The degradation of Ins(1,3,4,5)P-4 in Dictyostelium was investigated using a mixture of [H-3]Ins(1,3,4,5)P-4 and [3-P-32]Ins(1,3,4,5)P-4. After incubation of this mixture with a Dictyostelium homogenate the P-32/H-3 ratio found in the InsP(3) product was reduced to 24% of the ratio in the substrate. P-32-labelled inorganic phosphate was found as well, whereas hardly any InsP(2) was detected. This indicates that Ins(1,3,4,5)P-4 is mainly degraded by a 3-phosphatase. The other enzyme was characterized by identification of the P-32-labelled InsP(3) isomer. This isomer did not co-elute with Ins(1,3,3)P-3, indicating that no 5-phosphatase was present in Dictyostelium. The P-32-labelled InsP(3) could be oxidized using NaIO4. The only InsP(3) isomer that has these characteristics is Ins(3,4,5)P-3, indicating 1-phosphatase activity. The 1-phosphatase appeared to be dependent on MgCl2, whereas the 3-phosphatase was still active in the absence of MgCl2. An analogue of Ins(1,3,4,5)P-4 with a thiophosphate substitution at the 1-position was found to be almost completely resistant to hydrolysis by the 1-phosphatase, but was degraded by the 3-phosphatase.
|Number of pages||4|
|Publication status||Published - 15-May-1995|