Keith H. Byington

Inhibition of glutathione-S- aryltransferase from rat liver by organogermanium, lead and tin compounds

Abstract Compounds having three carbon—germanium, carbon—tin or carbon—lead bonds were found to be effective inhibitors of rat liver glutathione- S -aryltransferase activity with K i , values in the μM range. Classical competitive inhibition was observed with l,2-dichloro-4-nitrobenzene as the limiting substrate. However, when the second substrate, glutathione, was limiting, inhibition of the mixed type occurred. Within the metallic series IVb, triethylsilane chloride was not active, while the effectiveness as inhibitors increased through triethylgermanium chloride, triethyltin bromide and triethyllead chloride. Except in the case of sulfur, the fourth group or atom bonded to tin in a series of triphenyltin compounds had little effect on inhibitory activity. Sodium sulfide or 2,3-dimercapto-1-propanol (BAL) was capable of protecting glutathione- S -aryltransferase from inhibition by triphenyltin chloride. Glutathione conjugations at aromatic carbon atoms were much more sensitive to inhibition by organotins than were conjugations at alkyl carbons or epoxides.

The hemolytic activity of some trialkyltin and triphenyltin compounds

Abstract Triphenyltin chloride caused the rapid hemolysis of dog, hog, rabbit, or rat blood; however, human blood was resistant to hemolysis by triphenyltin chloride. Trialkytin compounds with alkyl groups having 3–6 carbon atoms were hemolytic to both human and rat blood. Tri-n-butyltin derivatives in which the fourth group or atom bonded to tin was acetate, methoxide, hydroxide, oxygen, bromide or chloride were all hemolytic; however, bis(triphenyltin) sulfide was not hemolytic. Butyltins having 1, 2 or 4 carbon-tin bonds, trimethyltin chloride and triethyltin bromide all exhibited low hemolytic activity. It was demonstrated that the rate of hemolysis of human blood by tri-n-butyltin chloride was reduced when the solutes of the medium were predominantly nonelectrolytes and that potassium leaked from the cells faster than hemoglobin during tri-n-butyltin induced hemolysis. A nitrogen atmosphere or the addition of low concentrations of dl -α-tocopherol also reduced the rate of hemolysis of human blood in the presence of tri-n-butyltin chloride. Human plasma, but not human serum albumin, was partially effective in protecting washed, human red cells from tri-n-butyltin induced hemolysis. Sodium sulfide and several sulfhydryl compounds, in concentrations about equal to that of the organotin, protected human and rat blood from the hemolytic activity of tri-n-butyltin chloride, tricyclohexyltin hydroxide, or triphenyltin chloride. It was concluded that the most hemolytic organotins were the trialkyltins with alkyl groups having 3–6 carbon atoms and that some sulfhydryl compounds can react chemically with the organotins to reduce hemolytic activity.

On the structure-activity relationships and mechanism of organotin induced, nonenergy dependent swelling of liver mitochondria

Abstract Organotin-induced, nonenergy dependent swelling of isolated rat liver mitochondria has been investigated. The organotins which were most active as swelling agents had three carbon-tin bonds, n -octanol: water partition coefficients greater than one, extracted chloride ions from water into benzene, and facilitated the diffusion of chloride ions through a benzene phase. Compounds having one, two, or four carbon-tin bonds exhibited low activity as swelling agents. Mitochondria were most sensitive to organotin-induced swelling when the major cation in the medium was the ammonium or triethylammonium ion; however, high concentrations of active organotins caused swelling which was independent of the nature of the medium. Bis-(triphenyltin)-sulfide exhibited low activity and sodium sulfide, BAL, or dithiothreitol protected mitochondria from organotin-induced swelling. The results suggest that low levels of active organotins may induce swelling by acting as foreign, anion specific ionophores in the inner mitochondrial membrane and that saturation of high capacity-low affinity mitochondrial binding sites by these compounds results in extensive and nonspecific alterations of the permeability of the mitochondrial membranes.

