Solubilization and partial purification of leukotriene C4 synthase from guinea-pig lung: a microsomal enzyme with high specificity towards 5,6-epoxide leukotriene A4.
Enzymic activities catalyzing allylic epoxide, leukotriene A4, to leukotriene C4 by conjugation with glutathione were present mainly in microsomal fractions of spleens and lungs of guinea pigs and rats. Leukotriene C4 (LTC4) synthase was solubilized from the microsomes of guinea-pig lung by the new procedures of a combination of 3-[3-cholamidopropyl)dimethylammonio)-1-propanesulfonate (CHAPS), digitonin and KCl. The enzyme was partially purified by two steps of column chromatography which resulted in a complete resolution of the enzyme from glutathione S-transferases (EC 2.5.1.18). The partially purified LTC4 synthase showed a Vmax value of 40 nmol/min per mg, and the apparent Km values for LTA4 and glutathione were 36 microM and 1.6 mM, respectively. The enzyme was unstable, and half of the activity was lost by incubation at 37 degrees C for 3 min. Glutathione at 10 mM completely protected the enzyme against this inactivation, while other sulfhydryl-group-reducing reagents were ineffective. The partially purified enzyme revealed a high specificity towards 5,6-epoxide leukotrienes (LTA4 and its methyl ester), while rat cytosolic glutathione S-transferases catalyzed conjugation of glutathione to various positional isomers of epoxide leukotrienes.
Izumi
T
T
Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, Japan.
Honda
Z
Z
Ohishi
N
N
Kitamura
S
S
Tsuchida
S
S
Sato
K
K
Shimizu
T
T
Seyama
Y
Y
D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D003600:Cytosol / Q000201:enzymology; D005260:Female; D005982:Glutathione Transferase / Q000302:isolation & purification* / Q000378:metabolism; D006168:Guinea Pigs; D007700:Kinetics; D017572:Leukotriene A4; D008168:Lung / Q000201:enzymology* / Q000648:ultrastructure; D008861:Microsomes / Q000201:enzymology; D012995:Solubility; D013379:Substrate Specificity; D014018:Tissue Distribution
D001095:Arachidonic Acids; D017572:Leukotriene A4; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase