The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells

We investigated the growth inhibitory effect of conjugated linoleic acid (CLA) on HepG2 (human hepatoma cell line), exploring whether the inhibitory action occurs via lipid peroxidation in the cells. When the cells were incubated up to 72 h with 5-40 microM of CLA (a mixture of 9c,11t-18:2 and 10t,12c-18:2), cell proliferation was clearly inhibited in a dose and time dependent manner but such an inhibition was not confirmed with linoleic acid (LA). In order to evaluate the possible contribution of lipid peroxidation exerted by CLA to cell growth inhibition, alpha-tocopherol (5-20 microM) and BHT (1-10 microM) as potent antioxidants were added to the medium with CLA (20 microM), which did not restore cell growth at all. Furthermore, after 72 h incubation, the membranous phospholipid hydroperoxide formation in the CLA-supplemented cells was suppressed respectively to 25% and 50% of that in LA-supplemented cells and control cells. No difference was observed by a conventional lipid peroxide assay, the TBA test, between CLA-supplemented cells and LA-supplemented cells. Although the cellular lipid peroxidation was not stimulated, lipid contents (triacylglycerol, total cholesterol and free cholesterol) and fatty acid contents (palmitic acid, palmitoleic acid and stearic acid) markedly increased in CLA-supplemented cells compared with LA-supplemented and control cells. Moreover, supplementation with 20 microM LA and 20 microM arachidonic acid profoundly interfered with the inhibitory effect of CLA in HepG2. These results suggest that the growth inhibitory effect of CLA on HepG2 is due to changes in fatty acid metabolism but not to lipid peroxidation.     

A role for transglutaminase in glucose-stimulated insulin release from the pancreatic beta-cell.

Preincubation of rat islets of Langerhans with the potent inhibitors of islet transglutaminase activity, monodansylcadaverine (30-100 microM) and N-(5-aminopentyl)-2-naphthalenesulphonamide (100-200 microM), led to significant inhibition of glucose-stimulated insulin release from islets. In contrast, the respective N'-dimethylated derivatives of these two compounds, which did not inhibit islet transglutaminase activity, were much less effective as inhibitors of glucose-stimulated insulin release. None of the compounds inhibited rat spleen protein kinase C activity at concentrations which gave rise to inhibition of glucose-stimulated insulin release. When tested for their effects on calmodulin-stimulated bovine heart phosphodiesterase activity, of the compounds that inhibited insulin release, only monodansylcadaverine did not act as an effective antagonist of calmodulin at concentrations (up to 50 microM) that gave rise to significant inhibition of glucose-stimulated insulin release. Furthermore, at 50 microM, monodansylcadaverine did not inhibit methylation of islet lipids. The inhibition of glucose-stimulated insulin release by monodansylcadaverine is therefore likely to be attributable to its interference with islet transglutaminase activity. The sensitivity of islet transglutaminase to activation by Ca2+ was investigated by using a modified assay incorporating dephosphorylated NN'-dimethylcasein as a substrate protein. The Km for Ca2+ obtained (approx. 3 microM) was an order of magnitude lower than previously reported for the islet enzyme [Bungay, Potter & Griffin (1984) Biochem. J. 219, 819-827]. Mg2+ (2 mM) was found to have little effect on the sensitivity of the enzyme to Ca2+. Investigation of the endogenous substrate proteins of islet transglutaminase by using the Ca2+-dependent incorporation of [14C]methylamine into proteins of islet homogenates demonstrated that most of the incorporated radiolabel was present in cross-linked polymeric aggregates which did not traverse 3% (w/v) acrylamide gels. The radiolabelled polymeric aggregates were present in 71 000 g-sedimented material of homogenates, and their formation was transglutaminase-mediated. These findings provide new evidence for the involvement of islet transglutaminase in the membrane-mediated events necessary for glucose-stimulated insulin release.     

Cadmium uptake in Escherichia coli K-12.

109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer. 109Cd2+ accumulation was both energy dependent and temperature sensitive. The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+. 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+. Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+. Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system. The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values. Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+. We were unable to demonstrate an active transport system for 65Zn2+ in E. coli.     

Inhibition of methanogenesis and carbon metabolism in Methanosarcina sp. by cyanide.

