Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes.
The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5'-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca(2+)-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the mental chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.
Parkin
E T
ET
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Turner
A J
AJ
Hooper
N M
NM
D015720:5'-Nucleotidase / Q000032:analysis; D000469:Alkaline Phosphatase / Q000032:analysis; D000626:Aminopeptidases / Q000032:analysis; D000818:Animals; D017302:Annexins / Q000032:analysis*; D018826:CD13 Antigens / Q000032:analysis; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000032:analysis; D018819:Dipeptidyl Peptidase 4 / Q000032:analysis; D005230:Fatty Acids, Nonesterified / Q000032:analysis; D043384:Glutamyl Aminopeptidase; D017261:Glycosylphosphatidylinositols / Q000032:analysis; D007425:Intracellular Membranes / Q000737:chemistry* / Q000201:enzymology; D008168:Lung / Q000737:chemistry* / Q000201:enzymology; D008563:Membrane Lipids / Q000032:analysis* / Q000737:chemistry; D008861:Microsomes / Q000201:enzymology; D007703:Peptidyl-Dipeptidase A / Q000032:analysis; D010743:Phospholipids / Q000032:analysis*; D011092:Polyethylene Glycols; D012995:Solubility; D013552:Swine
D017302:Annexins; D005230:Fatty Acids, Nonesterified; D017261:Glycosylphosphatidylinositols; D008563:Membrane Lipids; D010743:Phospholipids; D011092:Polyethylene Glycols; D002784:Cholesterol; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D000626:Aminopeptidases; D018826:CD13 Antigens; D043384:Glutamyl Aminopeptidase; C022750:X-Pro aminopeptidase; D018819:Dipeptidyl Peptidase 4; D007703:Peptidyl-Dipeptidase A