Cloning and expression of human 75-kDa inositol polyphosphate-5-phosphatase.

Inositol polyphosphate-5-phosphatase (5-phosphatase) hydrolyzes inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate and thereby functions as a signal terminating enzyme in cellular calcium ion mobilization. A cDNA encoding human platelet 5-phosphatase has been isolated by screening for beta-galactosidase fusion proteins that bind to inositol 1,3,4,5-tetrakisphosphate. The sensitivity of the screening procedure was enhanced 50- to 100-fold by amplification of "sublibraries" prior to carrying out binding assays. The sequences derived from the "expression clone" were used to screen human erythroleukemia cell line and human megakaryocytic cell line cDNA libraries. We obtained two additional clones which together consist of 2381 base pairs. The amino-terminal amino acid sequence from the 75-kDa 5-phosphatase purified from platelets is identical to amino acids 38-56 predicted from the cDNA. This suggests that the platelet 5-phosphatase is formed by proteolytic processing of a larger precursor. The cDNA predicts that the mature enzyme contains 635 amino acids (Mr 72, 891). Antibodies directed against recombinant TrpE fusion proteins of either an amino-terminal region or a carboxyl-terminal region immunoprecipitate the enzyme activity from a preparation of the 75-kDa form of platelet 5-phosphatase (Type II) but do not precipitate the distinct 47-kDa 5-phosphatase (Type I) also found in platelets. In addition, the recombinant protein expressed in Cos-7 cells has the same 5-phosphatase activity as the platelet 5-phosphatase.     

Identification and characterization of a novel inositol polyphosphate 5-phosphatase

We have identified a cDNA encoding a novel inositol polyphosphate 5-phosphatase. It contains two highly conserved catalytic motifs for 5-phosphatase, has a molecular mass of 51 kDa, and is ubiquitously expressed and especially abundant in skeletal muscle, heart, and kidney. We designated this 5-phosphatase as SKIP (Skeletal muscle and Kidney enriched Inositol Phosphatase). SKIP is a simple 5-phosphatase with no other motifs. Baculovirus-expressed recombinant SKIP protein exhibited 5-phosphatase activities toward inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol (PtdIns) 4,5-bisphosphate, and PtdIns 3,4, 5-trisphosphate but has 6-fold more substrate specificity for PtdIns 4,5-bisphosphate (K(m) = 180 microM) than for inositol 1,4, 5-trisphosphate (K(m) = 1.15 mM). The ectopic expression of SKIP protein in COS-7 cells and immunostaining of neuroblastoma N1E-115 cells revealed that SKIP is expressed in cytosol and that loss of actin stress fibers occurs where the SKIP protein is concentrated. These results imply that SKIP plays a negative role in regulating the actin cytoskeleton through hydrolyzing PtdIns 4,5-bisphosphate.     

Identification of a novel spliceoform of inositol polyphosphate 4-phosphatase type Ialpha expressed in human platelets: structure of human inositol polyphosphate 4-phosphatase type I gene

Inositol polyphosphate 4-phosphatases (IP4Ps) are enzymes involved in the regulation of phosphoinositide 3-kinase (PI3K) signaling. IP4Ps catalyze the hydrolysis of the D-4 position phosphoester of the PI3K generated lipid second messenger, phosphatidylinositol 3,4-bisphosphate. Western blot analysis detected the expression of a novel 110 kDa form of IP4P type Ialpha in mouse spleen, heart, lung, and uterus. In addition, the 110 kDa form of IP4P type Ialpha was found to be the major form of this enzyme expressed in human platelets, MEG-01 megakaryocytes and Jurkat T-cells. RT-PCR analysis of MEG-01 megakaryocytes and Jurkat T-cells indicates that the 110-kDa form of IP4P Ialpha is derived from an alternatively spliced mRNA that encodes an additional internal domain of 40 amino acids not present in the two previously described brain IP4P Ialpha spliceoforms. The predicted molecular mass of this spliceoform is 109,968 Da, consistent with its apparent molecular mass estimated by Western blot analysis. The novel domain is proline rich and contains a PEST sequence characteristic of proteins that are rapidly degraded by the calpain family of proteases. Analysis of genomic DNA sequence indicates that the IP4P type I gene consists of 25 exons and that this novel spliceoform is obtained as a result of an unusual type of differential splicing involving the use of an alternative 5'-GU donor splice site during the excision of intron 15. In addition, we show that all three known spliceoforms of IP4P Ialpha result from alternative splicing involving exon 15 and 16 indicating that structural variability in this region of the enzyme may be important for its function.     

