Histochemical technique for the demonstration of phosphofructokinase activity in heart and skeletal muscles

A histochemical multi-step technique for the demonstration of phosphofructokinase activity in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the non-structurally bound enzyme into the medium during incubation. In the histochemical system the enzyme converts the substrate D-fructose-6-phosphate to D-fructose-1,6-diphosphate, which in turn is hydrolyzed by exogenous and endogenous fructose diphosphate aldolase to dihydroxyacetone phosphate and D-glyceral-dehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into D-glyceraldehyde-3-phosphate by exogenous and endogenous triosephosphate isomerase. Next the D-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase into 1,3-diphospho-D-glycerate. Concomitantly the electrons are transported via NAD+, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.     

Histochemical technique for the demonstration of phosphofructokinase activity in heart and skeletal muscles

Summary A histochemical multi-step technique for the demonstration of phosphofructokinase activity in tissue sections is described. With this technique a semipermeable membrane is interposed between the incubating solution and the tissue sections preventing diffusion of the non-structurally bound enzyme into the medium during incubation. In the histochemical system the enzyme converts the substrate d-fructose-6-phosphate to d-fructose-1,6-diphosphate, which in turn is hydrolyzed by exogenous and endogenous fructose diphosphate aldolase to dihydroxyacetone phosphate and d-glyceraldehyde-3-phosphate. The dihydroxyacetone phosphate is reversibly converted into d-glyceraldehyde-3-phosphate by exogenous and endogenous triosephosphate isomerase. Next the d-glyceraldehyde-3-phosphate is oxidized by exogenous and endogenous glyceraldehyde-3-phosphate dehydrogenase into 1,3-diphospho-d-glycerate. Concomitantly the electrons are transported via NAD⁺, phenazine methosulphate and menadione to nitro-BT. Sodium azide and amytal are incorporated to block electron transfer to the cytochromes.     

Histochemical technique for the demonstration of pyruvate kinase activity

We describe an enzyme histochemical multistep technique for the demonstration of pyruvate kinase activity. In this technique, a semipermeable membrane is interposed between the incubation medium and the tissue sections, thus preventing diffusion of the enzyme into the medium during the incubation period. In this histochemical system, phosphoenolpyruvate (PEP) donates its phosphate group to ADP in a reaction catalysed by pyruvate kinase. Next, exogenous and endogenous hexokinase catalyses the reaction between ATP and D-glucose to yield D-glucose-6-phosphate and ADP. The D-glucose-6-phosphate is oxidized by exogenous and endogenous D-glucose-6-phosphate dehydrogenase, and concomitantly, the generated electrons are transported via NADP+, phenazine methosulphate and menadione to nitro-BT, which is finally precipitated as formazan. Sodium azide and amytal are included to block electron transfer to cytochromes. The method proved to be of value for the qualitative demonstration of pyruvate kinase activity in tissue sections of kidneys, heart muscle and skeletal muscle. For quantitative studies and for investigating the activity of this enzyme in liver sections, the method cannot be recommended.     

Histochemical technique for the demonstration of pyruvate kinase activity

Summary We describe an enzyme histochemical multistep technique for the demonstration of pyruvate kinase activity. In this technique, a semipermeable membrane is interposed between the incubation medium and the tissue sections, thus preventing diffusion of the enzyme into the medium during the incubation period. In this histochemical system, phosphoenolpyruvate (PEP) donates its phosphate group to ADP in a reaction catalysed by pyruvate kinase. Next, exogenous and endogenous hexokinase catalyses the reaction between ATP andd-glucose to yieldd-glucose-6-phosphate and ADP. Thed-glucose-6-phosphate is oxidized by exogenous and endogenousd-glucose-6-phosphate dehydrogenase, and concomitantly, the generated electrons are transported via NADP⁺, phenazine methosulphate and menadione to nitro-BT, which is finally precipitated as formazan. Schurin azide and amytal are included to block electron transfei to cytochromes. The method proved to be of value for the qualitative demonstration of pyruvate kinase activity in tissue sections of kidneys, heart muscle and skeletal arusele. For quantitative studies and for investigating the activity of this enzyme in liver sections, the method cannot be recommended.Dedicafed to Professor Dr. T.H. Schicbler on the occasion of his 65th birthday     

