Characteristics of spectrin-induced leakage of extruded, phosphatidylserine vesicles.
At neutral pH spectrin induces modest leakage of trapped calcein from reverse-phase or extruded, but not sonicated, vesicles composed of phosphatidylserine, but not phosphatidylcholine. The extent of leakage from extruded vesicles is not or is only slightly affected by magnesium ions at a physiological concentration or calcium ions at a greater than physiological concentration, respectively. In addition to accounting for several previously discrepant observations on the lytic effects of spectrin, these findings indicate that some proteins like spectrin may destabilize vesicles with low curvature more readily than vesicles of high curvature, in contrast to certain amphiphilic peptides. 60% less leakage is induced from phosphatidylserine vesicles by heat-denatured than by native spectrin. In contrast, both trypsin- and subtilisin-treated spectrins, if sufficiently digested, induce several-fold more leakage than undigested spectrin. Since spectrin prepared either by 1 M Tris dissociation of Triton-extracted cytoskeletons or by low ionic strength extraction of ghosts released the same amounts of calcein from vesicles of various compositions, these effects are unlikely to reflect artifacts of spectrin preparation. Furthermore, spectrin is unlikely to promote leakage in vivo, since vesicles composed of phosphatidylserine, cholesterol and/or phosphatidylethanolamine, which constitute the lipid composition of the inner monolayer of the red cell membrane, did not leak on addition of spectrin, whereas vesicles composed of phosphatidylserine and phosphatidylcholine, did leak in the presence of spectrin.
Subbarao
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NK
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208.
MacDonald
R I
RI
Takeshita
K
K
MacDonald
R C
RC
D002118:Calcium / Q000494:pharmacology; D002784:Cholesterol / Q000737:chemistry; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology; D008563:Membrane Lipids / Q000737:chemistry*; D009994:Osmolar Concentration; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry*; D011489:Protein Denaturation; D013049:Spectrin / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology
D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D013049:Spectrin; D002784:Cholesterol; D013381:Subtilisins; D014357:Trypsin; C017251:1,2-oleoylphosphatidylcholine; D008274:Magnesium; D002118:Calcium