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Biomedical subjects

V Stocchi

Publications and source records attributed to V Stocchi.

At least 109 records · Page 6Linked to original sources
PubMed1985

Regulatory properties of human erythrocyte hexokinase during cell ageing.

Human red blood cell hexokinase exists in multiple molecular forms with different isoelectric points but similar kinetic and regulatory properties. All three major isoenzymes (HK Ia, Ib, and Ic) are inhibited competitively with respect to Mg.ATP by glucose 6-phosphate (Ki = 15 microM), glucose 1,6-diphosphate (Ki - 22 microM), 2,3-diphosphoglycerate (Ki = 4 mM), ATP (Ki = 1.5 mM), and reduced glutathione (Ki = 3 mM). All these compounds are present in the human erythrocyte at concentrations able to modify the hexokinase reaction velocity. However, the oxygenation state of hemoglobin significantly modifies their free concentrations and the formation of the Mg complexes. The calculated rate of glucose phosphorylation, in the presence of the mentioned compounds, is practically identical to the measured rate of glucose utilization by intact erythrocytes (1.43 +/- 0.15 mumol h-1 ml red blood cells-1). Hexokinase in young red blood cells is fivefold higher when compared with the old ones, but the concentration of many inhibitors of the enzyme is also cell age-dependent. Glucose 6-phosphate, glucose 1,6-diphosphate, 2,3-diphosphoglycerate, ATP, and Mg all decay during cell ageing but at different rates. The free concentrations and the hemoglobin and Mg complexes of both ATP and 2,3-diphosphoglycerate with hemoglobin in the oxy and deoxy forms have been calculated. This information was utilized in the calculation of glucose phosphorylation rate during cell ageing. The results obtained agree with the measured glycolytic rates and suggest that the decay of hexokinase during cell ageing could play a critical role in the process of cell senescence and destruction.

Adenosine Triphosphate↗
PubMed1985

Hexokinase in human chorionic villi.

The level, intracellular distribution, and isozymic pattern of hexokinase (EC 2.7.1.1) were determined on human chorionic villi obtained by trophoblast biopsy in the first trimester of pregnancy. About 50% of total hexokinase activity was found to be particle-bound and 96% of this in the overt form (i.e. assayable without the addition of detergents). Both soluble and particulate hexokinase show the same affinities for glucose but differ in the affinity for MgATP2- and in their sensitivity to glucose 1,6-diphosphate inhibition. By chromatographic and kinetic methods the soluble hexokinase was found to be represented by isozymes I, II and traces of hexokinase III while most of the bound enzyme was hexokinase type I. These results provide evidence for the expression of at least three hexokinase isozymes in early human development and can partially explain the high rate of glucose utilization of the placenta.

Adenosine Triphosphate↗
PubMed1985

Human erythrocyte hexokinase deficiency: a new variant with abnormal kinetic properties.

A 14-month-old child who had a haemolytic episode when he was 5 years old, and with psychomotor retardation, was found to have decreased red cell hexokinase activity. The mutant enzyme was characterized by an increased affinity for glucose associated with an increased inhibition constant for glucose-1,6-diphosphate. Affinity for Mg ATP2-, heat stability and pH-optimum were normal. The isozymic pattern of the red cell enzyme was normal but all the molecular forms were present in reduced amounts. The kinetics of decay of hexokinase during cell ageing was also normal. Glucose consumption of the hexokinase deficient cells was 60-65% of the controls while the amount metabolized through the hexose monophosphate shunt was unchanged. Red cell 2,3-diphosphoglycerate and glucose-6-phosphate levels were normal in the proband but reduced in the erythrocytes of his parents, who were heterozygous for the defect but had normal haematological data. Comparison with the 13 previously reported cases of hexokinase deficiency confirms the broad phenotypic variability that characterizes this disorder.

Blood Glucose↗
PubMed1985

Hereditary nonspherocytic hemolytic anemia due to a new hexokinase variant with reduced stability.

