Pepsin fragmentation of botulinum type E neurotoxin: Isolation and characterization of 112, 48, 46, and 16 kD fragments

Controlled digestion of 150 kD single chain botulinum type E neurotoxin with pepsin atpH 6.0 produced 112, 48, 46, and 16 kD fragments. These were chromatographically purified; their locations in the 1300 amino acid residue long neurotoxin were determined by identifying the amino terminal 10 residues of 112 and 48 kD fragments, 50 residues of 46 kD fragment, and 59 residues of 16 kD fragment. The 48 and 112 kD fragments contain the N-terminal segment of the neurotoxin (i.e., residue no. 1 to 425 and 1 to 990, respectively), the 46 kD fragment corresponds to 407 residues of the C-terminal region, and the 16 kD fragment contains the 140 residues from a segment nearer to the C-terminus. The 48 kD fragment is similar to the 50 kD N-terminal light chain of the 150 kD dichain neurotoxin, which is generated by tryptic cleavage of the 150 kD single chain neurotoxin, and is separated from the 100 kD C-terminal heavy chain by dithiothreitol (DTT) reduction of an intrachain disulfide bond in the presence of 2 M urea (Sathyamoorthy and DasGupta,J. Biol. Chem. 260, 10461, 1985). The pepsin-generated 48 kD fragment, unlike the light chain, was isolated without exposure to DTT and urea. The single chain 112 kD fragment following trypsin digestion yielded 48 and 60 kD fragments that were separable after DTT reduction of the intrachain disulfide which links them. The N-terminal residues of the smaller fragment were identical to that of the single chain 150 kD neurotoxin; the single chain 112 kD fragment is therefore the neurotoxin minus the 50 kD C-terminal half of the heavy chain. The biological activities of the 48 and 112 kD fragments can be demonstrated in permeabilized PC12 cells (Lomnethet al., J. Neurochem. 57, 1413, 1991); they inhibit norepinephrine release.     

Botulinum neurotoxin type A: cleavage of the heavy chain into two halves and their partial sequences

The 145-kDa type A botulinum neurotoxin (NT) is produced by the bacteria Clostridium botulinum (strain, Hall). The heavy (H) and light (L) chains (97- and 53-kDa, respectively) of this protein are linked by at least one disulfide bond. The N- and C-terminal halves of the H chain appear to have different functions in the mechanism of action of the NT [1987) FEBS Lett. 226, 115-120). Well-characterized and highly purified preparations of the two halves of the H chain are needed for such studies. Two different approaches were taken to cut the H chain with trypsin and isolate the fragments. In one method the cleavage products were: (i) 94-kDa fragment made of the L chain linked to the N-terminal half of the H chain (49 kDa) by a disulfide bond(s), and (ii) the C-terminal 44-kDa fragment. The N-terminal half of H chain was separated from the L chain by reducing the disulfide bond(s) linking them and then purified by ion-exchange chromatography. The 1-27 residues of 49-kDa N-terminal half of the H chain were Ala-Leu-Asn-Asp-Leu-Cys-Ile-Lys-Val-Asn-Asn-Trp-Asp-Leu-Phe-Phe-Ser-Pro- Ser-Glu - Asp-Asn-Phe-Thr-Asn-Asp-Leu-. The sequence of the other half of the H chain (44 kDa) was X-Ile-Ile-Asn-Leu-X-Ile-Leu-Asn-Leu-Arg-Tyr-Glu-X-Asn-His-Leu-Ile-Asp-Le u-Lys- X-Tyr-Ala-Ser-. In the second method, the H chain was first separated from the L chain, purified, and then cleaved. One product of cleavage, the 44-kDa fragment, was partially sequenced; the first 25 residues were identical to the sequence of the 44-kDa fragment generated by the first method. The present work also demonstrated that (i) The cysteine residue(s) located on the N-terminal half of the H chain form the -S-S- link(s) with the L chain. (ii) The other half of the H chain (44-kDa fragment, apparently the C-terminal half) is not linked via -S-S- to the L-chain or to the N-terminal half (49-kDa fragment) of the H chain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Covalent structure of botulinum neurotoxin type A: Location of sulfhydryl groups, and disulfide bridges and identification of C-termini of light and heavy chains

