myo-Inositol oxygenase: molecular cloning and expression of a unique enzyme that oxidizes myo-inositol and D-chiro-inositol.

myo-Inositol oxygenase (MIOX) catalyses the first committed step in the only pathway of myo-inositol catabolism, which occurs predominantly in the kidney. The enzyme is a non-haem-iron enzyme that catalyses the ring cleavage of myo-inositol with the incorporation of a single atom of oxygen. A full-length cDNA was isolated from a pig kidney library with an open reading frame of 849 bp and a corresponding protein subunit molecular mass of 32.7 kDa. The cDNA was expressed in a bacterial pET expression system and an active recombinant MIOX was purified from bacterial lysates to electrophoretic homogeneity. The purified enzyme displayed the same catalytic properties as the native enzyme with K(m) and k(cat) values of 5.9 mM and 11 min(-1) respectively. The pI was estimated to be 4.5. Preincubation with 1 mM Fe(2+) and 2 mM cysteine was essential for the enzyme's activity. D-chiro-Inositol, a myo-inositol isomer, is a substrate for the recombinant MIOX with an estimated K(m) of 33.5 mM. Both myo-inositol and D-chiro-inositol have been implicated in the pathogenesis of diabetes. Thus an understanding of the regulation of MIOX expression clearly represents a potential window on the aetiology of diabetes as well as on the control of various intracellular phosphoinositides and key signalling pathways.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization.

A 25 kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na(2)CO(3), indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73-83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87-101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.

Expression, purification, and characterization of a recombinant 5-lipoxygenase from potato tuber.

We have isolated a full length 5-LOX cDNA clone from potato cDNA library using degenerate primers designed from conserved sequences of LOXs. Sequence analysis and comparison of the deduced amino acid sequence revealed high homology to other plant LOXs. We have expressed the cDNA in Escherichia coli and purified the recombinant protein to electrophoretic homogeneity by anion exchange liquid chromatography followed by HPLC on a Mono-Q column. Substrate specificity of the purified recombinant protein revealed LOX activity towards linoleic, linolenic acid, arachidonic acids as substrates with linoleic acid being the best substrate. The relative LOX activity as well as the product profiles for the recombinant L1 5-LOX are comparable to values determined for the purified potato tuber 5-LOX. When the recombinant L1 5-LOX and the native peak-2 5-LOX (the most abundant isozyme) were compared on SDS-PAGE, single bands of apparently identical mass 97,000 Da, was observed, which agrees well with the L1 molecular mass calculated from amino acid sequences.

Evidence for G-protein-dependent and G-protein-independent activation of phospholipase D in lymphocytes.

Previously we reported that tumor-promoting phorbol esters stimulate phospholipase D (PLD) independent of protein kinase C (PKC) activation in bovine lymph node lymphocytes. (Cao et al., Biochem. Biophys. Res. Commun. 171, 955-962, 1990; 217, 908-915, 1995). In the present study, we examined the effects of prostagladins (PGs), E2, F2 alpha, D2, and H2 on PLD activity as measured by conversion of [1-14C] arachidonic acid-labeled phospholipids into phosphatidylethanol (PEt) in bovine lymph node lymphocytes. Prostaglandins stimulated the formation of PEt at an optimal concentration of 10 microM with relative stimulatory effect on the order of PGE2 > PGF2 alpha > PGH2 > PGD2. The PGE2-stimulated formation of PEt was dose-dependent in the range of 0.1 to 10 microM and was not inhibited by PKC inhibitors staurosporine and K252a. When both PGE2 and 12-0-tetradecanoylphorbol-13-acetate (TPA) were included, their effect on the PLD activation was additive. Furthermore, NaF, a G-protein activator, stimulated the PEt formation. Interestingly, the stimulatory effects of PGE2 and NaF were not additive; however, the formation of PEt by NaF and TPA was additive. These results suggest that similar to TPA, PGs increase PLD activity independent of PKC and the stimulation by PGs and TPA in lymphocytes may involve both G-protein-dependent and G-protein-independent signaling pathways.

Glutathione-dependent protection against lipid peroxidation in sheep liver microsomes.

