Purification and properties of polyphosphoinositide phosphomonoesterase from rat brain.

1. On subcellular fractionation of rat brain homogenate, polyphosphoinositide phosphomonoesterase activity was greater in the cytosol than the membranous fractions. 2. The enzyme was purified from the cytosol by column chromatography on DEAE-cellulose, calcium phosphate gel and Sephadex G-100. 3. The final preparation of the enzyme showed a 430-fold purification over the whole homogenate and appeared to be homogeneous since it gave a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on isoelectric focusing. The enzyme has a relatively low molecular weight and an isoelectric point of 6.8. 4. The phosphatase showed a high affinity for triphosphoinositide. Without added Mg2+, the Km was 25 muM and V was 33 mumol Pi released/min/mg protein. 5. The enzyme hydrolysed diphosphoinositide at a slower rate than triphosphoinositide. In the presence of 10 mM Mg2+, the Km values for triphosphoinositide and diphosphoinositide were 5 muM and 25 muM respectively and V was the same for each substrate. 6. Both Mg2+ and Ca2+ activated the enzyme. While Ca2+ produced maximum activation at 100 muM, a much higher concentration of Mg2+ (10 mM) was required to elicit comparable activation. The enzyme did not show an absolute requirement for Mg2+ or Ca2+ as it exhibited low activity in the presence of 0.5 mM EDTA or EGTA. 7. The phosphatase showed maximum activity between 7.4 and 7.6. A drop in pH to 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity. 7.0 activated it almost completely, whereas an increase in pH to 8.0 halved the activity.     

Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes

Properties of phosphatidylinositol kinase activities in rabbit erythrocyte membranes were studied by measuring 32P incorporation into di- and triphosphoinositide from Mg-[gamma-32P]ATP. The Km's for 32P incorporation into di- and triphosphoinositide were 110 and 48 microM ATP, respectively. The optimal temperature for 32P incorporation into diphosphoinositide was at 32 degrees C, whereas the optimum for triphosphoinositide labeling occurred at 43 degrees C. Differences in the effects of pH on the rate of 32P incorporation into di- and triphosphoinositide were also found. At 37 degrees C but not at 25 degrees C 32P-labeled diphosphoinositide was phosphorylated to triphosphoinositide in the presence of Mg-ATP. Triton X-100 partially inhibited 32P incorporation into diphosphoinositide but completely inhibited the synthesis of triphosphoinositide. At physiological concentrations, 0.4 mM MgCl2 half-maximally activated di- and triphosphoinositide synthesis. Higher concentrations of MgCl2 (5 to 50 mM) decreased 32P incorporation into diphosphoinositide and greatly enhanced 32P incorporation into triphosphoinositide. NaCl or KCl (less than or equal to 100 mM) did not have any effects on polyphosphoinositide synthesis, whereas 150 to 300 mM NaCl or KCl decreased synthesis of diphosphoinositide and increased synthesis of triphosphoinositide. Further studies showed that 50 mM MgCl2 and 200 mM NaCl or KCl stimulate kinase-mediated phosphorylation of diphosphoinositide to triphosphoinositide. Triton X-100 inhibited the ability of 50 mM MgCl2 and neomycin to stimulate phosphorylation of diphosphoinositide to triphosphoinositide. The pathways for synthesis of di- and triphosphoinositides in erythrocyte membranes are discussed.     

