Intraluminal transport of iron from stomach to small-intestinal mucosa

Inorganic iron rarely exceeds 10⁻⁴ molar concentration in the stomach after a meal. Natural sugars, ascorbic acid, citric acid, and amino-acids form iron complexes, and if they are present in the meal complexing occurs when the gastric contents are neutralized. In their absence an iron complex is formed with gastric mucopolysaccharide, which acts as a carrier, stable at neutral pH. Iron can be detached from this carrier at neutral pH by some low molecular weight substances, of which citric acid, ascorbic acid, and cysteine are particularly effective at low concentrations. Under normal circumstances most of the iron released from food in the stomach becomes bound to the mucopolysaccharide carrier.     

Rapid generation of rotavirus-specific human monoclonal antibodies from small-intestinal mucosa

The gut mucosal surface is efficiently protected by Abs, and this site represents one of the richest compartments of Ab-secreting cells in the body. A simple and effective method to generate Ag-specific human monoclonal Abs (hmAbs) from such cells is lacking. In this paper, we describe a method to generate hmAbs from single Ag-specific IgA- or IgM-secreting cells of the intestinal mucosa. We found that CD138-positive plasma cells from the duodenum expressed surface IgA or IgM. Using eGFP-labeled virus-like particles, we harnessed the surface Ig expression to detect rotavirus-specific plasma cells at low frequency (0.03-0.35%) in 9 of 10 adult subjects. Single cells were isolated by FACS, and as they were viable, further testing of secreted Abs by ELISPOT and ELISA indicated a highly specific selection procedure. Ab genes from single cells of three donors were cloned, sequenced, and expressed as recombinant hmAbs. Of 26 cloned H chain Ab genes, 22 were IgA and 4 were IgM. The genes were highly mutated, and there was an overrepresentation of the VH4 family. Of 10 expressed hmAbs, 8 were rotavirus-reactive (6 with K(d) < 1 × 10(-10)). Importantly, our method allows generation of hmAbs from cells implicated in the protection of mucosal surfaces, and it can potentially be used in passive vaccination efforts and for discovery of epitopes directly relevant to human immunity.     

Metabolism of extracellular glutathione in rat small-intestinal mucosa

Metabolism of exogenous glutathione was investigated in suspensions of freshly isolated rat small-intestinal mucosal cells. The cells catalyzed the oxidation of reduced glutathione (GSH) to glutathione disulfide (GSSG). Neither serine . borate nor methionine significantly influenced this reaction. Formed GSSG was further metabolized as indicated by its disappearance from the medium. Degradation of GSSG was stimulated by methionine and inhibited by serine . borate. Separation and identification of GSSG metabolites were achieved by high performance liquid chromatography. The results indicate that the preferred route for GSSG metabolism to the constituent amino acids in small intestine, is by hydrolytic removal of the two gamma-glutamyl groups of GSSG to yield cystinyl-bisglycine which is subsequently hydrolyzed to cystine. gamma-Glutamyltransferase activity was compared in isolated intestinal, kidney and liver cells using gamma-glutamyl-p-nitrocarboxyanilide as substrate. Kidney cells were approximately 5-fold and 150-fold more active than intestinal and liver cells, respectively. Serine . borate markedly inhibited, and glycyl-glycine stimulated, hydrolysis of gamma-glutamyl-p-nitrocarboxyanilide in all cell types confirming the involvement of gamma-glutamyltransferase in the reaction. The hydrolysis of gamma-glutamyl-p-nitrocarboxyanilide was inhibited to approximately the same extent by either GSH or GSSG suggesting that both compounds interact at the donor site of gamma-glutamyltransferase. Comparison of the rates of glutathione metabolism by isolated intestinal and kidney cells suggests that the intestinal contribution to the degradation of extracellular glutathione may be physiologically more important than has previously been assumed.     

