The complete amino acid sequence of a prototype immunoglobulin-lambda light-chain-type amyloid-fibril protein AR.

The amino acid sequence of an amyloid-fibril protein of immunoglobulin light-chain type (AL) was elucidated. The sequence determination involved digesting the protein with trypsin, thermolysin and pepsin. The protein was found to consist of 154 amino acid residues and is thus missing about half of the constant region of a light chain. A certain heterogeneity in the length of the polypeptide was observed in the C-terminal region. The amino acid sequence from CDR (complementary-determining region) 1 and FR (framework region) 3 indicated an oligoclonal origin of the protein. By comparing the primary structure of protein AR with other lambda- and even kappa-chains, it was revealed that protein AR had an insertion of two residues of aspartic acid, namely residues 68 and 69, which has not been reported previously in light chains. The overall sequence homology in the variable region showed that protein AR is more similar to V lambda V than to the other subgroups [Kabat, Wu & Bilofsky (1979) Variable regions of Immunoglobulin Chains, Medical Computer Systems, Bolt, Beranek and Newman, Cambridge, MA].     

The amino acid sequence of a carbohydrate-containing fragment of hen ovotransferrin.

1. Hen ovotransferrin was treated with CNBr and fractionated by gel filtration. 2. After further treatment by reduction and carboxymethylation a carbohydrate-containing fragment of molecular weight 11990 was obtained (fragment BCd). 3. The amino acid sequence of this fragment was determined. It consists of a single chain of 94 residues. 4. The structure of a tryptic glycopeptide derived from whole ovotransferrin permitted a further eight residues to be assigned at the N-terminus of fragment BCd. 5. Heterogeneity was found at two positions. 6. Further evidence has been deposited as Supplementary Publication SUP 50045 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, W. Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1975), 145, 5.     

Structural studies of a carbohydrate-containing immunoglobulin-lambda-light-chain amyloid-fibril protein (AL) of variable subgroup III.

The amino acid sequence of the variable region of a carbohydrate-containing amyloid-fibril protein MOL of immunoglobulin-light-chain type (AL) was elucidated. The sequence determination involved cleaving the protein with CNBr, BNPS-skatole, thermolysin and trypsin. The sequenced protein consisted of about 130 amino acid residues; however, gel-filtration and N-terminal analysis studies revealed AL proteins ranging in Mr from about 10,000 to 25,000. The oligosaccharide chain was found to be bound in the hypervariable region. By sequence homology to other lambda chains the AL protein MOL was shown to be of the V lambda III subgroup.     

The amino acid sequence of porcine intestinal calcium-binding protein.

The complete amino acid sequence of the calcium-binding protein (CaBP) from pig intestinal mucosa has been determined: Ac-Ser-Ala-Gln-Lys-Ser-Pro-Ala-Glu-Leu-Lys-Ser-Ile-Phe-Glu-Lys-Tyr-Ala-Ala-Lys-Glu-Gly-Asp-Pro-Asn-Gln-Leu-Ser-Lys-Glu-Glu-Leu-Lys-Gln-Leu-Ile-Gln-Ala-Glu-Phe-Pro-Ser-Leu-Leu-Lys-Gly-Pro-Arg-Thr-Leu-Asp-Asp-Leu-Phe-Gln-Glu-Leu-Asp-Lys-Asn-Gly-Asn-Gly-Glu-Val-Ser-Phe-Glu-Glu-Phe-Gln-Val-Leu-Val-Lys-Lys-Ile-Ser-Gln-OH. The N-terminal octapeptide sequence was determined by mass spectrometric analysis by Morris and Dell. The first 45 residues of bovine CaBP differ only in six positions from the corresponding sequence of the porcine protein, except that the sequence starts in position two of the porcine sequence. The mammalian intestinal CaBP's belong to the troponin-C superfamily on the basis of an analysis by Barker and Dayhoff.     

The partial amino acid sequence of a non-histone chromosomal protein

The amino acid sequence of the first thirty nine residues of the nonhistone chromosomal protein HMG-17 has been determined. Results presented here give a molecular weight of 11,000 for the protein. Some interesting sequence homology with the trout specific histone, histone-T, is noted.     

Calcium-binding protein of bovine intestine. The complete amino acid sequence.