Botulinum neurotoxin Type A is synthesized byClostridium botulinum as a 150 kD single chain polypeptide. The posttranslational processing of the 1296 amino acid residue long gene product involves removal of the initiating methionine, formation of disulfide bridges, and limited proteolysis (nicking) by the bacterial protease(s). The mature dichain neurotoxin is made of a 50-kD light chain and a 100-kD heavy chain connected by a disulfide bridge. DNA derived amino acid sequencepredicted a total of 9 Cys residues (Binzet al., 1990,J. Biol. Chem. 265, 9153–9158; Thompsonet al., 1990,Eur. J. Biochem. 189, 73–81). Treatment of the dichain neurotoxin, dissolved in 6 M guanidine. HCl, with 4-vinylpyridine converted 5 Cys residues into S-pyridylethyl cysteine residues; but alkylation after mercaptolysis converted all 9 Cys residues in the S-pyridylethylated form. After confirming the predicted number of Cys residues by amino acid analysis, the positions of the 5 Cys residues carrying sulfhydryl groups and the 4 involved in disulfide bridges were determined by comparing the elution patterns in reversed-phase HPLC of the cyanogen bromide mixtures of the exclusively alkylated and the mercaptolyzed-alkylated neurotoxin. The chromatographically isolated components were identified by N-terminal amino acid sequence analysis. The HPLC patterns showed characteristic differences. The Cys residuespredicted in positions 133, 164, 790, 966, and 1059 were found in the sulfhydryl form; Cys 429 and 453 were found disulfide-bridged connecting the light and heavy chains, and Cys 1234 and 1279 were found in an intrachain disulfide-bridge near the C-terminus in the heavy chain. Ten amino acid residues, Thr 438-Lys 447,predicted to be present in the single chain neurotoxin were not found in the dichain neurotoxin. Nicking of single-chain neurotoxin by the protease(s) endogenous to the bacteria therefore appears to excise these 10 amino acid residues from the nicking region which leaves Lys 437 as the C-terminus of the light chain and Ala 448 as the N-terminus of the heavy chain. The N-terminal Pro 1 and C-terminal Leu 1295,predicted from the nucleotide sequence, remain conserved after nicking. Residues Pro 1-Lys 437 and Ala 448-Leu 1295 constitute the light and heavy chains, respectively. The C-termini were determined by isolation of short C-terminal peptide fragments and subsequent sequence analysis by Edman degradation. About 20% of the amino acid sequence predicted from DNA analysis was confirmed in these studies by protein-chemical methods.     

Human complement component C1s. Partial sequence determination of the heavy chain and identification of the peptide bond cleaved during activation

Human C1s proenzyme (Mr 83 000) was isolated by a rapid two-stage method involving affinity chromatography of C1 on IgG-Sepharose and isolation of subcomponent C1s by ion-exchange chromatography on DEAE-Sephacel. Single-chain C1s proenzyme was activated to two-chain C1s with self-activated C1r. After reduction and S-carboxamidomethylation the heavy chain of C1s (Mr 57 000) was isolated by ion exchange chromatography on DEAE-Sephacel. Cleavage of C1s heavy chain with CNBr yielded five fragments whose N-terminal sequences were determined. The alignment of the fragments within the heavy chain was established by tryptic peptides containing methionine. C1s heavy chain comprises about 470 amino acid residues and 42% of its sequence was determined. An intrachain sequence homology and a homology to the alpha 2 chain of human haptoglobin were identified. The C-terminal CNBr fragment comprising 44 amino acid residues was completely sequenced. From BNPS-skatole cleavage of reduced and alkylated C1s proenzyme a fragment was isolated which overlaps the C1s heavy and light chain parts and which contains the peptide bond cleaved during activation. The results show that this is an Arg-Ile bond and that under standard conditions of activation no peptide material is liberated from this portion of the molecule. The sequence data and homology to two-chain serine proteases indicate a single interchain disulfide bond in C1s.     

Covalent Structure of Botulinum Neurotoxin Type E: Location of Sulfhydryl Groups, and Disulfide Bridges and Identification of C-Termini of Light and Heavy Chains

Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an 150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992), Biochem. Biophys. Res. Commun. 183, 107–113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein—at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)—is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419–421 (Gly–Ile–Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1–Lys 418 constitute the 50–kDa light chain, Lys 422–Lys 1251 constitute the 100–kDa heavy chain; Cys 411–Cys 425 and Cys 1196–Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The 150–kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without ^I4C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nucleotide sequence.     

