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Phosphatidylinositol synthase from mammalian tissues1
Biochimica et Biophysica Acta 1348 Ž1997. 179–186
Chapter XIVB
Phosphatidylinositol synthase from mammalian tissues Bruno Antonsson
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GeneÕa Biomedical Research Institute, Glaxo Wellcome R & D S.A., 14 ch. des Aulx, 1228 Plan-les-Ouates, GeneÕa, Switzerland Received 12 March 1997; revised 14 April 1997; accepted 15 April 1997
Abstract Phosphatidylinositol synthase ŽCDP-diacylglycerol:myo-inositol 3-phosphatidyl-transferase, EC 2.7.8.11. is a 24-kDa membrane-bound enzyme. It is present in all mammalian cells and is localized predominantly to the endoplasmic reticulum. The enzyme performs the last step in the de novo biosynthesis of the phospholipid phosphatidylinositol by catalyzing the condensation of CDP-diacylglycerol and myo-inositol to form the products phosphatidylinositol and CMP. Phosphatidylinositol, apart from being an essential membrane phospholipid, is involved in protein membrane anchoring and is the precursor for the second messengers inositol-tri-phosphate and diacylglycerol. q 1997 Elsevier Science B.V. Keywords: Phosphatidylinositol synthase; Phosphatidylinositol; Inositol; Phospholipid
1. Introduction P hosphatidylinositol synthase Ž C D P -di acylglycerol:myo-inositol 3-phosphatidyl-transferase, EC 2.7.8.11. performs the last step in the de novo biosynthesis of phosphatidylinositol by catalyzing the condensation of CDP-diacylglycerol and myo-inositol to give the products, phosphatidylinositol and CMP w1x. In addition to de novo biosynthesis, phosphatidylinositol can be synthesized by an exchange reaction where free myo-inositol is incorporated into phosphatidylinositol in the presence of manganese. This reaction is catalyzed by a different enzyme and does not require CDP-diacylglycerol w2,3x. Phosphatidylinositol has been detected in membranes from all mammalian tissues that have been examined. It is a minor but essential membrane component which
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Fax: q41 22 7946965; E-mail: [email protected] Dedicated to Professor Eugene Kennedy.
constitutes between 2–12% of the total phospholipids in eucaryotic cells Žfor a review, see Ref. w4x.. Besides being an essential component of the cell membrane, phosphatidylinositol is involved in protein membrane anchoring and it is the precursor for the second messengers inositol-polyphosphate and diacylglycerol. Phosphatidylinositol is unique among phospholipids in that its head group, the inositol, can be further phosphorylated to phosphatidylinositol mono- or diphosphates. It has been proposed that two pools of phosphatidylinositol exist in the cell membranes w5,6x. One of which is sensitive to hormone-induced hydrolysis, and the other which is hydrolysis insensitive. Phosphatidylinositol-polyphosphates are essential parts of the Ca2qrPKC signal transduction pathways Žfor a review, see Ref. w7x.. A wide range of extracellular stimulations, including hormones and neurotransmitters, trigger the phospholipase C-mediated hydrolysis of the hormone-sensitive pool of phosphatidylinositol-di-phosphate. This gives rise to the two secondary messengers, inositol-1,4,5-triphos-
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phate and diacylglycerol. Inositol-tri-phosphate is liberated into the intracellular space and triggers Ca2q release from intracellular stores, which leads to the activation of the calcium-inducible signal transduction pathways. Diacylglycerol, which remains in the membrane, activates the protein kinase C ŽPKC.-signaling pathways. After receptor-triggered hydrolysis of phosphatidylinositol-polyphosphates the phospholipid must be resynthesised in order to maintain a constant level of phosphatidylinositol in the membranes, this requires phosphatidylinositol synthase activity.
2. Discovery, occurrence and localization Inositol-containing phospholipids were identified in mammalian tissues in the early 1940s. The enzyme activity responsible for the biosynthesis of phosphatidylinositol was first described by Agranoff et al. w8x and Paulus and Kennedy w1x about 10 years later. Paulus and Kennedy used the microsomal fraction of liver from guinea pig and chicken in their studies. Already these studies indicated that the enzyme activity was located in the membrane fraction. The first more detailed study of the occurrence and localization of the enzyme activity was carried out by Benjamins and Agranoff in various tissues from guinea pig w9x. They detected phosphatidylinositol synthase activity in all tissues tested, including brain, liver, kidney, heart, lung and spleen. Although, the specific activity of the enzyme varied between the various tissues. They also studied the subcellular localization in brain extract. Enzyme activity was predominantly co-localizing with the microsomal fraction, with less than 5% of the total activity in the soluble cell fraction. The localization of the enzyme activity to the microsomal fraction was confirmed in studies of rat brain extracts by Bishop and Strickland w10x and Rao and Strickland w11x. Studies of membrane fractions from rat liver by Williamson and Morre w12x showed that the enzyme was localized to both the rough and the smooth endoplasmic reticulum. They also found the enzyme to be present in the Golgi apparatus membrane. That the major part of phosphatidylinositol synthase is localized to the endoplasmic reticulum is not disputed. More recent studies, however, have indicated that the enzyme is also
present in other cellular membranes. The results, depending on the cells and tissues examined, are not unambiguous. Imai and Gershengorn w13x studied the distribution in rat pituitary tumor GH 3 cells. They showed that both the plasma membrane and the endoplasmic reticulum contained phosphatidylinositol synthase activity. When the two enzyme activities were characterized they displayed different kinetic characteristics. It was also shown that the enzyme residing in the plasma membrane could more easily be extracted from the membrane by high salt concentration compared to the endoplasmic reticulum enzyme w14x. These facts might indicate the existence of two different phosphatidylinositol synthases. Although, it is far from proven, and the differences observed could be due to differences in the lipid microenvironment of the enzyme in the two membranes. Phosphatidylinositol synthase activity was also detected in the plasma membrane from rabbit proximal tubule cells by Galvao and Shayman w15x. However, they could not detect any significant differences in the kinetic parameters between the endoplasmic reticulum and the plasma membrane enzyme. Conversely, in a study of the subcellular distribution in cultured glioma and neuroblastoma cells, Morris et al. w16x found that the enzyme was exclusively localized to the endoplasmic reticulum. They did not detect any activity in the plasma membrane. Baker and Chang w17x assayed phosphatidylinositol synthase activity in the nuclear and endoplasmic reticular fractions of immature rabbit cerebral cortex. They found that the nuclear membrane fraction contained the highest specific activity. As in all other studies, they also detected activity in the endoplasmic reticulum, with most activity found in the rough endoplasmic reticulum. In a study of the subcellular distribution in 1321 N1 astrocytoma cells Sillence and Downes w18x found activity in two distinct membrane fractions isolated by sucrose gradient centrifugation. One fraction co-migrated with the endoplasmic reticulum marker NADPH–cytochrome c reductase. The second peak however, did not co-migrate with the plasma membrane though they could not exclude the possibility of a small amount of activity being associated with the plasma membrane. Vaziri et al. w19x, in a study using a cell-free system from turkey erythrocytes, showed that the hormone-sensitive pool of phosphatidylinositol in the plasma membrane could
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be re-synthesized in the plasma membrane fraction itself. This suggests the presence of phosphatidylinositol synthase activity in the plasma membrane of the turkey erythrocytes. Taken together these results do not give a clear picture of the localization of the enzyme. The somewhat conflicting observations could reflect differences in different cell types and tissues. It cannot be excluded that the concentration of phosphatidylinositol synthase in different cellular membranes varies between cell types, which may for example enable the detection of activity in the plasma membrane of one cell type but prevent its detection in another. Differences in assay conditions is another possible reason for inconsistencies in the results. It has been suggested that the enzyme in different membranes requires more or less detergent for its detection w20x. What these results clearly show is that isolation of pure membrane fractions from subcellular structures is still not trivial and may be a contributing factor to the ambiguous results. The existence of two phosphatidylinositol synthase isozymes is an attractive idea, one responsible for synthesis of the hormone-sensitive pool in the plasma membrane, and the other responsible for the synthesis of the insensitive pool, primarily in the endoplasmic reticulum. However, conclusive evidence for the existence of different isozymes still has to be found.
