RESUMEN
The FMN-heme intraprotein electron transfer (IET) kinetics in a human inducible NOS (iNOS) oxygenase/FMN (oxyFMN) construct co-expressed with NCaM, a truncated calmodulin (CaM) construct that includes only its N-terminal globular domain consisting of residues 1-75, were determined by laser flash photolysis. The IET rate constant is significantly decreased by nearly fourfold (compared to the iNOS oxyFMN co-expressed with full length CaM). This supports an important role of full length CaM in proper interdomain FMN/heme alignment in iNOS. The IET process was not observed with added excess EDTA, suggesting that Ca(2+) depletion results in the FMN domain moving away from the heme domain. The results indicate that a Ca(2+)-dependent reorganization of the truncated CaM construct could cause a major modification of the NCaM/iNOS association resulting in a loss of the IET.
Asunto(s)
Calmodulina/metabolismo , Mononucleótido de Flavina/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Óxido Nítrico Sintasa de Tipo II/metabolismo , Sitios de Unión/genética , Calcio/metabolismo , Calmodulina/genética , Quelantes/farmacología , Clonación Molecular , Ácido Edético/farmacología , Transporte de Electrón/efectos de los fármacos , Mononucleótido de Flavina/química , Hemo/química , Hemo/metabolismo , Hemo Oxigenasa (Desciclizante)/genética , Humanos , Cinética , Modelos Biológicos , Ácido Nítrico/química , Ácido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Oxidación-Reducción/efectos de los fármacos , Fotoquímica , Fotólisis/efectos de los fármacosRESUMEN
Nitric oxide (NO) plays a critical role in a number of physiological processes and is produced in mammalian cells by nitric oxide synthase (NOS) isozymes. Because of the diverse functions of NO, pharmaceutical interventions which seek to abrogate adverse effects of excess NOS activity must not interfere with the normal regulation of NO levels in the body. A method has been developed for the control of NOS enzyme activity using the localized photochemical release of a caged isoform-specific NOS inhibitor. The caged form of an iNOS inhibitor has been synthesized and tested for photosensitivity and potency. UV and multiphoton uncaging were verified using a hemoglobin-based assay. IC(50) values were determined for the inhibitor (70+/-11 nM), the caged inhibitor (1098+/-172 nM), the UV uncaged inhibitor (67+/-26 nM) and the multiphoton uncaged inhibitor (73+/-11 nM). UV irradiation of the caged inhibitor resulted in a 86% reduction in iNOS activity after 5 min. Multiphoton uncaging had an apparent first order time constant of 0.007+/-0.001 min(-1). A therapeutic range exists, with molar excess of inhibitor to enzyme from 3- to 7-fold, over which the full dynamic range of the inhibition can be exploited.
