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.     

Cyanogen bromide cleavage of proteins in sodium dodecyl sulphate/polyacrylamide gels. Diagonal peptide mapping of proteins from epidermis.

A two-dimensional electrophoretic procedure employing CNBr has been devised for the analysis of proteins in sodium dodecyl sulphate/polyacrylamide gels. The technique allows the detection of an unusual class of epidermal proteins that lack methionine. The proteins have been identified by this method in newborn mouse, rat, and rabbit, because they are stable in the presence of CNBr and consequently lie on a diagonal. Adult human epidermis also contains CNBr-stable proteins, but in lesser amounts than in the newborn rabbit or newborn rodents. The methionine-containing proteins (i.e., the keratins) are degraded by CNBr into a series of unique and characteristics peptides which lie below the diagonal. Inter- and intra-species similarities and differences exist between the individual keratins, depending on the number and distribution of their methionine residues. The peptide-map patterns for the rodent and lagomorph proteins are more similar to each other than to that for the human proteins. The maps for rat and rabbit skin proteins are the most similar. We conclude that the epidermal keratins are a closely related, yet individually distinct, group of proteins that are found in conjunction with a class of proteins that lack methionine. The latter proteins are related to the histidine-rich basic protein, an epidermal structural protein that aggregates with keratin filaments.     

Cyanogen bromide cleavage at methionine residues of polypeptides containing disulfide bonds

A method for cleaving polypeptides at their methionine residues without affecting intramolecular disulfide bonds is described. This method may be applied for cleaving recombinant heterologous hybrid polypeptides with release of the interesting peptide. The method may also be applied to assign the correct positions of disulfide bonds in protein molecules.     

Cyanogen bromide cleavage of methionine residues as a control method for enkephalin immunocytochemistry

Serial semithin sections of rat neurohypophysis were immunostained with 2 antibodies to enkephalins using the peroxidase antiperoxidase method. One of the antibodies (R133) recognizes both met- and leu-enkephalin whereas the other (R26) reacts with met-enkephalin only. After cyanogen bromide pretreatment of the sections the antibody R133 stained only a subpopulation of nerve endings that were distinct from those stained with the latter antibody. R26-(met-enkephalin-like) immunoreactivity was totally abolished by cyanogen bromide pretreatment. This preincubation method which selectively interferes with the staining of met-enkephalin terminals may help to discriminate the two enkephalins in immunocytochemical preparations.     

Cyanogen bromide cleavage of methionin residues as a control method for enkephalin immunocytochemistry

Summary Serial semithin sections of rat neurohypophysis were immunostained with 2 antibodies to enkephalins using the peroxidase antiperoxidase method. One of the antibodies (R133) recognizes both met- and leu-enkephalin whereas the other (R26) reacts with met-enkephalin only. After cyanogen bromide pretreatment of the sections the antibody R133 stained only a subpopulation of nerve endings that were distinct from those stained with the latter antibody. R26-(met-enkephalin-like) immunoreactivity was totally abolished by cyanogen bromide pretreatment. This preincubation method which selectively interferes with the staining of met-enkephalin terminals may help to discriminate the two enkephalins in immunocytochemical preparations.     

Cyanogen bromide cleavage and partial sequence of the heavy chain of a pathological immunoglobulin G

The heavy chain of a pathological immunoglobulin G (Daw) of type L, subclass gamma(2b) (We) and Gm(a+)(f-), has been cleaved with cyanogen bromide. The five fragments resulting from the cleavage have been isolated and analysed. The papain-digest fragments, Fab and Fc, have also been cleaved with cyanogen bromide and the products analysed and compared with those from the heavy chain. The order of the five fragments in the heavy chain has been established and the location of some of the inter-chain and inter-fragment disulphide bonds has been determined. The sequence of the N-terminal fragment consisting of 34 residues is reported.     

Yeast phosphoglycerate mutate. Cyanogen bromide cleavage and amino acid sequence of an active-site peptide.

The molecular weight and amino acid composition of phosphoglycerate mutase from yeast were determined. CNBr cleavage produced a large (190-residue) fragment and a small (60-residue) fragment. Tryptic and chymotryptic peptides derived from the large fragment were fractionated by ion-exchange chromatography. Peptides from two histidine-containing regions were isolated and the amino acid sequences were determined. Correlation of these data with X-ray-crystallographic evidence shows that the histidine residue in the sequence Arg-Leu Asn-Glu-Arg-His-Tyr-Gly-Asp-Leu-Glu-Gly-Lys is located at the active site.     

Cyanogen bromide fragments of Akazara scallop Mr 52,000 troponin-I

Akazara scallop troponin-I of Mr 52,000 (52K) was cleaved into two fragments of 17K and 35K with cyanogen bromide. The 17K fragment, along with tropomyosin, inhibited weakly the rabbit actomyosin Mg-ATPase activity, however, the 35K fragment did not affect it at all. In the presence of Akazara scallop TnT (40K component), the 17K fragment, in turn, strongly inhibited the activity, while the 35K fragment did not. The amino acid composition and partial amino acid sequence suggested that the 17K and 35K fragments were derived from C- and N-terminal regions of the TnI, respectively, and that structural similarity to TnIs from other animals is present in the 17K region.     

Cyanogen bromide fragments of bovine cervical mucus glycoprotein.

Treatment of bovine cervical mucus glycoprotein with cyanogen bromide gives four fractions, with the molecular weights of the three major fractions being 230 000, 130 000, and 35 000. The results indicate that, as for other glycoproteins, there are different regions along the protein core, some of which have a high sugar content and others which contain considerably less carbohydrate; it seems likely that the regions of lower sugar content may be important to intermolecular linkages. The data suggest that the basic unit of the glycoprotein has a molecular weight of 550 000-600 000, with its protein core consisting of approx. 1200 amino acid residues.     

In situ cyanogen bromide cleavage of N-terminally blocked proteins in a gas-phase sequencer

Herein we describe a procedure for the in situ cyanogen bromide cleavage of N-terminally blocked proteins which have been immobilised onto the glass fiber sample disk of the gas-phase sequencer. In this manner, new amino terminii suitable for automated Edman degradation can be generated. Cytochrome C was cleaved using this method on the carboxyl side of methionine residues 65 and 80. This allowed sequence analysis to begin simultaneously at residues 66 and 81 (Table 1). This procedure offers an alternative sequencing tactic for methionine-containing proteins which are N-terminally blocked.