Regulatory effects of phospholamban on cardiac sarcoplasmic reticulum function

Thesis/Dissertation · Sun Jan 01 04:00:00 EST 1989

OSTI ID:5671223

Kim, Hae Won

In this thesis, the author reports the effect of phospholamban on: (a) Ca{sup 2+} release by cardiac SR and (b) the Ca {sup 2+}-ATPase activity in a purified reconstituted system. Phosphorylation of phospholamban by Ca{sup 2+} {center dot} calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca {sup 2+} release from cardiac SR vesicles loaded under passive conditions and on the apparent {sup 45}Ca{sup 2+} - {sup 40}Ca{sup 2+} exchange from cardiac SR vesicles loaded under active conditions. us, it appears that Ca{sup 2+} {center dot} calmodulin-dependent phosphorylation of phospholamban is not involved in the regulation of Ca{sup 2{plus}} release and {sup 45}Ca{sup 2{plus}{minus}40}Ca{sup 2{plus}} exchange. To determine the molecular mechanism by which phospholamban regulates the Ca{sup 2{plus}} pump, a reconstituted system was developed, using a freeze-thaw sonication procedure. The Ca{sup 2+}-ATPase was purified by a method which yields an active enzyme preparation essentially free of phospholamban. The best rates of Ca{sup 2+} uptake were obtained when cholate and phosphatidylcholine (PC) were used at a ratio of cholate/PC/Ca{sup 2 +}-ATPase of 2/80/1. The maximal rates of Ca{sup 2+} Uptake were 700 nmol/min/mg reconstituted vesicles compared to 800 nmol/min/mg SR vesicles. The EC{sub 50} values for Ca{sup 2+} were 0.05 {mu}M for both Ca{sup 2+} uptake and Ca{sup 2+}-ATPase activity in the reconstituted vesicles compared to 0.63 {mu}M Ca {sup 2+} in native SR vesicles. To determine the effect of phospholamban on the Ca {sup +}-ATPase activity in the reconstituted vesicles, purified phospholamban was added to the cholate/Ca {sup 2+}-ATPase mixture prior to combining it with liposomes.

• Research Organization: Cincinnati Univ., OH (United States)
• OSTI ID: 5671223
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ALKALINE EARTH ISOTOPES
ALKALINE EARTH METAL COMPOUNDS
ATP-ASE
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL FUNCTIONS
BODY
CALCIUM 40
CALCIUM COMPOUNDS
CALCIUM ISOTOPES
CARDIOVASCULAR SYSTEM
CELL CONSTITUENTS
ENDOPLASMIC RETICULUM
ENZYME ACTIVITY
ENZYMES
EVEN-EVEN NUCLEI
HEART
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
LIPIDS
LIPOPROTEINS
MEMBRANE TRANSPORT
NUCLEI
ORGANIC COMPOUNDS
ORGANS
PHOSPHOHYDROLASES
PROTEINS
REACTION KINETICS
SARCOPLASMIC RETICULUM
STABLE ISOTOPES
TRACER TECHNIQUES