Evidence for at least four different inositol bisphosphatases in bovine brain

Bovine brain supernatant contains at least four enzymes capable of hydrolysing inositol bisphosphates. These activities may be distinguished on the basis of their metal, salt and pH dependence, sensitivity to Li+ ions and thiol-modification reagents, and on their molecular sizes. In addition to Li+-sensitive Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase [Gee et al. (1988) Biochem. J. 253, 777-782] which has an absolute requirement for Mg2+, two (Li+-insensitive and Mg2+-independent) phosphatases, capable of hydrolysing Ins(3,4)P2 and Ins(1,3)P2, respectively, have been identified. Both enzymes were inhibited by only moderate concentrations of salt, although for the former there was no obvious correlation between inhibitory potency and either the nature of the anion/cation or the ionic strength of the buffer. Ins(3,4)P2 phosphatase had a pH optimum of 7.6 and this activity could be resolved on gel-filtration columns into a two overlapping peaks of molecular mass 170 kDa and 450 kDa. Mg2+-independent Ins(1,3)P2 phosphatase had a pH optimum of 7.1 and displayed a single broad activity peak on gel-filtration columns. However, if assays were performed in the presence of Mg2+, a second Ins(1,3)P2 phosphatase was revealed (35 kDa), which had a pH optimum of 8.8 Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase, Ins(3,4)P2 phosphatase, Mg2+-independent Ins(1,3)P2 phosphatase and inositol monophosphatase were all inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) with IC50 values of 34 microM, 65 microM and 560 microM and 1100 microM, respectively. The metabolism of Ins(1,3,4)P3 by brain supernatant was also examined. Product specificity was shown to be entirely dependent on the buffer conditions employed. In Mg2+-containing buffers, Ins(1,3,4)P3 was hydrolysed predominantly to Ins(3,4)P2, consistent with hydrolysis by Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase. In the presence of EDTA, Ins(1,3,4)P3 was degraded exclusively by a 4-phosphatase enzyme generating Ins(1,3)P2. Under these conditions, high concentrations of Ins(3,4)P2 blocked the hydrolysis of Ins(1,3,4)P3.