In the present studies we have described a glutathione-dependent system in sheep liver microsomes that protects against membrane lipid peroxidation initiated by either Fe+2/NADPH or Fe+2/ascorbate. Glutathione protected against lipid peroxidation in microsomes containing a wide range of alpha-tocopherol levels (0.02-0.11 microgram/mg protein). The addition of glutathione disulfide alone had no effect on microsomal lipid peroxidation, however, it prolonged the protection afforded by glutathione, particularly in assays containing Fe+2/NADPH. Whereas the glutathione-dependent protection was very labile, with loss of activity demonstrated in microsomes stored at 4 degrees C for 24 hours, the combined effect of glutathione and glutathione disulfide was not affected by storage. The glutathione S-transferase inhibitors, bromosulphothalein and S-hexylglutathione, reversed the protection observed with glutathione, indicating a possible role for microsomal glutathione S-transferases in this protection, but not that observed by the combination of glutathione and glutathione disulfide. In general, our findings support previous results observed with rat liver microsomes and suggest that microsomal glutathione S-transferases may be involved in the glutathione-dependent protection in sheep liver microsomes.

The mechanism of sphingosine enhancement of phorbol ester-mediated phospholipase D activation in lymphocytes.

Tumor-promoting phorbol esters stimulate the production of phosphatidylethanol (PEt) in bovine lymph node lymphocytes through the activation of Phospholipase D (PLD). Sphingosine enhanced the 12-O-tetradecanoylphorbol-13-acetate (TPA)-mediated formation of PEt in lymphocytes, while it had no effect by itself. Interestingly, the incorporation of labeled arachidonic acid into phospholipids was increased by sphingosine. Examination of the effect of sphingosine along with TPA on the incorporation of [1-14C]labeled-arachidonic acid into individual phospholipids revealed that they enhanced significantly the specific activity of only PC and lyso-PC fractions. These observations indicate that sphingosine does not activate PLD directly as TPA does. It appears that sphingosine increases the specific radioactivity of the substrate pool for PLD which results in an apparent increase in PEt when both sphingosine and TPA are present.

Increased thromboxane A2 synthesis by rat lung neutrophils during selenium deficiency.

Modulation of cellular hydroperoxide levels is considered one of the important physiological mechanisms for regulating the synthesis of prostaglandins (PGs) and leukotrienes (LTs) in mammalian cells. Both vitamin E and selenium (Se) have the potential to affect the concentration of peroxides and, thus, the biosynthesis of eicosanoids. To gain insight into some of the molecular mechanisms underlying the regulation of the arachidonic acid cascade by vitamin E and Se, we have investigated the influence of altered vitamin E and Se nutrition on the ability of polymorphonuclear leukocytes (PMNs) derived from endotoxin-challenged lung to secrete arachidonic acid metabolites. Selenium deficiency had no significant effect (p > 0.05) on lavage fluid levels of thromboxane (TX) B2, LTB4 or LTC4. Vitamin E deficiency, however, led to a significant increase in LTB4 recovered from lavage fluid while having no effect on TXB2. In contrast, Se deficiency, although producing no discernible effects on the production of LTB4, resulted in a significant increase in the release of TXB2 by PMNs. An increase in TXB2 release was seen in both in vitro-stimulated and nonstimulated PMNs. Vitamin E deficiency appeared to induce an enhancement of LTB4 release by PMNs but the increase was not statistically significant. No detectable levels of LTC4 were found in PMN cultures stimulated with either zymosan or A23187. Thus, these studies indicate that deficiencies of either Se or vitamin E lead to alterations in the metabolism of arachidonic acid in the lung.

Selenium deficiency alters the lipoxygenase pathway and mitogenic response in bovine lymphocytes.

