Assuntos
Adrenalectomia , Nucleotídeos de Guanina/farmacologia , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Guanilil Imidodifosfato/farmacologia , Fígado/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos/metabolismo , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Epinefrina/farmacologia , Feminino , Cinética , Prazosina/metabolismo , Ratos , Ratos Endogâmicos , Receptores Adrenérgicos alfa/efeitos dos fármacos , Ribonucleotídeos/farmacologiaAssuntos
Fígado/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos/metabolismo , Animais , Sítios de Ligação , Quimotripsina/farmacologia , Di-Hidroergotoxina/metabolismo , Feminino , Nucleotídeos de Guanina/farmacologia , Técnicas In Vitro , Modelos Químicos , Norepinefrina/metabolismo , Fenoxibenzamina/farmacologia , Prazosina/metabolismo , Ratos , Receptores Adrenérgicos alfa/efeitos dos fármacosAssuntos
Fígado/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Antagonistas Adrenérgicos alfa/farmacologia , Animais , Ligação Competitiva , Membrana Celular/metabolismo , Ativação Enzimática , Nucleotídeos de Guanina/farmacologia , Cinética , Fenoxibenzamina/metabolismo , Fosforilases/metabolismo , RatosAssuntos
Membrana Celular/metabolismo , Nucleotídeos de Guanina/metabolismo , Proteínas de Membrana/metabolismo , Norepinefrina/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Receptores Adrenérgicos/metabolismo , Animais , Hidrólise , Cinética , Fígado/metabolismo , Peptídeo Hidrolases/metabolismo , RatosAssuntos
Adenilil Ciclases/metabolismo , Ductos Biliares/fisiologia , Fígado/metabolismo , Receptores Adrenérgicos beta/metabolismo , Receptores Adrenérgicos/metabolismo , Animais , Membrana Celular/enzimologia , Colestase/metabolismo , Di-Hidroalprenolol/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Feminino , Glucagon/farmacologia , Isoproterenol/farmacologia , Cinética , RatosRESUMO
The beta 1-adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (--)-[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography. Binding of (--)-[3H]dihydroalprenolol to the membranes occurred to a single class of non-cooperative binding sites (0.2--0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (+/- 2) nM. These sites were identified as the functional, adenylate-cyclase-linked beta 1-adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30 degrees C; secondly, the stereospecific displacement of bound (--)-[3H]dihydroalprenolol by beta-adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol congruent to protokylol greater than norepinephrine congruent to epinephrine) similar to the one found for adenylate cyclase activation, and typical for beta 1-adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30--0.45 pmol/mg protein). Solubilized receptors were purified 500--2000-fold by affinity chromatography with a 25 to 35% yield, using an alprenolol-agarose affinity matrix. Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules. Membrane-bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N-ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.
Assuntos
Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Receptores Adrenérgicos beta/isolamento & purificação , Receptores Adrenérgicos/isolamento & purificação , Adenilil Ciclases/sangue , Adenilil Ciclases/isolamento & purificação , Animais , Ligação Competitiva , Cromatografia de Afinidade , Di-Hidroalprenolol/sangue , Cinética , Receptores Adrenérgicos beta/metabolismo , Relação Estrutura-Atividade , PerusAssuntos
Antagonistas Adrenérgicos beta/farmacologia , Fígado/efeitos dos fármacos , Propanolaminas/farmacologia , Adenilil Ciclases/metabolismo , Animais , Ligação Competitiva , Compostos Bicíclicos com Pontes/farmacologia , Membrana Celular/efeitos dos fármacos , Técnicas In Vitro , Isoproterenol/farmacologia , Fígado/enzimologia , Fígado/ultraestrutura , Ratos , Fatores de TempoAssuntos
