Purification of phospholamban, a 22,000-dalton protein from cardiac sarcoplasmic reticulum that is specifically phosphorylated by cyclic AMP-dependent protein kinase. [Dogs]

Journal Article · Sun Apr 25 04:00:00 EDT 1982 · J. Biol. Chem.; (United States)

OSTI ID:5212503

Bidlack, J M; Ambudkar, I S; Shamoo, A E

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

Related Subjects

550200* -- Biochemistry
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