The isolation and characterization of a cDNA encoding phospholipid-specific inositol polyphosphate 5-phosphatase

We report the cDNA cloning and characterization of a novel human inositol polyphosphate 5-phosphatase (5-phosphatase) that has substrate specificity unlike previously described members of this large gene family. All previously described members hydrolyze water soluble inositol phosphates. This enzyme hydrolyzes only lipid substrates, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 4,5-bisphosphate. The cDNA isolated comprises 3110 base pairs and predicts a protein product of 644 amino acids and M(r) = 70,023. We designate this 5-phosphatase as type IV. It is a highly basic protein (pI = 8.8) and has the greatest affinity toward phosphatidylinositol 3,4,5-trisphosphate of known 5-phosphatases. The K(m) is 0.65 micrometer, 1/10 that of SHIP (5.95 micrometer), another 5-phosphatase that hydrolyzes phosphatidylinositol 3,4,5-trisphosphate. The activity of 5-phosphatase type IV is sensitive to the presence of detergents in the in vitro assay. Thus the enzyme hydrolyzes lipid substrates in the absence of detergents or in the presence of n-octyl beta-glucopyranoside or Triton X-100, but not in the presence of cetyltriethylammonium bromide, the detergent that has been used in other studies of the hydrolysis of phosphatidylinositol 4,5-bisphosphate. Remarkably SHIP, a 5-phosphatase previously characterized as hydrolyzing only substrates with d-3 phosphates, also readily hydrolyzed phosphatidylinositol 4,5-bisphosphate in the presence of n-octyl beta-glucopyranoside but not cetyltriethylammonium bromide. We used antibodies prepared against a peptide predicted by the cDNA to identify the 5-phosphatase type IV enzyme in human tissues and find that it is highly expressed in the brain as determined by Western blotting. We also performed Western blotting of mouse tissues and found high levels of expression in the brain, testes, and heart with lower levels of expression in other tissues. mRNA was detected in many tissues and cell lines as determined by Northern blotting.     

The isolation and characterization of inositol polyphosphate 4-phosphatase

We previously identified an alternative pathway for the metabolism of inositol 1,3,4-trisphosphate (Ins(1,3,4)P3) in calf brain. The enzyme responsible for the degradation of Ins(1,3,4)P3 was designated as inositol polyphosphate 4-phosphatase (Bansal, V. S., Inhorn, R. C., and Majerus, P. W. (1987) J. Biol. Chem. 262, 9644-9647). We have now purified this enzyme 3390-fold from calf brain-soluble fraction. The isolated enzyme has an apparent molecular mass of 110 kDa as determined by gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme migrates as a protein of 105 kDa, suggesting that it is monomeric. Among various 4-phosphate-containing inositol polyphosphates, the enzyme hydrolyzes only Ins(1,3,4)P3 and inositol 3,4-bisphosphate (Ins(3,4)P2), yielding inositol 1,3-bisphosphate and inositol 3-phosphate as products. The inositol polyphosphate 4-phosphatase has apparent Km values of 40 and 25 microM for Ins(1,3,4)P3 and Ins(3,4)P2, respectively. The maximum velocities for these two substrates are 15-20 mumol of product/min/mg protein. Ins(1,3,4)P3 is a competitive inhibitor of Ins(3,4)P2 hydrolysis with an apparent Ki of 27 microM implying that the same active site is involved in hydrolysis of both substrates. The final enzyme preparation retained a small inositol polyphosphate 3-phosphatase activity (less than 2% of rate of inositol polyphosphate 4-phosphatase activity) which most likely reflects a contaminant. The enzyme displays maximum activity between pH 6.5 and 7.5. It is not inhibited by Li+, Ca2+, or Mg2+ except at 10 mM divalent ions. Mn2+ inhibits enzyme at high concentrations IC50 = 1.5 mM.     

Identification and isolation of vinculin from platelets

A vinculin-like protein was identified in chicken as well as in bovine platelets by ELISA competitive binding assay using antibodies against vinculin from chicken gizzard. By a modified procedure (J. Biol. Chem. (1980) 255, 1194–1199) we succeeded in isolating bovine platelet vinculin to apparent homogeneity. The structural identity of platelet and chicken gizzard vinculin was demonstrated by circular dichroism analysis. It was also shown that platelet vinculin induces a significant decrease in the low shear viscosity of F-actin. Vinculin, in all probability, plays an important role in the organization of actin filaments in platelets, especially in the linkages of microfilaments to the membrane.     

