Purification of phospholamban, a 22,000-dalton protein from cardiac sarcoplasmic reticulum that is specifically phosphorylated by cyclic AMP-dependent protein kinase. [Dogs]
Very low concentrations of the detergent, deoxycholate, have been used to isolate two functionally interesting proteins from canine cardiac sarcoplasmic reticulum. These two proteins are phospholamban, a 22,000-dalton protein, specifically phosphorylated by adenosine 3':5'-monophosphate-dependent protein kinase, and the (Ca/sup 2 +/ + Mg/sup 2 +/)-ATPase, the major protein of the sarcoplasmic reticulum, responsible for the active transport of calcium. The 22,000-dalton protein is first solubilized in a very low concentration of deoxycholate (over 2 orders of magnitude lower than normally employed), and then subjected to column chromatography. After gel filtration through Sephadex G-75, the 22,000-dalton protein appears as a single band on sodium dodecyl sulfate-polyacrylamide gels. The purified protein is specifically phosphorylated by cyclic AMP-dependent protein kinase to a level of 0.15 mol of phosphate/mol of protein. The (Ca/sup 2 +/ + Mg/sup 2 +/)-ATPase is purified by first solubilizing all of the extrinsic proteins with a low concentration of deoxycholate. An increasing amount of the deoxycholate is then added to yield the purified (Ca/sup 2 +/ + Mg/sup 2 +/)-ATPase. This protein is at least 95% pure as determined by sodium dodecyl sulfate-polyacrylamide gels and has an ATP hydrolytic activity of about 1.25 ..mu..mol of Pi/mg/min. Further addition of deoxycholate to the purified enzyme enhances the enzyme's ability to hydrolyze ATP to approximately 2.5 ..mu..mol of Pi/mg/min. The isolation of the 22,000-dalton protein and the (Ca/sup 2 +/ + Mg/sup 2 +/)-ATPase will aid in understanding how these two proteins function and if they specifically interact with one another.
- OSTI ID:
- 5212503
- Journal Information:
- J. Biol. Chem.; (United States), Journal Name: J. Biol. Chem.; (United States) Vol. 257:8; ISSN JBCHA
- Country of Publication:
- United States
- Language:
- English
Similar Records
Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum
Molecular mechanism by which cyclic amp regulates myocardial contractility
Related Subjects
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ADDITIVES
AMP
ANIMALS
ATP-ASE
BIOLOGICAL FUNCTIONS
BODY
CARDIOVASCULAR SYSTEM
CELL CONSTITUENTS
CHEMICAL REACTIONS
CHROMATOGRAPHY
DETERGENTS
DOGS
ELECTROPHORESIS
EMULSIFIERS
ENZYMES
FUNCTIONS
HEART
HYDROLASES
MAMMALS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PHOSPHOHYDROLASES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHORYLATION
PHOSPHOTRANSFERASES
PROTEINS
PURIFICATION
SARCOPLASMIC RETICULUM
SEPARATION PROCESSES
SURFACTANTS
TRANSFERASES
VERTEBRATES
WETTING AGENTS