QUANTITATIVE 2-DIMENSIONAL GEL-ELECTROPHORETIC ANALYSIS OF CLOCK-CONTROLLED PROTEINS IN CULTURED CHICK PINEAL CELLS - CIRCADIAN REGULATION OF TRYPTOPHAN-HYDROXYLASE

The progression of the circadian oscillator through its cycle and the circadian rhythm of melatonin production in dissociated chick pineal c ultures both require daily de novo protein synthesis during defined ci rcadian phases. To identify specific proteins involved in these two pr ocesses, we have performed a quantitative two-dimensional polyacrylami de gel electrophoretic screen of proteins that are synthesized at diff erent times of the day in chick pineal cell cultures. Out of similar t o 700 proteins analyzed, we have identified several proteins whose lev els of S-35 incorporation oscillate in a light/dark cycle. One protein of 56 kDa, pI 6 (p56) undergoes a diurnal oscillation that parallels the melatonin rhythm, reaching a peak early in the night and falling t o minimal levels during the day. A second protein of 22 kDa, pI 4.5 (p 22) also expresses a diurnal rhythm in S-35 incorporation; however, it peaks at the end of the night. The oscillations of both proteins pers ist, with a reduced amplitude, in constant darkness. Furthermore, the phases of the p56 and p22 rhythms are regulated by the light/dark cycl e. Both p56 and p22 appear to be under direct control of the chick pin eal circadian oscillator, and therefore can be described as ''clock-co ntrolled proteins. ''We have identified p56 as tryptophan hydroxylase by microsequencing and western blotting. Chick pineal tryptophan hydro xylase also expresses a 24-h oscillation in abundance both in vitro an d in vivo. The rhythm in tryptophan hydroxylase expression represents a newly discovered level of regulation of the melatonin synthesis path way by the circadian clock in chick pineal cells.