NaCN was tested for its inhibitory effects on growth of and metabolism by Methanosarcina barkeri 227. NaCN (10 microM) inhibited catabolism of acetate methyl groups to CH4 and CO2 but did not inhibit methanogenesis from methanol, CO2, methylamine, or trimethylamine. NaCN also inhibited the assimilation of methanol or CO2 (as the sole carbon source) into cell carbon and stimulated the assimilation of acetate. These results suggest that inhibition by NaCN was a result of its action as an inhibitor of in vivo CO dehydrogenase. The results also implicate CO dehydrogenase in the oxidation of acetate but not methanol methyl groups to CO2.     

Horse alpha 2-macroglobulin. Circular dichroism studies of conformational changes upon reaction with proteinases and methylamine

The interaction of horse alpha 2-macroglobulin with methylamine, trypsin and cathepsin D was studied by circular dichroism in the far and near UV region, by polyacrylamide gel electrophoresis and by determination of its inhibitory activity. The CD spectra of horse alpha 2-macroglobulin resemble those of bovine und human alpha 2-macroglobulin. The CD spectra were changed in a different manner after the interaction of alpha 2-macroglobulin with methylamine, trypsin and inactive or active cathepsin D, indicating that more than one conformational change occurs. Cathepsin D activity was not affected by complex formation with horse alpha 2-macroglobulin. In contrast to the action of trypsin, treatment with methylamine did not increase the electrophoretic mobility of alpha 2-macroglobulin.     

Experimental observations on the mechanism of hormonal imprinting: influence of actinomycin D, methylamine and colchicine on receptor memory in a unicellular model system

The first interaction between target cell and hormone gives rise to hormonal imprinting, which accounts for greater responsiveness of the cell at later interactions. The mechanism of hormonal imprinting is obscure; we based experimental approach to its closer study on combined treatment of Tetrahymena, as model cells, with diiodotyrosine (T2), which stimulates the division, and cell growth inhibitors, which interfere with different stages of cell reproduction, and methylamine, which inhibits cluster formation in the membrane. Of these, actinomycin D and methylamine inhibited the growth of the Tetrahymena, while colchicine did not, and all three suppressed the division stimulating action of T2, but could not prevent hormonal imprinting, as demonstrated on later re-exposure to T2 of cells preexposed and not preexposed to T2 in combination with the inhibitors. It appears that the underlying mechanism of hormonal imprinting is highly complex, and involves many subcellular mechanisms and structures, but suppression of, or gross interference with, one or another of these cannot delete, only quantitatively reduce, the consequence of the first interaction with the hormone, i.e. hormonal imprinting.     

Adenosine modulates cell growth in baby hamster kidney (BHK) cells

Adenosine is known to modulate cell growth in a variety of mammalian cells either via the activation of receptors or through metabolism. We investigated the effect of adenosine on Baby Hamster Kidney (BHK) cell growth and attempted to determine its mechanism of modulation. In wild-type BHK cells, adenosine evoked a biphasic response in which a low concentration of adenosine (1-5 microM) produced an inhibition of colony formation but at higher concentrations (up to 50 microM) this inhibition was progressively reversed. However, no biphasic response was observed in an "adenosine kinase" deficient BHK mutant, "5a", which suggests that adenosine kinase plays an important role in the modulation of growth response to adenosine. Adenosine receptors did not appear to have a role in regulating cell growth of BHK cells. Specific A1 and A2 receptor antagonists were unable to reverse the effect of adenosine on cell growth. Even though a specific A3 adenosine receptor antagonist MRS-1220 partly reversed the inhibition in colony formation at 1 microM adenosine, it also affected the transport of adenosine. Thus adenosine transport and metabolism appears to play the major role in this modulation of cell growth as 5'-amino-5'-deoxyadenosine, an adenosine kinase inhibitor, reversed the inhibition of cell growth observed at 1 microM adenosine. These results, taken together, would suggest that adenosine modulates cell growth in BHK mainly through its transport and metabolism to adenine nucleotides.     

