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PubMed1986

Stability of betamethasone sodium phosphate, hydrocortisone sodium phosphate, and prednisolone sodium phosphate injections submitted by U.S. hospitals.

The stability of betamethasone sodium phosphate, hydrocortisone sodium phosphate, and prednisolone sodium phosphate, injections stored in hospital pharmacies across the United States was studied. Through a voluntary drug stability program, FDA selected 58 samples (representing two manufacturers) from pharmacies representing a cross section of the country. The samples were analyzed for strength, identification, pH, and related impurities. All of the hydrocortisone sodium phosphate and prednisolone sodium phosphate samples met USP requirements for strength and pH. Assays of the betamethasone sodium phosphate injection samples yielded results that were in compliance with the manufacturer's strength and pH limits; there are no USP requirements for betamethasone sodium phosphate injection. All samples were within USP limits for related free steroids. Betamethasone sodium phosphate, hydrocortisone sodium phosphate, and prednisolone sodium phosphate injections obtained from hospital pharmacies appear to be stable after storage under actual marketplace conditions.

Betamethasone↗
PubMed2001

Crystal structure of histidinol phosphate aminotransferase (HisC) from Escherichia coli, and its covalent complex with pyridoxal-5'-phosphate and l-histidinol phosphate.

The biosynthesis of histidine is a central metabolic process in organisms ranging from bacteria to yeast and plants. The seventh step in the synthesis of histidine within eubacteria is carried out by a pyridoxal-5'-phosphate (PLP)-dependent l-histidinol phosphate aminotransferase (HisC, EC 2.6.1.9). Here, we report the crystal structure of l-histidinol phosphate aminotransferase from Escherichia coli, as a complex with pyridoxamine-5'-phosphate (PMP) at 1.5 A resolution, as the internal aldimine with PLP, and in a covalent, tetrahedral complex consisting of PLP and l-histidinol phosphate attached to Lys214, both at 2.2 A resolution. This covalent complex resembles, in structural terms, the gem-diamine intermediate that is formed transiently during conversion of the internal to external aldimine.HisC is a dimeric enzyme with a mass of approximately 80 kDa. Like most PLP-dependent enzymes, each HisC monomer consists of two domains, a larger PLP-binding domain having an alpha/beta/alpha topology, and a smaller domain. An N-terminal arm contributes to the dimerization of the two monomers. The PLP-binding domain of HisC shows weak sequence similarity, but significant structural similarity with the PLP-binding domains of a number of PLP-dependent enzymes. Residues that interact with the PLP cofactor, including Tyr55, Asn157, Asp184, Tyr187, Ser213, Lys214 and Arg222, are conserved in the family of aspartate, tyrosine and histidinol phosphate aminotransferases. The imidazole ring of l-histidinol phosphate is bound, in part, through a hydrogen bond with Tyr110, a residue that is substituted by Phe in the broad substrate specific HisC enzymes from Zymomonas mobilis and Bacillus subtilis. Comparison of the structures of the HisC internal aldimine, the PMP complex and the HisC l-histidinol phosphate complex reveal minimal changes in protein or ligand structure. Proton transfer, required for conversion of the gem-diamine to the external aldimine, does not appear to be limited by the distance between substrate and lysine amino groups. We propose that the tetrahedral complex has resulted from non-productive binding of l-histidinol phosphate soaked into the HisC crystals, resulting in its inability to be converted to the external aldimine at the HisC active site.

Amino Acid Sequence↗
PubMed1999

Oral phosphate binders: phosphate binding capacity of iron (III) hydroxide complexes containing saccharides and their effect on the urinary excretion of calcium and phosphate in rats.

