Penetration of C8 and C9 in the C5b-9 complex across the erythrocyte membrane into the cytoplasmic space.
We have developed a technique in which transglutaminase is used to measure the penetration of terminal complement proteins across the erythrocyte membrane into the cytoplasmic space. Penetration of a given terminal complement protein into the cytoplasmic space was assessed by labeling the protein of interest with radioactive iodine, forming the complement channel using the labeled protein, adding transglutaminase to only one side of the membrane, and allowing the enzyme to cross-link the susceptible proteins on that side of the membrane. Cross-linking was assessed by measuring the increase in molecular weight of the appropriate molecule on sodium dodecyl sulfate gels under reducing conditions. The results of these experiments indicate that C8 and C9 are rapidly cross-linked to high molecular weight from either the interior or the exterior of the membrane. In order to determine whether the cross-linking mediated by enzyme on the interior was occurring from within the ghosts and not via enzyme that had leaked into the extracellular medium, experiments were performed with dimethylcasein in the extracellular medium. In the presence of this protein, cross-linking of C8 and C9 from outside was negligible. Hence, if cross-linking occurs when transglutaminase is trapped inside the ghosts, it cannot be due to leakage of enzyme, but must be attributable to cross-linking from the inside. The results show that C9 definitely penetrated across the membrane into the intracellular space. With respect to C8, statistical evaluation indicates that C8 probably penetrated into the intracellular space.
Whitlow
M B
MB
Ramm
L E
LE
Mayer
M M
MM
D000217:Acyltransferases / Q000378:metabolism; D000906:Antibodies; D002364:Caseins / Q000494:pharmacology; D003185:Complement C8 / Q000378:metabolism*; D003186:Complement C9 / Q000378:metabolism*; D015938:Complement Membrane Attack Complex; D003165:Complement System Proteins / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005577:Forssman Antigen / Q000276:immunology; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000032:analysis*; D011503:Transglutaminases
D000906:Antibodies; D002364:Caseins; D003185:Complement C8; D003186:Complement C9; D015938:Complement Membrane Attack Complex; D008565:Membrane Proteins; D003165:Complement System Proteins; D005577:Forssman Antigen; D000217:Acyltransferases; D011503:Transglutaminases