Abstract
The bovine alpha and murine beta subunits of rod-photoreceptor cGMP-phosphodiesterase (PDE alpha and PDE beta) were expressed in adenovirus-transformed 293 human embryonic kidney cells. RNA blots from transfected cells showed transcripts of 3.0 and 2.8 kb corresponding to PDE alpha and PDE beta, respectively. Protein expression was analyzed by using affinity-purified antibodies against cGMP-PDE on immunoblots and by immunoprecipitation. PDE alpha and PDE beta exhibited the expected mobility (and thus apparent molecular size) and had cGMP hydrolytic activity. Reconstitution of the PDE alpha beta heterodimer with the expressed proteins increased by approximately 6-fold the activity of the individual alpha and beta subunits. Addition of expressed beta subunit to retinal extracts from 9- to 10-day-old rd/rd mice (which have only normal alpha and gamma subunits of rod cGMP-PDE and thus minimal activity) increased enzyme activity by approximately 3-fold. Our results therefore demonstrate that photoreceptor-specific cGMP-PDE can be synthesized in human kidney cells with consequent expression of enzymatic activity.
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