3. Substrate specificity and regulation In the presence of Mg 2q phosphatidylinositol synthase catalyzes the reaction w1x: CDP-diacylglycerolq myo-inositol ™ phosphatidylinositolq CMP Of the nine possible inositol isomers, only myo-inositol has been found in phospholipids isolated from cell membranes w21x. This suggests a high specificity for the inositol substrate. Some early in vitro studies performed on crude membrane fractions suggested that other inositol isomers could also be incorporated into the phospholipid w1,9x. However, this has not been confirmed in studies with purer enzyme preparations and such products have not been detected in cell membranes. The other substrate in the reaction, CDP-diacylglycerol, is composed of two subunits, the nucleotide and the lipid. Little is known about the
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specificity for the CDP part of the molecule. One study of phosphatidylserine synthase and phosphatidylglycerophosphate synthase from E. coli where various liponucleotides were tested as substrates might be of some relevance w22x. CDP-diacylglycerol and deoxy-CDP-diacylglycerol served as substrates for the enzymes. In contrast, UDP-, ADPand GDP-diacylglycerol were all inactive as substrates. Phosphatidylinositol synthase from human placenta used deoxy-CDP-diacylglycerol at least as efficiently as CDP-diacylglycerol as in vitro substrate w23x. CDP-diacylglycerol showed substrate inhibition, whereas this was not seen with the deoxy-derivative and the maximal activity was approximately 25% higher compared to CDP-diacylglycerol. Several studies have been performed to evaluate the influence of the lipid chain length and the degree of saturation on the efficiency of the CDP-diacylglycerol substrate w9,10,24x. Although, all the studies detected relative differences between the various lipid chains, the results are not conclusive as to which lipid chains make the better substrate. In bovine brain CDP-diacylglycerol has been shown to be present predominantly as 1-steroyl,2-archidonyl which is also the main composition of phosphatidylinositol w25x. Besides the two substrates, the enzyme has an absolute requirement for Mg 2q or Mn2q w8x. The regulation of phosphatidylinositol synthase expression and of its activity remains largely unclear. myo-Inositol depletion, induced by the inhibition of inositol monophosphatase by lithium, decreases the concentration of phosphatidylinositol in the cell membranes. Atack et al. showed that this decrease could be prevented by pre-incubation with exogenous myo-inositol w26x. In contrast the isomer scyllo-inositol had no effect. Under normal physiological conditions, however the extracellular inositol concentration does not change enough to have a regulatory effect on the synthesis of phosphatidylinositol. Models simulating diabetes have shown that, under pathological conditions, where the myo-inositol concentration is decreased, inositol becomes the limiting factor in the biosynthesis of phosphatidylinositol w27x. Phosphatidylinositol synthase activity has been shown to be upregulated after hormone-induced phospholipase C-mediated hydrolysis of phosphatidylinositol-polyphosphates, however the mechanism remains unknown w28x. It has been suggested that phosphatidyli-
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nositol synthase might be inhibited by its product, phosphatidylinositol w29x. A main unresolved issue regarding the regulation of phosphatidylinositol resynthesis after receptor triggered hydrolysis is whether the re-synthesis occurs in the plasma membrane or in the endoplasmic reticulum. Phosphatidylinositol synthase activity has been shown in the plasma membrane fractions of GH 3 cells, proximal tubule cells and turkey erythrocytes w13,15,19x. This would indicate that the re-synthesis could take place in the plasma membrane. However, other studies, using other cells and tissues, have failed to detect the enzyme in the plasma membrane. The other possibility would be that the re-synthesis takes place in the endoplasmic reticulum and phosphatidylinositol is transported to the plasma membrane by the wellcharacterized phosphatidylinositol transfer proteins w30x. These are soluble intracellular proteins that transport phosphatidylinositol between the various membrane compartments within cells. Thomas et al. showed that a soluble 35-kDa phosphatidylinositol transfer protein enhanced the G-protein-stimulated phospholipase C-b activity w31x. This was suggested to be achieved by transfer of phosphatidylinositol from intracellular membrane compartments to the plasma membrane, where phosphatidylinositol was converted to phosphatidylinositol-di-phosphate and hydrolysis by the phospholipase. Based on the available results, it is not possible to discriminate which of the two mechanisms are responsible for the resynthesis of phosphatidylinositol after receptor-triggered hydrolysis.
4. Purification The early studies of phosphatidylinositol synthase were performed on crude membrane fractions. Attempts to extract the enzyme from the membrane fraction using various detergents including Triton X-100, Lubrol, NP-40 or bile salts resulted in loss of the enzyme activity. Rao and Strickland w11x were the first to be successful in extracting active enzyme from rat brain microsomes using 0.3% Triton X-100. This preparation, however, was not free from membrane lipids and any attempt at further purification resulted in loss of the activity. The first partial purification of the enzyme was done by Parries and
Hokin-Neaverson from canine pancreas w32x. They used n-octyl glucopyranoside to solubilize the enzyme from the microsomal fraction. The ratio of detergent to protein was found to be important, and optimal solubilization was achieved at a ratio of 4–6:1 at a detergent concentration of 40 mM. Under these conditions 95–100% of the enzyme activity could be solubilized. The solubilized enzyme was stabilized by manganese. The enzyme was then purified by ion-exchange chromatography on DEAE-Cellulose. To preserve enzyme activity phosphatidylinositol and egg yolk phospholipids were required in the buffers. The overall purification was 10-fold over the microsomal fraction and the protein had a specific activity of 290 nmolrminrmg. More successful purifications have been achieved using affinity chromatography on CDP-diacylglycerol–Sepharose. This affinity resin had been used in the purification of several bacterial and yeast enzymes including phosphatidylinositol synthase from Saccharomyces cereÕisiae. The major disadvantage with this purification method is that mammalian phosphatidylinositol synthase has proven to bind very strongly to the affinity resin. Elution has thus required special buffer conditions and prolonged incubation times resulting in low recovery of enzyme activity. Despite these drawbacks this method has provided a means of obtaining purified mammalian enzyme. The first attempt to purify mammalian phosphatidylinositol synthase using this approach was made by Ghalayini and Eichberg w33x. The enzyme was solubilized with 0.6% Triton X-100 from rat brain microsomes. The soluble fraction was subsequently purified on a CDP-diacylglycerol–Sepharose column and the bound enzyme was eluted with CDP-diacylglycerol. Triton X-100 and phosphatidylcholine were also required in the elution buffer. The specific activity was increased 190-fold over the crude brain homogenate and the recovery of enzyme activity was 7%. The enzyme preparation however, was not homogeneous when analyzed by SDS–PAGE. Only in recent years have close to homogeneous preparations been obtained. Human phosphatidylinositol synthase was purified from placenta by Antonsson w23x. The enzyme was extracted to approximately 50% from the microsomal fraction with Triton X-100. The solubilized enzyme was subjected to affinity chromatography on CDP-diacylglycerol–Sepharose.
