No abstract

Haemagglutinin molecules from nine strains of A/Hong Kong/68 (H3N2) influenza virus, isolated between 1968 and 1977, were examined for changes in amino acid sequences. At least 18 changes, 9 of which were located precisely, occurred in the soluble tryptic peptides of the large haemagglutinin polypeptide (HA1) during this period. These peptides contained 262 residues (82% of HA1). In HA2, only two changes in 129 residues (58% of HA2) were detected. Sequential changes at a particular locus were not found; and as far as we can tell, once an amino acid changed, it did not change again in any subsequent variant examined.

A new simplified procedure for the purification of chorismate mutase/prephenate dehydratase, based on affinity chromatography on Sepharosyl-phenylalanine, has been developed. The method utilizes the effect of NaCl on the binding properties of the enzyme.NaCl inhibits both the mutase and dehydratase activities of the enzyme. In each case this inhibition is cooperative indicating homotropic interactions between NaCl binding sites on the enzyme. In addition NaCl induces homotropic cooperative effects between chorismate binding sites and between prephenate binding sites. NaCl also increases the sensitivity of the enzyme to inhibition by phenylalanine.In Escherichiu coli the biosynthesis of the aromatic amino acids phenylalanine, tyrosine and tryptophan, and the other aromatic compounds such as folate, vitamin K and ubiquinone proceeds from the common precursor chorismate [I, 21. Chorismate mutase/prephenate dehydratase is a single protein that catalyses the following reactions :

The amino acid sequence of component 0.62, a protein derived from wool keratin, with molecular weight of 6950, rich in glycine and aromatic residues, has been determined. The prot,ein contains no lysine, histidine, glutamic acid, isoieucine or methionine. Of the total residues, more than 5001, are glycine and aromatic amino acids. The sequence contains two sections rich in glycine, (Gly-X), and (Gly-X),, which comprise 22O/, of the sequence.The a-keratins, as exemplified by wool, hoof, horn and animal hairs and quills, consist of filamentous units, the microfibrils, embedded in a nonfilamentous matrix. The filaments have been identified with a set of highly a-helical proteins, containing it lower percentage of sulphur than the parent material, while the non-filamentous matrix between the microfibrils consists largely of a group of heterogeneous proteins, containing a higher percentage of sulphur than the parent material (for an up-to-date summary of the present position see [l]). Both of these types of protein have been investigated. A considerable amount is known about the primary structure of certain of their components [2--41.A third group of proteins, probably derived from the matrix, with an as-yet undefined function and structure is also present. This class is extremely rich in glycine and aromatic residues [5] which together may account for more than one half of the total amino acid residues in these proteins. These proteins are hereafter referred to as "tyrosine-rich proteins".This work describes the detailed study of the sequence of one of these tyrosine-rich components of wool obtained in a soluble form by the reduction of the disulphide bonds of wool and stabilization of the thiols by alkylation with iodoacetic acid MATERIALS AND METHODSThe tyrosine-rich protein, a gift from Dr J. M. Gillespie, was prepared from fine, non-peppin Merino wool as described in the accompanying paper Enzymic DigestionEnzymic digestion of the protein was performed at l o / , protein concentration with a 1/100 (w/w) ratio of enzyme to substrate. The buffer used for tryptic, chymotryptic and thermolytic digestions was N-ethylmorpholine acetate (0.1 M, pH 8.0). Carboxypeptidase hydrolysis of peptides was generally done on 0.05 pmol peptide in 100 pl 0.1 M NH,-HCO, with I0 pg carboxypeptidase A for various times a t 25 "C. Progress of the reaction was monitored by paper electrophoresis a t pH 1.9 or by amino acid analysis. Peptide PurificationAfter gel chromatography, peptides were further purified by high-voltage paper electrophoresis a t pH 1.9 or pH 3.5. Whatman paper 3 MM was used throughout a t 50 V/cm. The electrophoretic mobility a t pH 6.5 relative to aspartic acid ( ? A s p = -1.0 a t pH 6.5) [8] was the criterion used to determine the state of the single aspartic acid found in the protein.Paper chromatography in butanol-acetic acidwater-pyridine (15 : 3 : 12 : 10, v/v/v/v) was also used occasionally to separate peptide mixtures.

The amino acid sequence and oligosaccharide distribution for the haemagglutinin from the early Hong Kong influenza virus A/Aichi/2/68 (X-31) was investigated. The two polypeptide chains, HA1 and HA2, were fragmented by CNBr and enzymic digestion, and the amino acid sequence of each small peptide was deduced by comparing its chromatographic behaviour, electrophoretic mobility, amino acid composition and N-terminus with that of the corresponding peptide of the haemagglutinin of known structure from the influenza-virus variant A/Memphis/102/72. Those peptides in which changes were detected were sequenced fully. The complete amino acid sequence of the haemagglutinin HA1 chain (328 residues) and 188 of the 221 residues of the HA2 chain were established by this approach, and revealed only twelve differences between the amino acid sequences of variant-A/Aichi/68 and -A/Memphis/72 haemagglutinins. These occurred at positions 2, 3, 122, 144, 155, 158, 188, 207, 242 and 275 in the HA1 chain and 150 and 216 in the HA2 chain. The highly aggregated hydrophobic region (residues 180-121) near the C-terminal end of the HA2 chain was not resolved by peptide sequencing. The oligosaccharide distribution in variant-A/Aichi/68 haemagglutinin was identical with that found in that of A/Memphis/72, with sugar units attached at asparagine residues 8, 22 38, 81, 165 and 285 in the HA1 chain and 154 on the HA2 chain. The monosaccharide compositions of the individual carbohydrate units on variant-A/Aichi/68 haemagglutinin differed from those of the corresponding units in variant-A/Memphis/72 haemagglutinin, and evidence was found for heterogeneity in the oligosaccharide units attached at single glycosylation sites.