ABSTRACT
To determine the size of the functional catalytic unit of prostaglandin endoperoxide (prostaglandin H) synthase, radiation inactivation experiments were performed. Both microsomes from ovine seminal vesicles and purified enzyme were irradiated with 10 MeV electrons. The enzymic activities of prostaglandin H synthase, cyclooxygenase and peroxidase, showed mono-exponential inactivation curves dependent on radiation dose, indicating molecular masses of approximately 72 kDa. The enzyme in microsomes, in its native environment, as well as in its purified state after solubilisation with nonionic detergent showed identical molecular masses. The results clearly demonstrate that the monomer of the enzyme with an apparent molecular mass of 72 kDa (SDS/PAGE) is the functional unit for catalysis of both activities. Hence the two active sites of cyclooxygenase and peroxidase reside on the same polypeptide chain.
Subject(s)
Peroxidase/radiation effects , Prostaglandin-Endoperoxide Synthases/metabolism , Animals , Dose-Response Relationship, Radiation , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/radiation effects , Glucosephosphate Dehydrogenase/radiation effects , Male , Microsomes/enzymology , Microsomes/radiation effects , Molecular Weight , Oxygen Consumption , Prostaglandin-Endoperoxide Synthases/radiation effects , Seminal Vesicles/enzymology , Seminal Vesicles/radiation effects , Sheep , Structure-Activity RelationshipABSTRACT
Microsomes from ram seminal vesicles or purified prostaglandin H synthase supplemented with either arachidonic acid or prostaglandin G2 formed an unstable spectral intermediate with maxima at 430 nm, 525 nm and 555 nm and minima at 410 nm, 490 nm and 630 nm. At -15 degrees C the band at 430 nm disappeared within 4 min whereas the trough at 410 nm increased three fold. At higher temperatures (10-37 degrees C) spectral complex formation and decay were observed in less than 1 s. An apparent KS-value of about 3 microM was determined for the titration of purified prostaglandin synthase with prostaglandin G2 at -20 degrees C. Substrates for cooxidation reactions of prostaglandin synthase such as phenol, hydroquinone and reduced glutathione as well as the peroxidase inhibitors cyanide and azide inhibited the prostaglandin G2-induced spectral complex formation. The oxene donor iodosobenzene and hydrogen peroxide formed a spectral intermediate analogous to the complex observed with prostaglandin G2 or arachidonic acid in ram seminal vesicle microsomes as well as with the purified prostaglandin synthase. These results are interpreted as the formation of a ferryl-oxo complex (FeO)3+ of the heme of prostaglandin synthase with prostaglandin G2 analogous to the formation of compound I of horseradish peroxidase.
Subject(s)
Microsomes/enzymology , Peroxidases/metabolism , Seminal Vesicles/enzymology , Animals , Arachidonic Acid , Arachidonic Acids/metabolism , In Vitro Techniques , Luminescent Measurements , Male , Oxidation-Reduction , Peroxidases/antagonists & inhibitors , Prostaglandins G/metabolism , Sheep , Spectrophotometry , Substrate Specificity , TemperatureABSTRACT
To characterize further the prosthetic group of PGH synthase (EC 1.14.99.1), titrations of the apoenzyme with hemin were investigated by EPR. The first hemin bound per polypeptide showed an EPR signal at g = 6.7 and 5.3 (rhombicity 9%) and was tentatively assigned to the hemin effective as prosthetic group of PGH synthase. Additional hemin bound showed a less rhombic signal (g = 6.3 and 5.8, rhombicity 3%) presumably due to nonspecific hydrophobic binding sites not effective in catalysis.