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As already seen by Ghalayini and Eichberg the enzyme bound strongly to the affinity resin w33x. Active enzyme could be eluted from the column only in the presence of high salt Ž1.3 M NaCl. , 10% glycerol, 0.5% Triton X-100, phosphatidylcholine and diacylglycerol in addition to the affinity ligand CDP-diacylglycerol. The pool from the affinity column was far from pure and the eluted sample was applied to ion-exchange chromatography on Mono Q FPLC. By carefully adjusting pH and salt concentrations in the sample and the equilibration buffer a fraction of phosphatidylinositol synthase migrated with the flow-through fractions, whereas the remaining enzyme together with the impurities bound to the column and were eluted with a salt gradient. The gradient-eluted enzyme was resubmitted to this purification step and each time a fraction of phosphatidylinositol synthase emerged in the flow-through fractions. These results suggested that the enzyme was present in an equilibrium of free and complexed protein, presumably interacting with hydrophobic contaminating proteins. When analyzed by SDS– PAGE the pool was almost homogeneous with one protein Žover 90%. with an apparent molecular mass of 24 000 Da. The enzyme was purified 8300-fold over the microsomal fraction and the specific activity of the purified enzyme was 34 000 nmolrminrmg. A few months later Monaco et al. w34x reported the purification of the enzyme from rat liver. The enzyme was solubilized with the non-ionic detergent Hecameg. The soluble fraction was heated at 658C until a precipitate was formed. This step increased the specific activity 4-fold and the supernatant which contained phosphatidylinositol synthase was further purified on a CDP-diacylglycerol column. The enzyme was eluted with CDP-diacylglycerol in the presence of 20% glycerol, 1 mM MnCl 2 , and 1.6% Hecameg, and further purified on a PBE-94 column. The purified enzyme had a specific activity of 2800 nmolrminrmg and had been purified 5100-fold over the crude cell supernatant. When analyzed by SDS– PAGE the sample contained two main proteins, one at 51 000 and one at 21 000 Da. By correlating the band intensities with phosphatidylinositol synthase activity in the fractions eluted from the PBE-94 column, the 21 000-Da protein was shown to correlate with phosphatidylinositol synthase. These studies showed that mammalian phosphatidylinositol syn-
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thase has an apparent molecular mass of between 20 000 and 25 000 Da.
5. Cloning Unfortunately no amino acid sequence information has yet been obtained from the purified mammalian enzyme. Due to the instability of the enzyme once extracted from the membrane fraction only analytical amounts of the protein have been obtained in pure form. Attempts to use the known sequence for the Saccharomyces cereÕisiae enzyme to clone the mammalian enzyme have not been successful. However, recently Tanaka et al. have cloned the rat phosphatidylinositol synthase cDNA by functional complementation of a Saccharomyces cereÕisiae phosphatidylinositol synthase mutant, deficient in phosphatidylinositol synthase activity w35x. The cloned cDNA encodes a protein of 213 amino acids with a calculated molecular mass of 23 613 Da.
6. Structure and properties The amino acid sequence deduced from the cloned cDNA shows that the protein is pronounced hydrophobic with 60% hydrophobic amino acids w35x. The hydrophobic amino acids form three main clusters Žresidues 17–35, 77–98 and 165–195. with a hydropathy index greater then 1.5. One or more of these stretches could be involved in the membrane attachment of the protein. Comparison with the yeast phosphatidylinositol synthase amino acid sequence showed the two proteins to have 39% identity and 73% similarity. The three hydrophobic stretches present in the mammalian enzyme are less pronounced in the yeast enzyme. In the yeast enzyme the hydrophobic residues are distributed into smaller stretches. This might be a reason why the mammalian enzyme is more sensitive to removal of the membrane lipids, which might result in the exposure of the large hydrophobic regions to the aqueous solvent. In addition to microsomal preparations w9,15x the purified or partly purified preparations of the enzyme from canine pancreas w32x, rat brain w33x, human placenta w23x and rat liver w34x have been used to determine the kinetic parameters and properties of the
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enzyme. The optimal pH for the phosphatidylinositol synthase reaction was found to be between 8.5 and 9.0. For myo-inositol K m values between 0.3 and 4.5 mM have been reported. The fairly large variation is presumably due to differences in the enzyme preparations as well as variations in assay conditions, such as detergent, buffer compositions and pH. In purified preparations the K m was 0.28 mM for the enzyme from human placenta and 1.3 mM for the rat liver enzyme. The lipid substrate had K m values of 36 and 9.5 mM for the human and rat liver enzyme, respectively. With the human enzyme CDP-diacylglycerol concentrations above 0.5 mM showed substrate inhibition. The rat brain enzyme was also inhibited by CDP-diacylglycerol concentrations above 0.4 mM. The human enzyme was assayed with 2-deoxy-CDPdiacylglycerol as substrate. The deoxy-derivative had a K m of 45 mM, it showed almost no inhibition and the optimal activity obtained was 25% higher then with CDP-diacylglycerol. The enzyme has an absolute requirement for Mg 2q or Mn2q. Manganese was more efficient in activating the enzyme at lower concentrations, 0.1–1 mM w23,33,34x. Higher concentrations of manganese were inhibitory and at a concentration of around 50 mM the enzyme was completely inhibited w23,33x. To obtain optimal activation with magnesium concentrations between 10 and 100 mM were required. The optimal activity obtained with magnesium was between 2- and 4-fold higher then that obtained with manganese w23,36x. Manganese was shown to stabilize the enzyme once extracted from the membrane, in contrast magnesium had no stabilizing effect w23,32,34x. This might suggest that the enzyme has several ion-binding sites affecting stability and activity, respectively. Alternatively the effect seen on the activity may be due to ion binding to the liponucleotide substrate and not to direct interaction with the enzyme molecule. Calcium has been shown to inhibit the enzyme, and phosphatidylinositol synthase from human placenta was also inhibited by zinc w13,14,23x. Whether the inhibition by calcium has any physiological significance is unclear, in particular since the K i is in the mM region. Inhibition studies of the enzyme have been performed with modified substrates. Hexachlorocyclohexanes Ž HCCH. are chlorinated analogs of inositol which have been shown to inhibit the incorporation
