A Convenient Method For the Identification and Estimation of Soluble Elastin Synthesis in vitro

Abstract

A method is described by which newly synthesized soluble elastin can be routinely and conveniently identified and estimated in vitro without the need for unlabelled carrier tropoelastin. Aortic tissue from 11 day old chick embryos is incubated in the presence of [¹⁴C] L-proline and [³H] L-valine and the distributions of [¹⁴C] proline-, [¹⁴C] hydroxyproline- and [³H] valine-labelled proteins on SDS-polyacrylamide gels are determined. Soluble elastin is identified as a [¹⁴C] hydroxyproline-labelled peak of molecular weight approximately 70,000 daltons which also incorporates large quantities of [³H] valine and has a [¹⁴C] hydroxyproline/ [¹⁴C] proline ratio of about 0.2. Whereas the hydroxyproline label can be used to estimate newly synthesized soluble elastin even after several hours of incubation, similar use of the [³H] valine label is limited to short incubation times. Paradoxically, the quantity of [¹⁴C] hydroxyproline-labelled soluble elastin detected by the assay decreases with increased incubation time. This fall-off in labelled soluble elastin is not due to an increase in the rate of crosslinking of the protein over the course of the incubation. Soluble elastin is detectable both in tissue extract and medium fractions. The presence of hydroxyproline-labelled protein fragments in the medium fraction is evidence of proteolytic breakdown of collagen or elastin or both proteins. This proteolytic activity is augmented by the inclusion of serum in the incubation medium and is not inhibited by phenylmethylsulfonylfluoride. The method provides a convenient assay by which factors affecting the synthesis and secretion of soluble elastin may be studied.