alpha-chymotrypsin and methyl-4-mercaptobutyrimidate

Preformed phosphatidylethanolamine-containing liposomes were thiolated with 2-iminothiolane (Traut's reagent) and subsequently activated by mixed disulfide formation with 5,5'-dithiobis(2-nitrobenzoic acid). Up to 65% of amino groups of the outer liposomal lamella, corresponding to 230 SH-groups per vesicle, were modified. Covalent attachment of thiolated alpha-chymotrypsin to these thiol-liposomes via S-S bridges yielded a protein/lipid ratio of 3.6 X 10(-4) mol enzyme/mol lipid.

Topics: Chymotrypsin; Dithionitrobenzoic Acid; Imidoesters; Liposomes; Phosphatidylethanolamines; Sulfhydryl Compounds

Limited proteolysis and chemical cross-linking techniques have been used to study the interaction between alpha- and beta-tubulin subunits. Trypsin digestion of tubulin dimer resulted in the cleavage of the alpha-subunit into two fragments, whereas chymotrypsin cleaved the beta-subunit into two distinct fragments. All of these fragments have been mapped on the tubulin subunits by further proteolysis with formic acid. Cross-linking of trypsin- and chymotrypsin-cleaved subunits has been performed with two different cross-linker agents of different cross-linking distance. The addition of formaldehyde resulted in the cross-linking of the alpha-tubulin N-terminal fragment with beta-tubulin C-terminal domain. The same result was obtained when methyl 4-mercaptobutyrimidate was used.

Topics: Animals; Chymotrypsin; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Formaldehyde; Imidoesters; Peptide Fragments; Subtilisins; Swine; Trypsin; Tubulin