The NH2- and COOH-terminal amino acid sequence of nuclear protein A24.

The NH2- and COOH-terminal sequence of nuclear portein A24 has been determined by automatic Edman degradation and carboxypeptidase A and B digestion. Protein A24 is of interest because it is composed in part of histone 2A (Goldknofp, I.L., and Busch, H., (1975) Biochem, Biophys. Res. Commun. 65, 951-960). The sequence of the first 37 NH2-terminal residues is: Met-Gln-Ile-Phe-Val-Lys-Thr-Leu-Thr-Gly-Lys-Thr-Ile-Thr-Leu-Glu-Val-Glu-Pro-Ser-Asp-Thr-Ile-Glu-Asn-Val-Lys-Ala-Lys-Ile-Gln-Asp-Lys-Glu-Gly-Ile-Pro- This sequence is not homologous to any known histone sequence. It contains regions of internal homology (italics). The COOH-terminal amino acid sequence is the same as that of histone 2A, naely: -His-His-Lys-Ala-Lys-Gly-Lys-COOH.

This sequence is not homologous to any known histone sequence. It contains regions of internal homology (italics). The COOH-terminal amino acid sequence is the same as that of histone 2A, namely: -His-His-Lys-Ala-Lys-Gly-Lys-COOH.
Protein A24 is a non-histone chromatin protein with histonelike characteristics (1). The protein was initially observed by two-dimensional polyacrylamide gel electrophoresis in 0.4 N H,SO, extracts of nucleoli (2). It was subsequently found to decrease in amount in nucleoli undergoing hypertrophy induced by thioacetamide or liver regeneration (3,4). After purification and characterization (I), protein A24 was found to contain the tryptic and chymotryptic peptides of histone 2A as well as additional peptides (5). Furthermore, protein A24 contains the blocked acetyl-Ser-Gly-Arg-NH,-terminal sequence (6) identical to that of histone 2A (7). Since protein A24 also contains a single methionine NH, terminus (l), it was suggested that protein A24 contains an additional non-histone sequence linked to the histone 2A sequence (5). Because the NH, terminus of the histone 2A sequence was blocked by an acetyl group, the unblocked sequence was amenable to automatic Edman degradation without prior separation of the two polypeptides. This study was initiated to determine whether the NH,-terminal sequence of the unblocked portion of the protein is homologous to histone 2A or other histones and to determine the COOH-terminal sequence(s). It was found that the NH,-terminal sequence is unique and is not homologous to that of any known histone but that protein A24 contains a single COOH-terminal sequence which is identical to that of histone 2A.
Protein A24 and histone 2A purified from calf thymus as previously described (1,8)  In one run, glutamic acid was distinguished from glutamine and aspartic acid from asparagine by modification of free carboxyl groups with glycine ethyl ester (12). Free carboxyl groups were detected by the presence of increased quantities of glycine in addition to the parent amino acid as determined by amino acid analysis after hydrolysis with 57% HI. derivatives. Table I indicates that yields were variable for both   Rel eased  COOH-terminal  amino  acids were again  analyzed. systems even after correction for losses during hydrolysis, especially for the less stable amino acids serine, threonine, and lysine. Overlap between steps was generally less than 5% but RESULTS began to increase gradually at certain steps after cycle 9 (e.g. Protein A24 was subjected to automatic Edman degradation 12, 16, 19, 20, 21, 22, etc.). However, the overlap did not in three different runs with samples of 67 to 130 nmol of prevent positive identification of any of the 37 residues. protein. Repetitive yields were approximately 96% of the stable The amide functions of glutamine or asparagine were amino acids up to Step 30 but fell rapidly beyond that point.
assigned to residues 2, 25, and 31 by the presence of the Overlap between residues also became a problem at this point PTH-derivative, on isothermal gas chromatography at 235" and conclusive identification of residues was achieved only and by the absence of substantially increased glycine contents through Step 37.
by amino acid analyses. Conversely, the absence of glutamine Table I summarizes the results of 37 cycles of Edman or asparagine on the gas chromatograph and the large increase Protein A24 NH,-and COOH-terminal Sequence in glycine by amino acid analysis (greater than 0.6 mol/mol of parent amino acid) indicated the presence of glutamic or aspartic acid at residues 16, 18, 21, 25, 32, and 34. Some residues of glutamic or aspartic acid were confirmed by gas chromatography after silylation; however, problems of reproducibility and background limited the usefulness of this procedure.
Several PTH-derivatives are destroyed or converted to other amino acids by HI hydrolysis. Methionine is completely destroyed, but was positively identified at Step 1 by gas chromatography.
Threonine yields cY-aminobutyric acid, which was readily identified with the amino acid analyzer. Serine is converted to alanine but was identified on the gas chromatograph at residue 20. Isoleucine residues were identified as a mixture of isoleucine and alloisoleucine.  Fig. 1. Quantitative hydrazinolysis indicated that lysine is the sole COOH-terminal amino acid of protein A24 and its molar yield is identical to that of histone 2A (6). DISCUSSION This study indicates that protein A24 has a single COOHterminal amino acid sequence which is identical to histone 2A and a unique NH,-terminal sequence which is unlike that of histone 2A (7) or any known histone sequence (15). Protein A24 therefore appears to be a unique protein for which the linkage of the non-histone and histone polypeptides is presently unknown.
Interestingly, the A24 NH,-terminal sequence exhibits internal homology. Homologous tetrapeptides are present from residues 6 through 15 in which lysine and threonine residues are spaced by pentapeptide regularity: Ly6s-Thr-Leu Thr ;Iy L&Thr-Ile-Thr-& This suggests this portion of the sequence may have arisen from gene duplication as previously observed in protamines (16) and in haptoglobins (17).
This sequence presented contains an equal number of free carboxyl groups and amino groups (six of each), effectively resulting in a net charge of zero for this part of the molecule. It is of interest that no arginine was found in this NH,-terminal sequence. The total sequence of this structure and its linkage to the histone 2A portion of protein A24 requires further studies.
The results of studies on the COOH-terminal sequence by carboxypeptidase digestion (Table  II) and quantitative hydrazinolysis as noted above provide evidence that lysine is the sole COOH-terminal amino acid, and thus that protein A24 has a Y-shape (6). However, the possibility is not excluded that some unknown linkage could exist that is resistant to these procedures. Further studies on branch structures and the complete amino acid sequence of this molecule will be necessary to eliminate the possibility that it may have an H-shape.