The loci of the three amino acid residues that contribute their prosthetic groups to form the stable, nonreducible, trifunctional intermolecular cross-link histidinohydroxylysinonorleucine in skin collagen fibrils were identified. Two apparently homogeneous three-chained histidinohydroxylysinonorleucine cross-linked peptides were chromatographically isolated. They were obtained from a tryptic digest of denatured unreduced 6 M guanidine hydrochloride insoluble bovine skin collagen. Amino acid and sequence analyses demonstrated that the prosthetic groups of alpha 1(I)-chain Hyl-87, alpha 1(I)-chain Lys-16c, and alpha 2(I)-chain His-92 formed the cross-link. The latter results served to define the locus of the stable, nonreducible trifunctional moiety. Identical types of analyses were performed on the three-chained peptides isolated after bacterial collagenase digestion of the cross-linked tryptic peptides. This confirmed the initial identification and location of the three peptides linked by the cross-link. In addition, data reported here provide for a correction of the micromolecular structure for the alpha 2(I) chain. Stereochemical considerations concerning this trifunctional cross-link's specific locus indicate that the steric relationships between the alpha chains of skin and skeletal tissue collagens are fundamentally different and the intermolecular relationships in skin fibrils are specific for skin. The same molecular relationships also indicate that histidinohydroxylysinonorleucine links three molecules of collagen. The stereochemistry of cross-linking for skin collagen is in accordance with and explains the X-ray findings of a 65-nm periodicity found for this tissue [Stinson, R. H., & Sweeny, P. R. (1980) Biochim. Biophys. Acta 621, 158; Brodsky, B., Eikenberry, E. F., & Cassidy, K. (1980) Biochim. Biophys. Acta 621, 162].

A three-chained peptide from type I collagen, crosslinked by hydroxyaldolhistidine, has been isolated from a tryptic digest of 5 M guanidine.HCl-insoluble bovine skin collagen (a small but as yet unknown percentage of the total collagen in whole skin). OsO(4)/NaIO(4) specifically cleaved the crosslink at its double bond into a two-chained crosslink peptide and a single peptide. The sequence of the two-chained peptide containing the bifunctional crosslink was determined after amino acid analysis of the separated peptides. The crosslink consists of an aldehyde derived from hydroxylysine-87 in the aldehyde-containing cyanogen bromide fragment alpha1CB5(ald) and an aldehyde derived from the lysine in the COOH-terminal nonhelical region of the alpha1CB6(ald) fragment. The alpha1CB6(ald) portion of the peptide exhibited structural microheterogeneity, containing the inverted sequence Ala-Lys-His instead of the normal sequence Lys-Ala-His. This indicates that another structural gene exists for alpha1(I) chain. The original three-chained peptide did not contain any glycosylated hydroxylysine or glycosylated hydroxyaldolhistidine. The lack of glycosylation of hydroxylysine-87 in alpha1CB5, which is usually glycosylated, allowed formation of the aldehyde, and this, coupled with the sequence inversion, may have allowed formation of the nonreducible crosslink hydroxyaldolhistidine. We suggest that the role of glycosylation, a posttranslational modification, of specific hydroxylysine residues is to prevent their oxidative deamination to aldehydes, thereby precluding formation of complex stable crosslinks. Complex crosslinks would decrease the rate of collagen turnover. The decrease, with time, would increase the population of stable crosslinked collagen molecules, which would eventually accumulate with age.

The N-terminal sequence analyses were performed on the heavy (H) and light (L) chains of the idiotypically identical IgM kappa and IgG kappa paraproteins isolated from the serum of patient, Cam. The N-terminal 39 residues of the kappa chains of the IgM and IgG were identical and belonged to the human V kappa III subgroup. This sequenced stretch included the first L chain hypervariable region. The N-terminal 27 residues of the variable regions (VH) of the respective mu and gamma heavy chains were also identical and belonged to the human VHIII subgroup. These identical VH sequences were unique with lysine residues at positions 13 and 19. This dual lysine substitution has not been seen in 37 other human VHIII sequences reported in the literature. This N-terminal sequence homology in the V-regions of Cam IgM kappa and IgG kappa paraproteins and the shared idiotypy expressed by Cam IgM, IgG, and IgA proteins strongly suggest the existence of complete structural homology in the variable regions of the and L chains of these Ig molecules of three separate Ig classes. At the cellular and genetic level, these results point toward a common clonal origin for the idiotypically related Ig molecules and suggest that identical V-region (VH and VL) genes were utilized by the Cam lymphoid clone in the biosynthesis of the respective IgM, IgC, and IgA proteins.

