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Muscle expression of G6PD deficiency has been investigated in Mediterranean, Seattle-like and A-variants. G6PD activity was detected in samples obtained from biopsies on the quadriceps muscle of seven males and one female. The type of genetic variant was determined by molecular analysis of DNA, extracted from blood samples. All variants showed the enzyme defect in muscle. A statistically significant relationship was found in the activity of G6PD between erythrocytes and muscle of the male subjects (r = 0.968; p = 0.00008). The equation for the best fit line was: Y = 0.390X + 0.198. The results suggest that, for a given variant, the extent of the enzyme defect in muscle may be determined, using this equation, from the G6PD activity of erythrocytes.

Blood levels of acetaldehyde (ACh), ethanol and acetone were investigated in mice treated with ethanol for 6 months and receiving compatible erythrocytes (RBCs) overloaded with aldehyde dehydrogenase (AlDH). Following an acute dose of ethanol, ACh levels were significantly lower in these animals than in alcohol-treated mice receiving AlDH-unloaded RBCs, and were similar to the ACh levels of normal mice. The peak ethanol concentration was higher in normal mice than in both groups of alcohol-treated animals, while acetone concentrations were not significantly different in the three groups of animals.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency was found in erythrocytes, lymphocytes and muscle of an Italian male, whose family has lived for at least three generations in Lodi (Lombardy, northern Italy). The subject was hospitalized for myalgia and dark urine after intense physical exercise, but no sign of anemia and chronic hemolysis were present at rest. Family studies revealed that the mother and the maternal aunt had the same enzymopathy. The enzyme-specific activity in red blood cells was 15% of control and the kinetic properties were the following: slower electrophoretic mobility; biphasic pH activity curve; slightly reduced thermal stability, and increased utilization of the substrate analogs. The analysis of our patient's DNA showed a G----C mutation at nucleotide 844 which causes an Asp----His amino acid change in position 282. This is the same mutation found by De Vita et al. in the G6PD Seattle-like variant. However, by following a new convention, we labelled our variant as G6PD Lodi844C. As far as the muscle is concerned, we found that the enzyme-specific activity in this tissue was 14% of control values, but cultured myotubes and myoblasts revealed a normal level of G6PD as well as skin fibroblasts. On the contrary in the same type of cultured cells obtained from G6PD Mediterranean subjects, the G6PD activity was about 20% of normal. Our results complete the characterization of this mutant enzyme, demonstrate the expression of the deficit in muscle and describe the enzyme behaviour in cultured cells.

A procedure for rapid purification to homogeneity of glucose-6-phosphate dehydrogenase (G6PD) is herein presented. Our method is not new, but represents a simplification of the method of De Flora et al. (Arch. Biochem. Biophys. 169, 362-3, 1975) which consisted of three steps: DEAE-Sephadex, phosphocellulose (P11) and affinity chromatography on 2'5' ADP-Sepharose. These authors eluted the enzyme from the P11 with phosphate and from 2'5' ADP-Sepharose with KC1 and NADP. By our method, the DEAE-Sephadex step is omitted, the G6PD is eluted from P11 with citrate and NADP, and from 2'5' ADP-Sepharose with KC1, NADP and EDTA. The elution of the enzyme from the phosphocellulose was studied in detail and the temperature effect has been described. We report here an application of this method to a rapid microscale purification starting from 3.5-4 ml of rabbit blood, which can be performed in about 8 hours and a macroscale purification starting from 180-200 ml of human blood, which takes a day and a half.

