Molecular mechanism by which cyclic amp regulates myocardial contractility Page: 68 of 153
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Proteolipid Extraction of Phosphoryl ated Microsomes
As a means of separating the two phosphoryl ated protein, the
proteolipid extraction procedure of MacLennan (1974) was used. The
phosphoryl ated and non phosphoryl ated sarcoplasmic reticulun were
extracted with 90% methanol, followed by chi orofrom: methanol (2:1),
and finally acidified chi oroform: methanol (2:1:10 mMHCl).
Proteol ipi ds, proteins with lipids strongly associated with them,
are soluble in acidified chi oroform: methanol. Ethyl ether can
precipitate specifically the proteolipids from the acidified
chi oroform: methanol phase. Thin layer chromatography can separate
lipids and proteolipids. The various fractions were run on Silica
G thin layer plates with acidified chi orof orm:methanol as the
solvent. Fig. 5 shows an autoradiogram of the fractions from the
proteolipid extraction run on thin layer plated with acidified
chi orof orm: methanol as the solvent. In the methanol supernatant,
all counts remained at the origin. These counts represent the
"^P-ATP present in the reaction. This finding was. generally true
for the chi orof orm: methanol supernatant, with only a few counts
moving away from the origin. However, the supernatant from the
acidified chi orof orm:methanol extraction and its subsequent pellet,
precipitated with ethyl ether, did show J^P counts at the same
place that the proteolipid ran on the thin layer plates.
To rule out the possibility that a lipid might be the component
being phosphoryl ated, all the fractions from the proteolipid
extraction were run on thin layer plates with chi orof orm: methanol: .
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Bidlack, J. M. Molecular mechanism by which cyclic amp regulates myocardial contractility, report, January 1, 1979; United States. (https://digital.library.unt.edu/ark:/67531/metadc1085147/m1/68/: accessed July 17, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.