Antagonistas Adrenérgicos beta , Coração/efeitos dos fármacos , Propanolaminas/farmacologia , Adenilil Ciclases/metabolismo , Alprenolol/farmacologia , Animais , Compostos Bicíclicos com Pontes/farmacologia , Estabilidade de Medicamentos , Técnicas In Vitro , Isoproterenol/farmacologia , Masculino , Miocárdio/enzimologia , Propranolol/farmacologia , Ratos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologia , Fatores de TempoRESUMO
The adenylate cyclase [ATP pyrophosphatelyase (cyclizing); EC 4.6.1.1] and beta-adrenergic receptor of plasma membranes of turkey erythrocytes were solubilized in an active form by treatment with either NaF or guanylylimidodiphosphate and digitonin. The solubilized enzyme was no longer stimulated by catecholamines, NaF, or guanine nucleotides. The digitonin extract was chromatographed on an alprenolol-agarose derivative. While the bulk of protein and all the adenylate cyclase activity passed unretarded through the column, the receptor was retained. It eluted free of enzyme activity with an alprenolol solution containing 1 M NaCl; the yield was 25-30%. The protein content of the alprenolol eluates was too low to be estimated by the Lowry technique and was assessed by a more sensitive fluorometric method. Under these conditions, the beta-adrenergic receptor was purified approximately 2000-fold in a single step with retention of all its pharmacological properties. These experiments establish that the beta-adrenergic receptor and the adenylate cyclase are independent entities which may be separated on a functional basis.
Assuntos
Adenilil Ciclases/sangue , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Receptores Adrenérgicos beta , Receptores Adrenérgicos , Adenilil Ciclases/isolamento & purificação , Alprenolol/metabolismo , Animais , Ligação Competitiva , Cromatografia de Afinidade , Cinética , Receptores Adrenérgicos/isolamento & purificação , Receptores Adrenérgicos beta/isolamento & purificação , Receptores Adrenérgicos beta/metabolismo , PerusRESUMO
There are conflicting reports concerning the number of distinct delta5-3-oxosteroid isomerases present in various steroid producing tissues; this problem was reexamined by two techniques: (1) kinetics measurements with a mixture of androst-5-ene-3,17-dione and pregn-5-en-3, 20-dione. (2) thermal inactivation of the androst-5-ene-3,17-dione isomerase and pregn-5-ene-3,20-dione isomerase activities. The results are the following: (a) Kinetic data support the concept of a single delta5-3-oxosteroid isomerase with a low substrate specificity in the microsomes. (b) Thermal inactivation experiments also support the one enzyme theory, but strongly suggest an heterogeneity of the delta5-3-oxosteroid isomerase in the bovine adrenocortical microsomes. In view of these results, a tentative explanation of the conflicting reports is proposed.
Assuntos
Córtex Suprarrenal/enzimologia , Glândulas Suprarrenais/enzimologia , Isomerases/metabolismo , Microssomos/enzimologia , Esteroide Isomerases/metabolismo , Animais , Bovinos , Estabilidade de Medicamentos , Temperatura Alta , Cinética , Membranas/enzimologiaRESUMO
The role of phospholipid in the beef adrenal microsomal 3-oxosteroid delta4-delta5-isomerase (EC 5.3.1.1) has been investigated with the use of phospholipase A to alter the microsomal phospholipids. The byproducts of phospholipase A digestion have been removed with a wash solution containing bovine serum albumin. Removal of 80-85% of the phospholipid leads to loss of 80-90% of the 3-oxosteroid delta4-delta5-isomerase activity. Reconstitution experiments have been performed by introduction of lipid aqueous dispersions in the enzymatic assay. Asolectin, a commercially available preparation of soy phosphatides, is able to stimulate the enzymatic activity but does not restore the 3-oxosteroid delta4-delta5-isomerase activity in phospholipase-A-treated membranes. In contrast, the introduction of aqueous dispersions of microsomal total lipid mixtures in the enzymatic assay brings about a complete restoration of the 3-oxosteroid delta4-delta5-isomerase activity in the lipid-depleted membranes. It is concluded that the bovine adrenal microsomal 3-oxosteroid delta4-delta5-isomerase requires phospholipid(s) to exhibit its full catalytic activity.