Amino-Terminal Sequences and Sites of In Vivo Acetylation of Trout-Testis Histones III and IIb ₂

Abstract

The sequences of the first 25 residues of histone III, and the first 22 residues of histone Ib2, from trout testis have been determined on an automatic protein sequencer. The amino-terminal sequence of trout-testis histone III is identical to the corresponding region of calfthymus histone III, whereas the trout-testis histone IIb2 sequence differs from that of calf-thymus histone 11b2 at several positions in the amino-terminal region. Several in vivo sites of acetylation of these trout-testis histones have also been determined, by the same automated procedure. In addition to the two main sites at lysyl residues 14 and 23 acetylated in calf-thymus histone III, a lower degree of acetylation at two other sites, lysyl residues 9 and 18, has been detected. Four sites of acetylation have also been detected in trout-testis histone IIb2, at lysyl residues 5, 10, 13, and 18. When the amino-acid sequences around the acetylated lysyl residues of different histones are compared, striking similarities are seen. The methods used in these studies should prove useful in elucidation of the locations of chemically stable modifications in other proteins.We have reported the locations of sites of acetylation in trouttestis histones IV and JIb1 (1, 2) by isolation and characterization of [14C]acetyl-labeled tryptic peptides. We also showed (2) that histones III and IIb2 were acetylated in trout testis. In histones IIb1 and IV, the amino-terminal residue is Nacetylserine, so that automatic sequential Edman degradation is impossible, but in the case of histones III and IIb2, the amino termini consist of unblocked alanyl and prolyl residues, respectively.In view of the placement of the two acetylated tryptic peptides-obtained by DeLange et al. (3) (2) on a 3 X 320-cm column of Bio-Gel P-10 eluted with 0.01 N HCl. Purity of the samples was monitored by electrophoresis on urea-aluminum lactate starch gels (6). The histone III used for sequencing experiments contained a mixture of the reduced (monomer) and oxidized (dimer) forms.Sequencing of Histones III and 11b2. The sample of histone, containing 0.5-1.2 uimol of protein, was dissolved in 0.6 ml of distilled water and placed in the reaction cup of a Beckman 890 protein sequencer. A program similar to that of Edman and Begg (7) or, in some cases, that of Niall et al. (8) was used. The 2-anilino-5-thiazolinone derivatives of the amino acids from the sequencer were converted to their more stable 3-phenyl-2-thiohydantoin (PTH) derivatives, as described by Edman and Begg (7), except that the 1 N HCl contained 1 mM ethanethiol. The PTH derivatives were then extracted from the HCl phases with ethyl acetate (2 X 0.7 ml, Reagent Grade). Arginine was detected either after acid hydrolysis of the HCl phase after conversion to its PTH derivative (7), or else PTH arginine was first extracted from the HCl phase with ethyl acetate, after neutralization of the HC1 with 1 M Na2HPO4. Amino acids were identified on a Beckman 120C amino-acid analyzer after hydrolysis of their PTH derivatives in 6 N ...

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