Characterization by photoaffinity labelling of the human platelet thromboxane A2/prostaglandin H2 receptor: evidence for N-linked glycosylation.
Thromboxane A2 (TXA2) and prostaglandin H2 (PGH2) are potent proaggregatory and vasoconstrictor lipids acting through a receptor referred to as the TXA2/PGH2 receptor. The receptor was purified using a modification of a previously described method from human platelet membranes solubilized using the detergent (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and a combination of affinity chromatography and wheat germ lectin chromatography. This procedure resulted in a 1075 +/- 375-fold purification and a specific activity of 1.45 +/- 0.55 nmol/mg protein (n = 5). Repeating these chromatography steps on this partially purified receptor resulted in a preparation with a specific activity of 21 +/- 3 nmol/mg protein (n = 5). This represents the theoretical specific activity if one assumes a molecular weight of 50,000 for the receptor. The fold purification was 11,750 +/- 1250 based on crude membranes and an overall yield of 24%. To further the characterization of this receptor, we synthesized a new radioiodinated photoaffinity probe, 7-[(1R,2S,3S,5R)-6,6-dimethyl-3-(4-azido-3-iodobenzenesulfonylamino++ + )- bicyclo[3.1.1]hept-2-yl]-5(Z)-heptenoic acid (I-SAP-N3). [125I]l-SAP-N3 irreversibly incorporated into the purified receptor yielding a single band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography and indicated a molecular weight for the receptor of 50-51 kDa. The incorporation of the ligand could be inhibited by a variety of TXA2/PGH2 analogues. In addition, photoaffinity labelling was inhibited in a stereoselective manner as demonstrated by the pair of enantiomers (d)- and (l)-S145. Digestion of photoaffinity labelled receptor with N-glycosidase F demonstrated the presence of at least two N-linked glycosylation sites.(ABSTRACT TRUNCATED AT 250 WORDS)
Mais
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Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285.
True
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Martinelli
M J
MJ
D001792:Blood Platelets / Q000737:chemistry*; D001643:Bridged Bicyclo Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D005229:Fatty Acids, Monounsaturated; D006031:Glycosylation; D006801:Humans; D044262:Prostaglandin H2; D011463:Prostaglandins H / Q000502:physiology; D011869:Radioligand Assay; D011982:Receptors, Prostaglandin / Q000302:isolation & purification* / Q000502:physiology; D017482:Receptors, Thromboxane / Q000302:isolation & purification* / Q000502:physiology; D044244:Receptors, Thromboxane A2, Prostaglandin H2; D013928:Thromboxane A2 / Q000502:physiology
D001643:Bridged Bicyclo Compounds; D005229:Fatty Acids, Monounsaturated; D011463:Prostaglandins H; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D044244:Receptors, Thromboxane A2, Prostaglandin H2; C056566:S 145; D044262:Prostaglandin H2; D013928:Thromboxane A2