Expression and characterization of a murine enzyme able to cleave beta-carotene - The formation of retinoids

Because animals are not able to synthesize retinoids de novo, ultimately th ey must derive them from dietary provitamin A carotenoids through a process known as carotene cleavage. The enzyme responsible for catalyzing carotene cleavage (beta -carotene 15,15 ' -dioxygenase) has been characterized prim arily in rat intestinal scrapings. Using a recently reported cDNA sequence for a carotene cleavage enzyme from Drosophila, we identified a cDNA encodi ng a mouse homolog of this enzyme. When the cDNA was expressed in either Es cherichia coli or Chinese hamster ovary cells, expression conferred upon ba cterial and Chinese hamster ovary cell homogenates the ability to cleave be ta -carotene to retinal. Several lines of evidence obtained upon kinetic an alyses of the recombinant enzyme suggested that carotene cleavage enzyme in teracts with other proteins present within cell or tissue homogenates. This was confirmed by pull-down experiments upon incubation of recombinant enzy me with tissue 12,000 x g supernatants. Matrix-assisted laser desorption io nization-mass spectrometry analysis of pulled-down proteins indicates that an atypical testis-specific isoform of lactate de-hydrogenase associates wi th recombinant carotene cleavage enzyme. mRNA transcripts for the carotene cleavage enzyme were detected by reverse transcription-polymerase chain rea ction in mouse testes, liver, kidney, and intestine. In situ hybridization studies demonstrated that carotene cleavage enzyme is expressed prominently in maternal tissue surrounding the embryo but not in embryonic tissues at 7.5 and 8.5 days postcoitus. This work offers new insights for understandin g the biochemistry of carotene cleavage to retinoids.