Human erythrocyte ghosts crenated by salt can undergo conversion to smooth discs and cup-forms in a reaction requiring MgATP. In previous work, evidence against a requirement for protein or lipid phosphorylation was found, and inhibition of MgATP-dependent shape change by 1 MuM orthovanadate was shown to be associated with inhibition of approximately one-half of the MgATPase activity of the ghosts. This project will apply biochemical and biophysical techniques to study the mechanism of MgATP-dependent shape change and the possible role fo the vanadate-sensitive MgATPase. Experimental conditions will be optimized for shape change and MgATPase activity, and the relationship of these functions to ghost resealing and endocytosis will be studied. The mechanism of shape change will be addressed in studies fo MgATP-dependent, vanadate-sensitive changes in membrane protein organization: Fluorescence photobleaching recovery measurements, protein cross-linking and competition of membrane protein fragments and monovalent specific antibodies will be used to investigate these changes. The possible role of the membrane skeleton will be examined in a study of the compositions of Triton X-100 residues prepared after membrane incubations, and attempts will be made to detect MgATP-dependent volume changes in isolated skeletons under shape change conditions. In kinetic studies of the putative """"""""shape change ATPase"""""""", the effects o vanadate, ATPLambdaS and other agents will be compared with effects fo the same inhibitors on shape change. Photoaffinity labeling with (32p)8-azidoATP and analysis of (48V) vanadate binding patterns will be used to identify components of the shape change system and sites of vanadate inhibition. The gross localization of the MgATPase will be determined in membrane subfractionation experiments, and attempts will be made to isolate the vanadate-sensitive enzyme after detergent solubilization. The basis for the metabolic requirement in the maintenance of red cell biconcave shape is a fundamental unsolved problem in hematology. The long-term objective of this research is to use the ghost shape change model to elucidate it at the molecular level.
| Patel, V P; Fairbanks, G (1986) Relationship of major phosphorylation reactions and MgATPase activities to ATP-dependent shape change of human erythrocyte membranes. J Biol Chem 261:3170-7 |