Amino terminal sequence analyses were performed on the H and L chains of the idiotypically related IgMlambda and IgGkappa paraproteins isolated from the sera of a patient, Br. The N-terminal 41 residues of the Br k-chain belonged to the Vkiii subgroup, and L chains derived from BrIgMlambda revealed a blocked N-terminus characteristics of lambda-chains. Comparative sequence analysis of the Br mu- and gamma-chains indicated that, although both possessed an unblocked N-terminal glutamic acid, the respective VH regions belonged to seperate subgroups. The N-terminal 27 residues of the Br mu-chain reflected a typical VHiii subgroup sequence. The Br gamma-chain sequence demonstrated a VHI pattern with an unblocked N-terminus. These structural data stand in contrast to previously reported serologic evidence, which indicated that the BrIgMlambda and BrIgGkappa proteins possessed highly similar Vh-associated idiotypic determinants. Final interpretation of these findings of similar Vh idiotypic determinants expressed by H chains belonging to seperate Vh subgroups must await complete sequence analysis of hypervariable segments of the Br gamma- and mu-chains.

The covalent structure of apolipoprotein A-II, isolated from the serum high-density lipoprotein of a single male Rhesus monkey (Macaca mulatta), was determined. The amino acid sequence of this 77-residue polypeptide is: less than Glu-Ala-Glu-Glu-Pro5-Ser-Val-Glu-Ser-Leu10-Val-Ser-Gln-Tyr-Phe15-Gln-Thr-Val-Thr-Asp20-Tyr-Gly-Lys-Asp-Leu25-Met-Glu-Lys-Val-Lys30-Ser-Pro-Glu-Leu-Gln35-Ala-Gln-Ala-Lys-Ala40-Tyr-Phe-Glu-Lys-Ser45-Lys-Glu-Gln-Leu-Thr50-Pro-Leu-Val-Lys-Lys55-Ala-Gly-Thr-Asp-Leu60-Val-Asn-Phe-Leu-Ser65-Tyr-Phe-Val-Glu-Leu70-Arg-Thr-Gln-Pro-Ala75-Thr-Gln-COOH. A comparison of this structure to that of the monomeric form of human apolipoprotein A-II reveals a high degree of homology except for six conservative amino acid replacements (positions 3, 6, 40, 53, 59, and 71). Of particular structural significance is the replacement of cysteine by serine in position 6. This explaines why Rhesus A-II exists in monomeric form, contrary to the established dimeric nature of the human protein.

The amino acid sequence of the COOH-terminal cyanogen bromide fragment (residues 12 to 54) of the testis-specific basic protein of the rat has been determined. This analysis completes the primary structure of the whole protein by over-lapping the sequence of the 23 residues from the NH-2 terminus previously published (Kistler, W. S., Noyes, C., and Heinrikson, R.L. (1974) Biochem. Biophys. Res. Commun. 57, 341-347). The complete sequence of this small, highly basic protein is: (see article for formular).

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTAlignment and partial structural analysis of the cyanogen bromide fragments from yeast inorganic pyrophosphataseRichard Sterner, Claudia Noyes, and Robert L. HeinriksonCite this: Biochemistry 1974, 13, 1, 91–99Publication Date (Print):January 1, 1974Publication History Published online1 May 2002Published inissue 1 January 1974https://pubs.acs.org/doi/10.1021/bi00698a015https://doi.org/10.1021/bi00698a015research-articleACS PublicationsRequest reuse permissionsArticle Views22Altmetric-Citations14LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Automated Edman degradation of a testis-specific basic protein isolated from the rat gave the following NH2-terminal sequence of amino acids: Cleavage of the native protein with cyanogen bromide produced two fragments which were purified by gel filtration. Amino acid analysis of the smaller fragment revealed it to be the NH2-terminal undecapeptide resulting from cleavage at Met11. The partial sequence analysis of the intact protein coupled with compositional analyses of these cyanogen bromide peptides indicate that the basic testis protein contains 24 basic amino acids and a single methionine in a sequence of 54 amino acids.

