Topographic studies of microsomal and pure prostaglandin H synthase.
Prostaglandin H synthase catalyzes the first step in the conversion of polyunsaturated fatty acids to prostaglandins, thromboxanes, and prostacyclins. The enzyme is normally bound to the endoplasmic reticulum membrane, but can be purified to homogeneity after solubilization with detergent. The topologies of the microsomal and the pure detergent-solubilized forms of the synthase were compared by an examination of their sensitivity to degradation by proteases, of the effect of heme on this protease sensitivity, and of the sizes of proteolytic fragments produced. For the microsomal synthase, the localization of proteolytic fragments was also determined. Analysis of the microsomal proteins after proteolytic digests involved separation by polyacrylamide gel electrophoresis and selective detection of the synthase-derived polypeptides with a polyclonal antibody against the pure synthase. With both the microsomal and the pure synthase, incubation with trypsin led to a progressive loss of cyclooxygenase activity and cleavage of the synthase subunit (70K Da) into two fragments of 38K and 33K Da. Incubation of the detergent-solubilized form of the synthase with proteinase K and chymotrypsin also produced a very similar pair of fragments (38K and 33K Da). After incubation of the microsomes with trypsin both the 38K and 33K Da fragments from the synthase remained bound to the membrane; no cyclooxygenase activity was released in soluble form from the microsomes by trypsin. Further, neither trypsin nor proteinase K released soluble radiolabeled peptides from microsomes whose synthase had been labeled with [acetyl-14C]-aspirin. With the microsomal synthase the sensitivity to protease (66% of the cyclooxygenase activity was lost after 90 min incubation with proteinase K) was enhanced by depletion of heme (84% of activity lost) and was decreased by addition of heme (only 20% of activity lost), just as had been previously demonstrated for the detergent-solubilized synthase. At each of several intervals during an incubation of the pure synthase with trypsin the extent of cleavage of the synthase polypeptide correlated reasonably well with the extent of loss of cyclooxygenase activity; a similar relation between proteolytic cleavage and loss of activity was observed in digests of the pure synthase supplemented with differing amounts of heme.(ABSTRACT TRUNCATED AT 400 WORDS)
Kulmacz
R J
RJ
Department of Biological Chemistry, University of Illinois, Chicago 60612.
Wu
K K
KK
D000818:Animals; D001241:Aspirin / Q000494:pharmacology; D002918:Chymotrypsin / Q000494:pharmacology; D003902:Detergents; D019286:Endopeptidase K; D005914:Globins / Q000494:pharmacology; D006418:Heme / Q000494:pharmacology; D008297:Male; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D010449:Peptide Mapping; D011451:Prostaglandin-Endoperoxide Synthases; D011487:Protein Conformation; D012669:Seminal Vesicles / Q000201:enzymology; D012697:Serine Endopeptidases / Q000494:pharmacology; D012756:Sheep; D012995:Solubility; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000494:pharmacology
D003902:Detergents; D010446:Peptide Fragments; D006418:Heme; D005914:Globins; D011451:Prostaglandin-Endoperoxide Synthases; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; D019286:Endopeptidase K; D001241:Aspirin