Isolation of a phosphomonoesterase from human platelets that specifically hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate

Platelets, and a variety of other cells, rapidly hydrolyze the phosphoinositides in response to stimulation by agonists. One of the products of hydrolysis of phosphatidylinositol 4,5-diphosphate is inositol 1,4,5-trisphosphate, which recently has been suggested to mediate intracellular Ca2+ mobilization. We have found that human platelets contain an enzyme that degrades inositol 1,4,5-trisphosphate. We have isolated this soluble enzyme and find that it hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate (Km = 30 microM, Vmax = 5.3 microM/min/mg of protein). The products of the reaction are inositol 1,4-diphosphate and phosphate. The apparent molecular weight of the enzyme is 38,000 as determined both by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol. This enzyme is specific for inositol 1,4,5-trisphosphate. Other water soluble inositol phosphates as well as phosphorylated sugars are not hydrolyzed, while the only inositol containing phospholipid hydrolyzed is phosphatidylinositol 4,5-diphosphate at a rate less than 1% that for inositol 4,5-trisphosphate. The inositol 1,4,5-trisphosphate 5-phosphomonoesterase requires Mg2+ for activity and is inhibited by Ca2+, Ki = 70 microM. Li+, up to 40 mM, has no effect on enzyme activity. The duration and magnitude of any inositol 1,4,5-trisphosphate response in stimulated platelets may be determined by the activity of this enzyme.     

Phosphatidylinositol 3,4,5-trisphosphate is a substrate for the 75 kDa inositol polyphosphate 5-phosphatase and a novel 5-phosphatase which forms a complex with the p85/p110 form of phosphoinositide 3-kinase.

Agonist-stimulated production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], is considered the primary output signal of activated phosphoinositide (PI) 3-kinase. The physiological targets of this novel phospholipid and the identity of enzymes involved in its metabolism have not yet been established. We report here the identification of two enzymes which hydrolyze the 5-position phosphate of PtdIns(3,4,5)P3, forming phosphatidylinositol (3,4)-bisphosphate. One of these enzymes is the 75 kDa inositol polyphosphate 5-phosphatase (75 kDa 5-phosphatase), which has previously been demonstrated to metabolize inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. We have identified a second PtdIns(3,4,5)P3 5-phosphatase in the cytosolic fraction of platelets, which forms a complex with the p85/p110 form of PI 3-kinase. This enzyme is immunologically and chromatographically distinct from the platelet 43 kDa and 75 kDa 5-phosphatases and is unique in that it removes the 5-position phosphate from PtdIns(3,4,5)P3, but does not metabolize PtdIns(4,5)P2, Ins(1,4,5)P3 or Ins(1,3,4,5)P4. These studies demonstrate the existence of multiple PtdIns(3,4,5)P3 5-phosphatases within the cell.     

Cloning of the genomic locus of mouse SH2 containing inositol 5-phosphatase (SHIP) and a novel 110-kDa splice isoform, SHIPdelta

The SH2 domain containing inositol 5'-phosphatase (SHIP) was initially described as a 145-kDa protein phosphorylated on tyrosines upon growth factor and cytokine stimulation. It was shown to be phosphorylated after Fc and B cell receptor activation and plays a role in negative signaling. Different isoforms of the SHIP protein result from alternative mRNA splicing, proteolysis, or a combination of both. The expression of discrete SHIP isoforms changes with the potential developmental-dependent maturation state of myeloid cells, suggesting mechanisms for the regulation of SHIP interactions with other signaling molecules. A p135 (SHIPbeta) spliced isoform is known to be expressed in developing myeloid cells. Now we have identified a new SHIP isoform, SHIPdelta, which is the product of an out-of-frame splice with a deletion of 167 nucleotides in the C-terminal region, resulting in an approximately 110-kDa protein. Biochemically, SHIPdelta differs from SHIPalpha by exhibiting little or no tyrosine phosphorylation or association with the signaling protein Shc after M-CSF activation of FD-Fms cells. In addition, we have characterized the structure of the entire SHIP genomic locus, which provides a basis for understanding the alternative splicing events. SHIP is expressed in hematopoiesis and spermatogenesis, and we also describe the promoter for the SHIP gene, which has potential for explaining the tissue-specific expression pattern.     

