Abstract
Maximal activity of human leukocyte 5-lipoxygenase requires Ca2+, ATP, a microsomal membrane preparation, and two cytosolic stimulatory factors. We report here some effects of Ca2+ on the physical properties of the 5-lipoxygenase. When leukocytes were homogenized in the presence of 2 mM EDTA, 5-lipoxygenase was found to be a soluble enzyme. However, when Ca2+ was added to homogenization buffers at 0-1 mM in excess of EDTA, increasing quantities of the enzyme were recovered in the microsomal membrane fraction (100,000 X g pellet). The membrane-associated enzyme was resolubilized by washing pellet preparations in buffers containing 2 mM EDTA and was partially purified by anion-exchange chromatography. Studies of the stimulatory-factor requirements of the membrane-associated, resolubilized, and partially purified enzyme indicated that one of the cytosolic 5-lipoxygenase stimulatory factors exhibited a reversible, Ca2+-dependent membrane association, similar to that of the enzyme itself. Ca2+ also caused a destabilization of the 5-lipoxygenase. Homogenates prepared in the presence of Ca2+ contained lower total enzyme activity, and retention of activity in these samples over time was also diminished.
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