A 27-year-old woman with severe chronic hemolytic anemia was found to have reduced red cell hexokinase activity when the degree of reticulocytosis was considered. This enzyme had normal pH-dependent activity, normal Km for glucose, fructose, and mannose, normal Km for Mg adenosine triphosphate (ATP)2- and Ki for glucose-1,6-diphosphate. Furthermore, the pH-dependence and orthophosphate dependence of Ki for glucose-1,6-diphosphate were normal. However, this hexokinase was inactivated rapidly at 44 degrees C. No abnormalities were found in the red cell hexokinase isozymic pattern when it was compared with the profile obtained from cells of similar age. The hexokinase specific activity was reduced in all the red blood cell fractions obtained by density gradient ultracentrifugation; a marked difference in the distribution of cells through the gradient was evident. Among the glycolytic intermediates, a significant decrease of 2,3-diphosphoglycerate was evident. ATP and glucose 6-phosphate were also reduced when compared with cells of similar. Glucose consumption of the hexokinase-deficient cells decreased, but the rate of glucose metabolized through the hexose monophosphate shunt was unchanged. Although the total hexokinase activity in lymphocytes was only reduced by 37%, a marked hexokinase deficiency was detected in blood platelets (20% to 25% of normal activity). The parents and one of two siblings of the patient were heterozygous for the defect, with 66% to 74% of normal erythrocyte hexokinase activity and reduced heat stability of the enzyme. These results, when compared with those obtained in previously reported cases of hexokinase deficiency, provide further evidence of the broad phenotypic variability that characterizes this disorder. Furthermore, it is suggested that failure of energy generation is probably the primary cause of hemolytic anemia in hexokinase deficiency.

Adult↗
PubMed1984

Relationship between the rate of erythrocyte hexose monophosphate pathway and the glucose 6-phosphate concentration.

Erythrocytes of individuals with increased (+ 50%) or reduced (-35%) hexokinase activity contain respectively 70 and 17 nmole/ml RBC of glucose-6-phosphate (normal concentration 30 +/- 5nmole/ml RBC) and show comparable rates of the HMP (60 +/- 5nmole/hr/ml RBC). Similarly, in RBC of different ages, obtained by density gradient ultracentrifugation, the glucose-6-phosphate concentration range from 57 (young cells) to 18 (old cells) nmole/ml RBC but the rate at which glucose is utilized in the HMP is unchanged. These data exclude a regulatory role of glucose 6-phosphate in the HMP even if its concentration is under that required for maximal G6PD activity.

Chromosomes, Human, 6-12 and X↗
PubMed1984

Hexokinase in developing rabbit erythroid cells.

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits separated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Most of the falls in hexokinase activity take place when the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.

Anemia↗
PubMed1984

[Micromethods for the study of hexokinase in human erythrocytes].

A direct radioassay for the erythrocyte enzyme using U14C-glucose as substrate has been developed. With respect to the indirect spectrophotometric assay this method allows for the determination of true hexokinase activity. The assay proposed is sensitive, rapid and well suited for the determination of hexokinase activity in the erythrocyte lysate where the enzyme level is particularly low.

Erythrocytes↗
PubMed1984

[Determination of the energy load of erythrocytes in long-distance and medium-distance runners].

In this study erythrocytes drawn from well-trained athletes (middle- and long-distance runners) and from sedentary subjects have been compared for their adenine nucleotide contents. ADP and AMP appeared to be significantly (p less than 0,001) increased only in red cells from athletes in the rest state. After athletes' race this difference with control subjects become insignificant. Nevertheless, the observed ADP and AMP modifications are not great enough to influence the energy charge (CE) of the compared erythrocytes.

Adenine Nucleotides↗
PubMed1984

Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological.

The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37 degrees C (pH 7.2) was 10.23 +/- 1.90 mumol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg X ATP2-. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 microM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP4- and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94 +/- 0.02 mumol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92 +/- 0.3 mumol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.

