Research output: Contribution to Journal/Magazine › Journal article › peer-review
Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes. / Parkin, Edward; Turner, A J; Hooper, N M.
In: Biochemical Journal, Vol. 319 , No. 3, 1996, p. 887-896.Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes
AU - Parkin, Edward
AU - Turner, A J
AU - Hooper, N M
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
KW - 5'-Nucleotidase
KW - Alkaline Phosphatase
KW - Aminopeptidases
KW - Animals
KW - Annexins
KW - Antigens, CD13
KW - Cell Membrane
KW - Centrifugation, Density Gradient
KW - Cholesterol
KW - Dipeptidyl Peptidase 4
KW - Fatty Acids, Nonesterified
KW - Glutamyl Aminopeptidase
KW - Glycosylphosphatidylinositols
KW - Intracellular Membranes
KW - Lung
KW - Membrane Lipids
KW - Microsomes
KW - Peptidyl-Dipeptidase A
KW - Phospholipids
KW - Polyethylene Glycols
KW - Solubility
KW - Swine
M3 - Journal article
C2 - 8920995
VL - 319
SP - 887
EP - 896
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 3
ER -