Botulinum Neurotoxin Type A: Limited Proteolysis by Endoproteinase Glu-C and α-Chymotrypsin Enhanced Following Reduction; Identification of the Cleaved Sites and Fragments

Botulinum neurotoxin (NT) serotype A is a ～150-kDa dichain protein. Posttranslational nicking of the single-chain NT (residues Pro 1–Leu 1295) by the protease(s) endogenous to Clostridium botulinum excises 10 residues, leaving Pro 1–Lys 437 and Ala 448–Leu 1295 in the ～50-kDa light (L) and ～100-kDa heavy (H) chains, respectively, connected by a Cys 429–Cys 453 disulfide and noncovalent bonds [Krieglstein et al. (1994), J. Protein Chem. 13, 49–57]. The L chain is a metalloprotease, while the amino- and carboxy-terminal halves of the H chain have channel-forming and receptor-binding activities, respectively [Montecucco and Schiavo (1995), Q. Rev. Biophys. 28, 423–472]. Endoproteinase Glu-C and α-chymotrypsin were used for controlled digestion at pH 7.4 of the ～150-kDa dichain NT and the isolated ～100-kDa H chain (i.e., freed from the L chain) in order to map the cleavage sites and isolate the proteolytic fragments. The dichain NT appeared more resistant to cleavage by endoproteinase Glu-C than the isolated H chain. In contrast, the NT with its disulfide(s) reduced showed rapid digestion of both chains, including a cleavage between Glu 251 and Met 252 (resulting in ～30- and ～20-kDa fragments of the L chain) which was not noted unless the NT was reduced. Interestingly, an adjacent bond, Tyr 249–Tyr 250, was noted earlier [DasGupta and Foley (1989), Biochimie 71, 1193–1200] to undergo “self-cleavage” following reductive separation of the L chain from the H chain. The site Tyr–Tyr–Glu–Met (residues 249–252) appears to become exposed following reduction of Cys 429–Cys 453 disulfide. Identification of Glu 669–Ile 670 and Tyr 683–Ile 684 as protease-susceptible sites demonstrated for the first time that at least two peptide bonds in the segment of the H chain (residues 659–684), part of which (residues 659–681) is thought to interact with the endosomal membranes and forms channels [Oblatt-Montal et al., (1995), Protein Sci. 4, 1490–1497], are exposed on the surface of the NT. Two of the fragments of the H chain we generated and purified by chromatography are suitable for structure–function studies; the ～85- and ～45-kDa fragments beginning at residue Leu 544 and Ser 884, respectively (both extend presumably to Leu 1295) contain the channel-forming segment and receptor-binding segments, respectively. In determining partial amino acid sequences of 10 fragments, a total of 149 amino acids in the 1275-residue NT were chemically identified.     

Conformation and disulfide bond formation of the constant fragment of an immunoglobulin light chain: effect of truncation at the C-terminal region

Constant fragments with different carboxyl terminals, CL(109-211), CL(109-207), and CL-(109-200), were prepared by limited carboxypeptidase P or Y proteolysis of the constant fragment, CL-(109-214), of a type lambda immunoglobulin light chain, and their conformations and stabilities, and formation of the disulfide bond from the reduced fragments, were studied. No change in conformation or stability was observed on removal of three residues from the C-terminal end. Removal of seven or more residues from the C-terminal end destabilized the CL fragment. The rate of disulfide bond formation from reduced CL(109-207) was about 7 times faster than that for CL(109-214). These findings suggest that elongation of the polypeptide chain at least beyond the 207th residue is necessary for folding of the CL fragment into a definite conformation.     

Covalent Structure of Botulinum Neurotoxin Type B; Location of Sulfhydryl Groups and Disulfide Bridge and Identification of C-Termini of Light and Heavy Chains

