Metal-directed affinity labeling of zinc(II), cobalt(II) and cadmium(II) horse liver alcohol dehydrogenases

The active site metal in horse liver alcohol dehydrogenase has been studied by metal-directed affinity labeling of the native zinc(II) enzyme and that substituted with cobalt(II) or cadmium(II). Reversible binding of bromoimidazolyl propionic acid to the cobalt enzyme blueshifts the visible absorption band originating from the catalytic cobalt atom at 655 to 630 nm. Binding of imidazole to the cobalt(II) enzyme redshifts the 655 nm band to 667 nm. Addition of bromoimidazolyl propionic acid blueshifts this 667 nm band back to 630 nm. This proves direct binding of the label to the active site metal in competition with imidazole. The affinity of the label for the reversible binding site in the three enzymes follows the order Zn ? Cd ? Co. After reversible complex formation, bromoimidazolyl propionic acid alkylates cysteine-46, one of the protein ligands to the active site metal. The nucleophilic reactivity of this metal-mercaptide bond in each reversible complex follows the order Co ? Zn ? Cd.     

Ionization-linked cooperative interactions in the active site of horse liver alcohol dehydrogenase

Affinity labeling of horse liver alcohol dehydrogenase with iodoacetate in the presence of the activator imidazole has been studied from pH 6.1 to 10.5. The pH profiles for the dissociation constants of iodoacetate from the free enzyme and the enzyme-imidazole complex and of imidazole from the free enzyme and the binary enzyme-iodoacetate complex were determined. The variation with pH of the dissociation constants of iodoacetate (KI) and imidazole (KL) have in common a pKa of 8.6 assigned to the zinc-water ionization, and a pKa near 10. Lysine modification by ethyl acetimidate results in a higher affinity of iodoacetate to the enzyme at high pH as the pKa values of the lysine residues are increased. The binding of iodoacetate and imidazole at each enzyme subunit shows negative cooperativity at pH less than 9, with an interaction constant of 4.8 at pH 6.1. Positive cooperativity is observed at pH greater than 9, with an interaction constant of 0.5 at pH 10.5. The pH-dependent change in cooperativity results from the removal of the zinc-water ionization when imidazole becomes coordinated to the catalytic zinc ion. When iodoacetate binds at the anion binding site, a large perturbation of the zinc-water ionization is observed. Unlike imidazole, the binding of 1,10-orthophenanthroline and iodoacetate shows positive cooperativity at both pH 8.2 and 10.0 with an interaction constant as low as 0.06 at pH 10.0.     

Complex formation of zinc,cadmium, and mercury with penicillamine

The complex formation of zinc, cadmium, and mercury with D-penicillamine has been studied by pH titrations, using computer evaluation of the most likely complexes, which were found to be of the general formulas ML, MH₂L₂, MHL₂-, ML₂^2?, and ML₃^4?. The formation constants of the complexes were determined at 25 0°C in 0.1 M KNO₃. The magnitude of the respective constants cannot, by itself, account for the lack of effect of penicillamine treatment for mercury and cadmium poisoning.     

2,2-dipyridyl binding to metal substituted horse liver alcohol dehydrogenase

The binding of 2,2-dipyridyl to metal substituted horse liver alcohol dehydrogenase was measured by spectrophotometric titrations. Large changes in the visible absorption spectra were seen for the Co2+, Cu2+ and Ni2+ hybrids upon coordination of 2,2-dipyridyl, due to a change in coordination number. The formation constants for binding to the Co2+ and Cd2+ hybrids are of the order 10(6) M-1, which means that these hybrids have a 500-fold higher affinity for 2,2-dipyridyl than the native Zn2+ enzyme. 2,2-dipyridyl has a 100-fold higher affinity for enzyme bound Cd2+ than for aqueous Cd2+ ions, while for Cu2+ and Zn2+ the opposite is the case. None of the substituted metal ions were removed from the active site during titration with the chelator 2,2-dipyridyl.     

Effects of the high-molecular-weight cryoprotectant dextran on fluid secretion by Calliphora salivary glands

The rate of fluid secretion by isolated salivary glands of Calliphora was inhibited as a linear function of dextran and poly vinyl pyrrolidone concentrations in the range 15–35% w/w. This inhibition was not overcome by supramaximal concentrations of 5-hydroxytryptamine, nor was it caused by a decreased availability of K⁺ from the medium. Although the polymers caused large decreases of freezing point (and vapor pressure) of the incubation medium, the glands did not respond to this by secreting a more K ⁺-rich saliva. When dextran and polyvinyl pyrrolidone were added as powders to salt solutions, the total freezing-point depression of the mixture was equal to the sum of that exerted by the pure salt solution and that expected for the polymer concentration. The activities of K⁺ and Cl^?, as measured by ion-selective electrodes, were not increased in solutions by the addition of dextran. Dextran was demonstrated by electron microscopy to penetrate into the basal clefts and intercellular spaces of the isolated glands. These results demonstrate that addition of dextran (and probably of polyvinyl pyrrolidone) does not decrease the solvent activity of water in physiological salt solutions. The inhibition of fluid secretion by isolated salivary glands of Calliphora seems therefore due only to the altered physical characteristics of the medium.