Mitochondrial hexokinase and cardioprotection of the intact heart

The interaction of hexokinase with mitochondria has emerged as a powerful mechanism in protecting many cell types against cell death. However, the role of mitochondrial hexokinase (mitoHK) in cardiac ischemia-reperfusion injury has as of yet received little attention. In this review we examine whether increased binding of hexokinase to the mitochondrion is also an integral component of cardioprotective signalling. We discuss observations in cardiac mitochondrial activation that directed us to the hypothesis of hexokinase cellular redistribution with reversible, cardioprotective ischemia, summarize the data showing that many cardioprotective interventions, such as ischemic preconditioning, insulin, morphine and volatile anesthetics, increase mitochondrial hexokinase binding within the intact heart, and discuss similarities between mitochondrial hexokinase association and ischemic preconditioning. Although most data indicate that mitochondrial hexokinase may indeed be an integral part of cardioprotection, a definitive proof for a causal relation between the amount of mitoHK and cardiac ischemia-reperfusion injury in the intact heart is eagerly awaited. When such relationship is indeed observed, the association of hexokinase with mitochondria will offer an opportunity to develop new therapies to combat ischemic cardiac diseases.     

Mitochondrial hexokinase activity in a murine hybridoma

Mitochondrially bound hexokinases are often found in cells exhibiting high rates of glycolysis. These enzymes have kinetic and regulatory properties that differ from cytosolic hexokinases. In this work, we present preliminary findings for a mitochondrially bound hexokinase in a murine hybridoma that is relatively insensitive to glucose-6-phosphate inhibition when compared to the cytosolic hexokinase. Additional regulatory data on the effects of glucose-1,6-diphosphate and adenosine diphosphate on both the mitochondrial and cytosolic forms of this enzyme are also presented.     

Purification and properties of the cytoplasmic hexokinase from rabbit brain

About 90% of the total hexokinase activity in rabbit brain was found to be associated with mitochondria while the remaining part was found in the cytosolic fraction. The soluble enzyme was purified 4,700-fold to near homogeneity by a combination of ion-exchange chromatography, dye-ligand chromatography and affinity chromatography. The purified enzyme showed a specific activity of 110 units/mg of protein and was obtained in 70% yield. The molecular weight of the purified hexokinase was found to be approximately 98,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.2 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses. Mg . ATP2-, among the nucleotide substrates, was the most effective phosphate donor. The properties of the purified cytoplasmatic hexokinase were compared with those of the solubilized mitochondrial enzyme. No significant differences were found in molecular weight, isoelectric point, pH dependence of activity, electrophoretic mobility and affinity for glucose and Mg.ATP2-. However, the temperature dependence of activity, and the specificity for several hexose substrates were markedly different.     

Dexamethasone-induced alterations in the glycosphingolipids of rat proximal small-intestinal mucosa

Prior studies have demonstrated that glucocorticoids can influence the structure and function of several different organs, including the small intestine. However, to date, the effects of glucocorticoids on the glycosphingolipids of the rat small intestinal mucosa have not been examined. In the present experiments, male albino rats of the Sherman strain were subcutaneously administered dexamethasone (100 micrograms/100 g body wt. per day) or diluent for 4 days, and the ceramide, acidic and neutral glycosphingolipid compositions of the proximal small intestine of these animals were examined and compared. The results of these studies demonstrate that dexamethasone administration: (1) increased the content and relative percentage of hematoside (GM3) in this tissue; (2) increased the percentage of N-glycoylneuraminic acid of hematoside; (3) decreased the percentage of the long-chain base phytosphingosine of hematoside, glucosyl- and globotriaosylceramide; and (4) did not appear to influence significantly the concentration of the neutral glycosphingolipids or ceramide in this tissue. These data, therefore, indicate that dexamethasone administration induces alterations in the glycosphingolipids, particularly hematoside, of rat small-intestinal mucosa.     

Purification of nonbindable and membrane-bindable mitochondrial hexokinase from rat brain.

MgCl₂-induced binding of glucose-6-P solubilized rat brain hexokinase to rat liver mitochondria has been found to be markedly diminished by increasing ionic strength. Using a modified assay of binding ability, it has now been possible to demonstrate that purified preparations of brain hexokinase do retain appreciable ability to bind to mitochondria. A slight modification of the previous DEAE-cellulose chromatography procedure (4), permits resolution of the hexokinase into two major components designated as Type I_b and Type I_n based on their ability to bind and not bind, respectively, to mitochondria. I_b and I_n appear to be identical in molecular size and subunit composition, but differ slightly in net charge.     