Botulinum neurotoxin (NT) serotype B, produced by Clostridium botulinum (proteolytic strain), is a 150-kDa single-chain polypeptide of 1291 amino acids, of which 10 are Cys residues [Whelan et al. (1992), Appl. Environ. Microbiol. 58, 2345–2354] The posttranslational modifications of the gene product were found to consist of excision of only the initiating Met residue, limited proteolysis (nicking) of the 1290-residue-long protein between Lys 440 and Ala 441, and formation of at least one disulflde bridge. The dichain (nicked) protein, in a mixture with the precursor single-chain (unnicked) molecules, was found to have a 50-kDa light chain (Pro 1 through Lys 440) and a 100-kDa heavy chain (Ala 441 through Glu 1290). The limited in vivo nicking of the single-chain NT to the dichain form, by protease endogenous to the bacteria, and the nonfacile in vitro cleavage by trypsin of the Lys 440–Ala 441 bond appear to be due to the adjacent Ala 441–Pro 442 imide bond's probable cis configuration in a mixed population of molecules with cis and trans configurations. The two chains were found connected by an interchain disulfide formed by Cys 436 and Cys 445. Six other Cys residues, at positions 70, 195, 308, 777, 954, and 1277, were found in sulfhydryl form. In addition, a Cys at position 1220 or 1257 appeared to be in sulfhydryl form, hence our experimental results could not unambiguously identify presence of an intrachain disulfide bridge near the C-terminus of the NT. A total of 384 amino acid residues, including the 6 Cys residues at positions 70, 195, 308, 436, 445, and 1277, were identified by direct protein-chemical analysis; thus 29.7% of the protein's entire amino acid sequence predicted from the nucleotide sequence was confirmed. The 6 amino acids, residues 945–950, did not match with the sequence predicted in 1992, but did match with a later report of 1995. The above determinations were made by a combination of chemical (CNBr and acidic cleavage at Asp–Pro) and enzymatic (trypsin, clostripain, and pepsin) cleavages of the NT, and NT carboxymethylated with iodoacetamide (with or without ¹⁴C label), separation and isolation of the fragments by SDS–PAGE (followed by electroblotting onto PVDF membrane), and/or reversed-phase HPLC, and analyses of the fragments for the N-terminal amino acid sequences by Edman degradation and amino acid compositions.     

Involvement of C-terminal 14 residues of alkali light chain in binding to the heavy chain of myosin

The alkali light chain of rabbit skeletal muscle myosin, A1, was cyanylated with 2-nitro-5-thiocyanobenzoic acid, and the peptide bond at Cys 177 was subsequently cleaved in the presence of 0.05 M CaCl2. Two peptide fragments, from the N-terminal to the residue 176 (CF1) and from the residue 177 to the C-terminal (CF2), were obtained. The CD spectrum and the difference UV absorption spectrum induced by CaCl2 suggested that CF1 largely retained the higher order structure of A1. The CF1 fragment, however, could neither incorporate subfragment-1 (S-1) by an exchange reaction, nor bind with the renatured 20K fragment of S-1 heavy chain. On the other hand, the C-terminal fragment of 14 residues, CF2, could bind with the 20K fragment of S-1 heavy chain. These results indicate that the binding site of the alkali light chain for the heavy chain of myosin is located within the C-terminal 14 residues.     

Status of Cys Residues in the Covalent Structure of Botulinum Neurotoxin Types A, B, and E

Clostridium botulinum neurotoxin (NT) serotypes A, B, and E have 9, 10, and 8 Cys residues, respectively, as deduced from nucleotide sequences [Whelan et al. (1992), Appl. Environ. Microbiol. 48, 2345–2354]. Each of the 150-kDa NTs has at least one disulfide; but type B, like types A and E, may have two disulfides. Using two different chemical reagents, we studied the status of the Cys residues in these three proteins after (i) the final anion exchange chromatographic step in their purification (fresh NT), (ii) 24 hr storage at 8°C, (iii) precipitation with ammonium sulfate (precipitated NT), and (iv) dissolving the precipitated NT in 6 M guanidine·HCl. In all three NT serotypes the number of Cys residues titrated with 5,5-dithiobis-2-nitrobenzoic acid (DTNB) as free -SH groups varied, depending upon the absence or presence of EDTA added to the chromatography buffer, storage condition, age, and presence of the denaturant. Titration of 9.5–10 and 5.4–6.0 -SH groups in fresh NTs type B and E, respectively, indicated total and partial absence of disulfide bonds. Fewer titratable -SH groups in the precipitated NT than in the fresh NT suggested formation of disulfide and/or inaccessibility of the -SH groups due to protein's conformational change(s). When the precipitated NTs were dissolved in 6 M guanidine·HCl, in the absence of any added reducing agent, all Cys residues of types B and E, and 6.4–8.3 Cys in type A NT were titratable with DTNB. Iodoacetamide modification of precipitated NT types A, B, and E carboxymethylated 4, 2, and 2 Cys residues, respectively; these numbers rose to 6, 9.4, and 8 when these proteins were carboxymethylated after dissolving in 6 M guanidine·HCl in the absence of any added reducing agent. We propose that -S-S- cleavage mediated by the -SH/-S-S- exchange observed in vitro after unfolding the NTs (also unfolded by 2 M guanidine·HCl or urea) possibly mimicks a similar exchange process inside the endosomes, where the NTs are thought to undergo conformational changes, resulting in the reductive cleavage of the interchain disulfide between the 50-kDa light and 100-kDa heavy chain, which in turn releases the light chain and allows its egress out of the endosomes into the cytosol.     