Kidney membrane cyclic AMP receptor and cyclic AMP-dependent protein kinase activities: Comparison of plasma membrane and cytoplasmic fractions

Abstract Hog kidney cortex plasma membrane and cytosol fractions were utilized for comparison of cyclic AMP receptor and cyclic AMP-dependent protein kinase activities. Both preparations exhibited relatively high affinity for cyclic 3′5′ AMP and cyclic AMP activation of the phosphorylation of protamine. In addition cyclic AMP enhanced the phosphorylation of plasma membranes. The specificity of the membrane receptor appeared to be greater than the cytosol receptor as determined by competitive binding studies. The renal plasma membrane receptor could be solubilized with the detergents Lubrol-WX or sodium deoxycholate without inhibiting receptor activity. Triton X-100 solubilized membrane protein but also reduced 3H-cyclic AMP binding activity.

Effects of triphenyltin compounds on the adenosine triphophatase activity of beef heart submitochondrial particles

Abstract Low concentrations of several TPT compounds inhibit the ATPase activity of ETPH. Higher concentrations of these compounds cause a partial release of the inhibition and a concomitant loss of rutamycin sensitivity. Inhibition produced by low levels of TPT chloride is noncompetitive and the inhibitor constant is 2.8 × 10 −8 M. Several sulfur compounds are capable of partially protecting the ATPase from inhibition by low levels of TPT chloride. Heat treatment destroys the sensitivity of the ATPase to inhibition by TPT chloride and rutamycin.

Studies of kidney plasma membrane adenosine-3′,5′-monophosphate-dependent protein kinase

Abstract Porcine kidney cortex was utilized for the preparation of plasma-membrane-enriched and soluble cytoplasmic (cytosol) fractions for the purpose of examining the relative properties of cyclic [3H]AMP receptor and cyclic AMP-dependent protein kinase activities of these preparations. The affinity, specificity and reversibility of cyclic [3H]AMP interaction with renal membrane and cytosol binding sites were indicative of physiological receptors. Binding sites of cytosol and deoxycholate-solubilized membranes were half-saturated at approx. 50nM and 100 nM cyclic [3H]AMP. Native plasma membranes exhibited multiple binding sites which were not saturated up to 1 mM cyclic [3H]AMP. Modification of the cyclic phosphate configuration or 2′-hydroxyl of the ribose moiety of cyclic AMP produced a marked reduction in the effectiveness of the cyclic AMP analogue as a competitor with cyclic [3H]AMP for renal receptors. The cyclic [3H]AMP interaction with membrane and cytosol fractions was reversible and the rate and extent of dissociation of bound cyclic [3H]AMP was temperature dependent. With the plasma-membrane preparation, dissociation of cyclic [3H]AMP was enhanced by ATP or AMP. Assay of both kidney subcellular fractions for protein kinase activity revealed that cyclic AMP enhanced the phosphorylation of protamine, lysine-rich and arginine-rich histones but not casein. The potency and efficacy of activation of renal membrane and cytosol protein kinase by cyclic AMP analogues such as N6-butyryl-adenosine cyclic 3′,5′-monophosphate or N6,O2-dibutyryl-adenosine cyclic 3′,5′-monophosphate supported the observations on the effectiveness of cyclic AMP analogues as competitors with cyclic [3H]AMP in competitive binding assays. This study suggested that the membrane cyclic [3H]AMP receptors may be closely associated with the membrane-bound catalytic moiety of the cyclic AMP-dependent protein kinase system of porcine kidney.

Binding of triethyllead chloride by hemoglobin.

