Cyanogen bromide cleavage of bovine secretory component and its tryptic fragments

1. Five-domain bovine secretory component and its two-domain and three-domain tryptic fragments have been treated with cyanogen bromide. 2. N-terminal sequence analysis of the purified products showed that cleavage occurred within the disulphide bridged polypeptide loop of domain 2. The site lies within the region that binds IgM and IgA dimers. 3. The relative binding of the CNBr fragments to IgM has been measured and indicates that domains 1 and 3 are directly involved. 4. A possible role for domain 2 is less clear and domains 4 and 5 do not participate in binding.     

Active fragments obtained by cyanogen bromide cleavage of ovomucoid.

Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant O1, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11 000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56% of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol.wts. of approx. 12000 (fragment L) and 4700 (fragment S). Each of these peptides contained carbohydrate. Marked heterogeneity was observed in the hexose and hexosamine contents of fragment L. This may account for much of the heterogeneity in neutral carbohydrate occurring in ovomucoid preparations. It was found that fragment M was located at the N-terminal end, fragment S was in the centre and fragment L made up the C-terminal portion of the molecule.     

Lipid binding by fragments of apolipoprotein C-III-1 obtained by thrombin cleavage.

We have used thrombin to cleave apolipoprotein C-III-1 into two fragments constituting residues 1-40 (apoLP-C-III-A) and 41-79 (apoLP-C-III-B). The lipid binding properties of these fragments with dimyristoyl- and 1-palmitoyl-2-oleoylphosphatidylcholines have been determined using circular dichroic and intrinsic tryptophan fluorescence spectroscopy. The peptide-phospholipid mixtures were fractionated by density gradients of cesium chloride. ApoLP-C-III-A showed disordered structure in the absence and presence of DMPC and no significant amount of peptide-phospholipid complex was isolated. ApoLP-C-III-B showed conformational changes in the circular dichroic spectrum and a shift in the intrinsic tryptophan fluorescence spectrum. Ultracentrifugation in cesium chloride gradients yielded peptide-phospholipid complexes isolated between density 1.10 and 1.18. The molar ratio of lipid to protein was 12:1. The results of these studies and the examination of space filling models of apoLP-C-III provide evidence that an amphipathic alpha helix which contains a nonpolar face and a polar face is the basic structural unit for binding of phospholipid by the plasma apolipoproteins. These results also provide direct evidence that the hydrophobicity of the nonpolar face is important in lipid binding since the nonpolar face of residues 1-40 is considerably less hydrophobic than the nonpolar face of residues 41-79.     

Characterization of the lac repressor species produced by limited tryptic cleavage

Tryptic cleavage of native lac repressor under very mild conditions has been found to yield preparations suitable for detailed physical and chemical analysis. Sephadex G-200 chromatography of the digest produces one main protein peak followed by small peptides. The protein from the main peak was analyzed by automated Edman degradation and revealed two unique cleavage sites, one at residue 51 and the other at 59. The tryptic core protein under native conditions is tetrameric and exhibits a circular dichroism spectrum similar to that of native lac repressor.     

Characterization of a fully active N-terminal 37-kDa polypeptide obtained by limited tryptic cleavage of pig kidney D-amino acid oxidase

In order to obtain further information on the structure of D-amino acid oxidase (EC 1.4.3.3), limited proteolysis experiments have been carried out on its apo-, holo-, and holoenzyme-benzoate forms. The enzyme is unsensitive to 10% (w/w) chymotrypsin, while incubation with 10% (w/w) trypsin, under nondenaturating conditions, produces inactivation and proteolysis patterns which are different for the three forms of enzyme analyzed. These results confirm the previously reported conformational changes which occur upon binding of coenzyme to the apoprotein, and of benzoate to holoenzyme. The stable 37.0-kDa polypeptide, obtained from the apo- and holoenzyme-benzoate complex upon cleavage of a C-terminal 2.0-kDa fragment, retains full catalytic activity with unaltered kinetic parameters, and the coenzyme binding properties of the native enzyme. These results are in agreement with the tentative localization of the FAD-binding domain in the N-terminal region of the enzyme, and with the hypothesis that the function of the C-terminal region of D-amino acid oxidase could be related to the import of the enzyme into the peroxisomes, as suggested by Gould et al. (Gould, S. J., Keller, G. A., and Subramani, S. (1988) J. Cell. Biol. 107, 897-905).     

The disulfide bond pattern between fragments obtained by the limited proteolysis of bovine thyroglobulin.