Human erythrocytes were loaded with homogeneous acetaldehyde dehydrogenase (AcDH) purified from Alcaligenes Eutrophus (an enzyme species with an apparent Km for acetaldehyde similar to the mitochondrial enzyme), using an encapsulation procedure based on hypotonic haemolysis, isotonic resealing and reannealing. The AcDH-overloaded erythrocytes contained 1.55 +/- 0.25 I.U. of AcDH activity per ml of packed erythrocytes, a value 12-15 times higher than that of corresponding unloaded or native red cells. The AcDH-loaded erythrocytes were found to metabolize 4 +/- 0.8 mumol of acetaldehyde/hr/ml of red blood cells, whereas the glycolytic activity was almost unmodified. Estimates of intracellular adenine nucleotides showed 50% ATP decay in the AcDH-loaded cells when incubated in the presence of acetaldehyde concentrations higher than 50 microM, whereas the [NAD+]/[NADH] ratio was strongly decreased but to the same extent as in control cells, suggesting that this was due to the acetaldehyde itself and not to the presence of encapsulated AcDH. Similar results were obtained using mouse erythrocytes. AcDH-overloaded mouse red blood cells from donor animals were also injected intraperitoneally into compatible recipients (Balb/C) and 80 to 85% of these were found to enter into circulation within 24 hr and to circulate with a half-life of 6-7.3 days (normal half-life 11 days). Following an acute dose of ethanol (2g/kg intraperitoneally), blood levels of acetaldehyde were significantly lower in mice receiving the AcDH-loaded erythrocytes than in controls. Blood levels of ethanol were also lower in the treated mice compared to controls. These results show that AcDH-overloaded erythrocytes can perform in vitro and in vivo as bioreactors improving alcohol and acetaldehyde metabolism, and suggest that administration of these cells to alcoholic patients could be of value in restoring to normal, or improving, alcohol and acetaldehyde metabolism.

Reduced glutathione at 1 mM concentration is able to mantain rabbit red blood cell hexokinase (EC 2.7.1.1) in the reduced state with fully catalytic activity. At higher concentrations a marked inhibition is observed. In contrast, oxidized glutathione is a strong inhibitor of reduced erythrocyte hexokinase at all the concentration studied. Inactivation experiments show that some sulfhydryl groups reacting with oxidized glutathione are responsible for the enzyme inactivations. These findings suggest a cellular inter-relationship between redox and energetic metabolism coupled through glutathione at the hexokinase level.

In human erythrocytes, in the absence of specific enzymes, G1,6P2 synthesis and degradation are carried out by phosphoglucomutase PGM2 isoenzymes. The results presented, obtained by using partially purified preparations of these enzyme forms, suggest that erythrocyte G1,6P2 may play a crucial role in the physiological interconversion of several important sugar monophosphates.

In intact erythrocytes from normal adults, acetaldehyde, besides inducing metabolite modifications otherwise observed, markedly decreases the glucose 1,6-bisphosphate (G1,6P2) level. Pyruvate rapidly reverses the acetaldehyde effects. Also in vivo, the acetaldehyde that occurs in the blood stream after heavy alcohol intake produces a significant decrease of the erythrocyte G1,6P2 concentration. These changes support the role of 1,3-bisphosphoglycerate as the first substrate in the G1,6P2 synthesis. The significance of the glucose bisphosphate as glycolytic modulator is also discussed.

Vanadate causes a rapid breakdown of 2,3-bisphosphoglycerate in intact erythrocytes. This metabolite is nearly stoichiometrically transformed into pyruvate, which changes the cell redox state and enhances the glycolytic flux. The results show that the vanadate effect on 2,3-bisphosphoglycerate, also evident in hemolysates, is attributable to the stimulation of a phosphatase activity of the phosphoglycerate mutase. In agreement with others (J. Carreras, F. Climent, R. Bartrons, and G. Pons (1982) Biochim. Biophys. Acta 705, 238-242), vanadate is thought to destabilize the phosphoryl form of this enzyme which shows competitive inhibition between the ion and 2,3-bisphosphoglycerate in the mutase reaction. A competitive inhibition between vanadate and glucose 1,6-bisphosphate is also found for phosphoglucomutase, without evidence for phosphatase activity toward the bisphosphate cofactor.

Human erythrocytes incubated with various sugars lower their glucose 1,6-bisphosphate (Glc-1,6-P2) content, as do haemolysates containing exogenous Glc-1,6-P2 incubated with sugar monophosphates (sugar-P). Experiments performed with isolated erythrocyte phosphoglucomutase (PGM) isoenzymes indicate that only definite isoenzymatic forms, namely PGM2, are able to consume Glc-1,6-P2 during the mutation of sugar-P other than glucose-P. In this process a phosphate group is released from Glc-1,6-P2 and can be partially recovered in the biphosphate of the mutated sugar-P. The relevance of this mechanism of Glc-1,6-P2 degradation is discussed in regard to the physiological turnover of the biphosphate.

The influence on red cell metabolism of increasing the glucose concentration or the pH of the medium has been examined in order to compare the modification of the lactate/pyruvate ratio inside and outside the cell. In both situations, we evidenced a constancy in the intracellular lactate/pyruvate ratio and an increase in the extracellular one, thus suggesting that the cell efficiently opposes cytoplasmic modifications. In fact an increase of lactate efflux takes place when the intracellular pyruvate decreases. The implications of these changes in the two compartments are discussed on the basis of the available results.

