The reversible complex between the metalloenzyme bovine carbonic anhydrase B and the sulfonamide inhibitor acetazolamide can be "frozen" irreversibly by the addition of a covalent bond between the methyl group of the inhibitor and the tau-nitrogen of histidine-64. In both cases the inhibited enzyme is inactive as an esterase toward p-nitrophenyl propionate at physiological pH but retains activity controlled by an ionization in the protein exhibiting a pK-a greater than 10. Similarly, both the covalently and reversibly inhibited enzymes in which the catalytically essential Zn(II) ion has been replaced with Co(II) display the same visible absorption spectrum which is invariant over the pH range from 5 to 12. The evidence therefore indicates that the position of the acetazolamide moiety in the active site is independent of both pH and the presence of the covalent bond to histidine-64. Moreover, when reversibly bound, this inhibitor has been shown to replace the water molecule (or hydroxide ion) known to occupy the fourth coordination position of the metal ion and frequently implicated in the catalytic mechanism of carbonic anhydrases. Thus, the activity exhibited by the inhibited enzymes and consequently the second rise observed in the pH rate profile of the native enzyme above pH 0 cannot reflect the ionization of such a water molecule in contrast to what has been postulated previously (Pocker, Y., and Storm, D. R. (1968) Biochemistry 7, 1202-1214). Displacement of the zinc-bound solvent molecule rather than the alkylation of histidine-64 is suggested, however, as the cause of the inactivation of the alkylated enzyme round neutrality. Taken together, the biphasic pH rate profile of native bovine carbonic anhydrase B as well as the activity retained by the alkylated enzyme above pH 9 are best described by a model in which two groups in the enzyme ionize independently, thereby raising the possibility that the high pH activity is controlled by an ionization outside the active site region of the enzyme. Above pH 9.5 the pK; for the reversible interaction between native carbonic anhydrase and acetazolamide falls off linearly with increasing pH. The slope of --1.56 suggests that, among other factors, more than one ionization is responsible for the descending limb of the pH-i-pH profile.

Acetazolamide↗

PubMed1975

Aspects of the adrenal function in the domestic fowl.

Chromatographic purification of extracts of hen peripheral plasma on Florisil columns before measurement by spectrofluorometry showed a basal level for corticosterone of 1-3 mug/100 ml, which is much lower than concentrations previously reported using acid fluorescence. Neither handling, restraint nor repeated bleeding affected the concentration of this hormone or of glucose. Adrenal function tests with two preparations of synthetic corticotrophin showed that they caused a rapid rise in blood corticosterone and eventually of glucose. Results of studies using insulin or tolbutamide i.v. suggest that there is a threshold concentration of plasma glucose (about 70 mg/100 ml) below which hypoglycaemia stimulates the hypothalamic-pituitary-adrenal system in fowls. Prolonged treatment of laying hens with protamine zinc insulin led to aphagia and cessation of egg-laying; increased concentrations of corticosterone were observed 2 days after the administration of insulin ceased, coinciding with the return to normal plasma levels of glucose. 'Chemical adrenalectomy' with metyrapone showed that the restoration of plasma glucose to a normal concentration after insulin treatment is dependent upon fully functional adrenal cortical tissue. It appears likely that the adrenal medulla is a target for corticosterone which probably regulates the tissue levels of phenylethanolamine-N-methyltransferase, one of the enzymes necessary for the biosynthesis of adrenaline.

Adrenal Cortex Function Tests↗

PubMed1973