of inositol into phosphatidylinositol w37,38x. In a study of phosphatidylinositol synthase from dog pancreas microsomes the d-HCCH isomer, which has the stereochemical configuration of myo-inositol, was most inhibitory w37x. However, the inhibition was noncompetitive and not specific for inositol processing enzymes. The mode of inhibition is likely to be mediated through interactions with hydrophobic domains on the membrane-bound enzymes or with the lipids in their microenvironment. This is consistent with the fact that the inhibitory potency of the isomers correlates with their lipophilicity. Johnson et al. studied 3-substituted myo-inositol derivatives as substrates for phosphatidylinositol synthase and found that 3-NH 2-, 3-F-3-deoxy- and 3-keto all were used as substrate by the enzyme w39x. This indicates that the 3-position of the inositol substrate is probably not involved in the interaction with the enzyme. Another inhibitor that has been tested is an analog of CDP-diacylglycerol where the phosphate moiety was replaced by a methyl group. This liponucleotide cannot be hydrolyzed to act as a phosphatidyl donor, and it was found to be an efficient competitive inhibitor with a K i of 32 nM w40x. In the study of the enzyme from human placenta the effect of nucleotides and phosphate on the enzyme activity was examined w23x. In summary, the tri-phosphates Ž K i 1.4–1.8 mM. were more inhibitory then the di-phosphates Ž K i 3.8–4.1 mM. and the mono-phosphates did not inhibit. The nucleotide bases had no influence on the inhibition. Pyro- and ortho-phosphate were most inhibitory with K i values of 0.2 and 0.8 mM, respectively. Combined, the results suggest that the nucleotide base is not involved in direct interactions with the enzyme molecule. The interactions appear to be mediated through the phosphate and the ribose moieties
7. Unresolved issues Despite the increasing interest in the phosphatidylinositol-polyphosphates and their proven central role in signal transduction many issues about phosphatidylinositol synthase, which is an essential enzyme in the biosynthesis of these important molecules, remain unclear. The enzyme is membrane associated and has proven difficult to handle once
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extracted from its native environment. Although the enzyme has now been purified, purification of larger amounts for biochemical and structural work has not been possible. The recent cloning of the phosphatidylinositol synthase gene will hopefully provide better opportunities to study the enzyme from recombinant sources. In order to increase the solubility and make the protein more suitable for in vitro biochemical studies it might be possible to remove or modify the hydrophobic domains in the recombinant protein. An important question is the regulation of phosphatidylinositol synthase activity. How is the resynthesis after receptor-triggered hydrolysis of the phosphatidylinositol-polyphosphates regulated and controlled? Where does the synthesis take place, in the endoplasmic reticulum or in the plasma membrane? Even such an apparently trivial question as the subcellular localization of the enzyme remains unclear. The fact that the enzyme is located in the endoplasmic reticulum is undisputed, but is it also localized in, for example, the plasma, nuclear and Golgi membranes? The localization is a central question which awaits an answer. Here again, expression and purification of the recombinant protein, or parts of it, should make it possible to obtain antibodies against the protein which could help to answer this and other questions.
Acknowledgements I wish to thank Charles Bradshaw and Kathryn Radford for critical reading of the manuscript.
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tial role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signaling, Cell 74 Ž1993. 919–928. G.S. Parries, M. Hokin-Neaverson, Phosphatidylinositol synthase from canine pancreas: Solubilization by n-octyl glucopyranoside and stabilization by manganese, Biochemistry 23 Ž1984. 4785–4791. A. Ghalayini, J. Eichberg, Purification of phosphatidylinositol synthase from rat brain by CDP-diacylglycerol affinity chromatography and properties of the purified enzyme, J. Neurochem. 44 Ž1985. 175–182. M.E. Monaco, M. Feldman, D.L. Kleinberg, Identification of rat liver phosphatidylinositol synthase as a 21 kDa protein, Biochem. J. 304 Ž1994. 301–305. S. Tanaka, J. Nikawa, I. Hideaki, S. Yamashita, K. Hosaka, Molecular cloning of rat phosphatidylinositol synthase cDNA by functional complementation of the yeast Saccharomyces cereÕisiae pis mutation, FEBS Lett. 393 Ž1996. 89–92. N. Elabbadi, M.L. Ancelin, H.J. Vial, Characterization of phosphatidylinositol synthase and evidence of a polyphosphoinositol cycle in Plasmodium-infected erythrocytes, Mol. Biochem. Parasitol. 63 Ž1994. 179–192. G.S. Parries, M. Hokin-Neaverson, Inhibition of phosphatidylinositol synthase and other membrane-associated enzymes by sterioisomers of hexachlorocyclohexane, J. Biol. Chem. 260 Ž1985. 2687–2693. J.A. Pulido, N. del Hoyo, M.A. Perez-Albarsanz, The effects of different hexachlorocyclohexanes and cyclodienes on glucose uptake and inositol phospholipid synthesis in rat brain cortex, Life Sci. 50 Ž1992. 1585–1596. S.C. Johnson, J. Dahl, T.-L. Shih, D.J.A. Schedler, L. Anderson, T.L. Benjamin, D.C. Baker, Synthesis and evaluation of 3-modified 1D-myo-inositols as inhibitors and substrates of phosphatidylinositol synthase and inhibitors of myo-inositol uptake by cells, J. Med. Chem. 36 Ž1993. 3628–3635. L.A. Vargas, L.X. Miao, A.F. Rosenthal, Inhibition of platelet phosphatidylinositol synthetase by an analog of CDPdiacylglycerol, Biochim. Biophys. Acta 796 Ž1984. 123–128.