Phosphate binders that contain aluminum or calcium are frequently prescribed to treat hyperphosphatemia in patients with end-stage renal disease (ESRD), but an accumulation of aluminum can lead to encephalopathy, aluminum-related bone disease (ARBD) such as osteomalacia, anaemia, and resistance to erythropoietin, and calcium accumulation can lead to hypercalcaemia. High phosphate concentrations are reduced in vitro and in vivo by a phosphate adsorption pill, which is synthesized by hydrolyzing ferrous sulfate in the presence of saccharides, to form an iron (III)-saccharide complex that is acid resistant and binds phosphate greater than iron (III) hydroxide alone. Under in vitro conditions, containing 3.26 mg P/dL, the iron (III)-sucrose complex showed the highest phosphate adsorption capacity at pH 2 with artificial gastric juice, 58.9 mg P/g binder. For the 7 day in vivo study, 0% (Group 1), 1% (Group 2), 4% (Group 3), and 8% (Group 4) iron (III)-sucrose complex was admixed into the rodent chow by weight and fed to 15 male Wistar rats. The weight and volume of the feces and urine, and the calcium, iron, and phosphorus excretions in the feces and urine samples were monitored for any signs of irregularity. Total urine outflow was collected during a 24-h period to determine the amount of phosphate recovered, which indicates the ability of the phosphate binder to reduce gastrointestinal phosphate absorption. The fecal iron excretion was significantly effected by the amount of binder ingested throughout the study for Group 2 (p < 0.001), Group 3 (p < 0.01), and Group 4 (p < 0.001). The urinary calcium excretion (mg/rat/24-h) significantly increased by the 7th day for Group 2 (p < 0.05) and Group 4 (p < 0.01) in comparison to the control. Finally, after 7 days, there was a significant drop in the urinary phosphorus levels (mg P/rat/24-h) in a dose dependent manner for Group 2: from 7.82 +/- 1.46 to 1.98 +/- 0.10 mg P/rat/24-h (102 mg P/dL/24-h; p < 0.05); Group 3: from 6.70 +/- 1.14 to 0.16 +/- 0.09 mg P/rat/24-h (6.0 mg P/dL/24-h; p < 0.01); and Group 4: from 8.25 +/- 0.67 to 0.04 +/- 0.01 mg P/rat/24-h (0.9 mg P/dL/24-h; p < 0.01). The results show that this new adsorbent might provide an alternative to conventional aluminum and calcium containing phosphate-binding agents for combating hyperphosphataemia.

Administration, Oral↗
PubMed1983

Regulation of Spinach Leaf Sucrose Phosphate Synthase by Glucose-6-Phosphate, Inorganic Phosphate, and pH.

Sucrose phosphate synthase was partially purified from spinach leaves and the effects and interactions among glucose-6-P, inorganic phosphate (Pi), and pH were investigated. Glucose-6-P activated sucrose phosphate synthase and the concentration required for 50% of maximal activation increased as the concentration of fructose-6-P was decreased. Inorganic phosphate inhibited sucrose phosphate synthase activity and antagonized the activation by glucose-6-P. Inorganic phosphate caused a progressive increase in the concentration of glucose-6-P required for 50% maximal activation from 0.85 mm (minus Pi) to 9.9 mm (20 mm Pi). In the absence of glucose-6-P, Pi caused partial inhibition of sucrose phosphate synthase activity (about 65%). The concentration of Pi required for 50% maximal inhibition decreased with a change in pH from 6.5 to 7.5. When the effect of pH on Pi ionization was taken into account, it was found that per cent inhibition increased hyperbolically with increasing dibasic phosphate concentration independent of the pH. Sucrose phosphate synthase had a relatively broad pH optimum centered at pH 7.5. Inhibition by Pi was absent at pH 5.5, but became more pronounced at alkaline pH, whereas activation by glucose-6-P was observed over the entire pH range tested. The results suggested that glucose-6-P and Pi bind to sites distinct from the catalytic site, e.g. allosteric sites, and that the interactions of these effectors with pH and concentrations of substrate may be involved in the regulation of sucrose synthesis in vivo.

Journal Article↗
PubMed2005

Sodium-phosphate cotransporter in human salivary glands: molecular evidence for the involvement of NPT2b in acinar phosphate secretion and ductal phosphate reabsorption.

OBJECTIVE: In order to elucidate the cellular and molecular mechanisms of phosphate secretion by human salivary glands, the expression and intracellular distribution of sodium-phosphate cotransporters was investigated. DESIGN: Total RNA was extracted from 33 parotid gland (PG) and 35 submandibular gland (SMG) samples and RT-PCR was performed using gene specific primers for all known sodium-phosphate cotransporters. An antibody was raised against an NPT2b epitope and the cellular and intracellular distribution was investigated by immunohistochemistry. RESULTS: No mRNA for the type I cotransporter NPT1 was found. Out of the type II phosphate cotransporters only message for NPT2b but not for NPT2a or NPT2c could be detected in about the same number of samples (76% in PG versus 69% in SMG). Type III cotransporter mRNA was also found in both glands, PIT1 gave positive results for 93% of PG samples compared to 69% of SMG samples. For PIT2 also, a higher expression was found in PG than in SMG, although the difference was smaller (79% versus 51%). Immunostaining for NPT2b was found both in the acini and in the ducts, with a stronger reaction in the latter. In acinar cells, NPT2b was restricted to the basal-lateral plasma membrane, in duct cells, a broad band of reactivity was located in the apical part of the cell. CONCLUSIONS: These findings suggest a secondary active secretion of phosphate into the primary saliva. Ductal cells appear to be able to reabsorb phosphate, thereby modifying the phosphate concentration in the final saliva.