The phytohemagglutinin (PHAP) glycoproteins derived from Phaseolus vulgaris consist of five isomitogens that are tetrameric structures made up of two different glycoprotein subunits. Although identical in size (mol wt = 34,000), the subunits differ in their isoelectric points and amino acid sequences for six of the first seven amino-terminal residues, but are identical in primary structure from the 8th through the 24th amino acid residue. The isomitogen containing four L subunits (L-PHAP) is a potent leukoagglutinin and mitogen that lacks hemagglutinating properties. The isomitogen made up of four R subunits (4R H-PHAP) is a potent hemagglutinin. The hybrid isomitogens consisting of varying proportions of the two subunits (3L-1R, 2L-2R, 1L-3R) are capable of causing mixed erythrocyte-lymphocyte agglutination. These studies provide a structural basis for explaining the differences in biological activities of the various PHAP isomitogens.

Erythrocytic NADH methemoglobin diaphorase acquires NADH-dichlorophenolindophenol diaphorase activity when enzyme-associated NAD is removed. This transformation is reversible and can be mediated by membrane NAD glycohydrolase (EC 3.2.2.5) in hemolysates as well as in intact cells exposed to hydrogen peroxide. It is abolished either in NADH methemoglobin diaphorase deficiency or in NAD(P) glycohydrolase (EC 3.2.2.6) deficiency which is common in Afro-American but not in European-American adults. Activities of erythrocytic NADP glycohydrolase and NAD glycohydrolase appear to depend on a single membrane enzyme.

The partial amino acid sequences at the amino terminal of prothrombin and the intermediates of activation have been determined. These data indicate that the products of the first step of activation, whether derived from the action of factor Xa or thrombin, are identical. The data also show that the activation of prothrombin proceeds by the sequential cleavage of the amino terminal region of prothrombin and the intermediates, and confirm the mechanism of prothrombin activation as: NH2-Prothrombin-COOH Xa or thrombin NH2-Intermediate 3 + Intermediate 1-COOH; NH2-Intermediate 1-COOH Xa NH2-Intermediate 4 + Intermediate 2-COOH; NH2-Intermediate 2-COOH Xa NH2-A chain α-thrombin -S-S-B chain α-thrombin-COOH.

Evidence based upon a variety of experimental approaches is in accord with the conclusion that native yeast inorganic pyrophosphatase (EC 3.6.1.1, pyrophosphate phosphohydrolase) comprises two identical subunits approximately 32,000 in molecular weight with threonine and valine the NH2- and COOH-terminal residues, respectively. Gel filtration of reduced and carboxymethylated pyrophosphatase on a column of 6% agarose in 6 m guanidine hydrochloride revealed a single protein component of molecular weight 33,000; a value of 35,500 was obtained after exhaustive succinylation of the alkylated enzyme. The molecular weight thus derived for S-carboxymethylated pyrophosphatase was in close agreement with the values calculated from polyacrylamide gel electrophoresis in sodium dodecyl sulfate and amino acid compositional data which were, respectively, 33,000 and 30,250. Threonine was identified as the sole NH2-terminal residue by the dimethylaminonaphthalene-5-sulfonyl chloride end group procedure. Hydrolysis of alkylated pyrophosphatase by carboxypeptidase A was attended by the rapid release of COOH-terminal valine in quantities stoichiometric with the amount of monomeric protein present. Peptide maps of trypsin digests from S-carboxymethylated enzyme revealed a total of 30 peptides, five of which gave a positive test for arginine and three of which stained positively for tryptophan. These results are in accord with what would be expected on the basis of the lysine, arginine, and tryptophan content of the enzyme subunit. Finally, the partial sequence of NH2-terminal amino acid residues presented below was elucidated by Edman degradation with the aid of automated sequencing equipment. [see PDF for sequence] Yields of the phenylthiohydantoin amino acid derivatives obtained after each cycle of the degradative procedure were consistent with the quantity of monomeric protein subjected to analysis.

Evidence that the glutamine synthetase dodecamer from Escherichia coli is comprised of 12 identical subunits of molecular weight 49,000 has been obtained by end group analysis. Application of the carbamylation procedure to the NH2-terminal analysis revealed only serine. The yield was 98% of that expected from a polypeptide of the size proposed for the monomeric species. The following partial sequence of residues at the NH2 terminus of the molecule was elucidated with the aid of automated sequencing equipment: [see PDF for sequence] Hydrolysis of reduced and carboxymethylated glutamine synthetase by carboxypeptidase A yielded quantitative evidence for the following COOH-terminal sequence: -Leu-Tyr-Tyr-Ser-Val-COOH One mole of valine was released per mole of protein, assuming the subunit molecular weight to be 49,000.