Inhibition of Red Cell and Yeast Hexokinase by Triethyltin Bromide [(C\u3csub\u3e2\u3c/sub\u3eH\u3csub\u3e5\u3c/sub\u3e)\u3csub\u3e3\u3c/sub\u3eSnBr]

Triethyltin bromide was found to be a selective inhibitor of red cell hexokinase. When intact red cell suspensions were exposed to the reagent (0.025–0.5 mM), inhibition of hexokinase occurred without any hemolysis and without effects on the activity of other glycolytic enzymes. Yeast hexokinase was also inhibited by triethyltin. The red cell and yeast hexokinases were protected against inhibition by triethyltin when the sugar substrates of the enzymes were included in the incubation mixture. This work identifies hexokinase as another among a limited number of proteins that are known to interact with triethyltin

The Effects of Triethyltin Bromide on Red Cell and Brain Cyclic AMP-Dependent Protein Kinases

Triethyltin bromide activates the cyclic AMP-dependent protein kinases of human red cell membranes and of bovine brain. Additions of 25-500 microM triethyltin to red cell ghosts resulted in enhanced phosphorylation of ghost proteins. When added to partially purified cyclic AMP-dependent protein kinases from red cell ghosts or bovine brain, stimulation of the phosphorylation of calf thymus histone was observed. The enhancement of kinase activity was due to release of catalytic subunits from the intact protein kinase. Brief exposure of the partially purified enzymes to triethyltin, followed by DE52 chromatography, resulted in elution profiles for regulatory and catalytic subunits that were similar to the profile resulting after cyclic AMP activation. Triethyltin interacts with both regulatory and catalytic subunits. When it was added to the partially purified cyclic AMP-dependent protein kinases from human red cell ghosts or bovine brain, noncompetitive inhibition of cyclic AMP binding to the regulatory subunit of the enzyme was observed. It interacted with the catalytic subunit to produce slow inhibition of catalytic activity. The inhibition was non-competitive with respect to both histone and ATP. When intact red cells were subjected to brief exposure with triethyltin, enhanced phosphorylation of certain membrane proteins occurred, suggesting that the activation of the cyclic AMP protein kinases by triethyltin may be physiologically significant

Inactivation of Yeast Hexokinase B by Triethyltin Bromide and Reactivation by Dithiothreitol and Glucose

Binding of triethyltin bromide to yeast hexokinase B results in a rapid change in the reactivity of the sulfhydryl groups of the molecule. The change was characterized by an increased rate as well as extent of reaction of the -SH groups, and it preceded the onset of inhibition of the enzyme. Rapid gel filtration of the enzyme-triethyltin complex reversed this change in sulfhydryl reactivity, and when the eluted enzyme was subjected to short incubation periods, the slow inhibition that occurs with the unfiltered enzyme-triethytin complex was no longer manifested. With prolonged incubation, however, the gel-filtered sample demonstrated increased rate of loss of enzyme activity, indicating that the gel filtration step did not completely reverse the effects of triethyltin on the enzyme. Active enzyme was recovered, following the inactivation of yeast hexokinase with triethyltin, by incubation of the inactivated enzyme with a large excess of glucose and dithiothreitol. Near total recovery of enzyme activity with reversion to native enzyme conformation was achieved following incubation at 35 ℃ of the enzyme with glucose and dithiothreitol each 0.1 M. The possible involvement of either cysteine or histidine in the binding of triethyltin to the enzyme was probed, and it was concluded that neither of these amino acids are donor ligands for tin

Interactions of Triethyltin Bromide with Components of the Red Cell

Triethyltin bromide interacts with the cat hemoglobin and increases its oxygen affinity. The bound complex was visualized by isoelectric focusing on acrylamide gels. Human and trout hemoglobins showed no evidence of interaction with the reagent when examined in the same way. Triethyltin bromide was found, however, to cause hemolysis of trout, cat, and human red cells at physiological temperatures. The hemolytic effect was associated with inhibition of glycolysis as well as of membrane ATPase. Inhibition of glycolysis was identified with selective inhibition of hexokinase. Thus, in addition to cat hemoglobin, hexokinase and membrane ATPase are identified as red cell components that demonstrate functional consequences of interaction with triethyltin bromide. These three proteins are among the few that are known to bind the reagent

Inactivation of Yeast Hexokinase B by Triethyltin Bromide

Triethyltin bromide was found to demonstrate temperature-dependent inactivation of yeast hexokinase B. At temperatures of 20 °C or lower, little or no inactivation of the enzyme was detected after 2 h of reaction with 50-300 µM concentrations of the reagent. However, incubation at 25 °C or higher resulted in an increased rate and extent of loss of the enzyme activity with increasing incubation temperatures. The Arrhenius plot for the inactivation process showed a sharp break at approximately 30 °C, with a heat of activation (∆H*) above this temperature of 55.2 kcal, indicating that a triethyltin-induced conformational change occurred at the elevated temperatures. Sugar substrates provided protection against the inactivating effect by reducing the binding triethyltin to the enzyme. In the absence of glucose, two sites of different affinity for triethyltin to the enzyme was associated with a slower subsequent event. Comparative effects of various organotin compounds on the activity of the enzyme indicated that inhibitory potency was associated with increasing hydrophobicity of the alkyl groups attached to the tin

The Effect of Temperature on the Inhibition of Trout, Carp and Human Red Cell Hexokinase by Triethyltin Bromide

1. At temperatures below 20°C, brook and rainbow trout red cell hexokinases demonstrate sensitivity to the inhibitory effects of triethyltin bromide. 2. When the incubation temperature was raised to above 25°C, carp and human red cell enzymes were affected. 3. Arrhenius plots indicated that the ΔH∗ of the reaction was about 11 kcal for the trout enzymes and 40 kcal for the carp and human enzymes. 4. The differential temperature sensitivity of the trout and human enzymes to triethyltin is correlated with the temperature sensitivity of the corresponding red cells toward the hemolytic effect of the reagent

Interaction of Triethyltin with Cat Hemoglobin: Identification of Binding Sites and Effects on Hemoglobin Function

Binding of triethyltin to the cat hemoglobins (HbA and HbB) results in the “masking” of two of the freely reactive sulfhydryl groups (-SH) within the hemoglobin tetramer. That the “masked” -SH groups occur in position 13α of each α-subunit was demonstrated by the lack of labeling of cysteine 13α with [14C]N-ethylmaleimide when triethyltin is present. Studies with cat-human hybrid hemoglobins indicate that the α-subunit of the cat hemoglobins alone is involved in the formation of a complex with triethyltin. Using available data on the primary as well as three dimensional structures of animal hemoglobins, it is suggested the cysteine 13α and histidine 20α serve as axial ligands in the formation of a pentacoordinate triethyltin cat hemoglobin complex. The binding of triethyltin results in an increase in the oxygen affinity of the two cat hemoglobins