Chapter XIVB
Phosphatidylinositol synthase from mammalian tissues Bruno Antonsson
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GeneÕa Biomedical Research Institute, Glaxo Wellcome R & D S.A., 14 ch. des Aulx, 1228 Plan-les-Ouates, GeneÕa, Switzerland Received 12 March 1997; revised 14 April 1997; accepted 15 April 1997
Abstract Phosphatidylinositol synthase ŽCDP-diacylglycerol:myo-inositol 3-phosphatidyl-transferase, EC 2.7.8.11. is a 24-kDa membrane-bound enzyme. It is present in all mammalian cells and is localized predominantly to the endoplasmic reticulum. The enzyme performs the last step in the de novo biosynthesis of the phospholipid phosphatidylinositol by catalyzing the condensation of CDP-diacylglycerol and myo-inositol to form the products phosphatidylinositol and CMP. Phosphatidylinositol, apart from being an essential membrane phospholipid, is involved in protein membrane anchoring and is the precursor for the second messengers inositol-tri-phosphate and diacylglycerol. q 1997 Elsevier Science B.V. Keywords: Phosphatidylinositol synthase; Phosphatidylinositol; Inositol; Phospholipid
1. Introduction P hosphatidylinositol synthase Ž C D P -di acylglycerol:myo-inositol 3-phosphatidyl-transferase, EC 2.7.8.11. performs the last step in the de novo biosynthesis of phosphatidylinositol by catalyzing the condensation of CDP-diacylglycerol and myo-inositol to give the products, phosphatidylinositol and CMP w1x. In addition to de novo biosynthesis, phosphatidylinositol can be synthesized by an exchange reaction where free myo-inositol is incorporated into phosphatidylinositol in the presence of manganese. This reaction is catalyzed by a different enzyme and does not require CDP-diacylglycerol w2,3x. Phosphatidylinositol has been detected in membranes from all mammalian tissues that have been examined. It is a minor but essential membrane component which
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Fax: q41 22 7946965; E-mail: [email protected] Dedicated to Professor Eugene Kennedy.
constitutes between 2–12% of the total phospholipids in eucaryotic cells Žfor a review, see Ref. w4x.. Besides being an essential component of the cell membrane, phosphatidylinositol is involved in protein membrane anchoring and it is the precursor for the second messengers inositol-polyphosphate and diacylglycerol. Phosphatidylinositol is unique among phospholipids in that its head group, the inositol, can be further phosphorylated to phosphatidylinositol mono- or diphosphates. It has been proposed that two pools of phosphatidylinositol exist in the cell membranes w5,6x. One of which is sensitive to hormone-induced hydrolysis, and the other which is hydrolysis insensitive. Phosphatidylinositol-polyphosphates are essential parts of the Ca2qrPKC signal transduction pathways Žfor a review, see Ref. w7x.. A wide range of extracellular stimulations, including hormones and neurotransmitters, trigger the phospholipase C-mediated hydrolysis of the hormone-sensitive pool of phosphatidylinositol-di-phosphate. This gives rise to the two secondary messengers, inositol-1,4,5-triphos-
0005-2760r97r$17.00 q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 5 - 2 7 6 0 Ž 9 7 . 0 0 1 0 5 - 7
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phate and diacylglycerol. Inositol-tri-phosphate is liberated into the intracellular space and triggers Ca2q release from intracellular stores, which leads to the activation of the calcium-inducible signal transduction pathways. Diacylglycerol, which remains in the membrane, activates the protein kinase C ŽPKC.-signaling pathways. After receptor-triggered hydrolysis of phosphatidylinositol-polyphosphates the phospholipid must be resynthesised in order to maintain a constant level of phosphatidylinositol in the membranes, this requires phosphatidylinositol synthase activity.
2. Discovery, occurrence and localization Inositol-containing phospholipids were identified in mammalian tissues in the early 1940s. The enzyme activity responsible for the biosynthesis of phosphatidylinositol was first described by Agranoff et al. w8x and Paulus and Kennedy w1x about 10 years later. Paulus and Kennedy used the microsomal fraction of liver from guinea pig and chicken in their studies. Already these studies indicated that the enzyme activity was located in the membrane fraction. The first more detailed study of the occurrence and localization of the enzyme activity was carried out by Benjamins and Agranoff in various tissues from guinea pig w9x. They detected phosphatidylinositol synthase activity in all tissues tested, including brain, liver, kidney, heart, lung and spleen. Although, the specific activity of the enzyme varied between the various tissues. They also studied the subcellular localization in brain extract. Enzyme activity was predominantly co-localizing with the microsomal fraction, with less than 5% of the total activity in the soluble cell fraction. The localization of the enzyme activity to the microsomal fraction was confirmed in studies of rat brain extracts by Bishop and Strickland w10x and Rao and Strickland w11x. Studies of membrane fractions from rat liver by Williamson and Morre w12x showed that the enzyme was localized to both the rough and the smooth endoplasmic reticulum. They also found the enzyme to be present in the Golgi apparatus membrane. That the major part of phosphatidylinositol synthase is localized to the endoplasmic reticulum is not disputed. More recent studies, however, have indicated that the enzyme is also
present in other cellular membranes. The results, depending on the cells and tissues examined, are not unambiguous. Imai and Gershengorn w13x studied the distribution in rat pituitary tumor GH 3 cells. They showed that both the plasma membrane and the endoplasmic reticulum contained phosphatidylinositol synthase activity. When the two enzyme activities were characterized they displayed different kinetic characteristics. It was also shown that the enzyme residing in the plasma membrane could more easily be extracted from the membrane by high salt concentration compared to the endoplasmic reticulum enzyme w14x. These facts might indicate the existence of two different phosphatidylinositol synthases. Although, it is far from proven, and the differences observed could be due to differences in the lipid microenvironment of the enzyme in the two membranes. Phosphatidylinositol synthase activity was also detected in the plasma membrane from rabbit proximal tubule cells by Galvao and Shayman w15x. However, they could not detect any significant differences in the kinetic parameters between the endoplasmic reticulum and the plasma membrane enzyme. Conversely, in a study of the subcellular distribution in cultured glioma and neuroblastoma cells, Morris et al. w16x found that the enzyme was exclusively localized to the endoplasmic reticulum. They did not detect any activity in the plasma membrane. Baker and Chang w17x assayed phosphatidylinositol synthase activity in the nuclear and endoplasmic reticular fractions of immature rabbit cerebral cortex. They found that the nuclear membrane fraction contained the highest specific activity. As in all other studies, they also detected activity in the endoplasmic reticulum, with most activity found in the rough endoplasmic reticulum. In a study of the subcellular distribution in 1321 N1 astrocytoma cells Sillence and Downes w18x found activity in two distinct membrane fractions isolated by sucrose gradient centrifugation. One fraction co-migrated with the endoplasmic reticulum marker NADPH–cytochrome c reductase. The second peak however, did not co-migrate with the plasma membrane though they could not exclude the possibility of a small amount of activity being associated with the plasma membrane. Vaziri et al. w19x, in a study using a cell-free system from turkey erythrocytes, showed that the hormone-sensitive pool of phosphatidylinositol in the plasma membrane could
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be re-synthesized in the plasma membrane fraction itself. This suggests the presence of phosphatidylinositol synthase activity in the plasma membrane of the turkey erythrocytes. Taken together these results do not give a clear picture of the localization of the enzyme. The somewhat conflicting observations could reflect differences in different cell types and tissues. It cannot be excluded that the concentration of phosphatidylinositol synthase in different cellular membranes varies between cell types, which may for example enable the detection of activity in the plasma membrane of one cell type but prevent its detection in another. Differences in assay conditions is another possible reason for inconsistencies in the results. It has been suggested that the enzyme in different membranes requires more or less detergent for its detection w20x. What these results clearly show is that isolation of pure membrane fractions from subcellular structures is still not trivial and may be a contributing factor to the ambiguous results. The existence of two phosphatidylinositol synthase isozymes is an attractive idea, one responsible for synthesis of the hormone-sensitive pool in the plasma membrane, and the other responsible for the synthesis of the insensitive pool, primarily in the endoplasmic reticulum. However, conclusive evidence for the existence of different isozymes still has to be found.
