Aiko Kikuchi

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.

Fractionation of Whole Histone by Partition Chromatography on Sephadex G-25

Whole histone from calf thymus was fractionated by partition chromatography on the basis of distribution between an aqueous phase immobilized on Sephadex G-25 beads and mobile organic phases containing various concentrations of trichloroacetic acid. The chromatography was carried out by stepwise elution with five upper phases from water and butanol-2 solvent systems containing 4 M urea and 0.1-1.5% trichloroacetic acid, and finally water. Of the six peaks obtained, two (peaks 1 and 2) contained arginine-rich histones. Although these peaks were still heterogeneous electrophoretically, the band corresponding to F2al was observed only in the electrophoretic pattern of peak 1 and the main fraction in peak 2 was F3. A histone fraction having nearly equimolar amounts of arginine and lysine was obtained from peak 3. Its amino acid composition was similar to that of F2a2. Slightly lysine-rich histone obtained from peak 4 showed an amino acid composition typical of F2b. Peak 5 contained a histone fraction with a ratio of lysine/arginine of 6.14, showing a single band on gel-electrophoresis. Very lysine-rich histone (F1) was obtained from peak 6, and the electrophoretic pattern of this fraction showed a single band.