Polyphosphoinositide synthesis in rabbit erythrocyte membranes

Incubation of rabbit erythrocyte ghosts at 25 °C with 1 mm [γ-³²P]ATP and MgCl₂ results in incorporation of ³²P into diphosphoinositide and triphosphoinositide with initial rates of 15.6 and 1.8 nmol ³²P/mg/h, respectively. Incorporation of ³²P into diphosphoinositide plateaus after 20 min whereas incorporation into triphosphoinositide did not plateau until after 80 min. Diphosphoinositide and triphosphoinositide, prelabeled with ³²P, did not undergo significant breakdown when incubated at 25 °C for 15 to 20 min. Turnover of ³²P-labeled diphosphoinositide and triphosphoinositide was insignificant in the presence of MgCl₂ and cold ATP. Diphosphoinositide is not phosphorylated to triphosphoinositide in the presence of Mg-ATP under conditions in which synthesis of these polyphosphoinositides can occur. In the presence of neomycin and Mg-ATP, labeled diphosphoinositide was rapidly phosphorylated to triphosphoinositide. Neomycin had no effect on labeled di- and triphosphoinositide content in the absence of ATP. Freeze-thawing the ghosts or the addition of Triton X-100 does not produce the same effect as neomycin. The results of this investigation suggest that diphosphoinositide and triphosphoinositide are normally synthesized from endogenous phosphatidylinositol in rabbit ghosts by separate enzymatic pathways. Neomycin an aminoglycoside which interacts with polyphosphoinositides may perturb the organization of substrates and kinase activities involved in polyphosphoinositide metabolism and alter these pathways.     

Localization of enzymes involved in polyphosphoinositids metabolism on the cytoplasmic surface of the human erythrocyte membrane.

1. Impermeable inside-out and right-side-out vesicles were prepared from membranes of human erythrocytes. During preparation of each kind of impermeable vesicle, permeable vesicles were also obtained. 2. Incubation of vesicles with [gamma-32P]ATP at 37 degrees C for periods of up to 1 hr did not change the topography or the permeability of the vesicles. 3. Vesicles incorporated labeled phosphate from [gamma-32P]ATP into both diphosphoinositide and triphosphoinositide, but impermeable inside-out vesicles incorporated significantly more nuclide than did right-side-out vesicles. 4. Permeable vesicles derived during the preparation of inside-out vesicles were as active as impermeable inside-out vesicles in the incorporation of labeled phosphate into the polyphosphoinositides. However, permeable vesicles derived during the preparation of right-side out vesicles were not as active. 5. Impermeable right-side-out vesicles, treated with 0.01 percent saponin, incorporated labeled phosphate into the polyphosphoinositides at a level comparable to that of impermeable inside-out vesicles. 6. These data show that the enzymes involved in metabolism of diphosphoinositide and triphosphoinositide are located on the cytoplasmic surface of the erythrocyte membrane.     

The hydrolysis of triphosphoinositide by a phosphodiesterase in rat kidney cortex

The hydrolysis of triphosphoinositide by a phosphodiesterase has been demonstrated in rat kidney cortex. Subcellular fractionation studies revealed that the enzyme activity was predominantly found in the supernatant fraction. After acid precipitation and ammonium sulfate fractionation, the soluble enzyme was free from triphosphoinositide phosphomonoesterase activity.Although the partially purified enzyme did not require added divalent cations for activity, it was strongly inhibited by EDTA (0.1 mm). In the absence of EDTA, added MgCl₂ or CaCl₂ depressed the enzyme activity. The enzyme preparation was specific to polyphosphoinositides; it did not attack phosphatidylinositol and other phospholipids. It hydrolyzed both diphosphoinositide and triphosphoinositide with the formation of 1,2-diglyceride and organic phosphate.     

PHOSPHOINOSITIDE KINASES IN CHICK BRAIN AND SCIATIC NERVE, A DEVELOPMENTAL STUDY

Phosphatidylinositol kinase and diphosphoinositide kinase activities were measured in homogenates of brain and sciatic nerve of developing chick embryos and chicks. Characteristics of the chick nervous system enzymes were similar to those reported for rat brain. Diphosphoinositide kinase was inhibited by high concentrations of ATP and by low concentrations of triphosphoinositide. Both activities were greatly enhanced by the non-ionic detergent, Cutscum, and the ratio of detergent to protein in the reaction mixture was important. Optimum phosphatidylinositol kinase activity required a ratio of 7 : 1 for both tissues. The optimum ratio for diphosphoinositide kinase was 3:1 for nerve homogenates and 0.6:1 for brain. Cutscum increased the concentration of diphosphoinositide that is required for maximum diphosphoinositide kinase activity. Developmental changes were the same for both kinase activities, which were low in unmyelinated brain and sciatic nerve. The activities correlated with the concentration of polyphosphoinositides in chick brain where they increased 4-5 fold during the period of active myelination and remained high in the mature brain. The kinase activities correlated with the rate of triphosphoinositide deposition in sciatic nerve. Following a 2-3 fold increase during the initial phase of myelination the activities declined to values as low as those of embryonic nerve.     