3. Substrate specificity and regulation In the presence of Mg 2q phosphatidylinositol synthase catalyzes the reaction w1x: CDP-diacylglycerolq myo-inositol ™ phosphatidylinositolq CMP Of the nine possible inositol isomers, only myo-inositol has been found in phospholipids isolated from cell membranes w21x. This suggests a high specificity for the inositol substrate. Some early in vitro studies performed on crude membrane fractions suggested that other inositol isomers could also be incorporated into the phospholipid w1,9x. However, this has not been confirmed in studies with purer enzyme preparations and such products have not been detected in cell membranes. The other substrate in the reaction, CDP-diacylglycerol, is composed of two subunits, the nucleotide and the lipid. Little is known about the
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specificity for the CDP part of the molecule. One study of phosphatidylserine synthase and phosphatidylglycerophosphate synthase from E. coli where various liponucleotides were tested as substrates might be of some relevance w22x. CDP-diacylglycerol and deoxy-CDP-diacylglycerol served as substrates for the enzymes. In contrast, UDP-, ADPand GDP-diacylglycerol were all inactive as substrates. Phosphatidylinositol synthase from human placenta used deoxy-CDP-diacylglycerol at least as efficiently as CDP-diacylglycerol as in vitro substrate w23x. CDP-diacylglycerol showed substrate inhibition, whereas this was not seen with the deoxy-derivative and the maximal activity was approximately 25% higher compared to CDP-diacylglycerol. Several studies have been performed to evaluate the influence of the lipid chain length and the degree of saturation on the efficiency of the CDP-diacylglycerol substrate w9,10,24x. Although, all the studies detected relative differences between the various lipid chains, the results are not conclusive as to which lipid chains make the better substrate. In bovine brain CDP-diacylglycerol has been shown to be present predominantly as 1-steroyl,2-archidonyl which is also the main composition of phosphatidylinositol w25x. Besides the two substrates, the enzyme has an absolute requirement for Mg 2q or Mn2q w8x. The regulation of phosphatidylinositol synthase expression and of its activity remains largely unclear. myo-Inositol depletion, induced by the inhibition of inositol monophosphatase by lithium, decreases the concentration of phosphatidylinositol in the cell membranes. Atack et al. showed that this decrease could be prevented by pre-incubation with exogenous myo-inositol w26x. In contrast the isomer scyllo-inositol had no effect. Under normal physiological conditions, however the extracellular inositol concentration does not change enough to have a regulatory effect on the synthesis of phosphatidylinositol. Models simulating diabetes have shown that, under pathological conditions, where the myo-inositol concentration is decreased, inositol becomes the limiting factor in the biosynthesis of phosphatidylinositol w27x. Phosphatidylinositol synthase activity has been shown to be upregulated after hormone-induced phospholipase C-mediated hydrolysis of phosphatidylinositol-polyphosphates, however the mechanism remains unknown w28x. It has been suggested that phosphatidyli-
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nositol synthase might be inhibited by its product, phosphatidylinositol w29x. A main unresolved issue regarding the regulation of phosphatidylinositol resynthesis after receptor triggered hydrolysis is whether the re-synthesis occurs in the plasma membrane or in the endoplasmic reticulum. Phosphatidylinositol synthase activity has been shown in the plasma membrane fractions of GH 3 cells, proximal tubule cells and turkey erythrocytes w13,15,19x. This would indicate that the re-synthesis could take place in the plasma membrane. However, other studies, using other cells and tissues, have failed to detect the enzyme in the plasma membrane. The other possibility would be that the re-synthesis takes place in the endoplasmic reticulum and phosphatidylinositol is transported to the plasma membrane by the wellcharacterized phosphatidylinositol transfer proteins w30x. These are soluble intracellular proteins that transport phosphatidylinositol between the various membrane compartments within cells. Thomas et al. showed that a soluble 35-kDa phosphatidylinositol transfer protein enhanced the G-protein-stimulated phospholipase C-b activity w31x. This was suggested to be achieved by transfer of phosphatidylinositol from intracellular membrane compartments to the plasma membrane, where phosphatidylinositol was converted to phosphatidylinositol-di-phosphate and hydrolysis by the phospholipase. Based on the available results, it is not possible to discriminate which of the two mechanisms are responsible for the resynthesis of phosphatidylinositol after receptor-triggered hydrolysis.
4. Purification The early studies of phosphatidylinositol synthase were performed on crude membrane fractions. Attempts to extract the enzyme from the membrane fraction using various detergents including Triton X-100, Lubrol, NP-40 or bile salts resulted in loss of the enzyme activity. Rao and Strickland w11x were the first to be successful in extracting active enzyme from rat brain microsomes using 0.3% Triton X-100. This preparation, however, was not free from membrane lipids and any attempt at further purification resulted in loss of the activity. The first partial purification of the enzyme was done by Parries and
Hokin-Neaverson from canine pancreas w32x. They used n-octyl glucopyranoside to solubilize the enzyme from the microsomal fraction. The ratio of detergent to protein was found to be important, and optimal solubilization was achieved at a ratio of 4–6:1 at a detergent concentration of 40 mM. Under these conditions 95–100% of the enzyme activity could be solubilized. The solubilized enzyme was stabilized by manganese. The enzyme was then purified by ion-exchange chromatography on DEAE-Cellulose. To preserve enzyme activity phosphatidylinositol and egg yolk phospholipids were required in the buffers. The overall purification was 10-fold over the microsomal fraction and the protein had a specific activity of 290 nmolrminrmg. More successful purifications have been achieved using affinity chromatography on CDP-diacylglycerol–Sepharose. This affinity resin had been used in the purification of several bacterial and yeast enzymes including phosphatidylinositol synthase from Saccharomyces cereÕisiae. The major disadvantage with this purification method is that mammalian phosphatidylinositol synthase has proven to bind very strongly to the affinity resin. Elution has thus required special buffer conditions and prolonged incubation times resulting in low recovery of enzyme activity. Despite these drawbacks this method has provided a means of obtaining purified mammalian enzyme. The first attempt to purify mammalian phosphatidylinositol synthase using this approach was made by Ghalayini and Eichberg w33x. The enzyme was solubilized with 0.6% Triton X-100 from rat brain microsomes. The soluble fraction was subsequently purified on a CDP-diacylglycerol–Sepharose column and the bound enzyme was eluted with CDP-diacylglycerol. Triton X-100 and phosphatidylcholine were also required in the elution buffer. The specific activity was increased 190-fold over the crude brain homogenate and the recovery of enzyme activity was 7%. The enzyme preparation however, was not homogeneous when analyzed by SDS–PAGE. Only in recent years have close to homogeneous preparations been obtained. Human phosphatidylinositol synthase was purified from placenta by Antonsson w23x. The enzyme was extracted to approximately 50% from the microsomal fraction with Triton X-100. The solubilized enzyme was subjected to affinity chromatography on CDP-diacylglycerol–Sepharose.
