alpha-chymotrypsin and monobromobimane

We previously showed that the fluorescent inter-thiol cross-linker dibromobimane (DBB) [Kosower, N. S., Kosower, E. M., Newton, G. L., & Ranney, H. M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382-3386] cross-links two [50 and 20 kilodaltons (kDa)] of the three major fragments of myosin subfragment 1 (S-1); on intact S-1, DBB quenches tryptophans and inhibits all ATPases [Mornet, D., Ue, K., & Morales, M. F. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1658-1662]. Here we characterize the modification chemically: DBB cross-links Cys-522 (50 kDa) with Cys-707 (20 kDa), thereby sealing a large preexisting heavy-chain loop containing important functionalities. Cross-linking rate is insensitive to nucleotides, but apparently sterically, either monobromobimane or DBB reduces Ca2+-ATPase to low, nonzero levels.

Topics: Animals; Bridged Bicyclo Compounds; Bridged-Ring Compounds; Chymotrypsin; Cross-Linking Reagents; Cyanogen Bromide; Dithionitrobenzoic Acid; Fluorescent Dyes; Kinetics; Mercaptoethanol; Muscles; Myosin Subfragments; Myosins; Peptide Fragments; Rabbits

Highly active mouse cytotoxic T lymphocytes (CTL) generated in secondary mixed-lymphocyte responses were used to examine the manner in which adenosine derivatives, thiol-specific reagents, or protease-specific probes affected CTL-mediated lysis (CML). The adenosine deaminase inhibitor deoxycoformycin (dCF) enhanced inhibition by adenosine (AR) or by deoxyadenosine (AdR), but not by 7-deazaadenosine (tubercidin). L-Homocysteinethiolactone (L-Hcy) acted synergistically with AR, but not with AdR or tubercidin, to block CML. Thus, AR derivatives may act both by affecting cellular methylation reactions, as demonstrated by the synergism between AR and L-Hcy, and by inhibiting other events required for CML. Conditions were then established to determine whether these reagents preferentially affected either the Ca2+-independent initial stage of cytolysis or the subsequent Ca2+-dependent events. Methylation inhibitors blocked lysis most effectively if added before effector-target binding. Similarly, the nonpenetrating thiol-specific reagent quaternary ammonium monobromobimane (qBBr) was more inhibitory when added prior to the Ca2+-dependent stage. Protease inhibitors such as alpha-1-antichymotrypsin and protease substrates such as acetyltyrosine ethyl ester (ATEE) or tyrosine ethyl ester (TEE) also inhibited CML. But, in contrast to qBBr or methylation inhibitors, neither TEE nor ATEE was more effective when added prior to the initial effector-target interaction. Furthermore, TEE did not appreciably affect CTL binding to target cells at concentrations that nearly abrogated CML. Thus, the implicated protease step is unique in that it does not appear to participate in recognition or binding.

Topics: Adenosine; alpha 1-Antichymotrypsin; Animals; Bridged Bicyclo Compounds; Calcium; Chymotrypsin; Coformycin; Cytotoxicity, Immunologic; Deoxyadenosines; Methylation; Mice; Mice, Inbred Strains; Pentostatin; Peptide Hydrolases; Sulfhydryl Compounds; T-Lymphocytes, Cytotoxic; Time Factors; Tyrosine