J. Biol. Chem., Vol. 263, Issue 17, 8111-8119, Jun, 1988
Volume enlargement and recovery of Na+-dependent amino acid transport in proteoliposomes derived from Ehrlich ascites cell membranes
JI McCormick and RM Johnstone
McGill University, Department of Biochemistry, Montreal, Quebec, Canada.
Na+-dependent amino acid transport can be reconstituted from solubilized
Ehrlich cell plasma membranes by addition of asolectin vesicles, gel
filtration, and a freeze-thaw cycle. Removal of phosphatidic acid
(approximately 10% of the total lipid) by Ba2+ precipitation reduces the
efficiency of reconstitution of Na+-dependent amino acid transport by
approximately 73% and decreases intravesicular volume of the
proteoliposomes by approximately 43%. The loss of transport activity is not
due to exclusion of specific proteins during reconstitution. The
phosphatidic acid-free liposomes are less permeable and require more time
to attain an equilibrium distribution of solute. Transport activity and
intravesicular volume can be restored to Ba2+- precipitated asolectin
proteoliposomes by addition of egg-phosphatidic acid during reconstitution.
The extent of recovery of transport activity is proportional to the change
in intravesicular volume and depends on the amount of phosphatidic acid
present. Replacement of phosphatidic acid with 20% phosphatidylserine or
phosphatidylglycerol leads to increases in intravesicular volume with
little or no increase in amino acid transport. Generation of phosphatidic
acid in situ by treatment of Ba2+-precipitated proteoliposomes with
phospholipase D also restored transport. The observed increase in transport
activity (9- fold) is accompanied by a 46% increase in intravesicular
volume, presumably caused by vesicle fusion. Phosphatidic acid is also
required for successful reconstitution of Na+-dependent amino acid
transport from pure phosphatidylcholine:phosphatidylethanolamine (1:1)
mixtures with only a small change (approximately 16%) in intravesicular
volume. The results provide evidence for both indirect and direct effects
of phosphatidic acid on reconstitution of Na+-dependent amino acid
transport. The indirect effects occur through enlargement of intravesicular
volume, large vesicles showing higher rates of transport. However, there is
also evidence to indicate a specific effect of phosphatidic acid on the
Na+-dependent amino acid transporter, since other acidic lipids may change
intravesicular volume without a commensurate change in transport activity.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
