Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma.
A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.
Michalak
M
M
Department of Pediatrics, University of Alberta, Edmonton, Canada.
Wandler
E L
EL
Strynadka
K
K
Catena
R
R
Liu
H J
HJ
Olley
P M
PM
D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D015232:Dinoprostone / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D011982:Receptors, Prostaglandin / Q000302:isolation & purification* / Q000378:metabolism; D018078:Receptors, Prostaglandin E; D012508:Sarcolemma / Q000378:metabolism*
D000345:Affinity Labels; D011982:Receptors, Prostaglandin; D018078:Receptors, Prostaglandin E; D015232:Dinoprostone