after two recrystallizations from ethanol. The semicarbazone of PGA2, which to our knowledge is the first reported crystalline derivative of this substance, could be prepared in 90 % yield by reaction with semicarbazide at pH 3 in a 1:l mixture of ethanol-water (mp 124.5-126" from ethanol).* In contrast to prostaglandin synthetases isolated from mammalian source^,^ no cofactors have as yet been found for this prostaglandin synthetase. Glutathione, cysteine, coenzyme A, thioglycolic acid, and mercaptoethanol, each at M concentration, produced 20-80% inhibition. A concentration of M EDTA or citric acid had no effect; the addition of M Ca2+ or Mg2+, M Zn, V, Co, or Fe, or M Cu was also without effect. Addition of ATP M ) or NADH or NADPH (5 X M ) also had no effect. It is interesting that strong inhibition of the mammalian synthetase by Cu2+ and Zn2+ at these concentrations has been notedagaThe mammalian and gorgonian-derived prostaglandin synthetases show remarkably different behavior toward other inhibitors. Thus, indomethacin is a potent inhibitor of the mammalian synthetase lo (complete inhibition at 2.5 X M ) but displays no such interaction toward the synthetase from P . homomalla (no observable inhibition at 2.5 X IO-6 M ) . Similarly, no inhibition was noted with 5,8,11,14-eicosatetraynoic acid (6 X IO-5 M ) , a powerful inhibitor of the mammalian prostaglandin synthetase. l 1As expected, the gorgonian synthetase could also utilize 8,11,14-eicosatrienoic acid as substrate. Under the same conditions which allow the biosynthesis of PGAn from arachidonic acid, enzymic conversion of 2-tritio-8,11,14-eicosatrienoic acid to labeled PGAl was observed.Since PGE2 had been reported to be present in the Plexauru homomallu, we felt that the PGAz should be produced from the intermediary PGE2. 2 , 3 Surprisingly, incubations with labeled PGEl did not result in labeled PGA,. Likewise, incubations of 2-tritio-8,I 1,14-eicosatrienoic acid in the presence of 1 mg of cold PGEl resulted in only labeled PGAl formation. No labeled PGEl appeared. These results suggest that any intermediary PGE must be bound tightly to the enzyme complex. Furthermore, the enzyme responsible for converting PGE to PGA must not be accessible to exogenous PGE.Our initial efforts to purify the PGA2 synthetase of P . homomallu have thus far been foiled. Enzymic activity is lost upon storage at 0" for 24 hr, attempted fractionation by either ammonium sulfate or acetone (-20 ") precipitation, or column chromatography.Our investigations of prostaglandin biosynthesis are continuing.Acknowledgment. This work has been assisted (8) The pH of the reaction is extremely critical. Little or no crystalline semicarbazone o f PGAz could be obtained at pH 4 or 5 or pH 2. The semicarbazone o f PGA? methyl ester could also be prepared as a crystalline substance, mp 165.5-166".(9) (a)
Sir :The fungus Rhizoctonia Ieguminicolu utilizes the entire carbon skeleton of pipecolic acid (derived from the metabolism of lysine)2 in the biosynthesis of the parasympathomimetic alkaloi...