The amino acid sequence for vitamin D-dependent bovine intestinal calcium binding protein has been established. It contains 85 amino acids in a single chain and lacks cysteine, tryptophan, methionine, histidine, and arginine. The NH2-terminal lysine is blocked by an N-acetyl group. Enzymatic digestion with trypsin, chymotrypsin, and pepsin yielded a number of peptides which were purified by two-dimensional high voltage paper electrophoresis. These peptides were examined by end group analysis and sequenced by the dansyl procedure. The absence of tryptophan permitted by a single cleavage of the molecule by N-bromosuccinimide at the tyrosine residue at position 8 and the larger fragment was subjected to automated Edman degradation. By these means, the following sequence was established: N-Ac-Lys-Gln-Ser-Pro-Leu-Glu-Tyr-Ala-Ala-Glu-Lys-Ser-Ile-Gln-Lys-Glu-Ile-Glu-Lys-Gly-Phe-Phe-Lys-Gln-Leu-Leu-Val-Ser-Val-Gln-Lys-Ala-Gly-Asp-Lys-Glu-Ser-Leu-Gln-Pro-Leu-Phe-Thr-Leu-Leu-Lys-Ser-Gly-Pro-Glu-Glu-Asn-Leu-Lys-Glu-Ser-Gln-Asn-Gly-Pro-Asp-Leu-Ls7-Ser-Gly-Pro-Gly-Asn-Asp-Leu-Glu-Glu-Lys-Gly-Thr-Asp-Val-Phe-Ser-Leu-Lys-Gln. Microheterogeneity may exist in the molecule at residue 76 in which position threonine may be replaced by serine. Comparison of the sequence of calcium-binding protein to the "test" sequence of Tufty and Kretsinger ((1975) Science 187, 167-169) proposed to identify E-F hands in muscle proteins suggests that intestinal calcium-binding protein may likewise contain one or possibly two E-F hands which could account for calcium-binding property. Dayhoff alignment scores, however, calculated for calcium-binding protein against nine E-F hands in muscle proteins parvalbumin, troponin and alkali light chains do not indicate that intestinal calcium-binding protein is homologous to these muscle protein chains.     

The amino acid sequence of Limulus C-reactive protein. Evidence of polymorphism

The amino acid sequence, the positions of the disulfide bonds, and the site of glycosylation for the three subunits of Limulus C-reactive proteins (CRPs) 1.1, 1.4, and 3.3 have been established. The three subunits were shown to exist approximately in equimolar amount and are tightly associated. The hexagonal structure of Limulus CRP, as revealed by electron microscopic studies of Fernandez-Moran et al. (Fernandez-Moran, H., Marchalonis, J., and Edelman, G. M. (1968) J. Mol. Biol. 32, 467-469) might consist of two each of the subunits. The three subunits share an identical amino-terminal sequence of 44 residues and a carboxyl-terminal sequence from residues 206 to 218. Microheterogeneity exists to the extent of 10 to 11% for the entire protein. The positions of 6 half-cystines that form the three disulfide bonds and the site of glycosylation are constant in all subunits. Sequence analyses of peptides derived from enzymatic and chemical cleavages of affinity purified Limulus CRP indicate that subunits other than the three mentioned above exist in the hemolymph. Limulus CRP is therefore polymorphic. Topological analyses of Limulus CRPs, human CRP, rabbit CRP, human amyloid P-component, and Syrian hamster female protein indicate that the seven proteins may originate from the same ancestral gene. Using the topological data generated from the amino acid sequences of the proteins, we calculate that human and Limulus CRPs diverged about 500 million years ago. This figure is in general agreement with the evolutionary distance postulated by anthropological estimation of 400-500 million years.     

Putative amino acid sequence of chick calcium-binding protein deduced from a complementary DNA sequence.

Two DNA fragments coding for chick CaBP have been isolated and sequenced. cDNA was prepared from enriched intestinal mRNA and cloned in pUC12. The recombinant clones were screened by differential hybridisation with 32P-cDNA probes synthesized from vitamin D replete and deficient chick intestinal mRNA. Two clones had outstanding affinity with the +D probe. Hybrid-arrested and hybrid-selected translation systems showed that both clones hybridised to mRNA coding for immunoprecipitable CaBP. The mRNA for CaBP has a 100 bp G,C rich sequence before a 786 bp coding region followed by 1250 nucleotides 3' untranslated region. Nucleotides coding for the Ca-binding sites show a high degree of homology for Ca-binding sites in chick calmodulin and rat intestinal CaBP. The amino acid sequence specified by the longest open reading frame contains five Ca-binding sites but is too large for the native CaBP; post-translational modification must therefore occur.     

The amino acid sequence of a ribosome-inactivating protein from Saponaria officinalis seeds

The complete primary structure of saporin SO-6, a ribosome-inactivating protein extracted from Saponaria officinalis seeds, has been determined. The sequence was reconstructed following purification and analysis of peptides obtained after digestion of the protein with different proteolytic agents. The protein is composed of 253 amino acids, corresponding to a molecular weight of 28,621 Da. Comparison of the primary structure of SO-6 with the sequence deduced from cDNA, shows amino acid substitutions in 11 positions, suggesting a tissue-related genetic variability. When the sequence of saporin is compared to those of two related proteins, ricin A chain and trichosanthin, a low degree of similarity (12%) is found; nevertheless some considerations about structure-function relationships and evolution of RIPs are possible.     

Complete amino acid sequence of protein B

The complete amino acid sequence of protein B (= CAMP factor) of Streptococcus agalactiae has been determined. The sequence data were obtained mainly by manual sequencing of peptides derived from digestion with lysyl-peptidase, clostripain and Staphylococcus aureus protease and by solid phase sequencing of cyanogen bromide fragments. The protein contains 226 amino acids and has an Mr of 25,263. The sequence was compared with sequences of other Fc-binding proteins and partial sequence homology was found between protein B and the Fc-binding region of protein A.