The comparative analysis of the products of the limited proteolysis of bovine thyroglobulin with trypsin by SDS-polyacrylamide gel electrophoresis in non-reducing and reducing conditions revealed the presence of disulfide linkages between some of the fragments. In order to define the disulfide bond pattern between the proteolytic fragments of thyroglobulin, these were isolated by SDS-polyacrylamide gel electrophoresis in non-reducing conditions and electrophoretic transfer onto polyvinylidene difluoride membranes. Individual bands were desorbed from the membranes and re-analyzed by SDS-polyacrylamide gel electrophoresis in reducing conditions. The resulting peptides were identified by comparison with the peptides directly obtained by SDS-electrophoresis in reducing conditions, and characterized by amino-terminal peptide sequencing either in this study or in a previous investigation (Gentile F., Salvatore G., Eur. J. Biochem. 218 (1993) 603-621). The analysis revealed that several fragments, produced by cleavages within the context of various cysteine-rich repeats of type 1 and within cysteine-rich repeat 3b.1, did not separate in the absence of reduction. On the other hand, the products of the cleavages at the carboxy-terminal extremity of the linker between type 2 and type 3 cysteine-rich repeats, and in the middle of the acetylcholinesterase-similar domain of thyroglobulin separated freely, with no need for reduction. On the base of these data, a model is presented in which distinct subsets of cysteine-rich repeats and the carboxy-terminal, acetylcholinesterase-similar domain of thyroglobulin form sequentially aligned subdomains with internal disulfide linkages.     

Liberation of tryptic fragments from caseinomacropeptide of bovine kappa-casein involved in platelet function. Kinetic study.

Carbohydrate-free caseinomacropeptide (CMP) was purified from rennet-hydrolysed caseinate by trichloroacetic acid precipitation and DEAE-TSK Fractogel-650 ion-exchange chromatography. To study the liberation of 106-112, 106-116 and 113-116 fragments from carbohydrate-free CMP involved in platelet function, a quantitative study was made on the rate of hydrolysis of the three peptidic bonds that are susceptible to the action of trypsin. Data were obtained from reverse-phase (Ultrabase column) and cationic-exchange (Mono S column) h.p.l.c. On the basis of the disappearance of substrate, kcat. and Km were respectively 3.95 s-1 and 0.2 mM. The two 111-112 and 112-113 bonds were split according to similar kinetic parameters (kcat. = 1.97 s-1, Km = 0.2 mM) and much faster than the 116-117 bond. The difference in susceptibility of the bonds can probably be attributed to the nature of residues flanking the primary proteolytic sites rather than to their accessibility to the proteinase. On the basis of our results the 106-116 fragment cannot be formed.     

Chromatin core particle unfolding induced by tryptic cleavage of histones.

Chromatin 'core particles' have been digested with trypsin to varying extents. The resulting particles are homogeneous by the criterion of ultracentrifuge boundary analysis. Sedimentation coefficients are lowered as cleavages are introduced into the histones, showing that an unfolding of the core particle occurs. This unfolding is further characterised by a lower melting temperature together with a premelting phase, higher molar ellipticity in the circular dichroism spectra at 280 nm and increased kinetics of digestion by both micrococcal nuclease and DNase I. Differences are also observed in the products of nuclease digestion. The most consistent interpretation of the data involves an unfolding process whereby free rods of DNA are released to extend from a nucleoprotein core.     

Nuclear magnetic resonance studies on the solution conformation of histone IV fragments obtained by cyanogen bromide cleavage.

Two histone IV fragments obtained by cleavage at Met-84 by cyanogen bromide have been examined by proton magnetic resonance (PMR) spectroscopy as a function of temperature, peptide concentration, ionic strength, and pD. Sedimentation and gel electrophoresis studies on these peptides have also been carried out. The 220-MHz PMR spectrum of the N-peptide in both the high- and low-field regions was shown to be almost identical with that calculated for an extended coil N-peptide monomer. The calculated random coil and experimental C-peptide spectra, on the other hand, differ in many respects. Evidence was obtained for the presence of rigid secondary structure in the C-peptide. In addition, the Val, Leu, Ile CH3 resonance displays a prominent high-field satellite band which shifts downfield with increasing temperature. Sedimentation studies on the N-peptide reveal the formation of extremely large, remarkably homogeneous aggregates at ionic strengths larger than or equal to 0.01. The C-peptide, on the other hand, does not appear to form aggregates of sufficient size to be detectable in velocity sedimentation studies of a few hours duration. The relative area changes which have previously been noted in the PMR spectrum of histone IV with increasing ionic strength were also observed for the N-peptide but not the C-peptide. Interpretation of these relative area changes has been made in terms of the amino acid sequence of histone IV, and an effort was made to identify that segment of the polypeptide which undergoes secondary structural change with increasing ionic strength.     