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As already seen by Ghalayini and Eichberg the enzyme bound strongly to the affinity resin w33x. Active enzyme could be eluted from the column only in the presence of high salt Ž1.3 M NaCl. , 10% glycerol, 0.5% Triton X-100, phosphatidylcholine and diacylglycerol in addition to the affinity ligand CDP-diacylglycerol. The pool from the affinity column was far from pure and the eluted sample was applied to ion-exchange chromatography on Mono Q FPLC. By carefully adjusting pH and salt concentrations in the sample and the equilibration buffer a fraction of phosphatidylinositol synthase migrated with the flow-through fractions, whereas the remaining enzyme together with the impurities bound to the column and were eluted with a salt gradient. The gradient-eluted enzyme was resubmitted to this purification step and each time a fraction of phosphatidylinositol synthase emerged in the flow-through fractions. These results suggested that the enzyme was present in an equilibrium of free and complexed protein, presumably interacting with hydrophobic contaminating proteins. When analyzed by SDS– PAGE the pool was almost homogeneous with one protein Žover 90%. with an apparent molecular mass of 24 000 Da. The enzyme was purified 8300-fold over the microsomal fraction and the specific activity of the purified enzyme was 34 000 nmolrminrmg. A few months later Monaco et al. w34x reported the purification of the enzyme from rat liver. The enzyme was solubilized with the non-ionic detergent Hecameg. The soluble fraction was heated at 658C until a precipitate was formed. This step increased the specific activity 4-fold and the supernatant which contained phosphatidylinositol synthase was further purified on a CDP-diacylglycerol column. The enzyme was eluted with CDP-diacylglycerol in the presence of 20% glycerol, 1 mM MnCl 2 , and 1.6% Hecameg, and further purified on a PBE-94 column. The purified enzyme had a specific activity of 2800 nmolrminrmg and had been purified 5100-fold over the crude cell supernatant. When analyzed by SDS– PAGE the sample contained two main proteins, one at 51 000 and one at 21 000 Da. By correlating the band intensities with phosphatidylinositol synthase activity in the fractions eluted from the PBE-94 column, the 21 000-Da protein was shown to correlate with phosphatidylinositol synthase. These studies showed that mammalian phosphatidylinositol syn-
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thase has an apparent molecular mass of between 20 000 and 25 000 Da.
5. Cloning Unfortunately no amino acid sequence information has yet been obtained from the purified mammalian enzyme. Due to the instability of the enzyme once extracted from the membrane fraction only analytical amounts of the protein have been obtained in pure form. Attempts to use the known sequence for the Saccharomyces cereÕisiae enzyme to clone the mammalian enzyme have not been successful. However, recently Tanaka et al. have cloned the rat phosphatidylinositol synthase cDNA by functional complementation of a Saccharomyces cereÕisiae phosphatidylinositol synthase mutant, deficient in phosphatidylinositol synthase activity w35x. The cloned cDNA encodes a protein of 213 amino acids with a calculated molecular mass of 23 613 Da.
6. Structure and properties The amino acid sequence deduced from the cloned cDNA shows that the protein is pronounced hydrophobic with 60% hydrophobic amino acids w35x. The hydrophobic amino acids form three main clusters Žresidues 17–35, 77–98 and 165–195. with a hydropathy index greater then 1.5. One or more of these stretches could be involved in the membrane attachment of the protein. Comparison with the yeast phosphatidylinositol synthase amino acid sequence showed the two proteins to have 39% identity and 73% similarity. The three hydrophobic stretches present in the mammalian enzyme are less pronounced in the yeast enzyme. In the yeast enzyme the hydrophobic residues are distributed into smaller stretches. This might be a reason why the mammalian enzyme is more sensitive to removal of the membrane lipids, which might result in the exposure of the large hydrophobic regions to the aqueous solvent. In addition to microsomal preparations w9,15x the purified or partly purified preparations of the enzyme from canine pancreas w32x, rat brain w33x, human placenta w23x and rat liver w34x have been used to determine the kinetic parameters and properties of the
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enzyme. The optimal pH for the phosphatidylinositol synthase reaction was found to be between 8.5 and 9.0. For myo-inositol K m values between 0.3 and 4.5 mM have been reported. The fairly large variation is presumably due to differences in the enzyme preparations as well as variations in assay conditions, such as detergent, buffer compositions and pH. In purified preparations the K m was 0.28 mM for the enzyme from human placenta and 1.3 mM for the rat liver enzyme. The lipid substrate had K m values of 36 and 9.5 mM for the human and rat liver enzyme, respectively. With the human enzyme CDP-diacylglycerol concentrations above 0.5 mM showed substrate inhibition. The rat brain enzyme was also inhibited by CDP-diacylglycerol concentrations above 0.4 mM. The human enzyme was assayed with 2-deoxy-CDPdiacylglycerol as substrate. The deoxy-derivative had a K m of 45 mM, it showed almost no inhibition and the optimal activity obtained was 25% higher then with CDP-diacylglycerol. The enzyme has an absolute requirement for Mg 2q or Mn2q. Manganese was more efficient in activating the enzyme at lower concentrations, 0.1–1 mM w23,33,34x. Higher concentrations of manganese were inhibitory and at a concentration of around 50 mM the enzyme was completely inhibited w23,33x. To obtain optimal activation with magnesium concentrations between 10 and 100 mM were required. The optimal activity obtained with magnesium was between 2- and 4-fold higher then that obtained with manganese w23,36x. Manganese was shown to stabilize the enzyme once extracted from the membrane, in contrast magnesium had no stabilizing effect w23,32,34x. This might suggest that the enzyme has several ion-binding sites affecting stability and activity, respectively. Alternatively the effect seen on the activity may be due to ion binding to the liponucleotide substrate and not to direct interaction with the enzyme molecule. Calcium has been shown to inhibit the enzyme, and phosphatidylinositol synthase from human placenta was also inhibited by zinc w13,14,23x. Whether the inhibition by calcium has any physiological significance is unclear, in particular since the K i is in the mM region. Inhibition studies of the enzyme have been performed with modified substrates. Hexachlorocyclohexanes Ž HCCH. are chlorinated analogs of inositol which have been shown to inhibit the incorporation