Identification and characterization of a sac domain-containing phosphoinositide 5-phosphatase

We have characterized a novel Sac domain-containing inositol phosphatase, hSac2. It was ubiquitously expressed but especially abundant in the brain, heart, skeletal muscle, and kidney. Unlike other Sac domain-containing proteins, hSac2 protein exhibited 5-phosphatase activity specific for phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. This is the first time that the Sac domain has been reported to possess 5-phosphatase activity. Its 5-phosphatase activity for phosphatidylinositol 4,5-bisphosphate (K(m) = 14.3 microm) was comparable with those of Type II 5-phosphatases. These results imply that hSac2 functions as an inositol polyphosphate 5-phosphatase.     

Identification of protein-ribulosamine-5-phosphatase as human low-molecular-mass protein tyrosine phosphatase-A

Ribulosamines, which are substrates for the deglycating enzyme fructosamine-3-kinase-related protein, are presumably formed intracellularly through glycation of proteins with ribose 5-phosphate followed by dephosphorylation of resulting RN5Ps (ribulosamine 5-phosphates) by a putative RN5Pase (ribulosamine-5-phosphatase). Ribose 5-phosphate is known to be a potent glycating agent and we show in the present study that it reacts approximately 10 and 80-fold more rapidly with protein than ribose and glucose respectively. We also show that tissue extracts and, most particularly, erythrocyte extracts contain a protein-RN5Pase. We have purified this enzyme from human erythrocytes to near homogeneity and shown it to correspond to LMWPTP-A [low-molecular-mass ('weight') protein tyrosine phosphatase-A]. Human recombinant LMWPTP-A displayed an RN5Pase activity that was higher than its tyrosine phosphatase activity, indicating that this phosphatase may participate in protein deglycation, a new form of protein repair.     

Identification in extracts from AR4-2J cells of inositol 1,4,5-trisphosphate by its susceptibility to inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase.

Renal brush-border membrane vesicles from rat kidney cortex were irradiated in frozen state with a gamma-radiation source. Initial rates of influx into these vesicles were estimated for substrates such as L-glutamic acid, L-alanine, L-proline and L-leucine to establish the molecular sizes of their carriers. Transport was measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Initial rates of Na(+)-independent uptakes for those four substrates appeared unaffected in the dose range used (0-6 Mrad), indicating that the passive permeability of the membrane towards these substrates was unaffected. However, at higher doses of irradiation the Na+ influx and the intravesicular volume evaluated by the uptake of glucose at equilibrium were altered by radiation. Thus Na(+)-dependent influx values were corrected for volume changes, and the corrected values were used to compute radiation-inactivation sizes of the transport systems. Their respective values for L-glutamic acid, L-proline, L-leucine and L-alanine carriers were 250, 224, 293 and 274 kDa. The presence of the free-radicals scavenger benzoic acid in the frozen samples during irradiation did not affect the uptake of glucose, phosphate and alkaline phosphatase activity. These results indicate that freezing samples in a cryoprotective medium was enough to prevent secondary inactivation of transporters by free radicals. Uptakes of beta-alanine and L-lysine were much less affected by radiation. The radiation-inactivation size of the Na(+)-dependent beta-alanine carrier was 127 kDa and that of the L-lysine carrier was 90 kDa.     

Molecular characterization of an Arabidopsis gene encoding a phospholipid-specific inositol polyphosphate 5-phosphatase

Phosphoinositides are important molecules that serve as second messengers and bind to a complex array of proteins modulating their subcellular location and activity. The enzymes that metabolize phosphoinositides can in some cases serve to terminate the signaling actions of phosphoinositides. The inositol polyphosphate 5-phosphatases (5PTases) comprise a large protein family that hydrolyzes 5-phosphates from a variety of inositol phosphate and phosphoinositide substrates. We previously reported the identification of 15 putative 5PTase genes in Arabidopsis and have shown that overexpression of the At5PTase1 gene can alter abscisic acid signaling. At5PTase1 and At5PTase2 have been shown to hydrolyze the 5-phosphate from inositol phosphate substrates. We have examined the substrate specificity of the At5PTase11 protein, which is one of the smallest predicted 5PTases found in any organism. We report here that the At5PTase11 gene encodes an active 5PTase enzyme that can only dephosphorylate phosphoinositide substrates containing a 5-phosphate. In addition to hydrolyzing known substrates of 5PTase enzymes, At5PTase11 also hydrolyzes the 5-phosphate from phosphatidylinositol (3,5) bisphosphate. We also show that the At5PTase11 gene is regulated by abscisic acid, jasmonic acid, and auxin, suggesting a role for phosphoinositide action in these signal transduction pathways.     

