Incorporation of bovine enterokinase in reconstituted soybean phospholipid vesicles.

Incubation of the Ca2+, Mg2+-activated adenosine triphosphatase of Escherichia coli with phospholipid vesicles resulted in binding of the enzyme to the lipid. Binding was observed with vesicles of soybean phospholipid (asolectin), phosphatidylglycerol, phosphatidylserine, phosphatidylcholine, and cardiolipin. Binding was not affected by alterations in pH in the range of pH 6.5 to 8.5, by ionic strength, or by the presence of Mg2+. Loss of the δ subunit from the enzyme had no effect on binding. However, removal of the δ and ε subunits by treatment of the enzyme with trypsin prevented binding to phospholipid. This treatment also removed a small portion (<2000 daltons) of the α subunit. It is concluded that the ATPase of E. coli binds to phospholipid vesicles mainly by nonpolar interactions through the α and (or) ε subunits of the enzyme.

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A heteronuclear and homonuclear filtering strategy for studying the structure of membrane peptides in non-deuterated phospholipid vesicles

Journal de Chimie Physique ◽

10.1051/jcp:1998125 ◽

1998 ◽

Vol 95 (2) ◽

pp. 221-224

Author(s):

B. T. Doan ◽

C. Nezry ◽

L. Rene ◽

B. Badet ◽

J. C. Beloeil

Keyword(s):

Phospholipid Vesicles ◽

Membrane Peptides

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Thermally induced budding of phospholipid vesicles — a discontinuous process

Journal de Physique II ◽

10.1051/jp2:1993157 ◽

1993 ◽

Vol 3 (5) ◽

pp. 631-645 ◽

Cited By ~ 17

Author(s):

J. Käs ◽

E. Sackmann ◽

R. Podgornik ◽

S. Svetina ◽

B. Žekš

Keyword(s):

Phospholipid Vesicles ◽

Thermally Induced ◽

Discontinuous Process

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Plasma Factor V Activation Is Prevented by Activated Protein C in the Presence of Phospholipid Vesicles, Not Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651567 ◽

1993 ◽

Vol 69 (02) ◽

pp. 124-129 ◽

Cited By ~ 2

Author(s):

Susan Solymoss ◽

Kim Thi Phu Nguyen

Keyword(s):

Western Blotting ◽

Protein C ◽

Thrombin Generation ◽

Blood Platelets ◽

Activated Protein C ◽

Phospholipid Vesicles ◽

Factor V ◽

Two Stage ◽

One Stage ◽

Plasma Factor

SummaryActivated protein C (APC) is a vitamin K dependent anticoagulant which catalyzes the inactivation of factor Va and VIIIa, in a reaction modulated by phospholipid membrane surface, or blood platelets. APC prevents thrombin generation at a much lower concentration when added to recalcified plasma and phospholipid vesicles, than recalcified plasma and platelets. This observation was attributed to a platelet associated APC inhibitor. We have performed serial thrombin, factor V one stage and two stage assays and Western blotting of dilute recalcified plasma containing either phospholipid vesicles or platelets and APC. More thrombin was formed at a given APC concentration with platelets than phospholipid. One stage factor V values increased to higher levels with platelets and APC than phospholipid and APC. Two stage factor V values decreased substantially with platelets and 5 nM APC but remained unchanged with phospholipid and 5 nM APC. Western blotting of plasma factor V confirmed factor V activation in the presence of platelets and APC, but lack of factor V activation with phospholipid and APC. Inclusion of platelets or platelet membrane with phospholipid enhanced rather than inhibited APC catalyzed plasma factor V inactivation. Platelet activation further enhanced factor V activation and inactivation at any given APC concentration.Plasma thrombin generation in the presence of platelets and APC is related to ongoing factor V activation. No inhibition of APC inactivation of FVa occurs in the presence of platelets.

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Glycoproteins IIb and IIIa and Platelet Thrombospondin in a Liposome Model of Platelet Aggregation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661529 ◽

1986 ◽

Vol 55 (02) ◽

pp. 240-245 ◽

Cited By ~ 11

Author(s):

M E Rybak

Keyword(s):

Platelet Aggregation ◽

Direct Evidence ◽

Phospholipid Vesicles ◽

Aggregate Formation ◽

Membrane Glycoproteins ◽

Protein Orientation ◽

Phosphatidylcholine Liposomes ◽

Calcium Dependent ◽

Lentil Lectin ◽

Complete Reversal

SummaryPlatelet membrane glycoproteins IIb and IIIa and platelet thrombospondin were incorporated onto phosphatidylcholine liposomes, by freeze thawing and sonication. Protein orientation on the liposomes was confirmed by susceptibility to neuraminidase cleavage and binding to lentil lectin-Sepharose (GPIIb-IIIa liposomes) and to heparin-Sepharose (thrombospondin liposomes). Glycoproteins Ilb-IIIa bound 125I-fibrinogen with Kd of 7.5 × 10™7M. Binding was reversible and calcium-dependent. Ilb-IIIa liposomes underwent fibrinogen-dependent aggregation in the presence of 10 mM CaCl2. Maximal aggregate formation was observed with a combination of IIb-IIIa liposomes and thrombospondin liposomes. This aggregation was partially inhibited by preincubation with monoclonal antibodies to the IIb-IIIa complex. Addition of EDTA caused complete reversal of aggregates. Thrombospondin liposomes also underwent fibrinogen and calcium dependent aggregation, however, this aggregation was less than that observed with the GPIIb-IIIa liposomes. Maximal aggregate formation was observed with a mixture of IIb-IIIa liposomes and thrombospondin liposomes. These studies demonstrate that GPIIb-IIIa and thrombospondin can be incorporated into phospholipid vesicles with preservation of function. Direct evidence is provided to demonstrate that glycoprotein lib and Ilia and fibrinogen are sufficient for platelet aggregation and to demonstrate that thrombospondin may also contribute to platelet aggregation.

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