of inositol into phosphatidylinositol w37,38x. In a study of phosphatidylinositol synthase from dog pancreas microsomes the d-HCCH isomer, which has the stereochemical configuration of myo-inositol, was most inhibitory w37x. However, the inhibition was noncompetitive and not specific for inositol processing enzymes. The mode of inhibition is likely to be mediated through interactions with hydrophobic domains on the membrane-bound enzymes or with the lipids in their microenvironment. This is consistent with the fact that the inhibitory potency of the isomers correlates with their lipophilicity. Johnson et al. studied 3-substituted myo-inositol derivatives as substrates for phosphatidylinositol synthase and found that 3-NH 2-, 3-F-3-deoxy- and 3-keto all were used as substrate by the enzyme w39x. This indicates that the 3-position of the inositol substrate is probably not involved in the interaction with the enzyme. Another inhibitor that has been tested is an analog of CDP-diacylglycerol where the phosphate moiety was replaced by a methyl group. This liponucleotide cannot be hydrolyzed to act as a phosphatidyl donor, and it was found to be an efficient competitive inhibitor with a K i of 32 nM w40x. In the study of the enzyme from human placenta the effect of nucleotides and phosphate on the enzyme activity was examined w23x. In summary, the tri-phosphates Ž K i 1.4–1.8 mM. were more inhibitory then the di-phosphates Ž K i 3.8–4.1 mM. and the mono-phosphates did not inhibit. The nucleotide bases had no influence on the inhibition. Pyro- and ortho-phosphate were most inhibitory with K i values of 0.2 and 0.8 mM, respectively. Combined, the results suggest that the nucleotide base is not involved in direct interactions with the enzyme molecule. The interactions appear to be mediated through the phosphate and the ribose moieties
7. Unresolved issues Despite the increasing interest in the phosphatidylinositol-polyphosphates and their proven central role in signal transduction many issues about phosphatidylinositol synthase, which is an essential enzyme in the biosynthesis of these important molecules, remain unclear. The enzyme is membrane associated and has proven difficult to handle once
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extracted from its native environment. Although the enzyme has now been purified, purification of larger amounts for biochemical and structural work has not been possible. The recent cloning of the phosphatidylinositol synthase gene will hopefully provide better opportunities to study the enzyme from recombinant sources. In order to increase the solubility and make the protein more suitable for in vitro biochemical studies it might be possible to remove or modify the hydrophobic domains in the recombinant protein. An important question is the regulation of phosphatidylinositol synthase activity. How is the resynthesis after receptor-triggered hydrolysis of the phosphatidylinositol-polyphosphates regulated and controlled? Where does the synthesis take place, in the endoplasmic reticulum or in the plasma membrane? Even such an apparently trivial question as the subcellular localization of the enzyme remains unclear. The fact that the enzyme is located in the endoplasmic reticulum is undisputed, but is it also localized in, for example, the plasma, nuclear and Golgi membranes? The localization is a central question which awaits an answer. Here again, expression and purification of the recombinant protein, or parts of it, should make it possible to obtain antibodies against the protein which could help to answer this and other questions.
Acknowledgements I wish to thank Charles Bradshaw and Kathryn Radford for critical reading of the manuscript.
References w1x H. Paulus, E.P. Kennedy, The enzymatic synthesis of inositol monophosphatide, J. Biol. Chem. 235 Ž1960. 1303–1311. w2x T. Takenawa, K. Egawa, Phosphatidyl Inositol: myo-inositol exchange enzyme from rat liver: partial purification and characterization, Arch. Biochem. Biophys. 202 Ž1980. 601– 607. w3x A.E. Cubitt, M.C. Gershengorn, CMP activates reversal of phosphatidylinositol synthase and base exchange by distinct mechanisms in rat pituitary GH3 cells, Biochem. J. 272 Ž1990. 813–816.
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w4x D.A. White, The phospholipid composition of mammalian tissues, in: G.B. Ansell, J.N. Hawthorne, R.M.C. Dawson ŽEds.., Form and Function of Phospholipids, Vol. 3, Elsevier Scientific, Amsterdam, 1973, pp. 441–482. w5x K. Koreh, M.E. Monaco, The relationship of hormone-sensitive and hormone-insensitive phosphatidylinositol to phosphatidylinositol 4,5-biphosphate in the WRK-1 cell, J. Biol. Chem. 261 Ž1986. 88–91. w6x A.B. Cubbitt, B. Zhang, M.C. Gershengorn, Analysis by base exchange of thyrotropin-releasing hormone responsive and unresponsive inositol lipid pools in rat pituitary tumor cells, J. Biol. Chem. 265 Ž1990. 9707–9714. w7x M.J. Berridge, Inositol triphosphate and diacylglycerol: two interacting second messengers, Annu. Rev. Biochem. 56 Ž1987. 159–193. w8x B.W. Agranoff, R.M. Bradley, R.O. Brady, The enzymatic synthesis of inositol phosphatide, J. Biol. Chem. 233 Ž1958. 1077–1083. w9x J.A. Benjamins, B.W. Agranoff, Distribution and properties of CDP-diacylglycerol:inositol transferase from brain, J. Neurochem. 16 Ž1969. 513–527. w10x H.H. Bishop, K.P. Strickland, On the specificity of cytidine diphosphate diglycerides in monophosphoinositide biosynthesis by rat brain preparations, Can. J. Biochem. 48 Ž1970. 267–277. w11x R.H. Rao, K.P. Strickland, On the solubility, stability and partial purification of CDP-diacyl-sn-glycerol:inositol transferase from rat brain, Biochim. Biophys. Acta 348 Ž1974. 306–314. w12x F.A. Williamson, D.J. Morre, Distribution of phosphatidylinositol biosynthetic activities among cell fractions from rat liver, Biochem. Biophys. Res. Commun. 68 Ž1976. 1201– 1205. w13x A. Imai, M.C. Gershengorn, Independent phosphatidylinositol synthase in pituitary plasma membrane and endoplasmic reticulum, Nature 325 Ž1987. 726–728. w14x A.B. Cubitt, M.C. Gershengorn, Characterization of a saltextractable phosphatidylinositol synthase from rat pituitary tumor membranes, Biochem. J. 257 Ž1989. 639–644. w15x C. Galvao, J.A. Shayman, The phosphatidylinositol synthase of proximal tubule cells, Biochim. Biophys. Acta 1044 Ž1990. 34–42. w16x S.J. Morris, H.W. Cook, D.M. Byers, M.W. Spence, F.B.St.C. Palmer, Phosphoinositide metabolism in cultured glioma and neuroblastoma cells: subcellular distribution of enzymes indicate incomplete turnover at the plasma membrane, Biochim. Biophys. Acta 1022 Ž1990. 339–347. w17x R.R. Baker, H.-Y. Chang, Phosphatidylinositol synthetase activities in neuronal nuclei and microsomal fractions isolated from immature rabbit cerebral cortex, Biochim. Biophys. Acta 1042 Ž1990. 55–61. w18x D.J. Sillence, C.P. Downes, Subcellular distribution of agonist-stimulated phosphatidylinositol synthesis in 1321 N1 astrocytoma cells, Biochem. J. 290 Ž1993. 381–387. w19x C. Vaziri, C.P. Downes, S.C. Macfarlane, Direct labelling of hormone-sensitive phosphoinositides by a plasma-
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