Inhibition of glucose transport by cyclic GMP in cardiomyocytes

Recent evidence points to a potential role of cyclic GMP (cGMP) in the control of cardiac glucose utilization. The present work examines whether the glucose transport system of cardiac myocyte is a site of this cGMP-dependent regulation. Treatment of isolated rat cardiomyocytes (for 10 min) with the membrane-permeant cGMP analogue 8-(4-chlorophenylthio)-cGMP (8-p-CPT-cGMP, 200 microM) caused a decrease in glucose transport in non-stimulated (basal) myocytes, as well as in cells stimulated with insulin or with the mitochondrial inhibitor oligomycin B by up to 40%. An inhibitory effect was also observed with another cGMP analogue (8-bromo-cGMP), and in cells stimulated by hydrogen peroxide or anoxia. In contrast, 8-p-CPT-cAMP (200 microM), or the beta-adrenergic agonist isoprenaline (which increases cAMP levels) did not depress glucose transport, and even potentiated the effect of insulin. Blockade of endogenous cGMP formation with the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 microM) significantly increased basal and insulin-dependent glucose transport (by 25%), whereas addition of the guanylate cyclase activator 3-(5'-hydroxymethyl-2'furyl)-1-benzylindazol (YC-1, 30 microM) produced a depression of glucose transport (by 20%). Confocal laser scanning microscopic studies revealed that cGMP partially prevents the insulin-induced redistribution of the glucose transporter GLUT4 from intracellular stores to the cell surface. These observations suggest that the glucose transport system of cardiomyocytes represents a metabolic target of inhibition by cGMP, and that this regulation occurs at the level of the trafficking of glucose transporters.     

Beta-angonists modulate ACh-inhibition of a K current in intestinal smooth muscle cells.

ACh causes a long-lasting inhibition of STOCs via G proteins in intestinal smooth muscle cells. We examined the effects of isoproterenol (Iso) on the ACh-induced inhibition of STOCs in isolated ileal smooth muscle cells using the G omega-seal whole cell clamp technique. In control, ACh (1 microM) completely suppressed STOCs, which did not desensitize over a period lasting 20 minutes. When Iso (10 microM) was added to the bath in the presence of ACh, the ACh-induced inhibition of STOCs was gradually removed. This effect of Iso was prevented by propranolol (10 microM). Application of Db-cAMP (500 microM) mimicked the Iso effects. Intracellulary applied GTP-gamma S (100 microM) gradually suppressed STOCs in the absence of ACh, which could not be removed by either Iso or Db-cAMP. These results suggest that beta-adrenergic stimulation causes a removal of the muscarinic inhibition of STOCs via a cAMP-dependent process.     

Inhibition by cyclopropylamine of the quinoprotein methylamine dehydrogenase is mechanism-based and causes covalent cross-linking of alpha and beta subunits

Cyclopropylamine acted as a mechanism-based inhibitor of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans. The protein-bound quinone cofactor of this enzyme was rapidly reduced by addition of a stoichiometric amount of cyclopropylamine, but this compound did not serve as a substrate for the enzyme in the steady-state kinetic assay. Time-dependent inactivation of the enzyme by cyclopropylamine was observed only in the presence of a reoxidant. Saturation behavior was observed, and values of KI of 3.9 microM and K(inact) of 1.7 min-1 were determined. Enzyme inactivation was irreversible, as no restoration of activity was evident after gel filtration of methylamine dehydrogenase which had been incubated with cyclopropylamine in the presence of a reoxidant. The inactivated enzyme exhibited an altered absorption spectrum. Electrophoretic analysis of inactivated methylamine dehydrogenase indicated that covalent cross-linking of the alpha and beta subunits of this alpha 2 beta 2 oligomeric enzyme had occurred and that the quinone cofactor had been modified. A mechanism for this inhibition is proposed which is based upon the data presented and is consistent with the available structural information on methylamine dehydrogenase.     

Studies on the fate of receptor-bound 125I-interleukin 1 beta in porcine synovial fibroblasts

Binding of porcine interleukin 1, radiolabeled with Bolton-Hunter reagent (125I IL 1), to monolayers of porcine synovial fibroblasts (PSF) was found to be a temperature-dependent process. The rate of uptake and the amount of cell-associated ligand was higher at 37 degrees C than at 4 degrees C or 19 degrees C, and exceeded the apparent equilibrium binding capacity. The amount of bound 125I IL 1 that was removed by brief treatment with acidic buffers decreased from 80% at 4 degrees C to 35% for PSF incubated at 37 degrees C; this procedure was used to distinguish surface-bound from internalized ligand. In untreated PSF, surface binding was maximal at 1 hr and was maintained for at least 5 hr during which time the internal pool continued to increase. The lysosomotropic agent methylamine (20 mM) decreased surface binding by 50%; monensin (20 microM) decreased the rate and extent of internalization. Cycloheximide (10 micrograms/ml) did not affect ligand uptake, hence, continual expression of surface receptors could not be ascribed to their de novo synthesis. 40% of the radioactivity taken up by PSF during incubation at 37 degrees C subsequently appeared in the culture medium upon prolonged postincubation (5 hr) in the absence of added 125I IL 1: 60% of this fraction was trichloroacetic acid-soluble in untreated cultures, but the extent of degradation was halved by treatment with methylamine or monensin. Direct measurement of the rate of internalization of prebound 125I IL 1 was obtained by monitoring the formation of covalently cross-linked ligand-receptor complexes after warming PSF monolayers to 37 degrees C. By using gel electrophoresis we observed a decrease (t1/2 = 9 to 11 min) in labeling of the major cross-linkable species.     

