Treatment of SR membranes with mild acid (pH 5.6) (Berman, M.C., McIntosh, D.B. and Kench, J.E. (1977) J. Biol. Chem. 252, 994-1001) or incubation with millimolar concentrations of ethylene glycol bis (β-aminoethyl ether)-N ,N'-tetraacetic acid (EGTA) at neutral pH and 37°C (McIntosh, D. B. and Berman, M. C. (1978) J. Biol. Chem. 253, 5l40-5146) results in a progressive irreversible inhibition of calcium transport while (Ca²⁺, Mg²⁺)-ATPase activity is unimpaired. Possible conformational changes associated with this uncoupling were monitored by following alterations in kinetic mobility of sulphydryl (-SH) groups either by using 5, 5'-dithiobis- (2-nitrobenzoate) (DTNB) and stopped flow analysis or 1-¹⁴C-N-ethylmaleimide (NEM). Kinetic reactivity with DTNB revealed a total of 20 thiol groups/1.5 x 10⁵ g of SR protein (rontaining 1 mole of ATPase protein) in the presence of sodium dodecyl sulphate, which constitute four kinetic classes. In native control vesicles 4.5 thiol groups were unreactive, 0.4 represented the fast reacting class, 0.8 the moderately fast reacting class and 14.4 the slowly reacting class, displaying pseudo-first order rate constants, k, of 159.0-, 22.0- and 6.23 x 10⁻² sec⁻¹, respectively. Inactivation of calcium transport to the extent of 90%, using mild acid conditions, increased the number of fast and moderately fast reacting groups, each by 1.0 - 1.5 sulphydryl groups / mol ATPase. The number of slowly reacting groups decreased by approximately 3 .0 thiol groups/mol ATPase. The kinetics of the reaction with 1-¹⁴C-NEM was essentially similar to that with DTNB. EGTA inactivation of calcium transport, to the extent of 90% and subsequent 1-¹⁴C-NEM modification, resulted in an increase in the number of fast reacting thiol groups by 0.5-1.0 thiol groups/mol ATPase. The total number of reactive thiol groups decreased by 1.0 -2.0 thiol groups/ mol ATPase, probably due to autoxidation of the newly exposed sulphydryl group. Inactivation of transport carried out in the presence of N-ethylmaleimide to prevent autoxidation resulted in an increase of approximately one thiol group/mol ATPase. The rate constant for the increase in reactivity of this group was 1.45 min⁻¹. This thiol group was localized on the ATPase protein of molecular weight approximately 100 000 daltons. Trypsinization of the ATPase produced four fragments of molecular weights 55 000, 45 000, 30 000 and 20 000. More extensive cleavage resulted in a significant decrease in the 55 000 dalton fragment and increased amounts of the 30 000 and 20 000 dalton subfragments. There was increased labelling on all subfragments of EGTA-treated vesicles compared to control, untreated vesicles. However, the greatest relative increase in labelling appeared to be localized on the 55 000 dalton and 20 000 dalton subfragments. Peptide mapping of the purified ATPase revealed 24 ninhydrin-positive peptides. Five of these were labelled in control and EGTAtreated vesicles, four of which showed increased labelling in the latter preparation. Random labelling of the nonoverlapping fragments may be due to the enzyme being "trapped" in a number of intermediate conformations or due to heterogeneity within the ATPase populations. NEM modification of SR membranes did not affect the tryptic cleavage pattern or the mobilities of the tryptic subfragments. It did however, affect the extent of tryptic cleavage resulting in solubilization of NEM-labelled protein into the medium following centrifugation. This protein fraction was identified as consisting largely of the 55 000 dalton molecular weight species on sodium dodecyl sulphate gel electrophoresis. It is concluded that occupancy of high affinity K₀.₅(Ca²⁺)≈10⁻⁶M) calcium binding sites maintain the (Ca²⁺, Mg²⁺)- ATPase in a stable, coupled conformation. Displacement of this calcium induces a conformational change in the protein which results in the loss of the vectorial component of calcium transport.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uct/oai:localhost:11427/27256 |
| Date | January 1981 |
| Creators | Swiel, Denise |
| Contributors | Berman, Mervyn C |
| Publisher | University of Cape Town, Faculty of Health Sciences, Division of Chemical Pathology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Doctoral Thesis, Doctoral, PhD |
| Format | application/pdf |
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