Biomedical subjects

G Fornaini

Publications and source records attributed to G Fornaini.

At least 19 recordsLinked to original sources

PubMed1988

Improved metabolic properties of hexokinase-overloaded human erythrocytes.

Human erythrocytes were loaded with homogeneous hexokinase purified from human placenta (an enzyme species apparently identical to the erythrocyte enzyme), using a procedure of encapsulation based on hypotonic hemolysis, isotonic resealing and reannealing. The hexokinase-overloaded erythrocytes contained 4.77 +/- 0.75 IU of hexokinase activity per ml of packed erythrocytes, a value 15-times higher than that of corresponding unloaded or native red cells. The hexokinase-loaded erythrocytes were found to metabolize twice the amount of glucose consumed by the unloaded cells through a nearly doubled glycolytic activity, while the activity of the hexose monophosphate shunt pathway was unmodified. Estimates of glycolytic intermediates showed increased levels of most metabolites with respect to the unloaded erythrocytes, while the intracellular concentrations of adenine nucleotides and 2,3-bisphosphoglycerate were unaffected by entrapment of hexokinase. The new steady-state condition characterized by improved glycolytic function was demonstrated to be directly related to enhanced levels of hexokinase activity and not to the use of a rejuvenation solution during the procedure of entrapment. These results are consistent with suggestions by several investigators that glucose metabolism in human erythrocytes is regulated by hexokinase, and they open new perspectives for manipulating erythrocytes with the ultimate aim of improving their survival under different storage conditions.

Adenine Nucleotides↗

PubMed1988

Effect of phenylhydrazine on red blood cell metabolism.

In addition to the well known effect of phenylhydrazine on red blood cells (methaemoglobin and Heinz body formation, autologous IgG binding, lipid peroxidation, etc.) an increased glucose utilization was observed. Measurement of 14CO2 formation from [1-14C]-glucose showed a maximum value at 2mM phenylhydrazine followed by a progressive inhibition on increasing the drug concentration to 16 mM. Concomitantly we found a reduction in the reduced glutathione concentration but not a corresponding increase in the level of oxidized glutathione. Phenylhydrazine also causes ATP depletion. The ATP is in part dephosphorylated to ADP and AMP and in part converted to inosine monophosphate and hypoxanthine. Measurement of the cell content of reduced and oxidized pyridine nucleotides was also performed and showed a progressive increase in the reduced forms of these coenzymes. Thus phenylhydrazine promotes cellular ATP depletion followed by adenine nucleotide catabolism that is not efficiently counteracted by an increase in glucose utilization. The relevance of these data to the mechanism of phenylhydrazine-induced anemia is discussed.

Adenine Nucleotides↗

PubMed1988

Hexokinase microheterogeneity in rabbit red blood cells and its behaviour during reticulocytes maturation.

Hexokinase in rabbit reticulocytes is present in two molecular forms (hexokinase Ia and Ib) separable by ion-exchange chromatography on DE-52 columns. By the use of ion-exchange HPLC we have been able to show that the isozymic form we previously called hexokinase Ia can be resolved into two peaks of activity one of which is (Ia) soluble, the other (Ia*) particulate. Hexokinase Ia* can be solubilized by detergents like saponine and Triton X-100 and disappears during 'in vivo' reticulocytes maturation. This new hexokinase microheterogeneity is not caused by different oxidized forms of the enzyme nor influenced by the presence of proteolytic inhibitors during lysate preparation.

Animals↗

PubMed1988

Purification, properties, and evidence for two subtypes of human placenta hexokinase type I.

