ABSTRACT
Treatment of membranes from the dog caudate nucleus with sulfhydryl alkylating agents, N-ethylmaleimide and p-chloromercuribenzoate, results in selective inhibition of dopamine-sensitive adenylate cyclase activity that can be distinguished from effects on basal enzyme activity. Fifty per cent inhibition of dopamine-sensitive adenylate cyclase activity was observed in the presence of 10(-5) M N-ethylmaleimide and 3 X 10(-6) M p-chloromercuribenzoate. N-Ethylmaleimide (10(-5) M or less) also inhibited GTP- and NaF-stimulated adenylate cyclase activity, but had no effect on basal adenylate cyclase activity (assayed in the presence of magnesium) and on enzyme activity assayed in the presence of manganese. The reducing agents dithiothreitol, 2-mercaptoethanol, glutathione, and cysteine had no inhibitory effect on dopamine-sensitive adenylate cyclase activity. Pretreatment of membranes with guanyl-5'-yl imidodiphosphate or guanosine 5'-O-(3-thio)triphosphate prior to incubation with N-ethylmaleimide prevented the inhibitory effect of N-ethylmaleimide on adenylate cyclase activity. The results suggest that a reactive sulfhydryl group in the domain of the GTP-binding protein is important for the coupling of the components of the dopamine-sensitive adenylate cyclase complex in brain.
Subject(s)
Adenylyl Cyclases/metabolism , Dopamine/metabolism , Guanine Nucleotides/metabolism , Sulfhydryl Compounds/metabolism , Adenylyl Cyclase Inhibitors , Animals , Corpus Striatum , Dogs , Enzyme Activation/drug effects , Ethylmaleimide/pharmacology , Haloperidol/pharmacology , Sodium Fluoride/pharmacologySubject(s)
Dopamine/metabolism , Receptors, Dopamine/analysis , Sulfhydryl Reagents/pharmacology , Animals , Brain/metabolism , Cattle , Disulfides/metabolism , Dopamine Antagonists , In Vitro Techniques , Receptors, Dopamine/drug effects , Receptors, Dopamine/metabolism , Sulfhydryl Compounds/metabolismABSTRACT
Vitamin K-dependent carboxylation activity measured with pentapeptide substrate (Phe-Leu-Glu-Glu-Leu) gradually decreases upon in vivo injection of vitamin K to vitamin K-deficient rats. A decrease in pentapeptide carboxylation can also be observed by the in vitro addition of antibodies against prothrombin and other vitamin K-dependent proteins to the soluble system derived from vitamin K-deficient rat liver microsomes. In both cases, adding back in vitro partially decarboxylated vitamin K-dependent proteins or purified hepatic prothrombin precursor restores the level of pentapeptide carboxylation. After warfarin treatment, a 3-fold increase in carboxylation results, which can be abolished by giving cycloheximide along with the warfarin. However, the resulting decreased activity is restored by the in vitro addition of partially decarboxylated vitamin K-dependent proteins. These data are consistent with the hypothesis that (after warfarin treatment) increased peptide carboxylation is primarily due to activation of the system by precursor proteins, rather than synthesis of an increased amount of enzyme.
Subject(s)
Hypoprothrombinemias/metabolism , Microsomes, Liver/metabolism , Proteins/metabolism , Prothrombin/biosynthesis , Vitamin K Deficiency/metabolism , Animals , Antigen-Antibody Reactions , Carbon Dioxide/metabolism , Cycloheximide/pharmacology , Immune Sera , Kinetics , Male , Microsomes, Liver/drug effects , Oligopeptides/metabolism , Rats , Vitamin K/pharmacology , WarfarinSubject(s)
Vitamin K/metabolism , Animals , In Vitro Techniques , Microsomes, Liver/metabolism , RatsABSTRACT
The vitamin K-dependent carboxylating system has been solubilized by Lubrol PX or Triton X-100 treatment of vitamin K-deficient rat liver microsomes. As obtained from vitamin K-deficient rat liver, this soluble preparation is dependent upon the in vitro addition of vitamin K1 for carboxylating activity. The enzyme system is complex and is dependent upon NADH and dithiothreitol for maximum activity. While detergents used to solubilize the enzyme complex do markedly inhibit the activity of the system, the solubilized system is still highly responsive to vitamin K addition and can be used for further study of the carboxylating enzyme system. The requirement for dithiothreitol and the inhibition by p-hydroxymercuribenzoate indicate the involvement of an --SH enzyme in the carboxylating system.
