Location Of Disulfide Bonds Research Articles

Solution structure of murine epidermal growth factor: determination of the polypeptide backbone chain-fold by nuclear magnetic resonance and distance geometry.

The polypeptide backbone fold in the solution structure of murine epidermal growth factor has been determined by nuclear magnetic resonance spectroscopy and distance geometry calculations. The results are based on nearly complete sequence-specific resonance assignments and on 333 distance and dihedral-angle constraints; these were determined from nuclear Overhauser effect measurements, identification of hydrogen-bonded amide protons, the known locations of disulfide bonds, and backbone vicinal spin-spin coupling constants. The polypeptide chain of the protein is arranged into two distinct domains. The structures of these domains were determined independently in separate calculations and then combined to obtain an overall view of the protein. The backbone fold thus determined includes the regular backbone structure elements that were previously identified using different techniques for the analysis of the nuclear magnetic resonance data. The distance geometry calculations also provided additional details about the conformations of bends and loops and about the twists of the beta-sheets.

Topology and order of formation of interchain disulfide bonds in von Willebrand factor

Interchain disulfide bonds between the subunits in von Willebrand factor (vWf) dimers and in vWf multimers have been studied using some unique features of the cultured human umbilical vein endothelial cell system. Ammonium chloride inhibition of multimerization of vWf allowed selective examination of vWf dimeric molecules, and monoclonal antibody against the vWf propolypeptide was used to separate pro-vWf dimers from mature dimers. After cleavage of dimers and multimers with Staphylococcus aureus V-8 protease, the location of interchain disulfide bonds in amino (N)-terminal or carboxyl (C)-terminal fragments was determined by gel electrophoresis under reduced and nonreduced conditions. The first interchain disulfide bonds formed during dimerization are in the C-terminal region of the subunits, whereas interdimer disulfide bonds are located in the N-terminal portion. These data confirm recent electron microscopic projections of disulfide bond locations and provide support to the hypothetical role of the propolypeptide in the multimerization process.

Topology and Order of Formation of Interchain Disulfide Bonds in von Willebrand Factor

Interchain disulfide bonds between the subunits in von Willebrand factor (vWf) dimers and in vWf multimers have been studied using some unique features of the cultured human umbilical vein endothelial cell system. Ammonium chloride inhibition of multimerization of vWf allowed selective examination of vWf dimeric molecules, and monoclonal antibody against the vWf propolypeptide was used to separate pro-vWf dimers from mature dimers. After cleavage of dimers and multimers with Staphylococcus aureus V-8 protease, the location of interchain disulfide bonds in amino (N)-terminal or carboxyl (C)-terminal fragments was determined by gel electrophoresis under reduced and nonreduced conditions. The first interchain disulfide bonds formed during dimerization are in the C-terminal region of the subunits, whereas interdimer disulfide bonds are located in the N-terminal portion. These data confirm recent electron microscopic projections of disulfide bond locations and provide support to the hypothetical role of the propolypeptide in the multimerization process.

Location of the inter-chain disulfide bonds of the third component of human complement

Location of the disulfide bonds connecting three polypeptide chains (alpha 3, 27kd; 2, 43kd; beta, 75kd) of C3c has been investigated by partial reduction with cysteine followed by alkylation with 14C-monoiodoacetic acid. Treatment of C3c with cysteine produced a partially reduced fragment, composed of disulfide-linked beta and alpha 3 chains. A single thiol residue was detected on the alpha 3 chain but not on the beta chain of the fragment, suggesting that the alpha 2 chain in C3c is linked through a single disulfide bond to the alpha 3 chain but not to the beta chain.

Peptide studies using a fast atom bombardment high field mass spectrometer and data system. 2—characteristics of positive ionization spectra of peptides,m/z 858 tom/z 5729

Sixteen peptides ranging in molecular weights from 858 to 5729 were examined under positive ionization fast atom bombardment conditions employing a high field magnet mass spectrometer and data system. The contributions of the polyisotopic elements to the molecular protonated ion envelope become important in the interpretation of the data at higher mass. For masses less than 2000 u, the most abundant ion within this envelope is the monoisotopic molecular protonated ion. Above 2000 u, the most abundant ion in the envelope is a polyisotopic molecular protonated ion. Characterization of the peptide requires identification of both the molecular protonated ion envelope and significant fragment ions. Partial spectra displaying both the molecular ion and significant fragment ions are presented for mastoparan (mol. wt = 1478), somatostatin (mol. wt = 1637), bovine parathyroid hormone (1–34) (mol. wt = 4106), and bovine insulin (mol. wt = 5729). A partial spectrum for bovine ribonuclease A (mol. wt = 13673) displayed significant fragment ions that identified the protein. The types of fragment ions included those that indicated the amino acid sequence and the location of disulfide bonds. The abundance of these ions appears to be influenced by the characteristics of the noble gas fast atom beam and the sample matrix.

Chemical studies on actinoxanthin.

The antitumor protein actinoxanthin exhibits high inhibitory activity against a number of gram-positive bacteria and some strains of transplantable leucoses and related tumors. Actinoxanthin was shown to consist of a single polypeptide chain crosslinked by two disulfide bonds and to contain 107 amino acid residues. Reduced and alkylated actinoxanthin was digested with chymotrypsin, thermolysin and trypsin. Based on the sequence analysis of fragments so obtained the complete amino acid sequence and the location of disulfide bonds of actinoxanthin has been proposed. The high degree homology of some regions of actinoxanthin and the antitumor protein neocarzinostatin have been revealed.

Mitochondrial adenosine triphosphatase. Location of sulfhydryl groups and disulfide bonds in soluble enzyme from beef heart.

The soluble beef heart mitochondrial ATPase (F1) contains eight sulfhydryl groups and two disulfide bonds. N-Ethylmaleimide has been used to radioactively label the sulfhydryl groups before and after cleavage of the disulfide bonds by dithiothreitol. After subjecting the labeled protein to polyacrylamide gel electrophoresis in sodium dodecyl sulfate and measuring radioactivity in each of the separated subunits the location of all the sulfhydryl groups and the disulfide bonds may be specified. The conclusions are supported by direct examination of depolymerized, unreduced, enzyme by polyacrylamide gel electrophoresis. The results also indicate that current ideas regarding the overall subunit structure of this enzyme may be incorrect, and this is discussed in light of new data presented here.