Cloning and characterization of a 72-kDa inositol-polyphosphate 5-phosphatase localized to the Golgi network

The inositol-polyphosphate 5-phosphatase enzyme family removes the 5-position phosphate from both inositol phosphate and phosphoinositide signaling molecules. We have cloned and characterized a novel 5-phosphatase, which demonstrates a restricted substrate specificity and tissue expression. The 3.9-kb cDNA predicts for a 72-kDa protein with an N-terminal proline rich domain, a central 5-phosphatase domain, and a C-terminal CAAX motif. The 3. 9-kilobase mRNA showed a restricted expression but was abundant in testis and brain. Antibodies against the sequence detected a 72-kDa protein in the testis in the detergent-insoluble fraction. Indirect immunofluorescence of the Tera-1 cell line using anti-peptide antibodies to the 72-kDa 5-phosphatase demonstrated that the enzyme is predominantly located to the Golgi. Expression of green fluorescent protein-tagged 72-kDa 5-phosphatase in COS-7 cells revealed that the enzyme localized predominantly to the Golgi, mediated by the N-terminal proline-rich domain, but not the C-terminal CAAX motif. In vitro, the protein inserted into microsomal membranes on the cytoplasmic face of the membrane. Immunoprecipitated recombinant 72-kDa 5-phosphatase hydrolyzed phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3, 5-bisphosphate, forming phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3-phosphate, respectively. We propose that the novel 5-phosphatase hydrolyzes phosphatidylinositol 3,4, 5-trisphosphate and phosphatidylinositol 3,5-bisphosphate on the cytoplasmic Golgi membrane and thereby may regulate Golgi-vesicular trafficking.     

The inositol 5-phosphatase INPP5B regulates B cell receptor clustering and signaling

Upon antigen binding, the B cell receptor (BCR) undergoes clustering to form a signalosome that propagates downstream signaling required for normal B cell development and physiology. BCR clustering is dependent on remodeling of the cortical actin network, but the mechanisms that regulate actin remodeling in this context remain poorly defined. In this study, we identify the inositol 5-phosphatase INPP5B as a key regulator of actin remodeling, BCR clustering, and downstream signaling in antigen-stimulated B cells. INPP5B acts via dephosphorylation of the inositol lipid PI(4,5)P₂ that in turn is necessary for actin disassembly, BCR mobilization, and cell spreading on immobilized surface antigen. These effects can be explained by increased actin severing by cofilin and loss of actin linking to the plasma membrane by ezrin, both of which are sensitive to INPP5B-dependent PI(4,5)P₂ hydrolysis. INPP5B is therefore a new player in BCR signaling and may represent an attractive target for treatment of B cell malignancies caused by aberrant BCR signaling.     

The isolation, characterisation and kinetics of glutathione-S-transferase from human platelets

Glutathione S-transferase activity from human platelets was purified to homogeneity by affinity chromatography. The purified enzyme was found to be the acidic form and its molecular and catalytic properties were identical to acidic glutathione S-transferases purified from other human tissues. The purified platelet enzyme had no peroxidase activity and did not protect microsomes against peroxidation.     

A secreted salivary inositol polyphosphate 5-phosphatase from a blood-feeding insect: allosteric activation by soluble phosphoinositides and phosphatidylserine

Type II inositol polyphosphate 5-phosphatases (IPPs) act on both soluble inositol phosphate and phosphoinositide substrates. In many cases, these enzymes occur as multidomain proteins in which the IPP domain is linked to lipid-binding or additional catalytic domains. Rhodnius prolixus IPPRp exists as an isolated IPP domain which is secreted into the saliva of this blood-feeding insect. It shows selectivity for soluble and lipid substrates having a 1,4,5-trisphosphate substitution pattern while only poorly hydrolyzing substrates containing a D3 phosphate. With soluble diC8 PI(4,5)P(2) as a substrate, sigmoidal kinetics were observed, suggesting the presence of allosteric activation sites. Surprisingly, IPPRp-mediated hydrolysis of PI(4,5)P(2) and PI(3,4,5)P(3) was also stimulated up to 100-fold by diC8 PI(4)P and diC8 phosphatidylserine (PS). The activation kinetics were again sigmoidal, demonstrating that the allosteric sites recognize nonsubstrate phospholipids. Activation was positively cooperative, and analysis by the Hill equation suggests that at least three to four allosteric sites are present. In a vesicular system, hydrolysis of PI(4,5)P(2) followed a surface dilution kinetic model, and as expected, PS was found to be strongly stimulatory. If allosteric activation of type II IPPs by PI(4)P and PS is a widespread feature of the group, it may represent a novel regulatory mechanism for these important enzymes.     