2,3-Diphosphoglycerate↗
PubMed1984

Rabbit red blood cell hexokinase:intracellular distribution during reticulocytes maturation.

The intracellular localization and isozyme distribution of hexokinase were studied during rabbit reticulocyte maturation and aging. In reticulocytes 50% of the enzyme was particulate while in the mature erythrocytes all the hexokinase activity was soluble. The bound enzyme co-sediments with mitochondria and by column chromatography it was found to be hexokinase Ia. The cytosol of reticulocytes contains hexokinase Ia (38%) and hexokinase Ib (62%) while the mature erythrocytes contain only hexokinase Ia. The amount of bound hexokinase decreases very quickly during cell maturation and aging as was shown by following in vivo reticulocyte maturation or by analysis of hexokinase compartmentation in cells of different ages, obtained by density gradient ultracentrifugations. A role for this intracellular distribution of hexokinase is suggested.

Animals↗
PubMed1984

Rabbit red blood cell hexokinase. Evidences for an ATP-dependent decay during cell maturation.

Rabbit hexokinase (EC 2.7.1.1) has been shown to exist in reticulocytes as two distinct molecular forms, designated hexokinase Ia and Ib, but only one of these was consistently present in mature red cells. In vivo, hexokinase Ia and Ib show a decay rate of 3 and 8% a day, respectively, while in vitro they show a similar stability. The possibility that the proteolytic activities of the reticulocyte could be responsible for the fast decay of hexokinase was investigated. No differences were found in the decay rates of hexokinase Ia and Ib during in vitro reticulocyte maturation in presence or absence of proteolytic inhibitors. Contrariwise, many findings indicate the ATP-dependent proteolytic system of the reticulocyte as a possible mechanism. In fact, the decay of hexokinase and the degradation of 3H-globins are both stimulated by ATP and ubiquitin; they show similar kinetic properties and both disappear during reticulocyte maturation. The cellular localization of hexokinase Ia and Ib was shown to be responsible for the differences found between their decay rates.

Adenosine Triphosphate↗
PubMed1984

Red blood cell hexokinase in Fanconi's anemia.

The activity of ten red blood cell enzymes, including hexokinase, has been measured in 6 Fanconi's anemia patients. In disagreement with previous reports, in no instance were reduced or increased hexokinase levels found. Furthermore, the hexokinase isozymic pattern, thermostability, pH dependence of activity and kinetic properties were also in the normal range.

Adolescent↗
PubMed1983

Pig red blood cell hexokinase: evidence for the presence of hexokinase types II and III, and their purification and characterization.

Pig erythrocytes, in contrast to red blood cells from other mammals (M. Magnani, V. Stocchi, F. Canestrari, M. Dachà, and G. Fornaini (1982) Biochem. Int. 4, 673), have been shown to contain hexokinase (EC 2.7.1.1) types II and III. Hexokinase type III is the predominant form, accounts for 98% of the total glucose phosphorylating activity, and has been purified 290,000-fold by a combination of ion-exchange chromatography and affinity chromatography on Sepharose-N-hexanoylglucosamine. The enzyme was shown to be homogeneous by polyacrylamide and sodium dodecyl sulfate-gel electrophoresis. The highest specific activity obtained was 190 units/mg protein with a yield of 60%. Because the amount of hexokinase II was small, it was only partially purified by ion-exchange chromatography. The native proteins have the same molecular weight of 100,000 by gel filtration on Ultrogel AcA44. The apparent isoelectric point of hexokinase type II was shown to be 4.8 and 4.9 pH units, whereas hexokinase type III was shown to have a pI of 4.3 to 4.4 pH units by isoelectric focusing. Both hexokinases are able to phosphorylate several hexoses. However, while hexokinase II shows an apparent Km for glucose of 1.5 X 10(-4) M with negative cooperativity (nH = 0.4), hexokinase III shows an apparent Km for glucose of 1.5 X 10(-5) M and a positive cooperative effect (nH = 1.5). Furthermore, glucose at concentrations higher than 0.4 mM becomes an inhibitor of hexokinase III. Amino acid analysis of hexokinase type III revealed a low number of the aromatic residues Phe, Tyr, and Trp; this is in agreement with the low extinction coefficient of E1%280nm = 12.5.