We investigated the effect of altered selenium (Se) nutrition on arachidonic acid oxidation in immune cells. Experiments were conducted with peripheral blood lymphocytes obtained from dairy cattle fed diets either supplemented with or deficient in Se. The results indicate that the concanavalin A-stimulated lymphocyte proliferation was significantly lower in Se-deficient cows. When stimulated by calcium ionophore A23187, the lymphocytes derived from Se-deficient cows produced significantly less 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 (LTB4) than those obtained from Se-supplemented cows. When included in cell cultures from animals fed +Se diets, 5-HETE and LTB4 elicited a partial reversal of the inhibition of lymphocyte proliferation by either hydrocortisone or nordihydroguaiaretic acid. Based on this information, we postulate that dietary Se status, which in turn determines tissue Se concentration, plays an important role in the regulation of arachidonate metabolism by way of the 5-lipoxygenase pathway. This may be one of the biochemical mechanisms underlying the inhibition of lymphocyte proliferation and the decrease in resistance to infectious diseases observed in Se-deficient animals.

Increased 12-HETE production in bovine lymphocytes during selenium deficiency.

When peripheral blood lymphocytes were incubated with arachidonic acid in the presence of Ca++ ionophore (A23187), the cells from the selenium-deficient dairy cows produced significantly greater quantities of 12-hydroxyeicosatetraenoic acid (12-HETE) than the cells from the selenium-supplemented animals. The major product of reaction was verified as 12-HETE by cochromatography with a 12-HETE standard on an HPLC and structural analysis by GC-MS. Additionally, concanavalin A-stimulated lymphocyte proliferation was significantly decreased in cells from the Se-deficient cows. Furthermore, 12-HETE generated by the A23187-stimulated lymphocytes inhibited lymphocyte proliferation when added to Se-supplemented cell cultures. These observations suggest that self-regulation of lymphocyte proliferation might be mediated by 12-HETE production, especially during an altered nutritional state such as Se deficiency.

Inhibition of lipoxygenase and prostaglandin endoperoxide synthase by anacardic acids.

C22:1 omega 5-anacardic acid was found to be a good inhibitor of both potato lipoxygenase and ovine prostaglandin endoperoxide synthase with approximate IC50's of 6 and 27 microM, respectively. Very similar inhibition was seen with the crude exudate, rich in omega 5-anacardic acids, from glandular trichomes of an arthropod-resistant strain of geranium, Pelargonium xhortorum. The saturated anacardic acid (C22:0 sat), abundant in the trichome exudate of susceptible strains, was nearly as inhibitory toward both prostaglandin endoperoxide synthase and lipoxygenase as the omega 5-unsaturated compound. However, the dimethyl derivative of C22:1 omega 5-anacardic acid was a poor inhibitor of prostaglandin endoperoxide synthase and caused only moderate (32%) inhibition of lipoxygenase even at 135 microM. The possible role of prostaglandin endoperoxide synthase and lipoxygenase inhibition in the enhanced pest resistance of geraniums which produce the omega 5-AnAs is discussed.

Stereochemical nature of the products of linoleic acid oxidation catalyzed by lipoxygenases from potato and soybean.

When linoleic acid was incubated with the purified potato lipoxygenase under O2 atmosphere, a mixture of 9 and 13-hydroperoxyoctadecadienoic acids was formed. Stereochemical analysis of the respective methyl-hydroxyoctadecadienoic acids revealed that the 9-isomer was in S-configuration whereas 13-hydroxyoctadecadienoic acid was a mixture of S (39%) and R (61%). Exactly the opposite was the case with the soybean lipoxygenase products, where the 13-isomer was found to be in S-configuration and 9-hydroxyoctadecadienoic acid - a mixture of S (73%) and R (27%). A general scheme is proposed for the stereochemical nature of oxidation products of enzymes which are predominantly either [+2] or [-2] lipoxygenases.

Effects of inadequate vitamin E and/or selenium nutrition on the release of arachidonic acid metabolites in rat alveolar macrophages.