Purification and properties of cytoplasmic and mitochondrial hexokinase from the rabbit brain

The soluble Hexokinase from rabbit brain was purified 4,700-fold to near homogeneity by a combination of ion-exchange chromatography, dye-ligand chromatography and affinity chromatography. The purified enzyme showed a specific activity of 110 units/mg of protein and was obtained in 70% yield. The properties of the purified cytoplasmic hexokinase were compared with those of the solubilized mitochondrial enzyme. No significant differences were found in M.W., pI and electrophoretic mobility. However, the temperature dependence of activity and specificity for several hexose substrates were markedly different.     

Mitochondrial VDAC and hexokinase together modulate plant programmed cell death

The voltage-dependent anion channel (VDAC) and mitochondrially located hexokinase have been implicated both in pathways leading to cell death on the one hand, and immortalization in tumor formation on the other. While both proteins have also been implicated in death processes in plants, their interaction has not been explored. We have examined cell death following heterologous expression of a rice VDAC in the tobacco cell line BY2 and in leaves of tobacco plants and show that it is ameliorated by co-expression of hexokinase. Hexokinase also abrogates death induced by H₂O₂. We conclude that the ratio of expression of the two proteins and their interaction play a major role in modulating death pathways in plants.     

Purification and properties of rat brain hexokinase

Rat brain hexokinase has been purified to homogeneity as judged by disc-gel electrophoresis, isoelectric focusing, and analytical ultracentrifugation. More than 50% of the initial activity could be obtained in homogeneous form (sp act, 60 units/mg protein) by a simple procedure consisting essentially of two steps: relatively specific solubilization of the enzyme from the mitochondrial membrane by glucose-6-P, followed by DEAE-cellulose column chromatography. The molecular weight is approximately 98,000; this same molecular weight was observed when the denatured enzyme was examined by the SDS-polyacrylamide electrophoretic technique, strongly suggesting that the enzyme consists of a single polypeptide chain. In accord with this view, a single N-terminal amino acid, glycine, has been recovered in 80% yield based on a molecular weight of 98,000. The amino acid composition of the rat brain hexokinase has been determined and found to be very similar to that previously reported for the bovine brain enzyme (Schwartz, G. P., and Basford, R. E. (1967) Biochemistry6, 1070, suggesting extensive sequence homology. A notable feature of the brain hexokinases is a relatively low aromatic amino acid content, as judged by the amino acid composition and the relatively low molar extinction coefficient.     

Relationship between brain mitochondrial hexokinase and neuronal function

Mitochondrially bound brain hexokinase is solubilized by anesthetics and this effect has been suggested to contribute to anesthesia. In the present investigation the influence of the metabolic inhibitor 2-deoxy-D-glucose (2-DOG) was studied. An isolated rat brain preparation was used to avoid the contribution of peripheral reactions. Isolated rat brains were perfused for 45 min with media containing 4 mmol/l glucose, 10 mmol/l 2-DOG and/or 0.4 mmol/l thiopental. The EEG was monitored and acetylcholine, 2-DOG and its 6-phosphate, as well as the intracellular distribution of hexokinase activity were determined in brain tissue. Soluble hexokinase activity in brain cortex was enhanced by 2-DOG, as also by thiopental, and even more pronounced by both drugs used together. Results from in vitro experiments suggest that solubilization of mitochondrial hexokinase after 2-DOG is mediated by intracellularly accumulated 2-DOG-6-phosphate. 2-DOG produced a significant impairment of neuronal activity, revealing EEG patterns similar to those caused by thiopental anesthesia. Cortical acetylcholine levels were elevated by 2-DOG, as well as by thiopental, and again both drugs showed an additive effect when used in combination. This effect which may be the result of an inhibition of acetylcholine release, was also detectable in mice in vivo after 5 g 2-DOG/kg i.p., whereas the same dose of 3-O-methylglucose had no effect. The results provide further evidence that mitochondrial hexokinase may be involved in the relationship between cerebral metabolism and brain function.A preliminary report of these results has been made at the 22^nd spring meeting of the Deutsche Pharmakologische Gesellschaft at Mainz (38).