Partial amino acid sequence of a rabbit immunoglobulin light chain of allotype b5

The amino acid sequence of a rabbit immunoglobulin light chain of allotype b5 has been nearly completed. A comparison of its structure with that of light chains of allotypes b4, b6, and b9 confirms that the constant regions of these various kappa chains differ by 20-35%. The substitutions are clustered in parts of the second half of the chain, and the b5 form bears more resemblance to the b6 chain than to any other, in good agreement with previous serological data. The analysis of the variable region reveals the existence of certain allotype-associated residues which have also been reported in other b5 chains, but not in proteins of the other allotypes. An examination of the rabbit light chain sequences between positions 96 and 107 suggests that this portion of the chain may be encoded separately by a joining "J" DNA segment, as has been described previously for murine and human immunoglobulins. In the rabbit, however, these J kappa regions appear to differ from one allotype to another. Together with the extensive variations of the constant regions, these data suggest that the rabbit kappa gene organization more closely resembles the murine gamma system (four different C gamma genes each flanked by its J segment) than the murine kappa system (a single C kappa gene).     

Homogeneous rabbit immunoglobulin lacking group a allotypes: amino acid sequence analysis of the heavy chain.

The partial amino acid sequence of rabbit a-negative heavy chain has been determined for residues 1--43 as: less than EEQLEESGGGLVQPGGSLKLSCKGSGFDFSVYGVTWVRQAPGK; and for residues 64--120 as: MNGRFTISSDNAQNRLYLQLNSLTAADTATYFCARSMVVVAGVHSYFDVWGPGTLVTV. Comparison of this sequence with the human heavy chain subgroup III shows homology of 78% suggesting that a common ancestral variable region gene existed in mammals prior to speciation. The constant region of the a-negative chain is structurally identical with that of a-positive chains, whereas the variable region differs substantially between a-positive and a-negative molecules. These findings support the concept that two genes encode one immunoglobulin polypeptide chain and demonstrate the existence in the rabbit of variable region subgroups similar to those reported for humans and other species. A novel approach to the initial fragmentation of the heavy chain was developed in this study. This method, which involved digestion of the H chain with the protease V8, produced a free N terminus and should have wide application in future studies on heavy chains with blocked amino terminals.     

The amino acid sequence of rabbit cardiac troponin I.

The complete amino acid sequence of troponin I from rabbit cardiac muscle was determined by the isolation of four unique CNBr fragments, together with overlapping tryptic peptides containing radioactive methionine residues. Overlap data for residues 35-36, 93-94 and 140-145 are incomplete, the sequence at these positions being based on homology with the sequence of the fast-skeletal-muscle protein. Cardiac troponin I is a single polypeptide chain of 206 residues with mol.wt. 23550 and an extinction coefficient, E 1%,1cm/280, of 4.37. The protein has a net positive charge of 14 and is thus somewhat more basic than troponin I from fast-skeletal muscle. Comparison of the sequences of troponin I from cardiac and fast skeletal muscle show that the cardiac protein has 26 extra residues at the N-terminus which account for the larger size of the protein. In the remainder of sequence there is a considerable degree of homology, this being greater in the C-terminal two-thirds of the molecule. The region in the cardiac protein corresponding to the peptide with inhibitory activity from the fast-skeletal-muscle protein is very similar and it seems unlikely that this is the cause of the difference in inhibitory activity between the two proteins. The region responsible for binding troponin C, however, possesses a lower degree of homology. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50072 (20 pages), at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1976) 153, 5.     

The N-terminal sequence of the heavy chain of rabbit immunoglobulin IgG

The absence of an N-terminal amino acid with a free alpha-amino group from the heavy chain of rabbit immunoglobulin IgG has been confirmed and no evidence could be found of a blocking formyl, acetyl or propionyl group. The N-terminal amino acid appears to be pyrrolid-2-one-5-carboxylic acid (PCA) in all molecules. A mixed amino acid sequence follows in the approximate proportions: PCA-Ser-Val-Glu-Glu-Ser-Gly-Gly-Arg, 50%; PCA-Ser-Leu-Glu, 20%; PCA-Glu(NH(2)), 20%. The heavy chains of a purified antibody, namely anti-(human serum albumin), and of immunoglobulin IgG from a rabbit homozygous at the allotypic loci both showed a similar mixed N-terminal sequence.     