THE BIOSYNTHESIS OF TRIPHOSPHOINOSITIDE BY PURIFIED MYELIN OF PERIPHERAL NERVE

The distribution of diphosphoinositide kinase activity in homogenates and myelin of rabbit nerve was determined by measuring the synthesis of labelled triphosphoinositide. Evidence is presented to show that the biosynthesis of triphosphoinositide in peripheral myelin involves a membrane-bound diphosphoinositide kinase.     

Synthesis of diphosphoinositide by a supernatant fraction from Acanthamoeba castellanii.

Homogenates of the free-living amoeba Acanthamoeba castellanii incorporate phosphate from [gamma-32P]ATP into a lipid which co-chromatographs with diphosphoinositide on one- and two dimensional chromatography. Incorporation into lipids similar in mobility to triphosphoinositide is not detected. The product co-chromatographs with diphosphoinositide whether exogenous phosphatidylinositol or total amoeba lipid is the substrate. The inositide kinase is almost entirely located in the supernatant fraction after centrifugation at 100 000 g. Incorporation of phosphate from [gamma-32P]ATP is linear for at least 15 min in the presence of 0.5 mM phosphatidylinositol. The enzyme requires Mg2+ of Mn2+ as well as ATP and it is not affected by low concentrations of Ca2+. The apparent Km for phosphatidylinositol in 2 mM. Both ADP and cAMP inhibit the reaction.     

The deposition and metabolism of polyphosphoinositides in rat and guinea-pig brain during development

1. The deposition of triphosphoinositide and diphosphoinositide in rat and guinea-pig cerebral hemispheres during growth was measured. 2. The maximum increase in concentration of both of these phospholipids occurs during the period of myelination, but in the rat some di- and tri-phosphoinositide is present before significant myelination begins. 3. In guinea-pig cerebral hemispheres the polyphosphoinositides remaining after post-mortem breakdown are selectively enriched in dissected white matter compared with grey matter. 4. The polyphosphoinositides in the cerebral hemispheres of rats were labelled with injected (32)P very rapidly; the specific radioactivities were in the order triphosphoinositide>diphosphoinositide>monophosphoinositide>total lipid phosphorus. 5. The synthesis of triphosphoinositide in rat forebrain occurs at an appreciable rate before, and at the start of, myelination, but the amount formed per gram of tissue is four to five times greater in adult rat brains, thus maintaining a constant turnover time (about 1hr.) for the whole triphosphoinositide fraction. This indicates that the rapid turnover of triphosphoinositide is independent of myelin deposition. 6. The specific radioactivity of the brain acid-soluble phosphorus pool referred to a constant dose of (32)P/g. body wt. falls rapidly with age, reaching a minimum at 13-14 days, and then rises again. The specific radioactivities of the polyphosphoinositides reflect this change. 7. Part of the polyphosphoinositides in rat and guinea-pig cerebral hemispheres is rapidly hydrolysed post mortem leaving a stable portion resistant to further breakdown. 8. The rate and extent of post-mortem hydrolysis of the polyphosphoinositides in both species decrease with age. 9. After (32)P labelling, the specific radioactivity of the triphosphoinositide remaining in the cerebral hemispheres of the rat after post-mortem breakdown is lower than the original triphosphoinositide fraction, suggesting two metabolically distinct pools.     