In human placenta 85% of total hexokinase activity (EC 2.7.1.1) was found in a soluble form. Of this, 70% is hexokinase type I while the remaining 30% is hexokinase type II. All the bound hexokinase is type I. Soluble hexokinase I was purified 11,000-fold by a combination of ion-exchange chromatography, affinity chromatography, and dye-ligand chromatography. The specific activity was 190 units/mg protein with a 75% yield. The enzyme shows only one band in nondenaturing polyacrylamide gel electrophoresis that stains for protein and enzymatic activity; however, two components (with Mr 112,000 and 103,000) were constantly seen in sodium dodecyl sulfate-gel electrophoresis. Many attempts were made to separate these two proteins under native conditions; however, only one peak of activity was obtained when the enzyme was submitted to gel filtration (Mr 118,000), preparative isoelectric focusing (pI 5.9), anion-exchange chromatography, hydroxylapatite chromatography, and affinity chromatography on immobilized dyes and immobilized glucosamine. The high and low molecular weight hexokinases show the same isoelectric point under denaturing conditions as determined by two-dimensional gel electrophoresis. Each hexokinase subtype was obtained by preparative sodium dodecyl sulfate electrophoresis followed by electroelution. Monospecific antibodies raised in rabbits against electroeluted high and low molecular weight hexokinases were not able to recognize the native enzymes but each of them detected both hexokinases on immunoblots. Amino acid compositions and peptide mapping by limited proteolysis of the high and low molecular weight hexokinases were also performed and suggested a strong homology between these two subtypes of human hexokinase I.

Centrifugation, Density Gradient↗

PubMed1988

Effect of age on some properties of mice erythrocytes.

The hematological parameters of young (2-month-old) and old (2-year-old) mice were compared. No differences could be detected with the exception of an increased percentage of reticulocytes in the old animals suggesting that anemia in senescent mice does not occur. Red blood cell mean half-life in old mice was 8 +/- 0.8 days compared to 12 +/- 1 days in young mice. This reduced survival of red blood cell is not due to a different rate of cell phagocytosis in the reticulohistiocytic system of young and old animals since erythrocytes from young mice have the same mean half-life when injected both in young and old animals and vice versa. Thus, the old mice have a reduced red cell life-span but the same hematocrit of the young, suggesting that old animals possess a chronologically younger population of erythrocytes than do young animals. This has been confirmed by measuring the specific activities of some red blood cell age-dependent enzymes (hexokinase, glucose-6-phosphate dehydrogenase, pyruvate kinase) that were found to be higher in the older animals, and by the separation of erythrocytes into different density (age) groups by Percoll/albumin density gradient centrifugation. However, the erythrocytes osmotic fragility, and the cellular contents of adenine and pyridine nucleotides, as well as the content of 2,3-diphosphoglycerate and reduced glutathione, show that circulating erythrocytes in old animals constitute an heterogeneous cell population whose properties cannot be explained on the basis of a chronologically younger erythrocyte population. Furthermore, evaluation of cell components in hemopoietic tissues have shown an increased porportion of erythroid precursor cells in old animals confirming that old mice compensate for reduced red cell survival with an increased erythropoiesis.

Aging↗

PubMed1988

Simultaneous preparation from human placenta of several enzymes of glucose metabolism.

A procedure for the simultaneous purification to homogeneity of hexokinase, phosphoglucomutase 1 and 2, aldolase, phosphoglucose isomerase and glucose-6-phosphate dehydrogenase from human origin has been developed. Human placenta homogenate was first chromatographed on DE-52 column which retains hexokinase and glucose-6-phosphate dehydrogenase while the other enzymes are recovered in the unabsorbed protein fraction. The other steps in the purification involve Matrex gel and specific affinity chromatography for the DE-52 retained enzymes and phosphocellulose and Matrex gel chromatography for the other enzymes. All the enzymes mentioned were obtained in one week, with recoveries from 14 percent for glucose-6-phosphate dehydrogenase to 75 percent for hexokinase. Thus, the procedures utilized seem to be useful in obtaining large amounts of enzymes in a a homogeneous form from an easily available human tissue.

Chromatography, Affinity↗

PubMed1988

Membrane-bound immunoglobulins increase during red blood cell aging.

A flow cytofluorimetric method was used to detect the immunoglobulin molecules present on human red blood cells. Normal human erythrocytes were separated into seven fractions of increasing mean age by density centrifugation on discontinuous gradients. Some biochemical and morphological properties of these cells were determined as well as their IgG content. The results obtained suggest that IgG binding during red cell aging is a cumulative process.

Erythrocyte Aging↗

PubMed1987

A very fast ion-pair reversed-phase HPLC method for the separation of the most significant nucleotides and their degradation products in human red blood cells.

