Demonstration of leukotriene-C4 synthase in platelets and species distribution of the enzyme activity.
Human platelets have been demonstrated to possess leukotriene (LT)-C4 synthase activity and may thus be involved in transcellular 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic acid (LTC4) synthesis. In this study, platelets from seven different species were screened for LTC4 synthase activity. Very high enzyme activity was observed in suspensions of bovine platelets, with approximately 70% conversion of 5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid (LTA4) to LTC4. The capacity of equine platelets to produce LTC4 was similar to that of human platelets. In addition, ovine, rabbit, and rat platelets also produced LTC4 after incubation with LTA4. The results demonstrate that LTC4 synthase activity is a common feature among platelets from various species. In contrast, porcine platelets failed to transform LTA4 to LTC4. Instead, these cells produced 5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid (LTB4), indicating the presence of LTA4 hydrolase in porcine platelets. A protein with a molecular mass of approximately 18 kDa and LTC4 synthase activity was solubilised from lyophilised bovine platelet concentrates and purified to near homogeneity by affinity chromatography and gel filtration. The N-terminal amino acid sequence of this protein was analysed and found to be almost identical to the corresponding sequence of human LTC4 synthase (17 of 18 amino acid residues identical). Kinetic analysis of partially purified bovine platelet LTC4 synthase revealed Km (for LTA4) and Vmax values of 3.3 microM and 521 nmol x mg protein(-1) x min(-1), respectively. In addition, the presence of a mRNA transcript encoding LTC4 synthase was demonstrated in equine platelets by reverse transcription (RT) PCR using primers derived from the human LTC4 synthase cDNA sequence. Cloning and sequencing of the PCR fragment corresponding to a region near the N-terminus demonstrated very high identity between equine and human leukotriene-C4 synthase in this region. In summary, the present study establishes that platelets contain LTC4 synthase and indicates that this enzyme is widely distributed among platelets from various species.
Tornhamre
S
S
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Sjölinder
M
M
Lindberg
A
A
Ericsson
I
I
Näsman-Glaser
B
B
Griffiths
W J
WJ
Lindgren
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001792:Blood Platelets / Q000737:chemistry / Q000201:enzymology*; D002417:Cattle; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger; D011817:Rabbits; D051381:Rats; D013045:Species Specificity; D014158:Transcription, Genetic
D012333:RNA, Messenger; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase