Isolation and partial characterization of vesicles derived from the plasma membrane of the chicken gizzard muscle
Creators: name: Azad, Aristotle
Publisher: name: University of British Columbia
Year: 2010

description: Plasma membrane vesicles, isolated from the chicken gizzard using differential centrifugation and sucrose gradient centrifugation, were biochemically characterized. Two fractions obtained from the sucrose gradient, Fractions 4 and 5 (32% and 34% sucrose respectively), were judged to be the most pure in plasma membranes as based on 5' nucleotidase and iodination studies. Both fractions had the same coomassie blue and PAS staining profile when electrophoresed and under electron microscopy both fractions consisted of membrane vesicles of varying size. A Mg²⁺ stimulated ATPase activity was found to be present and highest in Fraction 5 while Fraction 4 exhibited little activity. This enzyme was inhibited in the presence of high concentrations of ATP and Mg. A similar ecto Mg²⁺ stimulated ATPase was observed in isolated smooth muscle cells. Phosphorylation using [γ-³²P] ATP was observed at 205,000, 165,000 and 145,000 daltons in Fraction 5 only. Mg promoted dephosphorylation of the 205,000 dalton band while Ca promoted phosphorylation of the 165,000 dalton band. All phosphorylated peaks were sensitive to hydroxylamine treatment. These results would seem to indicate that there is a difference in membrane orientation between Fraction 4 and Fraction 5. The membrane orientation in Fractions 4 and 5 was then examined using acetylcholinesterase and sialic acid was external plasma membrane markers. Fraction 4 was found to contain mainly inside-out vesicles in contrast to Fraction 5, which was thought to consist mainly of right-side-out plasma membrane vesicles. The orientation differences were further examined using lactoperoxidase catalyzed iodination using ¹²⁵I. Iodination of Fraction 4 resulted in the appearance of ¹²⁵I in a band migrating in SDS electrophoretigrams with an apparent molecular weight of 100,000 daltons, and minor labelling was seen at 205,000 and 55,000 daltons. 0.05% Triton X-100 significantly enhanced labelling of all three-bands. Iodination of Fraction 5 resulted in labelling of all three bands, but treatment of the membranes with Triton X-100 enhanced labelling only at 100,000 daltons. Iodination of intact single cells resulted in an iodination pattern similar to that of Fraction 5 in the absence of Triton X-100. Attempts were made to further purify the membranes using concanavalin A - Sepharose affinity chromatography. After Fraction 4 was applied to the column, four peaks of protein could be eluted. The first two peaks, eluted in the absence of α methyl-D-mannoside were thought to consist of inside-out vesicles as judged by iodination and acetylcholinesterase sidedness studies. The other two peaks, eluted in the presence of α methyl-D-mannoside were thought to contain unsealed plasma membrane vesicles. Over 90% of the originally applied protein was eluted, 20% being contained in the two peaks eluted in the presence of α methyl-D-mannoside. Fraction 5 behaved quite differently on the affinity columns. Approximately 90% of the originally applied protein could not be eluted even in the presence of α methyl-D-mannoside. Of the two peaks eluted, one peak obtained in the absence of α methyl-D-mannoside, was thought to consist of inside-out plasma membrane vesicles. The second peak, eluted in the presence of α methyl-D-mannoside was thought to contain unsealed membrane vesicles as indicated by sidedness studies. It was concluded that Fractions 4 and 5 represent plasma membrane preparations of differing orientation, Fraction 5 being predominantly right-side-out and Fraction 4 after affinity chromatography mainly insight-out. These two fractions may have some applicability in investigating the asymmetry of various membrane transport systems.