Ca~(2+)-MEDIATED CHANGE IN LIPID FLUIDITY MODULATES THE RECONSTITUTED SARCOPLASMIC RETICULUM Ca~(2+)-ATPase ACTIVITY
Ca~(2+)通过膜脂影响肌质网Ca~(2+)-ATP酶的活性与Ca~(2+)转运功能

Ca2 + -ATPase from sarcoplasmic reticulum was reconstituted into the soybean phospholipid vesicles with the freeze-thawing mathod.Four types of proteoliposomes containing sarcoplasmic reticulum Ca2+-ATPase have been prepared; Ca2+-"free" ,L.(Ca2+-ATPase) ;outside Ca2+-containing, L.(Ca2+-ATPase) ; inside Ca2+-containing,L. (Ca2+-ATPase) and two-side Ca2+-containing,L.(Ca2+-ATPase) proteoliposomes. L.(Ca2+-ATPase) was prepared by ATP driven Ca2+ pumping from outside into the interior of the proteoliposomes. Being chelated by EGTA the outside Ca2+ of the vesicles, the L. (Ca2+-ATPase) was transformed into the L. (Ca2+ -ATPase) .The fluidity of the proteoliposomes has been compared by fluorescence polarization probes diphenylhexatriene (DPH) or 7-, 12-, 16-(9-anthroyloxy)stearic acid (7, 12, 16-AS). The degree of polarization in these proteoliposomes decreases in the order.L.(Ca2+-ATPase) >L.(Ca2+-ATPase) >L.(Ca2+-ATPase) >L.(Ca2+-ATPase) .Concomitantly with the lipid fluidity measurements the ATP driven Ca2+ transport and Ca2+-ATPase activity were also monitored. It was interested to note that as the proteoliposomes were loaded with Ca2+ due to ATP driven Ca2+ pumping, the reconstituted Ca2+-ATPase activity as well as the Ca2+ transport was gradually inhibited. The ATP hydrolysis rate of Ca2+-ATPase containing proteoliposomes in the 4th minute was only 9% of that in the 1st minute. However, no similar inhibition was observed when the activity of purified non-incorporating Ca2+-ATPase was determined under the same experimental conditions. So, it seems that the reconstituted Ca2+-ATPase activity in liposomes may be modulated by the Ca2+-mediated changing fluidity of the lipid bilayer, resulting from the variation of transmembrane Ca2+ concentration gradient.