Phosphoramidate and phosphate prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine: synthesis, anti-HIV activity and stability studies

A series of phosphoramidate and phosphate prodrugs of DOT were synthesized via dichlorophosphate or H-phosphonate chemistry and evaluated for their anti-HIV activity against LAI M184V mutants in PBM cells as well as for their cytotoxicity. The antiviral and cytotoxic profiles of the prodrugs were compared with that of the parent compound (DOT), and it was found that four aryl phosphoramidates 5, 18, 20, and 26 showed a significant enhancement (8- to 12-fold) in anti-HIV activity without cytotoxicity. Chemical stability of these prodrugs was evaluated in phosphate buffer at pH values of biological relevance (i.e., pH 2.0 and 7.4). Enzymatic hydrolysis was also studied in esterase or lipase in buffer solution. Chemical stability studies indicate that the phosphoramidates have good chemical stability at pH 2.0 and at pH 7.4 phosphate buffer. Phosphoramidate prodrugs were hydrolyzed in vitro by esterase or lipase and found to be better substrates for lipases than for esterases. 1,3-Diol cyclic phosphates showed potent anti-HIV activity without increasing the cytotoxicity compared with that of DOT and have good chemical and enzymatic stability. Long-chain lipid phosphates, although showed potent anti-HIV activity, exhibited increased cytotoxicity.     

Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system.

Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble.     

ENZYMES OF PHOSPHOINOSITIDE METABOLISM DURING RAT BRAIN DEVELOPMENT

—The activities of four enzymes concerned with inositol lipid metabolism have been determined in homogenates of rat brains of different ages. The enzymes are CDP-diglyceride inositol phosphatidate transferase, phosphatidylinositol kinase, diphosphoinositide kinase and triphosphoinositide phosphomonoesterase. The activities of all the enzymes increased with age. Phosphatidylinositol kinase activity rose most sharply well before myelination, reaching a maximum at about 6 days of age. Diphosphoinositide kinase and triphosphoinositide phosphomonoesterase activities increased most rapidly during myelination. The increase in CDP-diglyceride inositol phosphatidate transferase showed no definite association with any period of development. It is concluded that triphosphoinositide metabolism is associated with myelin or a closely related structure.     

Calcium-activated hydrolysis of phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-bisphosphate in guinea-pig synaptosomes

1. Addition of the bivalent ionophore A23187 to synaptosomes isolated from guinea-pig brain cortex and labelled with [(32)P]phosphate in vitro or in vivo caused a marked loss of radioactivity from phosphatidyl-myo-inositol 4-phosphate (diphosphoinositide) and phosphatidyl-myo-inositol 4,5-bisphosphate (triphosphoinositide) and stimulated labelling of phosphatidate. No change occurred in the labelling of other phospholipids. 2. In conditions that minimized changes in internal Mg(2+) concentrations, the effect of ionophore A23187 on labelling of synaptosomal di- and tri-phosphoinositide was dependent on Ca(2+) and was apparent at Ca(2+) concentrations in the medium as low as 10(-5)m. 3. An increase in internal Mg(2+) concentration stimulated incorporation of [(32)P]phosphate into di- and tri-phosphoinositide, whereas lowering internal Mg(2+) decreased labelling. 4. Increased labelling of phosphatidate was independent of medium Mg(2+) concentration and apparently only partly dependent on medium Ca(2+) concentration. 5. The loss of label from di- and tri-phosphoinositide caused by ionophore A23187 was accompanied by losses in the amounts of both lipids. 6. Addition of excess of EGTA to synaptosomes treated with ionophore A23187 in the presence of Ca(2+) caused a rapid resynthesis of di- and tri-phosphoinositide and a further stimulation of phosphatidate labelling. 7. Addition of ionophore A23187 to synaptosomes labelled in vivo with [(3)H]inositol caused a significant loss of label from di- and tri-phosphoinositide, but not from phosphatidylinositol. There was a considerable rise in labelling of inositol diphosphate, a small increase in that of inositol phosphate, but no significant production of inositol triphosphate. 8. (32)P-labelled di- and tri-phosphoinositides appeared to be located in the synaptosomal plasma membrane. 9. The results indicate that increased Ca(2+) influx into synaptosomes markedly activates triphosphoinositide phosphatase and diphosphoinositide phosphodiesterase, but has little or no effect on phosphatidylinositol phosphodiesterase.     

