DEMONSTRATION OF LEUKOTRIENE-C-4 SYNTHASE IN PLATELETS AND SPECIES DISTRIBUTION OF THE ENZYME-ACTIVITY

Human platelets have been demonstrated to possess leukotriene (LT)-C-4 synthase activity and may thus be involved in transcellular -glutathi onyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4) synthesis. In th is study, platelets from seven different species were screened for LTC 4 synthase activity. Very high enzyme activity was observed in suspens ions of bovine platelets, with approximately 70% conversion of ans-5,6 -oxido-7,9-trans-11,14-cis-eicosatetraenoic acid (LTA(4)) to LTC4. The capacity of equine platelets to produce LTC4 was similar to that of h uman platelets. In addition, ovine, rabbit, and rat platelets also pro duced LTC4 after incubation with LTA(4). The results demonstrate that LTC4 synthase activity is a common feature among platelets from variou s species. In contrast, porcine platelets failed to transform LTA(4) t o LTC4. Instead, these cells produced (R)-dihydroxy-6,14-cis-8,10-tran s-eicosatetraenoic acid (LTB4), indicating the presence of LTA(4) hydr olase in porcine platelets. A protein with a molecular mass of approxi mately 18 kDa and LTC4 synthase activity was solubilised from lyophili sed bovine platelet concentrates and purified to near homogeneity by a ffinity chromatography and gel filtration. The N-terminal amino acid s equence of this protein was analysed and found to be almost identical to the corresponding sequence of human LTC4 synthase (17 of 18 amino a cid residues identical). Kinetic analysis of partially purified bovine platelet LTC4 synthase revealed K-m (for LTA(4)) and V-max values of 3.3 mu M and 521 nmol.mg protein(-1).min(-1), respectively. In additio n, the presence of a mRNA transcript encoding LTC4 synthase was demons trated in equine platelets by reverse transcription (RT) PCR using pri mers derived from the human LTC4 synthase cDNA sequence. Cloning and s equencing of the PCR fragment corresponding to a region near the N-ter minus demonstrated very high identity between equine and human leukotr iene-C-4 synthase in this region. In summary, the present study establ ishes that platelets contain LTC4 synthase and indicates that this enz yme is widely distributed among platelets from various species.