Molecular mechanism by which cyclic amp regulates myocardial contractility Page: 91 of 153
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59
EXPERIMENTAL PROCEDURE
' MATERIALS
Bovine heart cycl ic AMP-dependent protein kinase, sodium cyclic
AMP and tris-ATP were purchased from Signa Chemical Co.
O O
[y- P]ATP, ammonium salt (20 mCi per mmol) was obtained from
Amersham/Searle. Deoxychol ate was purified by the method of
MacLennan (1970).
METHODS
Isolation of Cardiac Microsomes
Cardiac microsomes were prepared from canine heart ventricle
according to Harigaya and Schwartz (1969) with the modifications
described in Bidlack and Shamoo (1979b). Briefly, cardiac muscle
was homogenized in 10 mM NaH(X>3 and centrifuged twice at 8700 x g
for 20 min. The supernatant was then centrifuged at 37,000 x g for
30 min. The pellet was suspended in 20 mM tris-maleate, 0.6 M KC1,
pH 6.8, and centrifuged at 100,000 x g for 20 min. The pellet,
containing microsomes, was suspended in 0.25 M sucrose.
Gel Electrophoresis
Sodium dodecyl sulfate gel el ectrophoresi s was run according to
Weber and Osborn (1969). Samples for electrophoresis were
suspended in the SDS-sample buffer consisting of 1% SDS, 20 mM
sodium phosphate (pH 7.2), 0.1 mM EDTA, 1% B-mercaptoethanol, 50%
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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/m1/91/: accessed July 17, 2024), University of North Texas Libraries, UNT Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.