Coisolation of glycophorin A and polyphosphoinositides from human erythrocyte membranes.

The lipid composition of purified erythrocyte membrane glycophorin was measured. Diphosphoinositide, triphosphoinositide, and phosphatidylserine are the major phospholipids in glycophorin preparation. Nearly all of the radioactive diphosphoinositide and triphosphoinositide extracted from erythrocyte membranes by lithium d?odosalicylate are recoverd in purified glycophorin. There appeared to be no significant enrichment of other acidic membrane phospholipids in the protein. The results do not permit a firm conclusion as to whether the polyphosphoinositides are associated specifically with the membrane protein or whether fortuitous binding has occurred during purification.     

The participation of phospholipids in the interaction of leucocidin and the cell membrane of the polymorphonuclear leucocyte

1. The interaction of the two components of leucocidin with various lipids has been studied by sedimentation, flotation, light-scattering and changes in the biological activity of leucocidin. 2. Phosphatidylserine, phosphatidylcholine, diphosphoinositide, triphosphoinositide and phosphatidic acid, but not phosphatidylethanolamine, lysophosphatidylcholine, cerebrosides, gangliosides or tristearin, induce aggregation of the F component of leucocidin. 3. The S component of leucocidin does not interact directly with these phospholipids, but interacts with the F component of leucocidin after its modification by lipids. 4. The increased sedimentation or light-scattering induced by low phospholipid concentrations is reversed at higher phospholipid concentrations. 6. The aggregates formed by phospholipids and leucocidin are due, not to adsorption of leucocidin alone, but also to the formation of leucocidin polymers. 7. It is concluded that the aggregation is due to the interaction of the F component with the fatty acid side chains in the lipid micelle. 8. The S component of leucocidin is inactivated by triphosphoinositide at physiological ionic strength; the F component of leucocidin is inactivated at low ionic strength by triphosphoinositide and remains inactive when the ionic strength is increased. 9. It is suggested that in the leucocyte cell membrane the S component of leucocidin interacts with the polar hydrophilic groups of triphosphoinositide and that the F component of leucocidin interacts with the hydrophobic parts of triphosphoinositide.     

Effect of fusaric acid on phosphoinositide metabolism in the erythrocyte membranes of rats with spontaneous hypertension

2- and 4-month-old male spontaneously hypertensive rats (SHR) were injected fusaric acid at a dose of 50 mg/kg body weight. Fusaric acid increased diphosphoinositide (DPI) and triphosphoinositide (TPI) levels in erythrocyte membranes of 4-month-old SHR by 41% and 20%, respectively. 32P incorporation into TPI decreased by 24% in 2- and by 20% in 4-month-old SHR. Phosphatidylinositol metabolism remained unchanged. The results also suggest that fusaric acid normalized DPI and TPI metabolism in erythrocyte membranes of SHR.     

The diphosphoinositide kinase of rat brain

1. The supernatant fraction of adult rat brain contains a diphosphoinositide kinase. 2. Formation of triphosphoinositide by the enzyme in the presence of ATP and Mg(2+) ions was shown with labelled ATP or labelled diphosphoinositide. 3. The kinase was also activated by Ca(2+), Mn(2+) and Co(2+) ions, but to a smaller extent than by Mg(2+) ions. 4. In the presence of optimum Mg(2+) ion concentration the enzyme was inhibited by Ca(2+) ions. 5. Activity did not depend on thiol groups and the pH optimum was 7.3. 6. The dialysed supernatant fraction had no diglyceride kinase activity and negligible phosphatidylinositol kinase activity. 7. Triphosphoinositide phosphomonoesterase was present but showed little activity under the conditions used to assay the kinase. 8. Diphosphoinositide kinase was purified by ammonium sulphate fractionation, ethanol treatment and chromatography on Sephadex G-200. 9. This purification removed much of the triphosphoinositide phosphomonoesterase.     