Efficient production of 2-deoxyribose 5-phosphate from glucose and acetaldehyde by coupling of the alcoholic fermentation system of Baker's yeast and deoxyriboaldolase-expressing Escherichia coli

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker's yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker's yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Lactose and D0galactose metabolism in Staphylococcus aureus: pathway of D-galactose 6-phosphate degradation

The pathway by which D-galactose 6-phosphate is degraded in Staphylococcus aureus has been elucidated. Galactose 6-phosphate is isomerized to tagatose 6-phosphate, which is phosphorylated with adenosine 5′-triphosphate, and the resulting tagatose 1,6-diphosphate is cleaved to dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The isomerase, kinase, and aldolase that catalyze these reactions are inducible and are distinct from the corresponding enzymes of glucose 6-phosphate metabolism.     

Antibacterial Activity of l-Lysine α-Oxidase against rec+ and rec− Strains of Bacillus subtilis

2-Deoxyribose 5-phosphate production through coupling of the alcoholic fermentation system of baker’s yeast and deoxyriboaldolase-expressing Escherichia coli was investigated. In this process, baker’s yeast generates fructose 1,6-diphosphate from glucose and inorganic phosphate, and then the E. coli convert the fructose 1,6-diphosphate into 2-deoxyribose 5-phosphate via D-glyceraldehyde 3-phosphate. Under the optimized conditions with toluene-treated yeast cells, 356 mM (121 g/l) fructose 1,6-diphosphate was produced from 1,111 mM glucose and 750 mM potassium phosphate buffer (pH 6.4) with a catalytic amount of AMP, and the reaction supernatant containing the fructose 1,6-diphosphate was used directly as substrate for 2-deoxyribose 5-phosphate production with the E. coli cells. With 178 mM enzymatically prepared fructose 1,6-diphosphate and 400 mM acetaldehyde as substrates, 246 mM (52.6 g/l) 2-deoxyribose 5-phosphate was produced. The molar yield of 2-deoxyribose 5-phosphate as to glucose through the total two step reaction was 22.1%. The 2-deoxyribose 5-phosphate produced was converted to 2-deoxyribose with a molar yield of 85% through endogenous or exogenous phosphatase activity.     

Quantitative cytochemical measurement of glyceraldehyde 3-phosphate dehydrogenase activity

Summary A system has been developed for the quantitative measurement of glyceraldehyde 3-phosphate dehydrogenase activity in tissue sections. An obstacle to the histochemical study of this enzyme has been the fact that the substrate, glyceraldehyde 3-phosphate, is very unstable. In the present system a stable compound, fructose 1, 6-diphosphate, is used as the primary substrate and the demonstration of the glyceraldehyde 3-phosphate dehydrogenase activity depends on the conversion of this compound into the specific substrate by the aldolase present in the tissue. The characteristics of the dehydrogenase activity resulting from the addition of fructose 1, 6-diphosphate, resemble closely the known properties of purified glyceraldehyde 3-phosphate dehydrogenase. Use of polyvinyl alcohol in the reaction medium prevents release of enzymes from the sections, as occurs in aqueous media. Although in this study intrinsic aldolase activity was found to be adequate for the rapid conversion of fructose 1, 6-diphosphate into the specific substrate for the dehydrogenase, the use of exogenous aldolase may be of particular advantage in assessing the integrity of the Embden-Meyerhof pathway.     

Quantitative cytochemical measurement of glyceraldehyde 3-phosphate dehydrogenase activity.

