Stimulation of a Cd-binding protein, and inhibition of the vitamin D-dependent calcium-binding protein, by zinc or cadmium in organ-cultured embryonic chick duodenum

Abstract

Embryonic chick duodenum maintained in organ culture responds to 1α,25-dihydroxy vitamin D 3 in the culture medium by de novo synthesis of a specific calcium-binding protein (CaBP). The addition of Cd 2+(3–5 × 10 −5 m) or Zn 2+(10 −5-10 −4 m) to the medium inhibited CaBP, but stimulated biosynthesis of a Cd-binding protein (CdBP). CdBP in duodenal homogenate supernatants was assessed in two ways: first, by its 109Cd-binding activity ( 109CdBA) using a competitive ion exchange procedure; and, second, by the extent of [ 35S]-cystine incorporation into a specific peak or band after gel filtration or analytical polyacrylamide disc gel electrophoresis, respectively. Regardless of whether cadmium- or zinc-stimulated, the 35S-labeled CdBP—the only protein significantly labeled under the conditions employed—migrated identically upon gel filtration and electrophoresis, and comigrated with purified chick liver Cd-metallothionein. Neither actinomycin D nor α-amanitin, in concentrations sufficient to severely inhibit CaBP, significantly reduced CdBP production. However, cycloheximide did inhibit either Cd 2+- or Zn 2+-stimulated CdBP by about 50% at an inhibitor concentration which abolished CaBP. The inhibitor studies, coupled with the observations of extensive incorporation of [ 35S]cystine into CdBP, suggest that the metals stimulated biosynthesis by a mechanism operating at the translational level. The organcultured duodenum seems well suited for studies of the regulation of CdBP biosynthesis especially since it responds predictably to the steroid hormone, 1α,25-dihydroxy vitamin D 3, in the induction of another specific protein, CaBP, at the transcriptional level. The biosynthesis of CaBP thus may serve as a convenient control in studies of CdBP production under various experimental conditions.