Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes

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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

Bibtex

@article{98dc85fca578428694a038b50b59a431,

title = "Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes",

abstract = "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.",

keywords = "5'-Nucleotidase, Alkaline Phosphatase, Aminopeptidases, Animals, Annexins, Antigens, CD13, Cell Membrane, Centrifugation, Density Gradient, Cholesterol, Dipeptidyl Peptidase 4, Fatty Acids, Nonesterified, Glutamyl Aminopeptidase, Glycosylphosphatidylinositols, Intracellular Membranes, Lung, Membrane Lipids, Microsomes, Peptidyl-Dipeptidase A, Phospholipids, Polyethylene Glycols, Solubility, Swine",

author = "Edward Parkin and Turner, {A J} and Hooper, {N M}",

year = "1996",

language = "English",

volume = "319 ",

pages = "887--896",

journal = "Biochemical Journal",

issn = "0264-6021",

publisher = "Portland Press Ltd.",

number = "3",

}

RIS

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 -

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