Inhibition of cell-plate formation by brefeldin A inhibited the depolymerization of microtubules in the central region of the phragmoplast

Treatment of tobacco BY-2 cells with 20 microM brefeldin A (BFA), which causes disassembly of the Golgi apparatus (Yasuhara et al. 1995), completely inhibited the formation of the cell plate when the treatment was started before the chromosomes had begun to to condense. In cells in which cell-plate formation was inhibited by BFA, the centrifugal development of the phragmoplast was also inhibited. In such cells, the depolymerization of microtubules in the central region of the phragmoplast did not occur at least for 1 h after the formation of the phragmoplast, while the centrifugal development of the phragmoplast and cell-plate formation were completed in almost all cells not treated with BFA. The inhibition of cell-plate formation seems to inhibit the centrifugal development of the phragmoplast by inhibiting the depolymerization of microtubules in the central region of the phragmoplast, which is required for the supply of free tubulin necessary for the polymerization of microtubules at the outer margins of the phragmoplast.     

Receptor-mediated internalization of Pseudomonas toxin by mouse fibroblasts

Pseudomonas exotoxin (PE) was used as a probe to study the mechanism by which protein ligands are internalized by mammalian cells. Both biochemical and electron microscopic methods were used to look at the internalization of PE by mouse LM cell fibroblasts. Our data suggest that PE enters cells by receptor-mediated endocytosis, a process previously thought to be restricted to the entry of biologically significant molecules such as lysosomal enzymes and peptide hormones. Biochemical studies showed that methylamine (20 mM) and chloroquine (10 microM) protected LM cells from the action of PE. Full protection was observed if methylamine or chloroquine was added to the monolayers simultaneously with toxin or if they were added up to 10 min after toxin binding. Later addition of amine or chloroquine afforded partial protection to the monolayers. With immunoelectron microscopy we observed that in the cold toxin bound diffusely to the cell surface but was rapidly internalized when cells were warmed to 37 degrees C. In the presence of methylamine, chloroquine or ammonium chloride, internalization did not occur. We propose that PE enters mouse fibroblasts by receptor-mediated endocytosis and that chloroquine and methylamine, agents which are known to block this process, prevent expression of toxicity.     

Food deprivation is not a prerequisite for the amoebal to plasmodial transition in Physarum polycephalum

The effect of food supply on the onset of asexual and sexual plasmodium formation in Physarum polycephalum was studied. Asexual differentiation occurs readily in amoebae carrying the matAh mating type allele. The density at which these amoebae begin to differentiate is influenced by the ind locus, which controls the production of a diffusible inducer. The alleles ind-1 and ind-2 are known. Strains carring the ind-1 allele begin plasmodium formation at a low amoebal density (rapid differentiation), while strains carring the ind-2 allele differentiate at a higher amoebal density (slow differentiation). The onset of differentiation is characteristic of the strain and did not change with a 20-fold variation in the number of food bacteria available. Sexual differentiation occurs between compatible amoebal strains. For a given pair of amoebal strains the onset of plasmodium formation occurs at a characteristic cell density that is determined by the genetic backgrounds of the strains. The ind locus is one of the genes that influences this cell density. Plasmodia are formed at a lower cell density in crosses involving compatible amoebae carrying the ind-1 allele than they are in crosses with strains carrying the ind-2 allele. As was found for asexual differentiation, an approximate 20-fold variation in the food supply did not affect the initiation of sexual plasmodium formation. These results suggest that in most cases starvation does not trigger the differentiation of amoebae into plasmodia. The time of onset of plasmodium formation is determined largely by genetic factors.     