Identification and characterisation of a new human glucose-6-phosphatase isoform

The liver endoplasmic reticulum glucose-6-phosphatase catalytic subunit (G6PC1) catalyses glucose 6-phosphate hydrolysis during gluconeogenesis and glycogenolysis. The highest glucose-6-phosphatase activities are found in the liver and the kidney; there have been many reports of glucose 6-phosphate hydrolysis in other tissues. We cloned a new G6Pase isoform (G6PC3) from human brain encoded by a six-exon gene (chromosome 17q21). G6PC3 protein was able to hydrolyse glucose 6-phosphate in transfected Chinese hamster ovary cells. The optimal pH for glucose 6-phosphate hydrolysis was lower and the K(m) higher relative to G6PC1. G6PC3 preferentially hydrolyzed other substrates including pNPP and 2-deoxy-glucose-6-phosphate compared to the liver enzyme.     

Up-regulation of phosphoinositide metabolism in tobacco cells constitutively expressing the human type I inositol polyphosphate 5-phosphatase

To evaluate the impact of suppressing inositol 1,4,5-trisphosphate (InsP(3)) in plants, tobacco (Nicotiana tabacum) cells were transformed with the human type I inositol polyphosphate 5-phosphatase (InsP 5-ptase), an enzyme which specifically hydrolyzes InsP(3). The transgenic cell lines showed a 12- to 25-fold increase in InsP 5-ptase activity in vitro and a 60% to 80% reduction in basal InsP(3) compared with wild-type cells. Stimulation with Mas-7, a synthetic analog of the wasp venom peptide mastoparan, resulted in an approximately 2-fold increase in InsP(3) in both wild-type and transgenic cells. However, even with stimulation, InsP(3) levels in the transgenic cells did not reach wild-type basal values, suggesting that InsP(3) signaling is compromised. Analysis of whole-cell lipids indicated that phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)), the lipid precursor of InsP(3), was greatly reduced in the transgenic cells. In vitro assays of enzymes involved in PtdInsP(2) metabolism showed that the activity of the PtdInsP(2)-hydrolyzing enzyme phospholipase C was not significantly altered in the transgenic cells. In contrast, the activity of the plasma membrane PtdInsP 5 kinase was increased by approximately 3-fold in the transgenic cells. In vivo labeling studies revealed a greater incorporation of (32)P into PtdInsP(2) in the transgenic cells compared with the wild type, indicating that the rate of PtdInsP(2) synthesis was increased. These studies show that the constitutive expression of the human type I InsP 5-ptase in tobacco cells leads to an up-regulation of the phosphoinositide pathway and highlight the importance of PtdInsP(2) synthesis as a regulatory step in this system.     

Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain.

Polyclonal antibodies to the major beta-naphthoflavone (BNF)-inducible form of cytochrome P-450 (P450IA) and to the major phenobarbitone (PB)-inducible form (P450IIB) have been used to quantify the contribution of these subfamilies to the total amount of cytochrome P-450 in rat livers and rat hepatocyte cultures treated with PB, BNF and metyrapone for 24 and 72 h. The P450IA and IIB subfamilies were not detectable (less than 5 pmol/mg of microsomal protein) in the livers of control rats, but administration of BNF resulted in the P450IA subfamily comprising more than 80% of the total hepatic cytochrome P-450. Administration of PB and metyrapone to rats did not elevate the level of this subfamily but elevated the levels of the P450IIB subfamily to 60% and 30% respectively of the total. Thus metyrapone is a ''PB-like'' inducer. However, in contrast with their effects in vivo, treatment with PB and metyrapone of rat hepatocytes did not elevate the proportion of the P450IIB subfamily relative to that in untreated cells but rather, like BNF, increased the P450IA subfamily. This would account for the ability of metyrapone to produce in hepatocyte culture, like BNF, a pronounced induction of ethoxyresorufin O-de-ethylase activity, but it does not account for why of all inducers studied only metyrapone can maintain the total cytochrome P-450 content of cultured hepatocytes, or the activity of ethylmorphine N-demethylase. This activity is generally considered to be associated with the P450IIB subfamily, but the lack of effect of metyrapone on this subfamily in hepatocyte culture must suggest that metyrapone is able to prevent the loss of the total amount of the cytochrome by increasing the expression of other cytochromes P-450.