Adult↗
PubMed1990

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: a model for the interconversion of D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate.

Based on experimental data, a model is proposed for the interconversion of either unlabelled hexose phosphates or D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate in the reaction catalyzed by phosphoglucoisomerase. This model takes into account the known differences in maximal velocity and affinity for each substrate, the intramolecular transfer of tritium between C1 and C2, and the isotopic discrimination between unlabelled and tritiated esters. This model reveals that, in a close system characterized by the progressive detritiation of hexose phosphates, the concentration ratio of D-glucose 6-phosphate to D-fructose 6-phosphate is much higher with the tritiated than unlabelled esters, a paradoxical increase in the specific radioactivity of D-glucose 6-phosphate above its initial value being even observed during the initial period of exposure of D-[2-3H]glucose 6-phosphate to phosphoglucoisomerase. The extension of this model to an open system may be essential for the correct interpretation of radioactive data collected in intact cells exposed to D-[2-3H]glucose.

Fructosephosphates↗
PubMed1976

Studies of phosphate transport in Escherichia coli. I. Reexamination of the effect of osmotic and cold shock on phosphate uptake and some attempts to restore uptake with phosphate binding protein.

1. The first stage of osmotic shock caused a slight reduction in the primary uptake of inorganic orthophosphate (Pi) in AB3311 cells of Escherichia coli which normally exhibit a biphasic type of phosphate uptake. The second stage of osmotic shock resulted in a marked reduction (a total of 80-85%) in the primary uptake phase and a lesser reduction (50%) in the secondary uptake. When osmotically shocked cells are allowed to recover in a phosphate-free, but otherwise complete medium sufficient repair occurs in the shocked cells to overcome growth lags and to restore the above losses in phosphate uptake almost to normal after 90-180 min of recovery. 2. Extensive investigation was made of the more mild cold shock procedure which involves the rapid disperion of Escherichia coli cells into 80 vol. of water at 2 degrees C. The most consistent cold shock effects, as evidenced by reductions in phosphate uptake, were obtained in cells after they were washed in appropriate buffered salts media, suspended in a minimal volume of water and shocked from 37 degrees C rather than 24 degrees C. Less severe shocks were obtained after washing in imidazole/salts/glucose or Tris/salts/glucose media than in NaCl/Tris. 3. A number of attempts were made to restore by the addition of phosphate binding protein the reduction in phosphate uptake of E. coli AB3311 cells caused by a variety of cold shocks. In no instance was good restoration of phosphate uptake achieved. Qualitatively, it appeared that a better restoration of uptake occurred in unstarved and starved cells washed in imidazole/salts/glucose where the cold shock effect was less severe.

Bacterial Proteins↗
PubMed1978

Isotopic (18O) shift in 31P nuclear magnetic resonance applied to a study of enzyme-catalyzed phosphate--phosphate exchange and phosphate (oxygen)--water exchange reactions.