Amino Acids↗
PubMed1983

Pig red blood cell hexokinase: regulatory characteristics and possible physiological role.

The regulatory properties of pig erythrocyte hexokinase III have been studied. Among mammalian erythrocyte hexokinases, the pig enzyme shows the highest affinity for glucose and a positive cooperative effect with nH = 1.5 at all the MgATP concentrations studied (for 0.5 to 5 mM). Glucose at high concentrations is also an inhibitor of hexokinase III. Similarly, the apparent affinity constant for MgATP is independent of glucose concentration. Uncomplexed ATP and Mg are both competitive inhibitors with respect to MgATP. Glucose 6-phosphate, known as a stronger inhibitor of all mammalian erythrocyte hexokinases, is a poor inhibitor for the pig enzyme (Ki = 120 microM). Furthermore, this inhibition is not relieved by orthophosphate as with other mammalian red blood cell hexokinases. A variety of red blood cell-phosphorylated compounds were tested and found to be inhibitors of pig hexokinase III. Of these, glucose 1,6-diphosphate and 2,3-diphosphoglycerate displayed inhibition constants in the range of their intracellular concentrations. In an attempt to investigate the role of hexokinase type III in pig erythrocytes some metabolic properties of this cell have been studied. The adult pig erythrocyte is able to utilize 0.27 mumol of glucose/h/ml red blood cells (RBC) compared with values of 0.56-2.85 mumol/h/ml RBC for the other mammalian species. This reduced capacity to metabolize glucose results from a relatively poor ability of the cell membrane to transport glucose. In fact, all the glycolytic enzymes were present and a low intracellular glucose concentration was measured (0.5 mM against a plasma level of 5 mM). Furthermore, transport and utilization were concentration-dependent processes. Inosine, proposed as the major energy substrate of the pig erythrocyte, at physiological concentrations is not as efficient as glucose in maintaining reduced glutathione levels under oxidative stress. Furthermore, newborn pig erythrocytes (fully permeable to glucose) possess hexokinase type II as the predominant glucose-phosphorylating activity. This fact and the information derived from the study of the regulatory characteristics of hexokinase III and from metabolic studies on intact pig erythrocytes permit the hypothesis that the presence of this peculiar hexokinase isozyme (type III) enables the adult pig erythrocyte to metabolize low but appreciable amounts of glucose.

Adenosine Triphosphate↗
PubMed1983

Red blood cell hexokinase in tumor bearing mice.

Red blood cell hexokinase of tumor-bearing BALB/c mice was found to be 35% higher than in the normal controls, whereas glucose 6-phosphate-dehydrogenase and other red blood cell glycolytic enzymes were in the normal range. This hexokinase increase cannot be explained by a mean younger red cell population because normal hematological data and normal red cell enzymes, known as red cell age-markers, have been found in tumor-bearing mice. The isozymic pattern of red cell hexokinase is not modified in the tumor-bearing mice.

Animals↗
PubMed1983

Relationship between erythrocyte hexokinase in cancer patients and red cell age.

A study of hexokinase isozymic pattern and age dependence of the enzyme was carried out on erythrocytes of 4 patients with adenocarcinoma of the gastrointestinal tract. On the basis of our results, we think that the increase in hexokinase activity previously reported and the increase in both subtypes (Ia and Ib) of the enzyme herein described are not due to a mean younger red cell population caused by secondary anemia, which is frequently present in cancer patients.

Adenocarcinoma↗