In vitro studies on the kinetics, composition, and homology of bovine keratohyalin

Abstract

Cattle hoof epidermis was incubated in Medium 199 supplemented with fetal calf serum and antibiotics, and lacking Tween 80 and the particular precursor studied. Specimens were removed at times ranging from 5 min to 24 h for autoradiography or for fractionation of isolated bovine keratohyalin (BKH) by polyacrylamide gel electrophoresis. Of the seven amino acids studied by autoradiography, tritiated histidine, arginine, serine, and glycine were concentrated in KH granules. In contrast, KH did not appear to be selectively labelled by 35S-cysteine, or by 3H-proline, 3H-tryptophan or 3H-uridine. Label appeared in BKH in vitro as early as 5 min after pulsing, and increased with time. At no time period was significant label found over the nucleus. The fractionated oligomeric species of BKH were labelled by continuous in vitro incubations with the same precursors that labelled KH granules in situ. BKH isolated after 15 min pulses demonstrated most of the label in the low molecular weight oligomers with progression to higher molecular weight oligomers with chasing as a function of time. Puromycin blocked the incorporation of tritiated histidine into BKH in situ and into the fractionated oligomers. These results indicate that BKH (1) is rapidly synthesized in the cytoplasm; (2) undergoes sequential polymerization into larger oligomeric species as a function of time; (3) is labelled in situ in accord with the known amino acid composition.