Effects of vitamin E and/or selenium (Se) deficiency on the secretion of arachidonic acid metabolites by zymosan-stimulated pulmonary alveolar macrophages (AM) were examined using cells from male Long-Evans hooded rats fed torula-yeast based diets with or without the supplementation of vitamin E (150 IU/kg) or Se (0.5 mg/kg). Alveolar macrophages obtained by lavage were purified by adherence and cultured for 4 h in Hank's balanced salt solution containing bovine serum albumin (0.1%) and zymosan (300 micrograms/ml). The arachidonic acid metabolites present in the culture supernatant were measured by radioimmunoassay. Altered vitamin E and Se nutrition had no effect on the number of cells or cell types recovered from the pulmonary airways. Alveolar macrophages derived from animals fed on diets deficient in vitamin E or Se or both nutrients secreted higher levels of prostaglandin E2 and thromboxane B2. Levels of both 5-hydroxyeicosatetraenoic acid and leukotriene B4 were significantly increased only in the group fed the diet adequate in Se but deficient in vitamin E. Our data suggest that vitamin E and Se might play an important role to control the levels of several physiologically and pathologically important arachidonic acid metabolites.

Endocytosis of bovine lactoperoxidase by two carbohydrate-specific receptors in rat liver.

A perfused rat liver took up bovine lactoperoxidase (LPO) by a Ca2+-dependent, saturable process. This endocytosis was accomplished by both hepatocytes and Kupffer or other nonparenchymal cells (NPCs). The mediating receptors were the Gal/GalNAc lectin of hepatocytes and the Man/GlcNAc lectin of NPCs. Blocking either one of these receptors caused a large shift in distribution of accumulated LPO into the cell type whose receptor was left unblocked, but the extent of uptake was unaffected and the rate was only moderately reduced. Effective inhibition of overall uptake into the perfused organ required the presence of competitors for both receptors. Conversely, LPO was an effective competitor of other ligands (asialoorosomucoid or mannan) for either of the two receptors. The major clearance capacity for LPO was associated with hepatocytes which in suspension took it up by a process completely inhibitable by asialofetuin (ASF) and at a rate more than three times greater than for ASF. A faster cycling time for Gal/GalNAc receptors when bound to LPO is suggested. The glycoprotein selectively lost its affinity for Man/GlcNAc receptors when digested by endoglycosidase H (endo H), suggesting that LPO contains mannose-rich oligosaccharides.

Uptake of asialoglycophorin-liposomes by the perfused rat liver.

Asialoglycophorin-containing liposomes and their contents (125I-labeled bovine serum albumin) were taken up by a perfused rat liver with subsequent digestion of their protein components. The uptake of these liposomes required Ca2+ as well as desialylation. The process was inhibited partially by asialofetuin and completely by further addition of asialoglycophorin to the perfusate.

Uptake of asialo-glycophorin by the perfused rat liver and isolated hepatocytes.

Perfused rat livers took up asialo-glycophorin, a glycoprotein derived from human erythrocyte membranes, with a t1/2 for clearance of 7 min. As a comparison, asialo-orosomucoid was taken up by this system with a t1/2 of 3.5 min. Both proiteins were digested and their 125I labels were released to the perfusate as free 125I-. EGTA completely inhibited uptake of these glycoproteins, but not uptake of denatured bovine serum albumin. Addition of Ca2+ reversed the inhibition nearly completely. Isolated hepatocytes had an uptake rate of approximately 3 ng/min per 10(6) cells for the asialo forms of glycophorin, orosomucoid and fetuin. Cellular uptake of each of these asialoglycoproteins could be inhibited by one of the other proteins. Asialo-fetuin caused a 95% inhibition of the uptake rate of asialo-orosomucoid by the perfused liver. This fetal calf glycoprotein had a similar inhibitory effect on asialo-glycophorin, but only after an initial 40% of the asialo-glycophorin had been taken up by the liver at an almost normal rate during the first 30 min of perfsuion. The possibility of an alternative hepatic removal system for asialo-glycophorin is suggested.

Pig reticulocytes: II. Characterization of density-fractionated maturing reticulocytes.