Abstract The interaction of triethyllead chloride with human and rat hemoglobin has been investigated using equilibrium dialysis. In addition the uptake of triethyllead by washed human and rat erythrocytes has been studied in vitro and the distribution of the organolead between blood cells and plamsa has been examined in vivo in the rat. Total lead was determined to atomic absorption spectrophotometry. Equilibrium dialysis demonstrated that human hemoglobin had a very low affinity and capacity for the binding of triethyllead. On the other hand, each tetramer of rat hemoglobin bound 3 mol of triethyllead at sites which did not exhibit cooperativity. ATP, EDTA, Na2S, and NaNO2 had no effect on the binding of triethyllead by rat hemoglobin while urea, glutathione, and N-ethylmaleimide inhibited the interaction. Rat red cells took up more triethyllead than did human red cells and the difference was not due to a higher binding capacity of the rat cell membrane for the organometal. For 80 min following the intravenous injection of triethyllead chloride, the total blood lead in rats was about 10 times the total plasma lead. It was concluded that triethyllead binds to the globin moiety of rat hemoglobin and that organometal binding sites occur on the hemoglobin tetramer but not on the monomers. The results demonstrated that the binding of triethyllead to hemoglobin is a significant determinant of the pharmacokinetics of the organometal in the rat and that the interaction between triethyllead and human hemoglobin would not have a significant effect on the pharmacokinetics of the organometal in the human.

The effects of acetaldehyde on the kinetics of the energy-dependent phosphate-induced swelling of rat liver mitochondria

Abstract Acetaldehyde in the range of 1 to 5 mM increases the rate and extent of swelling of rat liver mitochondria with either DL-3-hydroxybutyrate or succinate as substrate. Higher concentrations of acetaldehyde first increase the rate of swelling and then completely inhibit swelling. Exposure of mitochondria to 5 mM acetaldehyde does not alter the requirements of swelling for energy and inorganic phosphate. The results demonstrate that acetaldehyde is capable of altering the kinetics of swelling under conditions which prevent the oxidation of acetaldehyde.

p-Azidoclonidine: A photoaffinity label for the α2-adrenoceptor

Abstract We have synthesized and characterized p-azidoclonidine (AZC) as a putative α2-adrenoceptor photoaffinity label. [3H]AZC demonstrated high affinity (KD = 11.8 ± 2.5 nM, saturability (Bmax = 171 ± 21 fmol/mg protein), stereo-specificity, and rank order of potency expected of a specific α2-receptor label when used as a reversible ligand in the rat cerebral cortex. The pharmacologic profile of AZC was similar to p-aminoclonidine (PAC), an established α2-adrenoceptor partial agonist. Membranes covalently prelabeled with nonradioactive AZC showed a dose dependent decrease in the number of α2-receptor sites subsequently detected by [3H]PAC and [3H]yohimbine. Specific covalent [3H]AZC binding to rat cerebral cortical α2-receptors represented 35 ± 7% of the total [3H]AZC bound. These data indicate that AZC is a selective α2-adrenoceptor photoaffinity lavel which may be useful in the identification and purification of the α2-Adrenoceptor.

Interactions of acetaldehyde, ethyl alcohol and oxybarbiturates affecting mitochondrial functions☆

Abstract The effects of combinations of acetaldehyde, ethyl alcohol and an oxybarbiturate on the rate of oxygen consumption, the ADP/O ratio and energy-dependent swelling by rat liver mitochondria have been investigated. The effects of similar combinations on the rate of oxygen uptake by submitochondrial preparations, oxidizing several different substrates, have also been studied. Acetaldehyde at millimolar levels increased the rate of oxygen consumption by rat liver mitochondria which had been preincubated with ADP, inhibited NAD + -linked oxidations by mitochondrial and submitochondrial preparations, and increased the rate of the energy-dependent swelling of rat liver mitochondria with suceinate as substrate. Combinations of acetaldehyde, ethyl alcohol and an oxybarbiturate interacted to reduce the rate of oxygen consumption by rat liver mitochondria and the energy-dependent swelling of rat liver mitochondria when dl -3-hydroxybutyrate was the substrate. Ethyl alcohol was not involved in these interactions. The effects of all combinations tested could be ascribed to interactions) between acetaldehyde and an oxybarbiturate or to activities of acetaldehyde or the oxybarbiturate only. In the case of state 3 respiration and uncoupled mitochondrial respiration, acetaldehyde and the tested oxybarbiturates behaved as additive inhibitors. All of the results are consistent with the view that the NADH-ubiquinone segment (Complex 1) of the mitochondrial electron transport chain is the site of an acetaldehyde-oxybarbiturate interaction to inhibit electron transport.