gluconate 6-phosphate to D-ribulose 5-phosphate and CO 2 . No evidence has been found for the existence of separate enzymatic activities catalyzing the oxidative and the decarboxylative step. The crystalline enzyme preparation contains no detectable D-glucose 6-phosphate dehydrogenase activity and is also completely free of D-ribose 5-phosphate isomerase and D-xylulose 5-phosphate 3-epimerase.

Regulatory sulfhydryl groups and activation by homocystine in liver fructose diphosphatase

Abstract Incubation of rabbit liver fructose diphosphatase with l -homocystine leads to increases in activity at neutral pH of more than 8-fold when assayed with Mn 2+ and 3-fold when assayed with Mg 2+ . The specific activity of the activated enzyme at pH 8 was 45 units/mg, the highest yet observed for rabbit liver fructose diphosphatase under any assay conditions. Activation was correlated with the incorporation of 4 equiv of homocysteine per mole of enzyme and the disappearance of a corresponding number of enzyme sulfhydryl groups. No evidence for cooperative interaction of the subunits during activation was obtained. Activation followed saturation kinetics, suggesting that the enzyme contains specific binding sites for homocystine. Activation and binding of homocysteine were readily reversed by incubation with dithiothreitol. The activation reaction was specifically prevented by 10 −3 m fructose diphosphate, or by lower concentrations of substrate in the presence of the allosteric ligand, AMP. The same sulfhydryl groups appear to be involved in the reaction with homocystine as in the previously reported activation by CoA.