A system has been developed for the quantitative measurment of glyceraldehyde 3-phosphate dehydrogenase activity in tissue sections. An obstacle to the histochemical study of this enzyme has been the fact that the substrate, gylceraldehyde 3-phosphate, is very unstable. In the present system a stable compound, fructose 1, 6-diphosphate, is used as the primary substrate and the demonsatration of the glyceraldehyde 3-phosphate dehydrogenase activity depends on the conversion of this compound into the specific substrate by the aldolase present in the tissue. The characteristics of the dehydrogenase activity resulting from the addition of fructose 1, 6-diphosphate, resemble closely the known properties of purified glyceraldehyde 3-phosphate dehydrogenase. Use of polyvinyl alcohol in the reaction medium prevents release of enzymes from the sections, as occurs in aqueous media. Although in this study intrinsic aldolase activity was found to be adequate for the rapid conversion of fructose 1, 6-diphosphate into the specific substrate for the dehydrogenase, the use of exogenous aldolase may be of particular advantage in assessing the intergrity of the Embden-Meyerhof pathway.     

Synthesis of phosphono analogues of dihydroxyacetone phosphate and glyceraldehyde 3-phosphate.

The present paper describes the synthetic routes of six phosphono analogues of dihydroxyacetone phosphate and five phosphono analogues of glyceraldehyde 3-phosphate through alpha-, beta- and gamma-hydroxyphosphonate esters precursors containing a protected carbonyl group. In some situations, depending on the sequence used for the deprotection of the phosphonate and carbonyl groups, the aldol/ketol rearrangement allowed the synthesis of either dihydroxyacetone phosphate or glyceraldehyde 3-phosphate analogues from the same precursors. All these analogues are of interest both as active-site probes and as potential substrates for glycolytic enzymes such as fructose 1,6-diphosphate aldolases (EC 4.1.2.13).     

Photosynthesis by isolated chloroplasts. Reversal of orthophosphate inhibition by Calvin-cycle intermediates

1. The orthophosphate inhibition of photosynthesis by isolated spinach chloroplasts can be reversed by 3-phosphoglycerate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, fructose 6-phosphate and fructose 1,6-diphosphate. 2. Metabolically related compounds such as ribulose 1,5-diphosphate, glucose 6-phosphate, 6-phosphogluconate and phosphoenolpyruvate are ineffective. 3. The kinetics of reversal are characteristic of the intermediate used, but, in each instance, the onset of oxygen evolution is accompanied by a carbon dioxide fixation and except with 3-phosphoglycerate the stoicheiometry is close to unity. 4. The nature of orthophosphate inhibition and its reversal is discussed in relation to metabolic control of photosynthesis.     

Level of photosynthetic intermediates in isolated spinach chloroplasts

The level of intermediates of the photosynthetic carbon cycle was measured in intact spinach chloroplasts in an attempt to determine the cause of the induction lag in CO₂ assimilation. In addition, transient changes in the level of the intermediates were determined as affected by a light-dark period and by the addition of an excess amount of bicarbonate during a period of steady photosynthesis. Assayed enzymically were: ribulose 1,5-diphosphate, pentose monophosphates (mixture of ribose 5-phosphate, ribulose 5-phosphate and xylulose 5-phosphate, hexose monophosphates (mixture of glucose 6-phosphate, glucose 1-phosphate, and fructose 6-phosphate), glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, glycerate acid 3-phosphate, a mixture of fructose 1,6-diphosphate and sedoheptulose 1,7-diphosphate, adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP).     

Purification and properties of rabbit heart muscle aldolase.

Fructose diphosphate aldolase (D-fructose-1,6-biphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) from rabbit heart has been purified and obtained in crystalline form. The preparations are homogeneous on the basis of disc gel electrophoresis and ultracentrifugation. The catalytic and the molecular properties indicate that this is aldolase A. A comparison was made between rabbit heart aldolase and the rabbit muscle enzyme. The sedimentation coefficient, energy of activation and Michaelis constant for Fru-1,6-P2 were found to be identical with the values obtained for the muscle enzyme. As in case of the muscle enzyme, heart aldolase was found to have a broad pH optimum, remarkable stability over a wide pH range, and the ability to form a Schiff base intermediate with dihydroxyacetone phosphate upon reduction with borohydride. Cleavage of the methionyl bonds with CNBr yields the same pattern as obtained with the muscle enzyme.     

