Immunochemical evidence for the presence of a lipoxygenase 1 in isolated plasma membranes from soybean cotyledons

An antibody was raised against a synthetic oligopeptide, corresponding to the 19–31 interval of the polypeptide chain of soluble lipoxygenase 1 (LOX 1) from soybean seeds. Cross-reactivity of this antibody towards proteins of a soybean cotyledon plasma membrane (PM) fraction was detected. The anti-LOX 1 antibody cross-reacted with a protein of approximately 94 kDa, when the membrane proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The same protein, separated in non-denaturing conditions, exhibited LOX activity with an optimum at pH 9.0. The cross-reactivity of the 94 kDa protein was maintained also in Na 2CO 3 or NaCl-washed membranes, confirming that the enzyme appears to be tightly bound. Finally, the protein, showing LOX activity, was separated by immunoprecipitation using the anti-LOX 1 antibody bound to protein A-Sepharose CL-4B. Again, reactivity with the protein of 94 kDa was detected. These findings show that isolated soybean PMs exhibit a LOX activity, which depends on the presence of the LOX 1 isoform. It is suggested that soluble LOX 1 may be in a dynamic equilibrium with a part of the enzyme, which appears to be linked to PMs.