The reaction of prostaglandin H synthase with prostaglandin G2, the physiological substrate for the peroxidase reaction, was examined by rapid reaction techniques at 1 °C. Two spectral intermediates were observed and assigned to higher oxidation states of the enzymes. Intermediate I was formed within 20 ms in a bimolecular reaction between the enzyme and prostaglandin G2 with k1= 1.4 × 107 M−1 s−1. From the resemblance to compound I of horseradish peroxidase, the structure of intermediate I was assigned to [(protoporphyrin IX)+· FeIVO). Between 10 ms and 170 ms intermediate II was formed from intermediate I in a monomolecular reaction with k2= 65 s−1. Intermediate II, spectrally very similar to compound II of horseradish peroxidase or complex ES of cytochrome‐c peroxidase, was assigned to a two‐electron oxidized state [(protoporphyrin IX)FeIVO] Tyr+· which was formed by an intramolecular electron transfer from tyrosine to the porphyrin‐π‐cation radical of intermediate I. A reaction scheme for prostaglandin H synthase is proposed where the tyrosyl radical of intermediate II activates the cyclooxygenase reaction.

Purified prostaglandin H synthase (EC 1.14.994, reconstituted with hemin, was reacted with substrates of the cyclooxygenase and peroxidase reaction. The resulting EPR spectra were measured below 90 K. Arachidonic acid, added under anaerobic conditions, did not change the EPR spectrum of the native enzyme due to high-spin ferric heme. Arachidonic acid with 02, as well as prostaglandin G2 or H 2 0 2 , decreased the spectrum of the native enzyme and concomitantly a doublet signal at g = 2.005 was formed with maximal intensity of 0.35 spins/enzyme and a half-life of less than 20 s at -12°C. From the conditions for the formation and the effect of inhibitors,

Familial hypertrophic cardiomyopathy is a disease generally believed to be caused by mutations in sarcomeric proteins. In a family with hypertrophic cardiomyopathy linked to polymorphic markers on chromosome 11, we found a new mutation of a splice donor site of the cardiac myosinbinding protein-C gene. This mutation causes the skipping of the associated exon in mRNA from lymphocytes and myocardium. Skipping of the exon with a consecutive reading frame shift leads to premature termination of translation and is thus expected to produce a truncated cardiac myosinbinding protein-C with loss of the myosin-and titin-binding COOH terminus. However, Western blot analysis of endomyocardial biopsies from histologically affected left ventricular myocardium failed to show the expected truncated protein.These data show for the first time that a splice donor site mutation in the myosin-binding protein-C gene is transcribed to cardiac mRNA. Truncated cardiac myosin-binding protein-C does not act as a "poison polypeptide," since it seems not to be incorporated into the sarcomere in significant amounts. The absence of mutant protein and of significantly reduced amounts of wild-type protein in the presence of the mutated mRNA argues against the "poison protein" and the "null allele" hypotheses and suggests yet unknown mechanisms relevant to the genesis of chromosome-11-associated familial hypertrophic cardiomyopathy.

No 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.