Partial characterization of guanylyl cyclase activity in calf thyroid.
The aim of the present study was to perform the partial characterization of the enzyme guanylyl cyclase (GC) in bovine thyroid. The results obtained showed the presence of two types of GC: one is soluble and comprises around 79% of total activity, while the other is particulate. Treatment with 1% Triton X-100 increased both activities. When the kinetics of the enzyme was analyzed, using the complex MnGTP as a substrate, the results showed a Michaelis type kinetics for the soluble enzyme, with a Km of 0.037 mM, whereas the particulate GC showed a positive allosteric behavior with a S0.5 of 0.214 mM and a Hill coefficient of 1.9, indicating that the enzyme has at least two binding sites for the substrate. When the influence of different Mn2+ concentrations was studied, a positive allosteric behavior for the soluble GC was found, with a S0.5 of 1.2 mM and a Hill coefficient of 2.2. The kinetics of the particulate enzyme under similar conditions was of Michaelis type, with a Km of 0.752 mM. Although the enzyme is highly dependent on Mn2+, it was of interest to investigate the possible effects of other divalent cations, such as Ca2+ and Mg2+. The replacement of Mn2+ for Mg2+ caused a complete disappearance of the particulate enzyme activity, while the soluble activity decreased by 85%. Addition of Ca2+ had no effect on either GC. However, with suboptimum. concentrations of Mn2+, high Ca2+ concentration caused an increase in soluble activity, but it comprised only 20% of maximum activity with optimum Mn2+ concentrations. With the particulate enzyme a slight but significant inhibition was observed.
Bocanera
L V
LV
División Bioquímica Nuclear, Unidad de Actividad Radiobiologia, Comisión Nacional de Energía Atómica.
Martinetto
H
H
Flawiá
M M
MM
Pisarev
M A
MA
D000494:Allosteric Regulation; D000818:Animals; D009320:Atrial Natriuretic Factor / Q000494:pharmacology; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000378:metabolism*; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D009599:Nitroprusside / Q000494:pharmacology; D017830:Octoxynol / Q000494:pharmacology; D012995:Solubility / Q000187:drug effects; D013961:Thyroid Gland / Q000201:enzymology*
D002413:Cations, Divalent; D009599:Nitroprusside; D008345:Manganese; C037047:manganese GTP; D009320:Atrial Natriuretic Factor; D006160:Guanosine Triphosphate; D017830:Octoxynol; D006162:Guanylate Cyclase; D006152:Cyclic GMP