Properties of the phosphonomethyl isosteres of two phosphate ester glycolytic intermediates

The enzymic synthesis of the 1-phosphonomethyl isostere of fructose 1,6-diphosphate in which the 1-phosphate (-OPO(3)H(2)) is replaced by the phosphonomethyl group (-CH(2)PO(3)H(2)) is described. The kinetic properties of this fructose diphosphate isostere and of 4-hydroxy-3-oxobutylphosphonic acid, an isostere of dihydroxyacetone phosphate, with aldolase (EC 4.1.2.13), fructose diphosphatase (EC 3.1.3.11) and glycerol phosphate dehydrogenase (EC 1.1.1.8) are described (see Table 1).     

Inhibition of glucose phosphate isomerase by metabolic intermediates of fructose

1. Purified rabbit-muscle and -liver glucose phosphate isomerase, free of contaminating enzyme activities that could interfere with the assay procedures, were tested for inhibition by fructose, fructose 1-phosphate and fructose 1,6-diphosphate. 2. Fructose 1-phosphate and fructose 1,6-diphosphate are both competitive with fructose 6-phosphate in the enzymic reaction, the apparent K_i values being 1·37×10⁻³−1·67×10⁻³m for fructose 1-phosphate and 7·2×10⁻³−7·9×10⁻³m for fructose 1,6-diphosphate; fructose and inorganic phosphate were without effect. 3. The apparent K_m values for both liver and muscle enzymes at pH7·4 and 30° were 1·11×10⁻⁴−1·29×10⁻⁴m for fructose 6-phosphate, determined under the conditions in this paper. 4. In the reverse reaction, fructose, fructose 1-phosphate and fructose 1,6-diphosphate did not significantly inhibit the conversion of glucose 6-phosphate into fructose 6-phosphate. 5. The apparent K_m values for glucose 6-phosphate were in the range 5·6×10⁻⁴−8·5×10⁻⁴m. 6. The competitive inhibition of hepatic glucose phosphate isomerase by fructose 1-phosphate is discussed in relation to the mechanism of fructose-induced hypoglycaemia in hereditary fructose intolerance.     

A model of the pentose phosphate pathway in rat liver cells

A mathematical model based on kinetic data taken from the literature is presented for the pentose phosphate pathway in fasted rat liver steady-state. Since the oxidative and non oxidative pentose phosphate pathway can act independently, the complete (oxidative + non oxidative) and the non oxidative pentose pathway were simulated.Sensitivity analyses are reported which show that the fluxes are mainly regulated by D-glucose-6-phosphate dehydrogenase (for the oxidative pathway) and by transketolase (for the non oxidative pathway). The most influent metabolites were the group ATP, ADP, P₁ and the group NADPH, NADP⁺ (for the non oxidative pathway).Abbreviations GK Glucokinase, (E.C. 2.7.1.2.) - G6PDH D-glucose-6-phosphate dehydrogenase, (E.C. 1.1.1.49) - PLase 6-Phosphogluconelactonase, (E.C. 3.1.1.31.) - PGIcDH 6-Phosphogluconate dehydrogenase, (E.C. 1.1.1.44) - RPI D-ribose-5-phosphate keto-isomerase, (E.C. 5.3.1.6) - TK D-sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycol-aldehyde transferase, (E.C. 2.2.1.1.) - TA D-sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate dihydroxyacetone transferase, (E.C. 2.2.1.2) - EP D-ribulose-5-phosphate-3-epimerase, (E.C. 5.1.3.1) - PGI D-glucose-6-phosphate keto-isomerase, (E.C. 5.3.1.9) - TPI D-glyceraldehyde-3-phosphate keto-isomerase, (E.C.5.3.1.1)     

A simple approach to detect active-site-directed enzyme-enzyme interactions. The aldolase/glycerol-phosphate-dehydrogenase enzyme system

