| Summary: | The physical properties of native and deglycosylated glycophorin A and
of membrane protein reconstituted vesicles were investigated. The goals of
these studies were twofold. First, solutionscontaining glycophorin A were examined prior to and following removal of covalently attached carbohydrate
from the protein to determine the contribution of carbohydrate to the glycoprotein
amphiphilic properties. Second, the properties of glycophorin reconstituted
vesicles were studied to address the question: Does the incorporation
of a transmembrane protein into a membrane vesicle significantly increase
bilayer premeability to ions?
Glycophorin A, the major sialoglycoprotein of the human erythrocyte
membrane, is approximately 31,000 daltons: 55% by weight is carbohydrate
and 45% is protein. Neuraminidase was used to remove 99% of the sialic acid
producing asialoglycoprotein and endo-a-N-acetylgalactosaminidase was used to
remove 90% of the serine and threonine linked carbohydrate chains producing
apoglycophorin A.
Removal of carbohydrate results in increased binding of sodium dodecyl
sulfate (SDS) to the polypeptide chain at saturating SDS concentrations. This increased binding indicates that sialic acid residues exhibit little or no
SDS binding and actually inhibit SDS binding to the peptide chain. In detergent
free solutions, native, asialo, and apoglycophorin are heterogeneously
aggregated. SDS associates with aggregated glycophorin below its critical
micelle concentration indicating the presence of noncooperative monomer binding
sites; however, as with other membrane proteins, the association of SDS
to glycophorin occurs principally as a cooperative transition above the critical
micelle concentration. The additional binding of SDS to the deglycosylated
forms does not produce significant changes in the peptide conformation
as measured by circular dichroism.
Removal of carbohydrate also produces corresponding changes in molecular
weight, electrophoretic mobility on SDS gels, and distribution coefficients
on gel chromatographic columns. At saturating levels of SDS binding,
glycophorin and its deglycosylated derivatives were determined to be predominantly
monomeric by sedimentation equilibrium. The relative mobility of
the three forms on SDS gels increases with increasing carbohydrate removal.
Relative mobility depends upon ionic strength, SDS concentration, and protein
concentration. Native glycophorin and apoglycophorin in solution as a
1:1 molar mixture behave as noninteracting species by sedimentation equilibrium
and gel filtration.
Glycophorin incorporated vesicles were prepared by removal of detergent
from solutions containing mixed micelles of egg phosphatidylcholine, octyl
glucoside, and purified glycophorin. Glycophorin was not uniquely oriented
in the membrane as shown by trypsin and neuraminidase treatment of vesicles.
Vesicles that were formed in the presence or absence of glycophorin were similar
in size and morphology (2300 ± 400 A in diameter) as imaged in the electron
microscope. The permeability of these large unilamellar vesicles to Cl-, Na+, and Rb+ followed first order kinetics with rate constants of 2.1 x
-5 -7 -7
10 , 2.6 x 10 , and 9.0 x 10 , respectively. The presence of glycophorin
in the membrane at levels up to 220 copies per vesicle increased the permeability
by less than a factor of five. This demonstrated that incorporation
of membrane protein into vesicles does not necessarily lead to dramatic increases
in membrane ion permeability, and therefore; this methodology may
provide a useful control in the reconstitution of ion pumps into vesicles.
To determine whether some biological activities can be maintained or
restored under these conditions two integral membrane proteins, dopamine-8-
hydroxylase and hepatic asialoglycoprotein receptor,were incorporated using
similar procedures. These proteins were found to either retain or regain activity
upon incorporation into vesicles. Both proteins are oriented in these
vesicles such that all substrate binding sites are exposed on the vesicle exterior.
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