The amino acid sequence of rabbit muscle triose phosphate isomerase.

The amino acid sequence of rabbit muscle triose phosphate isomerase was deduced by characterizing peptides that overlap the tryptic peptides. Thiol groups were modified by oxidation, carboxymethylation or aminoen. About 50 peptides that provided information about overlaps were isolated; the peptides were mostly characterized by their compositions and N-terminal residues. The peptide chains contain 248 amino acid residues, and no evidence for dissimilarity of the two subunits that comprise the native enzyme was found. The sequence of the rabbit muscle enzyme may be compared with that of the coelacanth enzyme (Kolb et al., 1974): 84% of the residues are in identical positions. Similarly, comparison of the sequence with that inferred for the chicken enzyme (Furth et al., 1974) shows that 87% of the residues are in identical positions. Limited though these comparisons are, they suggest that triose phosphate isomerase has one of the lowest rates of evolutionary change. An extended version of the present paper has been deposited as Supplementary Publication SUP 50040 (42 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1975) 145, 5.     

The amino acid sequence of the C- terminal cyanogen bromide peptide of human J chain

The carboxyl-terminal peptide obtained from human J chain treated with cyanogen bromide (CNBr)¹ has been isolated and the amino acid sequence determined as Val - Glx - Thr - Ala - Leu - Thr - Pro - Asx - Ala - CMCys - Tyr - Pro - Asx. Comparisons with the primary structures of human lambda, kappa, alpha, or mu chains failed to disclose analogous regions with these immunoglobulins.     

The complete amino acid sequence of rabbit muscle phosphoglucomutase

The complete amino acid sequence of rabbit muscle phosphoglucomutase has been determined by isolating the 11 peptide fragments produced by the cyanogen bromide cleavage reaction and subjecting these to automated sequencing procedures. Products produced by treatment of some of these fragments with hydroxylamine, iodosobenzoic acid, mild acid, cyanogen bromide in formic and heptafluorobutyric acids, Staphylococcus aureus V8 protease, and trypsin (with or without blocking at lysine residues) were used to complete the sequence for each of the cyanogen bromide fragments. The cyanogen bromide fragments were ordered by isolating the four tryptic peptides produced by a limited tryptic digest of the native enzyme in the presence of its substrates and its bivalent metal ion activator, Mg2+, degrading these by means of trypsin, after blocking digestion at lysine residues, and isolating and identifying all fragments thus produced that contained 10 or more residues. The 561-residue sequence thus obtained is one of the longest that has been determined by chemical means. There is excellent agreement between this sequence and published compositions after appropriate normalization. The absorbance of the enzyme is about 7.0 at 278 nm for a 1% solution; this value is 9% lower than that previously used.     

The amino acid sequence of rabbit skeletal muscle glycogenin

The amino acid sequence of glycogenin from rabbit skeletal muscle has been determined. The N-acetylated protein consists of 332 amino acids and has a molecular mass of 37278 Da. The novel tyrosyl-glucose linkage between glycogenin and glycogen [Smythe, C., Caudwell, F. B., Ferguson, M. & Cohen, P. (1988) EMBO J. 7, 2681-2686] is shown to occur at a single site, tyrosine-194. Although glycogenin is a UDP-Glc utilising glucosyltransferase that self-glucosylates [Pitcher, J., Smythe, C. & Cohen, P. (1988) Eur. J. Biochem. 176, 391-395], following addition by an unknown enzyme of the first glucose to tyrosine-194, it is not homologous to either human glycogen synthase or other UDP-Glc-requiring enzymes.     