An isotopic shift of the (31)P nuclear magnetic resonance due to (18)O bonded to phosphorus of 0.0206 ppm has been observed in inorganic orthophosphate and adenine nucleotides. Thus, the separation between the resonances of (31)P(18)O(4) and (31)P(16)O(4) at 145.7 MHz is 12 Hz and, in a randomized sample containing approximately 50% (18)O, all five (16)O-(18)O species are resolved and separated from each other by 3 Hz. Not only does this yield the (18)O/(16)O ratio of the phosphate but, more important, the (18)O-labeled phosphate in effect can serve as a double label in following phosphate reactions, for oxygen in all cases and for phosphorus, provided the oxygen does not exchange with solvent water. Thus, it becomes possible to follow labeled phosphorus or labeled oxygen continuously as reactions proceed. Rate studies involving (i) phosphorus and (ii) oxygen are illustrated by continuous monitoring of the exchange reactions between (i) the beta phosphate of ADP and inorganic phosphate catalyzed by polynucleotide phosphorylase and (ii) inorganic orthophosphate and water catalyzed by yeast inorganic pyrophosphatase. In the ADP-P(i) exchange, the P(i) ((18)O(4)) yielded an alpha P((16)O(3) (18)O) and a beta P((18)O(4)), proving that bond cleavage occurs between the alpha P and the alpha-beta bridge oxygen. Among the many additional potential uses of this labeling technique and its spectroscopic observation are: (i) different labeling of each phosphate group of ATP, (ii) to follow rate of transfer of (18)O from a nonphosphate compound such as a carboxylic acid to a phosphate compound, and (iii) to follow the rate of scrambling (for example, of the beta-gamma bridge oxygen of ATP to nonbridge beta P positions) and simultaneously the rate of exchange of the gamma P nonbridge oxygens with solvent water in various ATPase reactions.

Adenosine Diphosphate↗
PubMed1997

Affinity labeling of Escherichia coli glucosamine-6-phosphate synthase with a fructose 6-phosphate analog--evidence for proximity between the N-terminal cysteine and the fructose-6-phosphate-binding site.

Glucosamine-6-phosphate synthase (GlcNP-synthase) catalyzes the formation of glucosamine 6-phosphate from fructose 6-phosphate using the gamma-amide functionality of glutamine as the nitrogen source. In the absence of glutamine, GlcNP-synthase was recently found to catalyze the formation of glucose 6-phosphate corresponding to a phosphoglucoisomerase-like activity. Here we report active-site directed, irreversible inhibition of Escherichia coli GlcNP-synthase (k(inact) = 0.60 +/- 0.05 min(-1), Kirr = 1.40 +/- 0.20 mM) by anhydro-1,2-hexitol 6-phosphates previously known as irreversible inhibitors of phosphoglucoisomerase. Enzyme inactivation with the tritiated affinity label, followed by tryptic digestion and purification of the radioactive fragments, allowed identification of three peptides. Two of them, accounting for 54% of the recovered radioactivity, are believed to result from the nucleophilic attack of side-chain carboxylates of Glu255 and Glu258 and thiol of Cys300 of the fructose-6-phosphate-binding site on the epoxide functionality of the inhibitor. The major peptide corresponds to derivatization of the N-terminal cysteine from the glutamine-binding site by the inhibitor. These results provide evidence for the close proximity of glutamine and fructose-6-phosphate-binding sites recently suggested by Bearne [Bearne, S. L. (1996) J. Biol. Chem. 271, 3052-3057].

Affinity Labels↗
PubMed1988

Acholeplasma laidlawii B-PG9 adenine-specific purine nucleoside phosphorylase that accepts ribose-1-phosphate, deoxyribose-1-phosphate, and xylose-1-phosphate.

An adenylate-specific purine nucleoside phosphorylase (purine nucleoside:orthophosphate ribosyltransferase, EC12.4.2.1) (PNP) was isolated from a cytoplasmic fraction of Acholeplasma laidlawii B-PG9 and partially purified (820-fold). This partially purified PNP could only ribosylate adenine and deribosylate adenosine and deoxyadenosine. The A. laidlawii partially purified PNP could not use hypoxanthine, guanine, uracil, guanosine, deoxyguanosine, or inosine as substrates, but could use ribose-1-phosphate, deoxyribose-1-phosphate, or xylose-1-phosphate as the pentose donor. Mg2+ and a pH of 7.6 were required for maximum activity for each of the pentoses. The partially purified enzyme in sucrose density gradient experiments had an approximate molecular weight of 108,000 and a sedimentation coefficient of 6.9, and in gel filtration experiments it had an approximate molecular weight of 102,000 and a Stoke's radius of 4.1 nm. Nondenaturing polyacrylamide tube gels of the enzyme preparation produced one major and one minor band. The major band (Rf, 0.57) corresponded to all enzyme activity. The Kms for the partially purified PNP with ribose-1-phosphate, deoxyribose-1-phosphate, and xylose-1-phosphate were 0.80, 0.82, and 0.81 mM, respectively. The corresponding Vmaxs were 12.5, 14.3, and 12.0 microM min-1, respectively. The Hill or interaction coefficients (n) for all three pentose phosphates were close to unity. The characterization data suggest the possibility of one active site on the enzyme which is equally reactive toward each of the three pentoses. This is the first report of an apparently adenine-specific PNP activity.