Structure of heavy and light chain subunits of type A botulinum neurotoxin analyzed by circular dichroism and fluorescence measurements

Summary The secondary and tertiary structural features of botulinum neurotoxin (NT) serotype A, a dichain protein (M_r 145 000), and its two subunits, the heavy (H) and light (L) chains (M_r 97 000 and 53 000, respectively) were examined using circular dichroism and fluorescence spectorscopy. Nearly 70% of the amino acid residues in each of the three polypeptide preparations were found in ordered structure (sum of helix, sheet and turns). Also, the helix, sheet, turns and random coil contents of the dichain NT were nearly equal to the weighted mean of each of these secondary structure parameters of the L and H chains; e.g., sum of helix of L chain (22%) and H chain (18.7%), as weighted mean, 19.8% was similar to that of NT (20%). These agreements suggested that the secondary structures of the subunits of the dichain NT do not significantly change when they are separated as isolated L and H chains. Fluorescence emission maximum of L chain, 4 nm less (blue shift) than that of H chain, suggested relatively more hydrophobic environment of fluorescent tryptophan residue(s) of L chain. Tryptophan fluorescence quantum yields of L chain, H chain and the NT, 0.072, 0.174 and 0.197, respectively, suggested that a) an alteration in the micro-environment of the tryptophan residues was possibly caused by interactions of L and H chain subunits of the NT and b) quantum yields for L and H chains were altered when they are together as subunits of the NT. Possible implications of structural features of the L and H chains, their interactions and the molecular mechanism of action of botulinum NT are assessed.     

Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.     

Botulinum neurotoxin type E fragmented with endoproteinase Lys-C reveals the site trypsin nicks and homology with tetanus neurotoxin

Botulinum neurotoxin type E, a 150 kDa single chain protein, cleaved with endoproteinase Lys-C yielded 113, 73, and 50 kDa fragments. The N-terminal sequence of the 113 kDa fragment, Gly-Ile-Arg-Lys-Ser-Ile-Cys-Ile, overlaps the N-terminal sequence, Lys-Ser-Ile-Cys-Ile, of the 103 kDa heavy chain produced by nicking the neurotoxin with trypsin. The -Arg-Lys- bond is therefore the site on the single chain type E NT where trypsin nicks generating the 50 kDa light and 103 kDa heavy chains of the dichain NT. The sequence of the first 50 N-terminal residues of the 73 kDa fragment were determined. This fragment is a segment of the heavy chain; 50% of the 50 residues are present in identical positions in a similar segment of the heavy chain of tetanus neurotoxin.     

Purification and characterization of β-structural domains of β-lactoglobulin liberated by limited proteolysis

Incubation of β-lactoglobulin with immobilized trypsin at 5–10°C results in a time-dependent release of several fragments of the core domain in yields approaching 15%. Digests were fractionated by ion-exchange chromatography with a Mono Q HR5/5 column and analyzed after disulfide reduction by polyacrylamide gel electrophoresis in sodium dodecylsulfate. Three fragments with approximate molecular weights of 13.8, 9.6, and 6.7 kD were identified. The fraction from ion-exchange chromatography yielding the 6.7 kD fraction after disulfide reduction was further characterized because it was most homogeneous and gave the highest yield. The C-terminal cleavage site of the 6.7 kD core fragment appeared to be Lys₁₀₀ or Lys₁₀₁ as determined by C-terminal amino acid analysis. The exact masses, after reduction with dithiothreitol, are 6195 and 6926 as determined by laser desorption mass spectrometry, corresponding to residues 48–101 and 41–100. Prior to reduction, β-lactoglobulin C-terminal residues 149–162 are connected to these core domain fragments as shown by C-terminal analysis and mass spectrometry. Structural studies indicate that these 7.9 and 8.6 kD core domain fragments released by immobilized trypsin retain much of their native structure. CD spectra indicate the presence of antiparallel β-sheet structure similar to the native protein but the α-helix is lost. Spectra in the aromatic region indicate the existence of tertiary structure. Moreover, structural transitions in urea are completely reversible as measured by CD spectra, although the extrapolated ΔG _D ^H20 and the urea concentration at the transition midpoint are lower than for the native protein. The core domain fragments also display apH-dependent binding to immobilizedtrans-retinal as does intact protein. A single endotherm is obtained for both core domain fragments and native protein upon differential scanning calorimetry, but again, the domain is less stable as indicated by a transition peak maxima of 56.9°C as compared with 81.1°C for native protein.     

