Incorporation of [Me-3H]Methionine into Wool Follicle Proteins: A Biochemical and Ultrastructural Study

Abstract

The formation of the ‘low-sulphur’ group of proteins which constitutes more than half the cortical cell content of wool was investigated. Of the amino acids present in the low- and high-sulphur proteins isolated from wool, methionine is the only one which is exclusive to the low-S group. The incorporation of [Me-3H]methionine, administered intradermally to a sheep, was examined using both biochemical and autoradiographic procedures. During the first hour after injection, most of the [3H]methionine appeared in the blood, but some was incorporated into the wool follicle proteins at the injection sites. Most of the radioactivity incorporated by the fibre during this period was subsequently retained. Initially, more than 90% of the activity was present in the prekeratin (8 M urea-soluble) portion of the fibre; 50 h elapsed before half of the radioactive material appeared in the keratinized (8 M urea-insoluble) part of the fibre. Methionine and its oxidation products accounted for more than 80% of the radioactivity present in hydrolysates of wool removed from injection sites after 4 h or 22 days. Analysis of labelled wool showed that the specific radioactivities of those fractions low in sulphur (α- and β-keratose) were 7 and 14 times greater than that of the high-S (λ-keratose) fraction. Autoradiographic studies showed that the main site of formation of the low-S proteins was in the bulb region of the follicle. Here there was no specific intracellular site of low-S protein synthesis, the [3H]methionine being incorporated into the nucleus, cytoplasm, the various cell organelles and intercellular membranes. The amount of radioactivity incorporated by the cortical cells diminished at progressively higher levels in the fibre. In the keratinization zone, the various cell components showed limited protein synthesis, but 1-8 h after injection there was a localization of tritium over the keratin fibrils. It is suggested that a large proportion of the low-S proteins formed within the developing cortical cell ultimately contribute to the keratin microfibrils. The results provide further evidence for the formation of the low-S and high-S proteins at different sites in the follicle.