A Simple Method for the Preparation of d-Ribulose 5-Phosphate

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Rose Bengal as a Specific Photosensitizer for a Histidine Residue at the Triphosphopyridine Nucleotide Binding Site of 6-Phosphogluconate Dehydrogenase

Rose Bengal is a potent inhibitor of 6-phosphogluconate dehydrogenase from Candida utilis and mediates the photoinactivation of the enzyme. The experiments reported in this paper indicate that photoinactivation occurs only when the dye is bound to the TPN binding site of the enzyme. Since the photoinactivation is correlated to the specific oxidation of only 2 residues of histidine, it can be assumed that this amino acid is located at the TPN binding site of 6-phosphogluconate dehydrogenase. These results are a new application of the technique of active site-specific photooxidation

Fructose 1,6-Diphosphatase from Rabbit Liver XII. EFFECT OF SUBSTRATE AND ADENOSINE MONOPHOSPHATE ON THE IONIZATION OF THE TYROSYL RESIDUES

Abstract The effects of substrate and AMP on the state of ionization of tyrosyl residues of fructose 1,6-diphosphatase have been investigated. In the native enzyme, 5 to 6 tyrosyl residues were titrated with a pK of about 8.4, and 7 to 8 residues were titrated with a pK of 9 to 9.2. The remaining tyrosyl residues were not titrated below pH 10. In the presence of 10-5 m fructose 1,6-diphosphate, when 4 moles of substrate are bound per mole of enzyme, approximately 4 of the low pK tyrosyl residues are shifted from pK 8.4 to pK 9.7. However, all of the pK values are modified, and under these conditions 2 tyrosyl residues show a pK of 8.7, and 10 or 11 residues show a pK of 9.7. At a high concentration of fructose 1,6-diphosphate, 10-3 m, all 12 to 13 of the tyrosyl residues are titrated with a pK of 9.8. The results can be correlated with previous studies on the acetylation of fructose 1,6-diphosphatase. The tyrosyl residues with low pK are more reactive and are associated with loss of sensitivity to AMP. However, after acetylation, only 10 tyrosyl residues are titrated with a pK of 9.3 to 9.4, and the pK is shifted to 9.7 when the substrate is bound. A method of analyzing the titration data is presented which permits simultaneous evaluation of number of residues and pK value of each titrated group. This has revealed changes in tyrosyl residues that would otherwise have been overlooked

THE NATURE OF THE AMINO ACID RESIDUES INVOLVED IN THE INACTIVATION OF GLUCONATE 6-PHOSPHATE DEHYDROGENASE BY IODOACETATE.

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Studies on the mechanism of action of the gluconate 6-phosphate dehydrogenase. The presence of a cysteine residue in the active center.

Abstract The role of the sulfhydryl groups in the gluconate 6-phosphate dehydrogenase has been studied. Nine cysteine residues have been detected by spectrophotometric titration with p-hydroxymercuribenzoate, but the reaction of only 3 of them is sufficient to inactivate the enzyme completely. Chlorodinitrobenzene acts even more specifically. Incorporation of 1.6 dinitrobenzene residues leads to enzyme inactivation. The amino acid residues involved in dinitrophenylation have been identified as cysteine. Gluconate 6-phosphate completely protects the enzyme against inactivation by chlorodinitrobenzene

Molecular and Functional Properties of a Calpain Activator Protein Specific for μ-Isoforms

A natural calpain activator protein has been isolated from bovine brain and characterized in its properties and molecular structure. The protein is a homodimer with a molecular mass of about 30 kDa and results in being almost identical to UK114 goat liver protein. Significant similarities with mouse HR12 protein were also observed, whereas a lower degree of similarity was found with a family of heat-responsive proteins named YJGF and YABJ from Haemophilus influenzae and Bacillus subtilis, respectively. The brain activator expresses a strict specificity for the mu-calpain isoform, being completely ineffective on the m-calpain form. As expected, also UK114 was found to possess calpain-activating properties, indistinguishable from those of bovine brain activator. A protein showing the same calpain-activating activity has been also isolated from human red cells, indicating that this factor is widely expressed. All these activators are efficient on mu-calpain independently from the source of the proteinase. The high degree of specificity of the calpain activator for a single calpain isoform may be relevant for the understanding of sophisticated intracellular mechanisms underlying intracellular proteolysis. These data are indicating the existence of a new component of the Ca2+-dependent proteolytic system, constituted of members of a chaperonin-like protein family and capable of promoting intracellular calpain activation

Conformational states of rabbit liver fructose 1,6-diphosphatase.

Abstract An analysis of the conformational states of fructose 1,6diphosphatase by the circular dichroism technique has been performed. It has been shown that the enzyme presents a strong rigidity of its over-all conformation. The addition of either the substrate or the allosteric inhibitor, AMP, or changes in the pH of the medium, do not produce significant modifications of the secondary and tertiary structure of the protein. The same situation holds even in 0.05 m sodium dodecyl sulfate, a condition in which the quaternary structure of the enzyme undergoes a profound modification. Small conformational changes have been detected, however, after the addition of the substrate and of AMP. These changes appear to be restricted to a limited number of tyrosyl and, at maximum, to a few vicinal residues. It is concluded that the catalytic and regulatory functioning of the enzyme do not require substantial alterations in the secondary and tertiary structure of the protein, but are related to changes in the ionization of amino acid residues and to subtle variations of the quaternary structure

CLEAVAGE OF SEDOHEPTULOSE 1,7-DIPHOSPHATE BY A PURIFIED RAT LIVER DIPHOSPHATASE.

SUMMARY treatment. A simple procedure is described for the purification, from rat None of a variety of other monophosphate and diphosphate liver, of a diphosphatase activity which cleaves both fructose esters tested as substrates was cleaved by the purified enzyme diphosphate and sedoheptulose diphosphate to fructose 6-phos- at significant rates. These results suggest an absolute require- phate and sedoheptulose 7-phosphate, respectively. ment for the presence of two phosphate groups and also for the Evidence that a single enzyme is responsible for both reactions configuration found in FDP and SDP. n-Bibulose 1,5-diphos- is derived from the results of the purification steps, from kinetic phate differs in the configuration at C-3 and is not attacked. measurements, and from other properties. The enzyme has no With respect to the physiological role of the diphosphatase, activity with a variety of monophosphate esters and other di- although it has been generally recognized that fructose diphos- phosphate esters. phatase may represent a key enzyme in the synthesis of glycogen Both activities are present in the liver of all mammalian species from 3 carbon fragments, no direct evidence for a role of sedo- studied. heptulose diphosphatase has yet been obtained. REFERENCES Three possibilities may be considered with respect to the physiological significance of the latter activity. (a) It may be 1. GOMORI, G., J

Fructose diphosphatase from rabbit liver. 3. Nature of the groups reactive with dinitrofluorobenzene.

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A Direct Interconversion: d-Fructose 6-Phosphate Sedoheptulose 7-Phosphate and d-Xylulose 5-Phosphate Catalyzed by the Enzymes Transketolase and Transaldolase

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