A novel approach has been elaborated to identify the mechanism of intermediate transfer in interacting enzyme systems. The aldolase/glycerol-3-phosphate-dehydrogenase enzyme system was investigated since complex formation between these two enzymes had been demonstrated. The kinetics of dihydroxyacetone phosphate conversion catalyzed by the dehydrogenase in the absence and presence of aldolase was analyzed. It was found that the second-order rate constant (kcat/Km) of the enzymatic reaction decreases due to the formation of a heterologous complex. The decrease could be attributed to an increase of the Km value since kcat did not change in the presence of aldolase. In contrast, an apparent increase in the second-order rate constant of dihydroxyacetone phosphate conversion by the dehydrogenase was observed if the triose phosphate was produced by aldolase from fructose 1,6-bisphosphate (consecutive reaction). Moreover, no effect of dihydroxyacetone phosphate on the dissociation constant of the heterologous enzyme complex could be detected by physico-chemical methods. The results suggest that the endogenous dihydroxyacetone phosphate produced by aldolase complexed with dehydrogenase is more accessible for the dehydrogenase than the exogenous one, the binding of which is impeded due to steric hindrance by bound aldolase.     

Light modulation of the activity of carbon metabolism enzymes in the crassulacean Acid metabolism plant kalanchoë

When intact Kalanchoë plants are illuminated NADP-linked malic dehydrogenase and three enzymes of the reductive pentose phosphate pathway, ribulose-5-phosphate kinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and sedoheptulose-1,7-diphosphate phosphatase, are activated. In crude extracts these enzymes are activated by dithiothreitol treatment. Light or dithiothreitol treatment does not inactivate the oxidative pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase. Likewise, neither light, in vivo, nor dithiothreitol, in vitro, affects fructose-1,6-diphosphate phosphatase. Apparently the potential for modulation of enzyme activity by the reductively activated light effect mediator system exists in Crassulacean acid metabolism plants, but some enzymes which are light-dark-modulated in the pea plant are not in Kalanchoë.     

Detection of precursors of quinolinic acid in Escherichia coli.

A technique is described which allows the detection of precursors of quinolinic acid produced by Escherichia coli, independent of a bioassay. This is based on a double autoradiogram utilizing radioactive aspartic acid and fructose-1,6-diphosphate (as a source of dihydroxyacetone phosphate). Six radioactive spots are derived from aspartic acid and four are derived from fructose-1,6-diphosphate. The latter four spots correspond to four of the former spots in migration in each of two solvent systems. The significance of these data relative to genetic and enzymatic data is discussed.     

Hepatic fructose-metabolizing enzymes and related metabolites: role of dietary copper and gender

The purpose of this study was to further examine the hypothesis that variations in hepatic fructose-metabolizing enzymes between males and females might account for the differences in the severity of copper (Cu) deficiency observed in fructose-fed male rats. Weanling rats of both sexes were fed high-fructose diets either adequate or deficient in copper for 45 days. Cu deficiency decreased sorbitol dehydrogenase activity and dihydroxyacetone phosphate levels and increased glyceraldehyde levels in both sexes. Gender effects were expressed by higher activities of glycerol 3-phosphate dehydrogenase and aldehyde dehydrogenase in male than in female rats and higher levels of dihydroxyacetone phosphate and fructose 1,6-diphosphate (F1,6DP) in female than in male rats. The interactions between dietary Cu and gender were as follows: alcohol dehydrogenase activities were higher in female rats and were further increased by Cu deficiency in both sexes; aldehyde dehydrogenase activities were decreased by Cu deficiency only in male rats; sorbitol levels were higher in male rats and were further increased by Cu deficiency in male rats; fructose 1-phosphate (F1P) levels were increased by Cu deficiency in both sexes, but to a greater extent in male rats; glyceraldehyde 3-phosphate levels were higher in female rats, but were decreased by Cu deficiency in female and increased in male rats. Though most of the examined hepatic fructose-metabolizing enzymes and metabolites showed great differences between rats fed diets either adequate or deficient in Cu, it is the activity of fructokinase and aldolase-B, and the concentrations of their common metabolites, F1P and notably F1,6DP, that could be in part responsible for differences in the severity of pathologies associated with Cu deficiency observed between female and male rats.