Acholeplasma laidlawii↗
PubMedDate not supplied

The SO4-.-induced oxidation of 2'-deoxyuridine-5'-phosphate, uridine-5'-phosphate and thymidine-5'-phosphate. An ESR study in aqueous solution.

Reactions of photolytically generated SO4-. with 2'-deoxyuridine-5'-phosphate (5'-dUMP), uridine-5'-phosphate (5'-UMP) and thymidine-5'-phosphate (5'-dTUMP) were studied by ESR spectroscopy in aqueous solution under anoxic conditions. From 5'-dUMP and 5'-UMP the 5',5-cyclic phosphate-6-yl radicals 10 and 11 were generated (pH 2-11) whereas from 5'-dTMP at pH 3-8 the 5,6-dihydro-6-hydroxy-5-yl radical 14 and at pH 7-11 the 5-methylene-2'-deoxyuridine-5'-phosphate radical 15 was produced. In the experiments with 5'-UMP in addition to radical 11 the signals of sugar radicals 12 and 13 were detected. It is assumed that the base radical cations act as intermediates in the SO4-.-induced radical reactions. The 5'-phosphate group adds intramolecularly to the C(5)-C(6) bond of the uraclilyl radical cation whereas the thymidyl radical cation of 5'-dTMP reacts with H2O at pH less than 8 to yield the 6-OH-5-yl adduct 14 and deprotonates at pH greater than 7 thus forming the allyl-type radical 15. In 5'-UMP transfer of the radical site from the base to the sugar moiety competes with intramolecular phosphate addition.

Deoxyuracil Nucleotides↗
PubMed1980

Lactose and D-galactose metabolism in Staphylococcus aureus. II. Isomerization of D-galactose 6-phosphate to D-tagatose 6-phosphate by a specific D-galactose-6-phosphate isomerase.

The inducible D-galactose-6-phosphate isomerase that functions in the metabolism of lactose and D-galactose in Staphylococcus aureus was partially purified from extracts of D-galactose-grown cells. It was shown to catalyze specifically the reversible isomerization of D-galactose 6-phosphate to D-tagatose 6-phosphate, the apparent Km values being 9.6 mM and 1.9 mM, respectively. At equilibrium, the ratio of D-galactose 6-phosphate to D-tagatose 6-phosphate was 9.0. The enzyme was not simulated by mono- or divalent cations and was not inhibited by EDTA, but it was inactivated reversibly by the thiol reagent N-ethylmaleimide. Its molecular weight was estimated to be about 100,000 both by gel filtration and by sedimentation in a sucrose density gradient. Data on stability, pH optimum, and inducibility of the enzyme are also presented. An improved procedure for the chemical synthesis of D-tagatose 6-phosphate is described, and resolution of the anomers of D-tagatose 6-phosphate by gas-liquid chromatography is reported.

Aldose-Ketose Isomerases↗
PubMed1984

Phosphate uptake in the yeast Candida tropicalis: purification of phosphate-binding protein and investigations about its role in phosphate uptake.

The purification of a phosphate-binding protein (PiBP2) by immunoadsorption is described. The entire anti phosphate-binding protein 2 antibodies as well as the Fab fragments obtained from these antibodies inhibit Pi uptake by whole cells. The inhibition is a mixed type of inhibition (Vm and Km are affected). These results should be regarded as a possible involvement of phosphate-binding protein 2 in Pi uptake. The binding of 125I-labelled fragments prepared from anti phosphate-binding protein 2 antibodies to whole cells, to shocked cells and to protoplasts has been investigated. The results confirm the release of phosphate-binding protein by osmotic shock and during protoplast formation. From these findings, a cell-wall localisation, near the cell surface of the phosphate-binding protein should be proposed.

Antibodies↗
PubMed2000

Characterization of a phosphate binding domain on the alpha-subunit of chloroplast ATP synthase using the photoaffinity phosphate analogue 4-azido-2-nitrophenyl phosphate.

