alpha-chymotrypsin and 2-3-dimethylmaleic-anhydride

Modification with succinic anhydride (SA) of Clostridium perfringens epsilon prototoxin or toxin resulted in a loss of activation by trypsin or lethal activity, respectively. The prototoxin was more sensitive to succinylation than the toxin. On the other hand, the succinylated prototoxin was activated and cleaved by chymotrypsin, but not by trypsin. The lethal activity of the toxin was also lost after treatment with 2,3-dimethylmaleic anhydride (DMA) or 2,4,6-trinitrobenzenesulfonic acid (TNBS). When the DMA-treated toxin treated with SA or TNBS was incubated under acidic condition, it regained lethal activity. Thus modification of amino groups (lysine residues) prevented activation of the prototoxin by trypsin, and abolished lethal activity of the toxin.

Topics: Bacterial Toxins; Chymotrypsin; Clostridium perfringens; Lysine; Maleic Anhydrides; Structure-Activity Relationship; Succinic Anhydrides; Trinitrobenzenesulfonic Acid; Trypsin

Bovine band 3 which had been cleaved into two membrane intercalated fragments by extracellular chymotryptic digestion was partially purified and the behavior of fragmented band 3 was compared with that of intact band 3 in nonionic detergent, C12E9, solution. The two fragments were always co-eluted with nearly a 1:1 molar ratio from ion-exchange and gel filtration columns, confirming the observation of Reithmeier ((1979) J. Biol. Chem. 254, 3054-3060) that the fragments are bound together. However, as shown by the molecular size of fragmented band 3 and its reactivity with dimethylmaleic anhydride and/or a cross-linking reagent, the fragmentation caused some change in gross conformation of the protein. Dissociation of the two fragments, though not complete, was found to occur on treatment of fragmented band 3 with dimethylmaleic anhydride, which is a dissociating reagent for band 3 oligomers into the monomers. The disruption of self-associated band 3 was accompanied with a disruption of helical structure. The results suggest that a mutual interaction between the chymotryptic fragments is of importance to form the folded structure maintaining a band 3 dimer.

Topics: Animals; Anion Exchange Protein 1, Erythrocyte; Blood Proteins; Cattle; Chemical Phenomena; Chemistry; Chymotrypsin; Detergents; Erythrocyte Membrane; Erythrocytes; Maleic Anhydrides; Peptide Fragments; Protein Conformation; Solutions

The state of association of bovine Band 3, which is a major intrinsic protein of erythrocyte membranes, was examined in nonaethyleneglycol dodecyl ether (C12E9) solution by ultracentrifugation, gel filtration, gel electrophoresis and cross-linking studies. The molecular weight of bovine Band 3 was 107,000 +/- 5% as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. When Band 3 was purified in C12E9 solution with the aid of 2,3-dimethylmaleic anhydride (DMMA), which is a known dissociating reagent for peripheral proteins from erythrocyte membranes and for Band 3-Band 4.2 complex, the protein was present as a monomer and was not oxidatively cross-linked. On the other hand, DMMA-untreated Band 3 was present as oligomeric forms composed mainly of the dimer and tetramer, and the oligomer in C12E9 was a susceptible to oxidative cross-linking as Band 3 in ghosts. The oligomeric form apparently retained a more ordered conformational state than the monomeric form. These results indicate that the bovine Band 3 oligomer is a stable form in C12E9, but the present result showing the coexistence of dimer and tetramer in C12E9 contrasts with the reported observation that human Band 3 is present as a stable dimer in a nonionic detergent, Triton X-100. It was shown that polyacrylamide gradient gel electrophoresis in the presence of C12E9 gave better resolution of the associated species of Band 3 than ultracentrifugation or gel filtration, and this method made it possible to determine the Stokes radii of Band 3-C12E9 complexes. This result suggests the usefulness of electrophoretic methods in the presence of nonionic detergent for studies of the state of association of other membrane proteins.

Topics: Animals; Cattle; Chymotrypsin; Erythrocyte Membrane; Erythrocytes; Macromolecular Substances; Maleic Anhydrides; Membrane Proteins; Molecular Weight; Peptide Fragments; Polidocanol; Polyethylene Glycols; Sodium Dodecyl Sulfate; Solubility; Spectrometry, Fluorescence