The early time course of calcium-binding protein induction by 1,25-dihydroxyvitamin D3 as determined by computer analysis of two-dimensional electrophoresis gels

The time course of calcium-binding protein induction by 1,25-dihydroxyvitamin D3 was examined in embryonic chick duodena by single-label autoradiography. Duodena were excised from 19-day-old embryos, cultured for 24 h in defined medium, and exposed to 1,25-dihydroxyvitamin D3 at 1/2, 1, 1 1/2, 2, 2 1/2, 4, 6, and 20 h before the end of the culture period. Control duodena were identically handled but were not exposed to the hormone. All duodena were pulsed with 14C]leucine during the final 30 min of incubation. Cytosolic proteins, extracted from each radiolabeled tissue, were resolved by two-dimensional electrophoresis and autoradiographs were prepared from the resulting gels. Calcium-binding protein was detectable by autoradiography in duodena cultured with hormone for 1 h or more but not in the control duodena. The autoradiographs were converted to digital images by a scanning densitometer and entered into the Man-computer Interactive Data Access System. The total radioactivity incorporated into calcium-binding protein during the 30-min pulse was determined by computer analysis of the digitized autoradiographs, and the rate of calcium-binding protein biosynthesis was calculated. Duodena cultured with hormone for 1-20 h synthesized calcium-binding protein at rates of 129 +/- 17 to 8800 +/- 110 pg/mg of cytosolic protein/30 min. These rates demonstrate that calcium-binding protein is induced within the first hour of exposure to 1,25-dihydroxyvitamin D3, but in amounts too small to be detected by commonly used immunoassays.