Structure of the tryptic glycopeptide isolated from rabbit transferrin.

The structure of the tryptic glycopeptide isolated from rabbit transferrin was elucidated by use of sequential Edman degradations, specific exoglycosidases, endo-beta-N-acetylglucosaminidases, methylation analyses, and periodate oxidation studies. The glycopeptide consists of a heteropolysaccharide, AcNeualpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 3[AcNeualpha2 leads to 6Galbeta1 leads to 4GlcNAcbeta1 leads to 2Manalpha1 leads to 6]-Manbeta1 leads to 4GlcNAcbeta1 leads to 4GlcNAc, attached to a peptide, Asn-Ser-Ser-Leu-Cys, via a linkage involving N-acetyl-glucosamine and asparagine. The stoichiometry of this glycopeptide is 2 mol/mol of protein, indicating that rabbit transferrin contains two structurally identical glycopeptide segments.     

The sites of tryptic cleavage in bovine secretory component: structural and functional implications

Tryptic fragments from bovine secretory component and sIgA have been separated by HPLC and/or SDS polyacrylamide gel electrophoresis and electroblotting. Their N-terminal amino acid sequences have been determined and their positions in the secretory component molecule deduced by homology with the amino acid sequences of human secretory component and rabbit polyimmunoglobulin receptor. Taken in conjunction with the known binding affinities of the tryptic fragments, the results imply that the three most N-terminal domains of secretory component are directly involved in binding IgM and IgA dimers. The results also favour the concept of an extended 'zig-zag' structure for the secretory component molecule.     

Isolation and biochemical characterization of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density.

Relatively homogeneous fractions of proteoglycan fragments were prepared from tryptic digests of the 4M-guanidinium chloride extract of bovine nasal cartilage. Glycosaminoglycan-containing fragments were separated from non-proteoglycan contaminants by ion-exchange chromatography and fractionated by equilibrium density-gradient centrifugation under dissociative conditions. The fractions of highest buoyant density were chromatographed on a column of Sepharose 4B, digested with chondroitinase ABC and chromatographed on a column of Sepharose 6B, yielding two distinct fractions: fraction B/6B-4 contained fragments from the chondroitin sulphate-bearing region of the proteoglycan monomer, and fraction B/6B-2 fragments from the keratan sulphate-rich region, most probably including a chondroitin sulphate-bearing monomer segment. By dansyl chloride analysis, fraction B/6B-2 had alanine and leucine as sole and fraction B/6B-4 had isoleucine and leucine as greatly predominant N-terminal amino acids, indicative of the relative homogeneity of these preparations of cartilage proteoglycan monomer fragments.     

Mössbauer studies of electrophoretically purified monoferric and diferric human transferrin

Summary Electrophoretically purified⁵⁷Fe-enriched monoferric and diferric human transferrins and selectively labeled complexes ([C-⁵⁶Fe,N-⁵⁷Fe]transferrin and [C-⁵⁷Fe,N-⁵⁶Fe]transferrin) were studied by Mössbauer spectroscopy. The data were recorded at 4.2 K over a wide range of applied magnetic fields (0.05–6 T) and were analyzed by a spin-Hamiltonian formalism. Characteristic hyperfine parameters were found and the obtained zero-field splitting parameters (D=0.25±0.05 cm^–1 andE/D = 0.30 ± 0.02) agree with previous electron paramagnetic resonance (EPR) findings. The weak-field spectra of the [N-⁵⁷Fe]transferrin are slightly broader than those of the [C-⁵⁷Fe]transferrin, indicating that the N-terminal iron site may be more heterogeneous. However, the absorption line positions and the relative intensities of the subspectra originating from the three Kramers doublets of each Fe³⁺ site are identical. Thus the electronic structures of the two iron sites can be described by the same set of spin- Hamiltonian parameters, indicating that the ligand environments for the two sites are the same, as suggested by the recent X-ray crystallographic studies. This suggestion is further supported by the observation that the strong-field spectra of the two monoferric transferrins are indistinguishable. The selectively labeled mixed-isotope transferrins exhibit spectra that are identical to those of the corresponding monoferric⁵⁷Fe-enriched transferrins, implying that the occupation of one iron site has little or no effect on the immediate envirnoment of the other site, a finding that is not surprising since the two sites are separated by approximately 4.2 nm.