Kinetic properties of PGM1 and PGM2 phosphoglucomutase "primary" isoenzymes from human erythrocytes were studied. The two enzyme forms share a "ping-pong" kinetic mechanism and show similar Km for substrate (glucose 1-P) and cofactor (glucose 1,6-P2). Micromolar concentrations of fructose 1,6-P2 and glycerate 2,3-P2 inhibit both PGM1 and PGM2 isoenzymes to a similar extent. The sole PGM2 form is affected by ribose monophosphates (ribose 1-P and ribose 5-P) that act as mutase inhibitors vs. glucose 1,6-P2 and as apparent activators vs. glucose 1-P. The interaction between PGM2 isoenzyme and ribose monophosphates is discussed in the light of the ability of this form to also display phosphoribomutase activity.

Rabbit red blood cell hexokinase (EC 2.7.1.1.) has been purified 300,000-fold by a combination of ion exchange chromatography, affinity chromatography, and preparative polyacrylamide gel electrophoresis. The hexokinase activity has been isolated in 35% yield as a protein that is homogeneous by polyacrylamide and sodium dodecyl sulfate gel electrophoresis. The highest specific activity obtained was 145 units/mg of proteins. The native protein has a molecular weight of 110,000 by gel filtration on Ultrogel AcA 44 and 112,000 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 110,000 indicating that hexokinase is a monomer. The enzyme had a pI of 6.20 to 6.30 pH units by isoelectric focusing. The enzyme was specific for Mg . ATP and Mg . ITP as the nucleotide substrates. Several hexokinase with different affinities.

Glucose 1,6-biphosphate (G1,6P2) was measured in human, pig, cow, rabbit, rat and sheep red blood cells. Mean values are variable among the species and range from 33 to 122 nmol/ml RBC for pig and rabbit erythrocytes, respectively. The activities of G1,6P2 synthase, phosphoglucomutase (PGM) and phosphoribomutase (PRM) have also been assayed in red cell haemolysates of the same species. The correlations between the biphosphate content and the occurrence of the three enzymatic activities have been studied in order to gain an insight into the regulation of the G1,6P2 turnover in mammalian erythrocytes.

The metabolic effect of a lactate or pyruvate load and methylene blue (Mb) on hexose monophosphate shunt (HMPS) and Embden-Meyerhof pathway (EMP) were investigated in intact erythrocytes in the presence of glucose. Pyruvate enhances glucose consumption and 14CO2 production, lactate reduces 14CO2 production but glucose consumption is unchanged. These effects are dependent on the lactate and pyruvate concentrations in the buffer. A pyruvate load does not results to influence the net production of pyruvate, which is conversely markedly increased by Mb. The lactate load raises slightly the total pyruvate level but its rate of production is unchanged, with respect to that of the control. A considerable effect on the intracellular to extracellular pyruvate ratio is observed in these different conditions, as well as modifications in the levels of inorganic phosphate (Pi).

This paper reports the effect of acetaldehyde on erythrocytes (RBC) of human adults and newborns. Acetaldehyde increases glucose consumption in adult RBC, but has no effect on that of newborn RBC. The compound stimulates the hexose monophosphate shunt and decreases the pyruvate production of the two RBC suspensions. In newborn RBC, acetaldehyde slightly modifies triose-P and fructose-1,6-bisphosphate but has no effect on the ATP/ADP ratio and glucose-1,6-bisphosphate content, which change markedly in adult RBC. Analysis of aldehyde dehydrogenase reveals nearly one half of enzyme activity in newborn RBC. The data indicate that in both adult and newborn RBC, acetaldehyde causes an intracellular reduced state, but the newborn cells take advantage of their greater pyruvate production for complete removal of the exceeding NADH equivalents.

In this study human erythrocytes have been incubated with various effectors most of which able to decrease the G1, 6P2 content. By using haemolysates and partially purified phosphoglucomutase isoenzymes we provide evidence that the G1, 6P2 decrease can be attributed to the ability of phosphoglucomutase PGM2 isoenzymes to mutate various sugar monophosphates. These isoenzymes, phosphorylated by G1, 6P2, may transfer the phosphate group to monophosphate sugars thus releasing the respective bisphosphate.