Arachidonic acid metabolites, hydrogen peroxide, and EDHF in cerebral arteries

We tested the hypotheses that EDHF in rat middle cerebral arteries (MCAs) involves 1) metabolism of arachidonic acid through the epoxygenase pathway, 2) metabolism of arachidonic acid through the lipoxygenase pathway, or 3) reactive oxygen species. EDHF-mediated dilations were elicited in isolated and pressurized rat MCAs by activation of endothelial P2Y(2) receptors with either UTP or ATP. All studies were conducted after the inhibition of nitric oxide synthase and cyclooxygenase with N(omega)-nitro-l-arginine methyl ester (10 microM) and indomethacin (10 microM), respectively. The inhibition of epoxygenase with miconazole (30 microM) did not alter EDHF dilations to UTP, whereas the structurally different epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanoic acid (20 or 40 microM) only modestly inhibited EDHF at the highest concentration of UTP. An antagonist of epoxyeicosatrienoic acids, 14,15-epoxyeicosa-5(Z)-enoic acid, had no effect on EDHF dilations to UTP. Chronic inhibition of epoxygenase in the rat with 1-aminobenzotriazol (50 mg/kg twice daily for 5 days) did not alter EDHF dilations. The inhibition of the lipoxygenase pathway with either 10 microM baicalein or 10 microM nordihydroguaiaretic acid produced no major inhibitory effects on EDHF dilations. The combination of superoxide dismutase (200 U/ml) and catalase (140 U/ml) had no effect on EDHF dilations. Neither tiron (10 mM), a cell-permeable scavenger of reactive oxygen species, nor deferoxamine (1 or 10 mM), an iron chelator that blocks the formation of hydroxyl radicals, altered EDHF dilations in rat MCAs. We conclude that EDHF dilations in the rat MCA do not involve the epoxygenase pathway, lipoxygenase pathway, or reactive oxygen species including H(2)O(2).     

Inhibition of SIN-1–Induced Change in Mitochondrial Membrane Permeability in PC12 Cells by Dopamine

Dopamine (50 or 100 microM) attenuated the nuclear damage and cell death due to 500 microM SIN-1, a donor of superoxide and nitric oxide, in differentiated PC12 cells whereas 200 microM dopamine did not depress cell death. Dopamine at 50-100 microM for a 4-h treatment did not show a significant cytotoxic effect on PC12 cells. Dopamine (100 microM) inhibited the decrease in mitochondrial transmembrane potential, cytochrome c release, activation of caspase-3, formation of reactive oxygen species, and depletion of glutathione (GSH) due to 500 microM SIN-1 in PC12 cells. The reaction of dopamine with peroxynitrite reduced an amount of peroxynitrite. The results suggest that dopamine exhibits a biphasic effect against the cytotoxicity of SIN-1 depending on concentrations. Dopamine at 50-100 microM may attenuate the reactive nitrogen species-induced viability loss in PC12 cells by suppressing the mitochondrial membrane permeability change through inhibition of the formation of reactive species, including peroxynitrite.     

The beta-cell glibenclamide receptor is an ADP-binding protein.

Pathways of bulk protein degradation controlled by insulin and isoprenaline (isoproterenol) were distinguished in Langendorff-perfused rat hearts. Proteins were biosynthetically labelled in vitro with [3H]leucine, followed by addition of 2 mM non-radioactive leucine to competitively prevent reincorporation. Rapidly degraded proteins were eliminated during a 3 h preliminary perfusion period without insulin. One third of bulk myocardial protein degradation was inhibited by isoprenaline as described previously. An insulin concentration of 5 nM maximally inhibited proteolysis, beginning within 2 min. Inhibition reached 32% within 1.25 h and 35% after 1.5 h. The minimum effective insulin concentration was approx. 10-50 pM, which caused 10-20% inhibition. Following 3 h of perfusion without insulin, the lysosomal inhibitor, chloroquine (30 microM), inhibited 38% of bulk degradation. The 35% proteolytic inhibition caused by insulin was followed by very little further inhibition on subsequent concurrent infusion of chloroquine, i.e. the inhibitory effects of insulin and chloroquine were not additive. In contrast, prior inhibition of lysosomal proteolysis by insulin or chloroquine did not prevent the subsequent additive inhibition caused by isoprenaline. Insulin and beta-agonists additively inhibited approx. two-thirds of bulk degradation. The biguanide antihyperglycaemic agent phenformin (2 microM) inhibited 35% of bulk degradation, beginning at 2 min and reaching a near maximum at approx. 1.25-1.5 h. Following inhibition of proteolysis with phenformin (20 microM), subsequent infusion of chloroquine (30 microM) produced only a slight additional inhibition. Following inhibition of 35% of degradation by 1.5 h of perfusion with insulin (5 nM), subsequent exposure to phenformin (2 microM) produced only a slight additional inhibition which did not exceed 38% of basal proteolysis. Thus insulin and phenformin both inhibit lysosomal proteolysis; however, the adrenergic-responsive pathway is distinct.     

