Kensuke Shimura

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.

The occurrence and synthesis of female- and egg-specific proteins in the silkworm, Bombyx mori

The occurrence of a specific protein, famale-specific protein, was demonstrated in the haemolymph of female pupae and egg yolk, but not in the haemolymph of male pupae and larva of either sex of the silkworm Bombyx mori. The existence of an egg-specific protein was shown only in the egg yolk by column chromatographic and immunochemical methods. Incorporation experiments of labeled amino acid in vivo and in vitro into the egg yolk and the proteins released from the fat body have shown that the female-specific protein was synthesized in the fat body of female pupae, secreted into the haemolymph, then transported to the oocyte, and incorporated into the egg yolk, whereas the egg-specific protein was synthesized solely in the oocyte. The synthesis of both the specific proteins was stimulated by ecdysterone.

Molecular cloning of the fibroin light chain complementary DNA and its use in the study of the expression of the light chain gene in the posterior silk gland of Bombyx mori

Fibroin light chain (L-chain) mRNA (mol. wt 4.0×105 daltons) was purified from the posterior silk gland of the silkworm,Bombyx mori (J-131 strain). Double-stranded complementary DNA was synthesized and inserted into the PstI site of pBR322 employing the oligo(dC)-oligo(dG) tailing method. Several recombinant plasmids containing the inserts of about 800 base pairs were isolated. Hybridization-translation assay demonstrated that these clones hybridized specifically with the fibroin L-chain mRNA. One of these clones (pLA23) was used as a probe to investigate relative concentrations of the fibroin L-chain gene and mRNA in the posterior silk glands at different stages of late larval development.

Biosynthesis of bacitracin: II. Incorporation of 14C-labeled amino acids into bacitracin by a cell-free preparation from Bacillus licheniformis

Abstract 1. 1. A cell-free system prepared from the lysate of protoplasts from Bacillus licheniformis, consisting of a 12 000 × g supernatant, incorporated the constituent 14C-labeled amino acids into bacitracin. Using this system, the basal conditions for the incorporation and some properties of its process were investigated. 2. 2. The reaction of the incorporation of l -[14C]histidine into bacitracin required ATP and component amino acids, and was optimal at approx. pH 7.2. 3. 3. The incorporation of l -[14C]histidine into bacitracin was decreased by the addition of any one of d -phenylalanine, d -glutamic acid, d -asparagine and d -ornithine to the reaction system, though they are present as d -isomers in the bacitracin molecule. This result suggests that d -amino acids are not direct precursors for bacitracin synthesis. 4. 4. The incorporation of l -[14C]histidine into bacitracin was not affected by ribonuclease, chloramphenicol or puromycin. The centrifugation of the 12 000 × g supernatant at 105 000 × g for 90 min did not cause any decrease in incorporating activity. These results indicate that the biosynthetic process of bacitracin is different from that of proteins.

The presence of 4′-phosphopantetheine in the bacitracin synthetase

A soluble enzyme complex participates in the biosynthesis of bacitracin which is an antibiotic dodecapeptide produced by a strain of Bacillus licheniformis and the peptide is synthesized from amino acids bdund to enzyme protein through thioester linkages [ 1,2] . These features of bacitracin synthetase resemble those in the biosynthesis of gramicidin S and tyrocidine [3]. Recently, it was reported that gramicidin S and tyrocidine synthetases contained one and two moles of 4’-phosphopantetheine pr mole of synthetase, respectively [4,5], and that phosphopantetheine arm participated in peptide chain elongation [6]. Bacitracin synthetase has beeri separated into two complementary fractions (Peaks I and II) by column chromatography on hydroxyapatite [2]. The present work demonstrates that Peak II fraction was further separated into two components and that each of the three components responsible for bacitracin biosynthesis contains approximately one equivalent of 4’-phosphopantetheine.

