Manual precyclization of fiberglass filters for microsequence analysis of peptides and proteins.

A manual procedure for precyclization of fiberglass filters has been described. The quality of the manually precycled filter is as good for microsequence analysis of proteins and peptides as a filter that has been precycled by an automatic system. The manually precycled filter is convenient to use and saves time and money.

hv1 is an evolutionarily conserved H2A variant that is preferentially associated with active genes.

Polyclonal antibodies to the Tetrahymena macronuclear-specific histone variant hv1 cross-react with histone-like molecules from yeast, wheat, and mouse. A novel purification scheme has allowed isolation of sufficient hv1 to enable determination of the sequence of 61 amino-terminal residues as well as 27 additional internal residues. These data clearly demonstrate that hv1 shares a number of conserved sequence elements with the H2A family of histones. Comparison of hv1 with H2A.F (= H2A.Z = M1), another evolutionarily conserved H2A variant whose sequence is known, reveals that they share an unblocked amino-terminal alanine (instead of acetylserine) and a distinctive structure in a "variant box" region that distinguishes them from major H2As. In addition, 10 residues have been identified which are identical (or highly similar) in hv1 and H2A.F, but are different from residues conserved in the major H2As. Therefore, in many ways hv1 resembles chick H2A.F more than the major Tetrahymena H2A. The sites of acetylation of hv1 also differ from those of the major Tetrahymena H2As. In spite of their similarities, hv1 and H2A.Z differ significantly in their amino termini, and antibodies against hv1 do not react with H2A.Z. Interestingly, the nucleolar staining pattern reported with anti-hv1 serum is similar to that reported for an antiserum to another H2A variant, mouse testes-enriched H2A.X. Since both H2A.Z and hv1 appear to be enriched in transcriptionally active chromatin, these results suggest that there may be a number of different, functionally distinct, nonallelic variants in the H2A family of histones and that hv1 is a hybrid H2A variant with properties of both vertebrate H2A.Z and H2A.X.

Covalent structure of human haptoglobin: a serine protease homolog.

The complete amino acid sequences and the disulfide arrangements of the two chains of human haptoglobin 1-1 were established. The alpha 1 and beta chains of haptoglobin contain 83 and 245 residues, respectively. Comparison of the primary structure of haptoglobin with that of the chymotrypsinogen family of serine proteases revealed a significant degree of chemical similarity. The probability was less than 10(-5) that the chemical similarity of the beta chain of haptoglobin to the proteases was due to chance. The amino acid sequence of the beta chain of haptoglobin is 29--33% identical to bovine trypsin, bovine chymotrypsin, porcine elastase, human thrombin, or human plasmin. Comparison of haptoglobin alpha 1 chain to activation peptide regions of the zymogens revealed an identity of 25% to the fifth "kringle" region of the activation peptide of plasminogen. The probability was less than 0.014 that this similarity was due to chance. These results strongly indicate haptoglobin to be a homolog of the chymotrypsinogen family of serine proteases. Alignment of the beta-chain sequence of haptoglobin to the serine proteases is remarkably consistent except for an insertion of 16 residues in the region corresponding to the methionyl loop of the serine proteases. The active-site residues typical of the serine proteases, histidine-57 and serine-195, are replaced in haptoglobin by lysine and alanine, respectively; however, aspartic acid-102 and the trypsin specificity, residue, aspartic acid-189, do occur in haptoglobin. Haptoglobin and the serine proteases represent a striking example of homologous proteins with different biological functions.

Characterization of the cyanogen bromide fragments of the beta chain of human haptoglobin.

Characterization of the cyanogen bromide (CNBr) fragments of the beta chain of human haptoglobin revealed five major fragments resulting from cleavage of four methionyl residues. The fragments were isolated by gel filtration in guanidine-HCl on Sepharose 6B and Bio-Gel P10 and P60. Compositional analyses of the five cyanogen bromide fragments accounted for 248-253 amino acid residues in agreement with the number of residues determined for the intact beta chain. Most of the carbohydrate was attached to CNBr II. Automated amino-terminal sequence analysis and carboxyl-terminal hydrolysis with carboxypeptidase of the haptoglobin beta chain and cyanogen bromide fragments identified 139 residues, or about 55% of the beta-chain molecule. The placement of the fragments within the beta-chain molecule was established by sequence analysis of whole beta chain and a plasmin cleavage fragment. The position of CNBr V was confirmed by the absence of homoserine or homoserine lactone. Cyanogen bromide reaction of intact haptoglobin 1-1 resulted in the isolation of a beta-chain fragment, CNBr III, covalently attached to the intact alpha1 chain by a single disulfide bond. The beta chain was shown to have primary structural similarities to the chymotrypsin family of serin eproteases. Partial sequence analysis of CNBr V established the region which is comparable to the serine-195 active-site region: /Asp-Thr-Cys-Tyr-Gly-Asp-Ala-Gly-Ser-Ala-Phe/ (residues 189-199, chymotrypsinogen A numbering). The active-site serine-195 is replaced by alanine; however, the specificity residue of the trypsin-like enzymes, Asp-189, is preserved. Several minor cyanogen bromide cleavage products were also identified in yields of up to 15%. These minor cleavage products give evidence that tryptophanyl residues in proteins, or glycoproteins, are also susceptible to cyanogen bromide cleavage.

Chemical characterization of the structure of cholera toxin and its natural toxoid.

Acrylamide gel electrophoresis demonstrated that the toxin of Vibrio cholerae is comprised of three polypeptide chains, alpha, beta, and gamma, of molecular weights 24,000, 9,700, and 9,700 daltons, respectively. Amino acid sequence analysis of intact toxin indicated a molecular composition of alpha gamma beta4. Acrylamide gel electrophoresis and sequence analysis confirmed that the natural toxoid (choleragenoid) is identical to the toxin beta-chain. The alpha- and gamma-chains of the toxin are disulfide-linked (fragment A) but are noncovalently bound to the beta-chains. About 50% of the primary structure of the N-terminal portion of the beta-chain has been identified and a small segment of the D-terminus has also been characterized. Twenty residues of the N-terminal portions of the alpha- and gamma-chains have been tentativly identified. The amino acid composition of the beta-chain was determined and compared to that of the natural toxoid.