RESUMO
CEL I, isolated from celery, is the first eukaryotic nuclease known that cleaves DNA with high specificity at sites of base-substitution mismatch and DNA distortion. The enzyme requires Mg(2+) and Zn(2+) for activity, with a pH optimum at neutral pH. We have purified CEL I 33 000-fold to apparent homogeneity. A key improvement is the use of alpha-methyl-mannoside in the purification buffers to overcome the aggregation of glycoproteins with endogenous lectins. The SDS gel electrophoresis band for the homogeneous CEL I, with and without the removal of its carbohydrate moieties, was extracted, renatured, and shown to have mismatch cutting specificity. After determination of the amino acid sequence of 28% of the CEL I polypeptide, we cloned the CEL I cDNA. Potential orthologs are nucleases putatively encoded by the genes BFN1 of Arabidopsis, ZEN1 of Zinnia, and DSA6 of daylily. Homologies of CEL I with S1 and P1 nucleases are much lower. We propose that CEL I exemplifies a new family of neutral pH optimum, magnesium-stimulated, mismatch duplex-recognizing nucleases, within the S1 superfamily.
Assuntos
Apiaceae/enzimologia , Endonucleases/isolamento & purificação , Proteínas de Plantas/isolamento & purificação , Sequência de Aminoácidos , Apiaceae/genética , Pareamento Incorreto de Bases , Sequência de Bases , Clonagem Molecular , DNA Complementar/isolamento & purificação , DNA de Plantas/isolamento & purificação , Endonucleases/química , Endonucleases/genética , Endonucleases/metabolismo , Concentração de Íons de Hidrogênio , Magnésio/química , Dados de Sequência Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Renaturação Proteica , Substâncias Redutoras/químicaRESUMO
The rabbit retina contained histamine, showed well expressed histamine-methyltransferase activity and marginal histidine decarboxylase activity. Slices of the rabbit retina and hypothalamus took up [(3)H]histamine against a concentration gradient in a temperature-dependent process. [(3)H]Histamine uptake by the rabbit retina was inhibited by ouabain, and was unaffected by imipramine, fluoxetine, nisoxetine and nomifensine (all at 10 ?M concentration). [(3)H]Histamine taken up by the retina was metabolized to tele-methylhistamine and methylimidazoleacetic acid. It is suggested that histamine in the retina may be of some functional importance.