A simple and fast ion pair reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of ATP, ADP, AMP, GTP, GDP, IMP, NADP+, NADPH+, NAD+, NADH, ADP-ribose, inosine, adenosine, hypoxanthine, and xanthine. This method allows us to have a complete picture of the most important nucleotides present in fresh human erythrocytes. Furthermore it is particularly useful in the study of the erythrocyte adenine nucleotide catabolism allowing the detection of degradation products such as IMP, inosine, adenosine, hypoxanthine, and xanthine. The separation of the compounds under investigation is achieved in less than 15 min using a reversed-phase 3-micron Supelcosil LC-18 column and adding tetrabutylammonium, as ion-pair agent, to the buffers. The short time of analysis, the high reproducibility of the system, and the accurate evaluation of the compounds of interest make this method particularly suitable for routine analysis. Finally it is possible to use this assay as an alternative method of measuring activities of enzymes which catalyze reactions involving some of these compounds, as in the case of Na+-K+ ATPase, AMP deaminase, and adenosine deaminase.

Chromatography, High Pressure Liquid↗

PubMed1987

Rabbit bone marrow glucose-6-phosphate dehydrogenase during erythroid cell development.

Studies were carried out on glucose-6-phosphate dehydrogenase (G6P-DH) during the differentiation of rabbit bone marrow erythroid cells. It was found that G6P-DH, although displaying a 7-fold activity decrease, did not change the relative amounts of its three dimeric forms. Using homogeneous enzyme preparations, we observed that from dividing to non-dividing erythroblasts the following properties remained constant: V max dependence on pH and temperature, Km for G6P dependence on pH, heat stability, 2-deoxy glucose-6-phosphate utilization, molecular weight, while the Km for NADP significantly increased in non-dividing erythroblasts. These results indicate that no shift towards the oxidized form of the enzyme and no substantial modifications of the protein take place during cell differentiation.

Animals↗

PubMed1987

Adenine and pyridine nucleotides during rabbit reticulocyte maturation and cell aging.

In this paper we have used a new method which allows the simultaneous extraction and HPLC determination of ATP, ADP, AMP, NADP, NADPH, NAD and NADH to evaluate the changes in concentration of these compounds during maturation of rabbit reticulocytes and cell aging. The results show a significant increase of ATP concentration, higher ATP/ADP, ATP/AMP ratios and lower NADP+/NADPH, NAD+/NADH ratios in rabbit reticulocytes when compared to mature cells. Similar results were also obtained when the whole red blood cell population was separated into fractions of increasing mean age on discontinuous gradients of Percoll-BSA. These metabolic modifications are discussed in relation to the age-dependent metabolic decline of the erythrocyte.

Adenine Nucleotides↗

PubMed1987

Action of acetaldehyde on glucose metabolism of newborn and adult erythrocytes.

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.

2,3-Diphosphoglycerate↗

PubMed1987

Red blood cell glucose metabolism in Down's syndrome.

The specific activity of red blood cell glycolytic enzymes was determined in 20 Down's syndrome patients and compared with 20 normal controls. According to previous evidence, a 50% increase of phosphofructokinase and a 30% increase of glucose-6-phosphate dehydrogenase and glutathione peroxidase activity was found. Metabolic studies of the patients' erythrocytes revealed a decrease in fructose-6-phosphate and 2, 3-diphosphoglycerate concentrations, while fructose-1, 6-diphosphate and ADP both increased. Glucose utilization by intact erythrocytes from Down's syndrome patients did not differ from that of normal controls. However, addition of methylene blue or inorganic phosphate produced a higher stimulation of erythrocyte glycolysis in patients with Down's syndrome compared to controls. These metabolic abnormalities could be, at least in part, ascribed to the increased phosphofructokinase activity which is due to a gene-dosage effect.

Blood Glucose↗

PubMed1987

Adenine and pyridine nucleotides in the erythrocyte of different mammalian species.