The photoaffinity phosphate analogue 4-azido-2 nitrophenyl phosphate (ANPP) was shown previously (Pougeois, R., Lauquin, G. J.-M., and Vignais, P. V. (1983) Biochemistry 22, 1241-1245) to bind covalently and specifically to a single catalytic site on one of the three beta-subunits of the isolated chloroplast coupling factor 1 (CF(1)). Modification by ANPP strongly inhibited ATP hydrolysis activity. In this study, we examined labeling of membrane-bound CF(1) by ANPP by exposing thylakoid membranes to increasing concentrations of the reagent. ANPP exhibited saturable binding to two sites on CF(1), one on the beta-subunit and one on the alpha-subunit. Labeling by ANPP resulted in the complete inhibition of both ATP synthesis and ATP hydrolysis by the membrane-bound enzyme. Labeling of both sites by ANPP was reduced by more than 80% in the presence of P(i) (> or = 10 mM) and ATP (> or = 0.5 mM). ADP was less effective in competing with ANPP for binding, giving a maximum of approximately 35% inhibition at concentrations > or = 2 mM. ANPP-labeled tryptic peptides of the alpha-subunit were isolated and sequenced. The majority of the probe was contained in three peptides corresponding to residues Gln(173) to Arg(216), Gly(217) to Arg(253), and His(256) to Arg(272) of the alpha-subunit. In the mitochondrial F(1) (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628), all three analogous peptides are located within the nucleotide binding pocket and within close proximity to the gamma-phosphate binding site. The data indicate, however, that the azidophenyl group of bound ANPP is oriented at approximately 180 degrees in the opposite direction to the adenine binding site with reference to the phosphate binding site on the alpha-subunit. The study has confirmed that ANPP is a bona fide phosphate analogue and suggests that it specifically targets the gamma-phosphate binding site within the nucleotide binding pockets on the alpha- and beta-subunits of CF(1). The study also indicates that in the resting state of the chloroplast F(1)-F(0) complex both the alpha- and beta-subunits are structurally asymmetric.

Azides↗
PubMed1985

Enzymic determination of inorganic phosphates, organic phosphates and phosphate-liberating enzymes by use of nucleoside phosphorylase-xanthine oxidase (dehydrogenase)-coupled reactions.

Coupled enzyme assays are described for measuring inorganic phosphates, organic phosphates and phosphate-liberating enzymes in biological material. The assays all determine Pi by its reaction with inosine, catalysed by nucleoside phosphorylase; this yields ribose 1-phosphate and hypoxanthine. The hypoxanthine is oxidized to uric acid by xanthine oxidase, and may be measured either by the absorbance of the uric acid, or by the formazan formed when a tetrazolium salt is used as the oxidant. The coupled enzyme assays are characterized by high sensitivity, quantitative utilization of phosphates and stoichiometric formation of the measurable products, measurement at pH 6.0-8.5, determination of phosphates within a single analytical step, and continuous measurement of phosphohydrolase activity in a corresponding rate assay. Examples include determinations of substrates such as Pi, PPi and AMP, and of enzymes such as 5'-nucleotidase, inorganic pyrophosphatase and glucose-6-phosphatase. Directions for further examples are given.

5'-Nucleotidase↗
PubMed1992

Mammalian cells that express Bacillus cereus phosphatidylinositol-specific phospholipase C have increased levels of inositol cyclic 1:2-phosphate, inositol 1-phosphate, and inositol 2-phosphate.

Phosphatidylinositol-specific phospholipase C (PtdIns-PLC) of Bacillus cereus catalyzes the conversion of PtdIns to inositol cyclic 1:2-phosphate and diacylglycerol. NIH 3T3, Swiss mouse 3T3, CV-1, and Cos-7 cells were transfected with a cDNA encoding this enzyme, and the metabolic and cellular consequences were investigated. Overexpression of PtdIns-PLC enzyme activity was associated with elevated levels of inositol cyclic 1:2-phosphate (2.5-70-fold), inositol 1-phosphate (2-20-fold), and inositol 2-phosphate (3-20-fold). The increases correlated with the levels of enzyme expression obtained in each cell type. The turnover of phosphatidylinositol (PtdIns) was also increased in transfected CV-1 cells by 13-fold 20 h after transfection. The levels of PtdIns, phosphatidic acid, diacylglycerol, or other inositol phosphates were not detectably altered. Expression of bacterial PtdIns-PLC decreased rapidly after 20 h implying that either the increased PtdIns turnover or the accumulation of inositol phosphates was detrimental to cells and that by some adaptive mechanism enzyme expression was suppressed.

3T3 Cells↗