High affinity of acid phosphatase encoded by PHO3 gene in Saccharomyces cerevisiae for thiamin phosphates

The enzymatic properties of acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) encoded by PHO3 gene in Saccharomyces cerevisiae, which is repressed by thiamin and has thiamin-binding activity at pH 5.0, were investigated to study physiological functions. The following results led to the conclusion that thiamin-repressible acid phosphatase physiologically catalyzes the hydrolysis of thiamin phosphates in the periplasmic space of S. cerevisiae, thus participating in utilization of the thiamin moiety of the phosphates by yeast cells: (a) thiamin-repressible acid phosphatase showed Km values of 1.6 and 1.7 microM at pH 5.0 for thiamin monophosphate and thiamin pyrophosphate, respectively. These Km values were 2-3 orders of magnitude lower than those (0.61 and 1.7 mM) for p-nitrophenyl phosphate; (b) thiamin exerted remarkable competitive inhibition in the hydrolysis of thiamin monophosphate (Ki 2.2 microM at pH 5.0), whereas the activity for p-nitrophenyl phosphate was slightly affected by thiamin; (c) the inhibitory effect of inorganic phosphate, which does not repress the thiamin-repressible enzyme, on the hydrolysis of thiamin monophosphate was much smaller than that of p-nitrophenyl phosphate. Moreover, the modification of thiamin-repressible acid phosphatase of S. cerevisiae with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in the complete loss of thiamin-binding activity and the Km value of the modified enzyme for thiamin monophosphate increased nearly to the value of the native enzyme for p-nitrophenyl phosphate. These results also indicate that the high affinity of the thiamin-repressible acid phosphatase for thiamin phosphates is due to the thiamin-binding properties of this enzyme.     

Synthesis of a water-soluble prodrug of entacapone

Entacapone was reacted with phosphorous oxychloride in dry pyridine to yield a phosphate ester. The phosphate promoiety increased aqueous solubility of the parent drug by more than 1700- and 20-fold at pH 1.2 and 7.4, respectively. The phosphate ester provides adequate stability (t(1/2) = 2227 h; pH 7.4) towards chemical hydrolysis, and allowed for release of the parent drug via enzymatic hydrolysis in liver homogenate.     

Interactions of human matrix metalloproteinase 7 (matrilysin) with the inhibitors thiorphan and R-94138

The effects of the metalloproteinase inhibitors thiorphan and R-94138 on the matrilysin-catalyzed hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diamino-propionyl]-L-Ala-L-Arg-NH(2) [MOCAc-PLGL(Dpa)AR] were examined. The inhibitor constants (K(i)) of thiorphan and R-94138 for matrilysin at pH 7.5, 25 degrees C were determined to be 11.2 and 7.65 microM, respectively. From the temperature dependence of the K(i) values at pH 7.5, the standard enthalpy change (Delta H degrees ') values for the binding of matrilysin with thiorphan and R-94138 were determined to be -(18.2 +/- 0.9) and (1.65 +/- 1.07) kJ x mol(-1), respectively. The binding of matrilysin to thiorphan is exothermic and the free energy change in the complex formation depends mainly on the change in enthalpy, while the binding to R-94138 is endothermic and typically entropy-driven. Hydrophobic interactions are suggested to contribute significantly to the binding of matrilysin to R-94138 as well as to the substrate. The pH dependence of the K(i) value suggests that at least two ionizing groups with pK(a) values of 4.5 and 9.1--9.3 are involved in the binding. The matrilysin activity is regulated by ionizing groups with pK(a) values of 4.3 and 9.6. Both inhibition and hydrolysis are suggested to be controlled by the same residues in matrilysin, most likely Glu 198 and Tyr 219, respectively.