Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). InsP3 3-kinase was purified from rat brain by Blue-Sepharose, phosphocellulose and calmodulin (CaM)-Sepharose affinity chromatography. The purified enzyme was stimulated by Ca2+/CaM by 3-6-fold as compared with the activity measured in the presence of EGTA. Rat brain InsP3 3-kinase activity was associated with two silver-stained bands of about equal activity which migrated with an apparent Mr of 50,000 on SDS/polyacrylamide gels. InsP3 3-kinase activity from rat brain could be immunoprecipitated by an antiserum against the SDS/PAGE-purified 50,000-Mr protein doublet. InsP3 kinase activity from bovine brain and the InsP3 5-phosphatase activity from rat brain were not immunoprecipitated. On Western blot, the human brain crude InsP3 3-kinase reacted specifically, but less strongly than the rat brain enzyme, with the antiserum.

Ins(1 ,4,5)P3 5-phosphatase catalyses the dephosphorylation of Ins(1,4,5)P3 in the 5 position. At 1 pM Ins(1,4,5)P3, 10-15% of total activity of a bovine brain homogenate was measured in the soluble fraction, whereas 85 -90% was in the particulate fraction. Particulate activity could be solubilized by cholate or, to a lower extent, by 2 M KCI. Two soluble enzymes (type I and type 11) could be fractionated by DEAE-Sephacel chromatography. Soluble activities have been further purified by blue-Sepharose, Sephacryl S-200 and phosphocellulose chromatography. Specific activities reached 10-30 pmol . min-' mg protein-' for type I and were 10-20 times lower for type 11. Type I and type 11 Ins(1,4,5)P3 5-phosphatase displayed different K,,, values and molecular masses, as estimated by gel filtration. Type I dephosphorylated both Ins(1,4,5)P3 and Ins(1 ,3,4,5)P4; in contrast, type I1 specifically dephosphorylated Ins(1 ,4,5)P3 but not Ins(1,3,4,5)P4. Type 1 lns(1,4,5)P3 5-phosphatase eluted as a single peak of activity with an apparent molecular mass of 51 kDa when gel filtration was performed in the presence of cholate. This molecular mass is identical to the molecular mass estimated for the particulate Ins(1 ,4,5)P3 5-phosphatase that was solubilized by cholate. K , values for Ins(1,4,5)P3 and Ins(1 ,3,4,5)P4 obtained with type I Ins(1,4,5)P3 5-phosphatase were 11 pM and 1 pM, respectively. Similar values were obtained with particulate Ins(1 ,4,5)P3 5-phosphatase. In conclusion, the catalytic domains of type I and particulate Ins(1 ,4,5)P3 5-phosphatase activity may be very similar, if not identical, but different from type I1 phosphatase.Agonist-stimulated hydrolysis of phosphatidylinositol 4,s-bisphosphate produces two signal molecules, Ins(1 ,4,5)P3 and diacylglycerol. Ins( 1 ,4,5)P3, a second messenger for mobilizing intracellular calcium [l, 21, has been shown to be metabolized to Ins(1,4)P2 or phosphorylated to Ins(1,3,4,5)P4 [3]. These reactions are catalyzed by an Ins(1,4,5)P3 5-phosphomonoesterase or phosphatase and an Ins(1 ,4,5)P3 3-kinase, respectively. These two enzymes represent potential control points at which both Ins(1 ,4,5)P3 and Ins(1,3,4,5)P4 concentrations could be regulated.Ins(1,4,5)P3 5-phosphatase was originally described in human erythrocyte membranes, where its activity is M g Z fdependent and inhibited by 2,3-bisphosphoglycerate [4]. The product of dephosphorylation is Ins(1 ,4)P2. The same enzyme specifically removes the 5 phosphate from Ins( 1,3,4,5)P4 to form lns(1,3,4)P3, the most abundant InsP3 isomer in many cells [S]. The Ins(1,4,5)P3 5-phosphatase is found in the soluble fractions of most tissues, although the membrane-bound activity is often quantitatively more important 16-91. A single enzyme form was purified from the soluble fraction of human platelets by the group of Majerus [lo]. Its apparent molecular mass was 38 kDa as determined by gel filtration. Both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 appeared to be substrates for the platelet Ins(1 ,4,5)P3 5-phosphatase [I I]. In platelets, ...

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A variety of mutations have been identified in the X-linked adrenoleukodystrophy (X-ALD) gene, none of which is prevalent. In this work we describe a reverse transcription polymerase chain reaction (RT-PCR)-based strategy specially suited to the molecular characterisation of mutations in index cases. After RT-PCR amplification of the X-ALD transcript a conformation-sensitive gel electrophoresis analysis is performed followed by sequencing of the fragments with altered mobility. Two X-ALD patients were studied using this strategy. In both cases, splice site mutations were found. The first patient studied has a single base substitution at the first position of the invariant GT dinucleotide donor splice site of intron 8. In spite of this alteration, small quantities of correctly spliced mRNA molecules were easily detected. In agreement with these data, a small amount of ALDP was found by western blotting analysis. An alteration at the -1 position of the donor splice site of exon 1 was detected in the second patient. This mutation results in the utilisation of a cryptic 5' splice site within intron 1. Nevertheless, this transition also allows for some correct splicing. Western blotting analysis revealed the existence of normal-migrating ALDP. However, as expected, the levels of this protein were greatly decreased. Taken together, our data suggest that some less severe or late-onset forms of X-ALD associated with splice mutations result from the production of small amounts of normal ALDP. It is proposed that the quantification of ALDP levels in these patients could provide important insights concerning the correlation between clinical phenotype and amount of normal ALDP.

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