Adenine (ATP, ADP, AMP) and pyridine nucleotides (NADP+, NADPH, NAD+, NADH) concentrations have been determined by HPLC in the erythrocytes from five different mammalian species (pig, rat, mouse, rabbit and cow) and compared to those in human red blood cells. Two different extraction procedures have been used and the results obtained are compared and discussed. A good correlation between the different abilities of the erythrocytes of the six species to utilize glucose and the NAD+/NADH ratio was found, with high NAD+/NADH ratio in the red blood cell of the species with high glucose utilization rates. The levels of all the glycolytic enzymes and some of the pentose phosphate shunt enzymes were also determined.

Adenine↗

PubMed1987

Decay mechanisms of rabbit hexokinase during reticulocyte maturation.

In rabbit erythrocytes hexokinase (EC 2.7.1.1) specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by an in vitro made policlonal antibody shows that this maturation dependent hexokinase decay is not due to the accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system made from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinase activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. 125I-hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mecaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugate to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP and ubiquitin dependent and involves both the hexokinase molecular forms (hexokinase Ia and Ib) present in reticulocytes. "In vivo", hexokinase Ia is mitochondrial bound while hexokinase Ib is soluble. The energy dependent degradation system of reticulocytes is active only on the soluble enzyme, namely hexokinase Ib. As the cell mature mitochondria are degradated, hexokinase Ia becomes soluble but there is a concomitant decay also of the proteolytic system resulting in a mature erythrocyte that contains only hexokinase Ia in a soluble form.

Adenosine Triphosphate↗

PubMed1986

Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation.

In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.

Adenosine Triphosphate↗

PubMed1986

Red blood cell phagocytosis and lysis following oxidative damage by phenylhydrazine.

Red blood cells exposed in vitro to phenylhydrazine acquired Heinz bodies, bound autologous IgG and were then phagocytized when incubated with autologus mononuclear phagocytes. In vivo, phenylhdyrazine administered to rabbits, caused the appearance of high plasma hemoglobin levels and hemoglobinuria as well as Heinz body formations and IgG binding to erythrocytes. This suggests that while in vitro the main mechanism of red cell removal seems to be phagocytoses, in vivo both intravascular hemolysis and phagocytosis are active processes. Preliminary biochemical studies on phenylhydrazine-exposed erythrocytes showed that together with the well-known appearance of Heinz bodies, methemoglobin and a drop in reduced glutathione, this drug also causes ATP depletion. This is initially concomitant with the appearance of ADP and AMP and subsequently hypoxantine. Thus, irreversible ATP depletion may contribute to the genesis of the hemolytic process observed in vivo.

Erythrocytes↗

PubMed1986

Glucose metabolism in fibroblasts from patients with erythrocyte hexokinase deficiency.

Four different hexokinase (HK) isoenzymes are distributed in different proportions in human tissues. Fibroblasts contain HK type I as the predominant glucose phosphorylating activity, the same isoenzyme that predominates in red blood cells (RBC). We have established cell lines from two patients homozygous for RBC HK deficiency but carrying different mutations. In one case (HK-Melzo) the residual RBC enzyme shows a marked heat instability but possesses normal kinetic and regulatory properties; in the other (HK-Napoli), the enzyme is characterized by an increased Ki for glucose-1,6-diphosphate. These properties are also retained by the fibroblasts' hexokinase. Glucose utilization by cultured fibroblasts from these patients was markedly reduced in the cell lines where HK deficiency was more pronounced. However, cells with only 30% HK activity retained their full ability to utilize glucose in the hexose monophosphate pathway. This was shown to be true not only under basal conditions but also in the presence of oxidative agents such as methylene blue. Significant reduction of the ATP level was also found in HK-Melzo fibroblasts. Thus, HK deficiency is associated with reduced glucose utilization and normal hexose monophosphate shunt rates. Results previously obtained on RBC support similar conclusions.

Adenosine Triphosphate↗

PubMed1986

Human erythrocyte hexokinases are immunologically related.

Human erythrocytes contain three major hexokinase isoenzymes eluted by DE-52 chromatography between hexokinase type I and type II. Cross-reactivities of these isoenzymes were studied by means of a monospecific rabbit antibody against purified human placenta hexokinase type I. It was shown that the three hexokinase isoenzymes were immunologically related, supporting the concept of a postsynthetic mechanism(s) as their origin.

Animals↗