The amino acid sequence of rabbit slow-muscle troponin I

Troponin I was isolated from six red muscles in the hind leg of the rabbit. Soleus, semi-tendinosus, vastus intermedius and adductor longus muscles contained primarily slow-muscle troponin I, vastus lateralis contained fast-muscle troponin I and quadratus femoris contained a mixture of the two. The complete amino acid sequence of the troponin I from slow muscle was determined. Seven CNBr fragments were isolated and sequenced by using the dansyl-Edman technique after digestion with proteolytic enzymes. The CNBr fragments were ordered by isolation of tryptic peptides containing carboxy[(14)C]methyl-methionine. Direct evidence for the conjunction of residues 8 and 9 has not been obtained, and one of the carboxyl groups between residues 71 and 79 may carry an amide group. Slow-muscle troponin I is a single polypeptide chain of 184 residues with a mol.wt. of 21146. It has a net overall positive charge of 18 at pH7, and an absorption coefficient, A(1%,1cm) (280), of 5.43. The protein was isolated with both a blocked and an unblocked N-terminus, although the nature of the blocking group was not determined. Proline was found to be the N-terminal amino acid. Two forms of the protein could also be distinguished by the presence of an extra two residues at the C-terminus. Comparison of sequences of troponin I from rabbit slow, fast and cardiac muscle shows that homology is most marked in the C-terminal half of the molecules. Towards the N-terminus the homology becomes much less marked. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50079 (32 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained in the terms given in Biochem. J. (1977), 161, 1.     

The amino acid sequence of troponin I from rabbit skeletal muscle.

The complete amino acid sequence of rabbit skeletal muscle troponin I was determined by the isolation of the cyanogen bromide fragments and the tryptic methionine-containing peptides. Troponin I contains 179 amino acid residues and has a molecular weight of 20864. Its N-terminus is acetylated. Detailed evidence on which the sequence is based has been deposited as Supplementary Publication SUP 50055 (23 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7QB, U.K., from whom copies may be obtained on the terms given in Biochem. J. (1975) 145, 5.     

The amino acid sequence of the rabbit lutropin beta subunit

The amino acid sequence of the beta subunit of rabbit lutropin (lLH) has been determined. The amino terminus of about 97% of the beta subunit has a two amino acid extension (pyro-Glu-Pro) compared to other lutropin beta sequences. Overlapping peptides from trypsin and chymotrypsin digestions of the performic acid-oxidized beta subunit and trypsin digestion of the S-aminoethylated cysteine beta subunit were isolated by chromatography on TSK Fractogel 40F and high-pressure liquid chromatography (HPLC). Sequencing was by a combination of the dansyl-Edman method and the direct Edman method. Amide placements were established by HPLC analysis of the PTH amino acid derivatives. The proposed sequence of lLH subunit is: This sequence is highly homologous to the other known lutropin beta subunits, especially rat and pig lutropin beta (91%). Partial cleavage of the peptide bond between Asp-79 and Pro-80 was observed during cyanogen bromide treatment. Rabbit thyrotropin and thyrotropin beta subunit copurified with lLH and lLH except at a final chromatography on Sephadex G-100.     

The amino acid sequence of a human κ light chain

The amino acid sequence of the light chain of the myeloma protein Dee was studied. The light chain is of the kappa type and of subgroup I. The variable part contains some substitutions that are unique and also some that have been observed already in other kappaI chains (repeated variants). Based on these repeated variants a subdivision of the kappaI subgroup is proposed.     

The amino acid sequence of rabbit skeletal alpha-tropomyosin. The NH2-terminal half and complete sequence

The amino acid sequence of the large cyanogen bromide fragment (residues 11 to 127) derived from the NH2-terminal half of alpha-tropomyosin has been determined. This was achieved by automatic sequence analysis of the whole fragment as well as manual sequencing of fragments derived from tryptic digestion of the maleylated fragment and thermolytic, Myxobacter 495 alpha-lytic and Staphylococcus aureus protease digestion of the unmodified fragment. Methionine-containing overlap peptides have been isolated from tryptic digests of the maleylated protein as well as from S. aureus protease digests of the unmodified protein. Coupled with previously published information on the small cyanogen bromide fragments and methionine sequences of tropomyosin, these analyses have permitted the completion of the primary structure of the protein. The complete sequence differs by only 1 residue (Gln-24 instead of Glu-24) from that previously reported. Analysis of the sequence by several authors has permitted rational explanations for the stabilization of its coiled-coil structure, for the existence of its two chains in a nonstaggered arrangement, for a head-to-tail overlap of molecular ends of 8 to 9 residues, for the existence of 14 actin-binding sites on each tropomyosin molecule, and a suggestion for the site of binding of troponin-T.