Human immunoglobulin subclasses. Partial amino acid sequence of the constant region of a γ4 chain

The heavy chain of a human myeloma protein (Vin) belonging to the gamma4 subclass was subjected to tryptic digestion after reduction and carboxymethylation. Cyanogen bromide fragments were also prepared and all 19 tryptic peptides that account for one of them (the Fc-like fragment) were studied. Selected peptic peptides were isolated and provided evidence for the order of 15 of the tryptic peptides. In addition the sequence of two large peptic peptides derived from two sections of the molecule including all the interchain bridges is presented. Comparison with published data on other chains allows us to propose a sequence of gamma4 chains that extends from just before the presumed starting point of the invariable region (at about residue 113) to the C-terminal end of the chain (approx. residue 446), except for a section of about 50 residues. The results of the comparison suggest that the immunoglobulin subclasses have a recent independent evolutionary origin in different species. Implications for complement fixation and for the evolutionary origin of antibody diversity are also discussed.     

Primary structure of cyanogen bromide fragments of sei whale (Balaenoptera borealis) pituitary somatotropin]

5 fragments are isolated after the degradation of somatotropin from sei whale pituitary glands with cyanogen bromide: N-terminal 4-segmented; C-terminal 12-segmented with the internal disulfide bond; middle 25- and 30-segmented and a high molecular weight fragment following N-terminal tetrapeptide and bound with disulfide bond to 30-segmented fragment. Complete amino acid sequence of three shortest cyanogen bromide fragments is deciphered and N- and C-terminal sequence is investigated in two large fragments after their uncoupling under performic acid oxidation. Amino acid sequence is deciphered of a peptide obtained after trypsine hydrolysis of 30-segmented cyanogen bromide fragment. Comparison of amino acid sequence of whale somatotropin fragments with that of sheep, beef and human somatotropin has revealed that 57 out of 61 identified amino acid residues of whale somatotropin repeat amino acid residues in similar regions of beef somatotropin, 56--of sheep and only 42--of human somatotropins. Besdies, 4 of 5 revealed amino acid substitutions in whale hormone, as compared with sheep somatotropin, are amino acids which are present at the same positions in human hormone.     

Effect of the light chain C-terminal serine residue on disulfide bond susceptibility of human immunoglobulin G1λ

The light chain cysteine residue that forms an interchain disulfide bond with the cysteine residue in the heavy chain in IgG1κ is the last amino acid. The cysteine residue is followed by a serine residue in IgG1λ. Effect of the serine residue on the susceptibility of disulfide bonds to reduction was investigated in the current study using a method including reduction, differential alkylation using iodoacetic acid with either natural isotopes or enriched with carbon-13, and mass spectrometry analysis. This newly developed method allowed an accurate determination of the susceptibility of disulfide bonds in IgG antibodies. The effect of the serine residue on disulfide bond susceptibility was compared using three antibodies with differences only in the light chain last amino acid, which was either a serine residue, an alanine residue or deleted. The results demonstrated that the presence of the amino acid (serine or alanine) increased the susceptibility of the inter light and heavy chain disulfide bonds to reduction. On the other hand, susceptibility of the two inter heavy chain disulfide bonds and intrachain disulfide bonds was not changed significantly.     

Localisation of light chain and actin binding sites on myosin

A gel overlay technique has been used to identify a region of the myosin S-1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I-labelled myosin light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S-1 prepared from these myosins and the C-terminal tryptic fragments from them (i.e. the 20-kDa or 24-kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S-1 respectively). MgATP abolished actin binding to myosin and to S-1 but had no effect on binding to the C-terminal tryptic fragments of S-1. The light chains and actin appeared to bind to specific and distinct regions on the S-1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50-100 molar excess of unlabelled competing protein. The skeletal muscle C-terminal 24-kDa fragment was isolated from a tryptic digest of Mg2+/papain S-1 by CM-cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5-I-AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N-terminal and C-terminal sequence analyses. Electron microscopical examination of this S-1 C-terminal fragment revealed that: it had a strong tendency to form aggregates with itself, appearing as small 'segment-like' structures that formed larger aggregates, and it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24-kDa fragment with Staphylococcus aureus V-8 protease produced a 10-12-kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10-12-kDa peptide was derived from the region between the SH1 thiol residue and the C-terminus of S-1. It was further shown that the C-terminal portion, but not the N-terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three-dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S-1, it appears that these sites are all located within a length of the S-1 heavy chain of about 100 amino acid residues.