A simple centrifugation technique initially developed for fractionation of human red cells in their own plasma has been found to be readily applicable to the separation of reticulocytes normally appearing in the pig soon after birth as well as to those induced in the adult pig by phenylhydrazine administration. The density of the naturally occurring reticulocytes, maximally amounting to 10-15% of total cells derived from a 7-day-old pig, varied from 1.073 to 1.101, in contrast to the mature cells, presumed to be the fetal cell type, whose density ranged in a narrower limit, from 1.101 to 1.106. In adult animals, the reticulocytosis amounting to more than 70% takes place in response to daily administration of phenylhydrazine for 7 days. The density of these reticulocytes varied widely from 1.068 to 1.094; in contrast, the density of the mature adult red cells ranged from 1.095 to 1.106. Among various physiological parameters examined, a marked decrease in cell size, cell water content, RNA, Na+ -K+ -ATPase, and Ca2+ -ATPase activities was observed in the course of reticulocyte maturation in vivo in the adult animal. All these physiological parameters were found to undergo a similar reduction from the top to the bottom fractions of the reticulocytes separated according to their density, suggesting that the separation technique employed here represents separation of reticulocytes according to their age.

Observations on the (Ca2+ plus Mg2+)-ATPase activator found in various mammalian erythrocytes.

1. A soluble activator of membrane (Ca2+ plus Mg2)-ATPase is present in hemolysates of the newborn calf and cow, the new born and adult pig as well as human erythrocytes. 2. The activator is also found in reticulocytes of the adult pig. 3. The activator obtained from any of the above species is capable of stimulating the membrane (Ca2+ plus Mg2+)-ATPases of the other species, regardless of the age of the animals. 4. The results obtained from density fractionation of human erythrocytes revealed that the soluble factor has little simulatory effect on membranes of young erythrocytes from which it is derived but caused a marked stimulation on (Ca2+ plus Mg2+)-ATPase activity of the intermediate aged and old erythrocyte membranes. 5. The above observations support the following conclusions: (a) the extremely low levels of (Ca2+ plus Mg2+)-ATPase in cow erythrocytes is not due to the lack of a (Ca2+ plus Mg2+)-ATPase activator; (b) the distribution of (Ca2+ plus Mg2+)-atpase activator is not species specific and the differences in the level of membrane (Ca2+ plus Mg2+)-ATPase activity in various species of cells is an inherent property of that particular membrane (c) the (Ca2+ plus Mg2+)-ATPase activator is present at least from the time of reticulocyte formation and remain during tthe life span of the erythrocyte.

Studies on an activator of the (Ca2+ plus Mg2+)-ATPase of human erythrocyte membranes.

1. An activator of the (Ca2+ plus Mg2+)-stimulated ATPase present in the human erythrocytes (membrane) has been isolated in soluble form from hemolysates of these cells. Partial purification has been achieved through use of carboxymethyl-Sephadex chromatography. The resulting activator fraction contained no hemoglobin and only 0.3% of the total adenylate kinase activity of the cell. 2. Whereas the activator was released from erythrocytes subjected to hemolysis in 20 miosM buffer at pH 7.6 or at pH 5.8, only the membranes prepared at pH 7.6 were affected by it. 2. Whereas the activator was released from erythrocytes subjected to hemolysis in 20 miosM buffer at pH 7.6 or at pH 5.8, only the membranes prepared at pH 7.6 were affected by it. 3. When (Ca2+ plus Mg2+)-ATPase activity was measured by 32Pi release from (gamma-32P)ATP, freeze-thawed erythrocytes, as well as membranes prepared at pH 5.8 and at pH 7.6, expressed lower values than noted by assay for total Pi release. When ADP instead of ATP was used as substrate, significant amount of Pi were released by these erythrocyte preparations. Further study revealed (a) production of ATP and AMP from ADP with membranes and hemolysate alone, and (b) exchange of the gamma-and B-position phosphate on (gama-32P)ATP in the presence of membranes plus hemolysates. These observations established the presence of adenylate kinase activity in the (membrane-free) hemolysates and in membranes. It further supports the conclusion that Pi release from ADP by human erythrocytes (freeze-thawed) and by their isolated membranes is due to formation of ATP by adenylate kinase and hydrolysis of this generated ATP by (Ca2+ plus Mg2+)-ATPase. 4. The following points were also established: (a) absence of an ADPase in human erythrocytes; (b) the (Ca2+ plus Mg2+)-ATPase activator enhanced cleavage only of the gama-position of ATP and (c) the (Ca2+ plus Mg2+)-ATPase activator is neither adenylate kinase nor hemoglobin.