• Korean Journal of Fisheries and Aquatic Sciences
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• v.34 no.4
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• pp.370-377
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• 2001

Flounder brain cytosol contains protein inhibitors that markedly inhibit the activity of partially purified brain membrane phospholipase D (PLD) which is dependent on phosphatidylinositol 4,5-bisphosphate ($PIP_2$) but insensitive to ADP-ribosylation factor (ARF), The PLD inhibitors have been enriched through several chromatographic steps and characterized with respect to size and mechanism of inhibition. Sequential chromatography of the brain cytosol yielded six inhibitor fractions, Two (IIA and IIB) of six inhibitor fractions showed the $PIP_2$-phosphatase activities. IIA was identified as synaptojanin, a nerve terminal protein that has known to be a member of the inositolpolyphosphate 5-phosphatase family, by immunoblot analysis. IIB showed an apparent molecular mass of 158 kDa by Superose 12 gel filtration chromatography and was immunologically distinct from synaptojanin. IIB hydrolyzed $PIP_2$, yielding only phosphatidylinositol phosphate (PIP) as product, suggesting that IIB hydrolyzes only one phosphate from either the 4- or 5-position of PI (4,5)$P_2$. These studies demonstrate that the existence of multiple $PIP_2$-phosphatases have been implicated in the negative regulation of $PIP_2$-dependent PLD activity within flounder brain.