Diurnal rhythmicity in cytosolic control of cell‐free hepatic protein synthesis

Abstract

Protein synthesis was investigated in a hepatic in vitro system containing microsomes and, besides other components, Sephadex G-25 eluate. [ 3 H] -leucine incorporation was measured in total protein as well as in a fraction of purified integral membrane proteins prepared from microsomal membranes by treatment with EDTA and 2M LiCl. Incubations were carried out combining microsomes and Sephadex G-25 eluates from postmicrosomal supematants isolated at 8 different diurnal phases.When microsomal fractions from various phases were incubated with Sephadex G-25 eluate from a single time of day, the rates of translation exhibited diurnal rhythmicity, with a major maximum at 3 00 h. A maximum also appeared in the same phase of the 24-h cycle, when Sephadex G-25 eluates from various phases were combined during incubation with a single microsomal fraction.The influence of cytosolic components on protein synthesis was further investigated by fractionation of Sephadex G-25 eluates by Sephacryl S-300 filtration and subsequent SDS-PAGE. Highest stimulatory capacity was found in two fractions of G-25 eluate from 3 00 h; 4 protein bands in these fractions showed diurnal variations. Our results suggest that the diurnal rhythms of protein synthesis are not only depending on mRNA concentration, but also on the concerted action of a cytosolic control mechanism.

Circadian rhythms and the role of regulation mechanisms at the macromolecular level

“…These findings may be seen in relation to a cycle in the capacity of cellular extracts to stimulate protein synthesis in vitro (7). A similar rhythmicity of stimulatory capacity was found in extracts from rat liver (4). Gel electrophoretic separation of extract components revealed large temporal variations of at least 4 protein bands.…”