Isolation of nonhistone chromosomal protein from calf thymus

1. 1. A method for the preparation of nonhistone chromosomal proteins from calf thymus under mild conditions was proposed. 2. 2. Calf thymus chromatin was dissolved in 0.1 M Tris HCl (pH 7.9) containing 2 M NaCl and 5 M urea to dissociate it into the chromosomal proteins and DNA. Then, the DNA was precipitated selectively by adding LaCl3 at a concentration of 0.0135 M, leaving the proteins in the supernatant. The nonhistone chromosomal proteins in the supernatant were separated from histone by CM-cellulose column chromatography with 0.01 M Tris-HCl (pH 7.9) containing 5 M urea. 3. 3. The amino acid composition of the protein was analyzed.

Biosynthesis of bacitracin III. Partial purification of a bacitracin-synthesizing enzyme system from Bacillus licheniformis

1. 1. A bacitracin-synthesizing enzyme system was partially purified from the crude extract of Bacillus licheniformis by (NH4)2SO4 fractionation, treatment of calcium phosphate gel, Sephades G-200 gel filtration and hydroxyapatite column chromatography. About a 25-fold purification of the system was achieved. 2. At the step of column chromatography on hydroxyapatite the partially purified system was separated into two distinct components, Peaks I and II, both of which were required for bacitracin synthesis. 3. The enzyme system catalyzed ATP-PPi exchange reactions dependent on the l-isomers of all the bacitracin constituent amino acids. Among the four component d-amino acids, only d-phenulalanine promoted the exchange reaction. 4. The Peak I fraction activated the amino acids constituting the acyclic peptide side-chain of bacitracin and the Peak II fraction activated the amino acids contained in the cyclopeptide moeity. 5. Mixed-substrate experiments suggests that l-isoleucine, l-leucine and l-valine are activated at the same active site(s) as that for l-isoleucine or l-leucine. 6. The activated amino acids seems to be linked as thioesters to the enzyme protein in an intermediate state of bacitracin biosynthesis.

Biosynthesis of bacitracin: I. Formation of bacitracin by a subcellular fraction of Bacillus licheniformis

Abstract 1. 1. The formation of bacitracin by a subcellular fraction prepared from a strain of Bacillus licheniformis has been studied. 2. 2. Under the conditions described here, the formation of antibiotic was greatly decreased in the absence of cysteine. The single omission of any of the other amino acids which are constituents of bacitracin did not greatly affect the amount of antibiotic formed. In addition, no stimulatory effect of ATP for the reaction was observed. 3. 3. The antibiotic formed has been demonstrated to incorporate [ 35 S]cystine and [ 14 C]isoleucine actively, but [ 14 C]lysine which is only contained in the cyclic peptide part of bacitracin very slightly. 4. 4. A possible explanation for these results is that the reactions carried out in the present system are mainly composed of the addition of cysteine and isoleucine to a peptide precursor of bacitracin.

Further evidence for the presence of a thiazoline ring in the isoleucylcysteine dipeptide intermediate in bacitracin biosynthesis

Isoleucylcysteine dipeptide, a first intermediate peptide in bacitracin biosynthesis, was liberated from the enzyme protein and oxidized with manganese dioxide in dimethylsulfoxide. The resulting oxidation product was identified by thin‐layer chromatography as 2‐(2‐methyl‐l‐oxobutyl)‐thiazole‐4‐carboxylic acid which has been isolated from the hydrolysate of bacitracin F. This result shows that the intermediate dipeptide contains a thiazoline ring, and that the thiazoline ring is synthesized at the dipeptide stage in the process of peptide chain elongation in bacitracin biosynthesis. Improbability of non‐enzymatic dehydrative cyclization of the dipeptide is discussed.

Thiazoline ring formation in bacitracin biosynthesis

Bacitracin A contains a thiazoline ring in the N-terminal portion of the molecule. The thiazoline ring has been thought to be synthesized by a cyclic condensation of isoleucylcysteine portion of the peptide [I 1. However, it has not been known when the thiazoline ring is formed in the process of bacitracin biosynthesis. The work described here shows that the enzymebound dipeptide, a first intermediate peptide in peptide chain elongation in bacitracin biosynthesis 121, contained a thiazoline ring. This suggests that the thiazoline ring is formed at the first step of peptide chain elongation in bacitracin biosynthesis.