Prevention of staurosporine-induced cell death in embryonic chick cardiomyocyte is more dependent on caspase-2 than caspase-3 inhibition and is independent of sphingomyelinase activation and ceramide generation

Apoptosis was induced in embryonic chick cardiomyocytes by staurosporine. Treatment of cardiomyocytes with the preferential caspase-2 inhibitor, z-VDVAD-fmk (100 microM), produced a significant (P < 0.05) although small reduction in the amount of cell death. Ac-DVED-cmk (100 microM), which preferentially inhibits caspase-3 but inhibits to a lesser extent caspase-6, -7, -8, and -10, produced a minimal decrease in cell death. The combination of the caspase-3 and -2 inhibitors produced an additive reduction in cell death after staurosporine (1 microM for 6 h) from 80.4 +/- 0.7 to 54.6 +/- 1.3%. The ability of staurosporine to activate caspase-3 was confirmed in these cardiomyocytes by measurement of caspase-3 activity. A role for ceramide formation, from sphingomyelin to induce caspase activation was unlikely, as there were no changes in cellular ceramide or sphingomyelin after staurosporine treatment of cardiomyocytes when sphingomyelin was labeled by [(3)H]palmitate for 24 h. Neither were there any changes in sphingomyelinase activity. While staurosporine effectively suppressed PKC activity, phorbol 12-myristate 13 acetate did not alter staurosporine-induced cell death or DNA fragmentation. These results demonstrate that, in this model of cardiac cell death, caspase-2 inhibition is of considerable importance, caspase-3 inhibition is of lesser significance but may produce additional effects in the combination with caspase-2 inhibition, and ceramide production from sphingomyelin is not operative in the pathway leading to caspase activation and cell death.     

Ascorbate protects against tert-butyl hydroperoxide inhibition of erythrocyte membrane Ca2+ + Mg2(+)-ATPase

The incubation of erythrocyte suspensions or isolated membranes containing a residual amount of hemoglobin (0.04% of original cellular hemoglobin) with tert-butyl hydroperoxide (tBHP, 0.5 mM) caused significant inhibition of basal and calmodulin-stimulated Ca2+ + Mg2(+)-ATPase activities and the formation of thiobarbituric acid reactive products measured as malondialdehyde. In contrast, the treatment of white ghosts (membranes not containing hemoglobin) with tBHP (0.5 mM) did not lead to appreciable enzyme inhibition within the first 20 min and did not result in malondialdehyde (MDA) formation. However, the addition of either 10 microM hemin or 100 microM ferrous chloride + 1 mM ADP to white ghosts produced hydroperoxide effects similar to those in pink ghosts (membranes with 0.04% hemoglobin). The concentrations of hemin and ferrous chloride which caused half-maximal inhibition of Ca2+ + Mg2(+)-ATPase activity at 10 min were 0.5 and 30 microM, respectively. The effects of several antioxidants (mannitol, thiourea, hydroxyurea, butylated hydroxytoluene, and ascorbate) were investigated for their protective effects against oxidative changes resulting from tBHP treatment. Over a 30-min incubation period only ascorbate significantly reduced the enzyme inhibition, MDA formation, and protein polymerization. Thiourea and hydroxyurea decreased MDA formation and protein polymerization but failed to protect against the enzyme inhibition. Butylated hydroxytoluene was similar to thiourea and hydroxyurea but with better protection at 10 min. Mannitol, under these conditions, was an ineffective antioxidant for all parameters tested.     

Kinetin inhibits protein oxidation and glycoxidation in vitro

We tested the ability of N(6)-furfuryladenine (kinetin) to protect against oxidative and glycoxidative protein damage generated in vitro by sugars and by an iron/ascorbate system. At 50 microM, kinetin was more efficient (82% inhibition) than adenine (49% inhibition) to inhibit the bovine serum albumin (BSA)-pentosidine formation in slow and fast glycation/glycoxidation models. Kinetin also inhibited the formation of BSA-carbonyls after oxidation significantly more than adenine did. However both compounds inhibited the advanced glycation end product (AGE) formation to the same extent (59-68% inhibition). At 200 microM, kinetin but not adenine, limited the aggregation of BSA during glycation. These data suggest that kinetin is a strong inhibitor of oxidative and glycoxidative protein-damage generated in vitro.