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[55] Phosphoglyceride synthesis in endoplasmic reticulum
[55]
P H O S P H O G L Y C E R I D E S Y N T H E S I S IN E R
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[55] P h o s p h o g l y c e r i d e S y n t h e s i s in E n d o p l a s m i c R e t i c u l u m By THOMAS S. MOORE, JR.
The endoplasmic reticulum of plants is thought to play a major role in membrane phosphoglyceride synthesis, L2 similar to the situation in animals, 3 although it is known that the Golgi apparatus, mitochondria, and plastids 1,2 also play a role. The production of phospholipids by an in vitro microsomal fraction was first demonstrated by Sastry and Kates in 1966,4 and such fractions, probably consisting of endoplasmic reticulum (ER) plus various other membranes, have been reported to synthesize a variety of phospholipids.l,2 The purity of the organelle fractions in many studies is uncertain, and so the relative roles of the ER, Golgi apparatus, and other membranes cannot be clearly argued for most plant systems. The castor bean endosperm system has the advantage of a reduced contribution from the Golgi apparatus as well as clean separation of the other organelles present on sucrose density gradients, and the clear compartmentation of most of the phospholipid synthesis into the endoplasmic reticulum of that tissue supports a major role for that organelle in synthesis of many organelle membranes. 5 For these reasons, the enzyme assays described below are based primarily on work with castor bean endosperm (see Fig. 1). EC 2.7.8.2 Cholinephosphotransferase (CDPcholine: 1,2-Diacylglycerol Cholinephosphotransferase) CDPcholine + sn-l,2-diacylglycerol~ phosphatidylcholine+ CMP This activity usually is the most active of the phospholipid synthesizing enzymes, 6 which may be a reflection of its product being the most T. S. Moore, Annu. Rev. Plant Physiol. 33, 235 (1982). 2 T. S. Moore, in "Structure, Function and Metabolism of Plant Lipids" (P.-A. Siegenthaler and W. Eichenberger, eds.), p. 83. Elsevier Science, Amsterdam, New York, 1984. 3 R. M. Bell and R. A. Coleman, Annu. Rev. Biochem. 49, 459 (1980). 4 p. S. Sastry and M. Kates, Can. J. Biochem. 44, 459 (1966). 5 T. S. Moore, this series, Vol. 71, p. 596. 6 T. S. Moore and G. D. Troyer, in "Biosynthesis and Function of Plant Lipids" (W. W. Thomson, J. B. Mudd, and M. Gibbs, eds.), p. 16. Am. Soc. Plant Physiol., Rockville, Maryland, 1983.
METHODS IN ENZYMOLOGY, VOL. 148
Copyright © 1987by Academic Press, Inc. All rights of reproduction in any form reserved.
586
NUCLEI, ER, AND PLASMA MEMBRANE
[55]
CMP CDPCho ,~ ~PtdCho
DAG~Aqz''''''7
Ptd
"
,
CDP-DAGe G ~ CMPPtdGroP~PtdGro Pi FIG. 1. Outline of the pathways of phosphoglyceride synthesis in plant endoplasmic reticulum.'The enzymes are indicated by their nomenclature numbers, which correlate with the text. The phosphatidate phosphatase (EC 3.1.3.4) has not been adequately described in plant endoplasmic reticulum, and so is not discussed. The two exchange enzymes (producing Ptdlns and PtdSer) have not been assigned a number. Abbreviations: CDP-DAG, CDPdiacylglycerol; DAG, diacylglycerol; Ptd, phosphatidate; PtdCho, phosphatidylcholine; PtdEth, phosphatidylethanolamine; PtdGro, phosphatidylglycerol; PtdGroP, phosphatidylglycerol-P; PtdIns, phosphatidylinositol; PtdSer, phosphatidylinositol.
abundant phospholipid of many membranes.7,8 The activity was first characterized from plants in 1971.9
Assay Method Principle. The chloroform-soluble product radiolabeled from the water-soluble CDPcholine is extracted from the reaction mixture following the assay and its radioactivity determined.~° Reagents Tris-HCl, 50 mM, pH 7.5 Dithiothreitol, 5 mM MgCI2, 100 mM Diacylglycerol, from egg phosphatidylcholine, 2 mM 7 C. Hitchcock and B. W. Nichols, "Plant Lipid Biochemistry." Academic Press, New York, 1971. 8 M. Kates, Adv. Lipid Res. 8, 225 (1970). 9 K. A. Devor and J. B. Mudd, J. Lipid Res. 12, 403. 10T. S. Moore, Plant Physiol. 57, 382 (1976).
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PHOSPHOGLYCERIDE SYNTHESIS IN E R
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CDP[1,2A4C]choline, 1 mM, 5 mCi/mmol Chloroform-methanol-H20 (1 : 2 : 0.3, by volume) Chloroform KC1, 1.0 M Assay Procedure. Equal volumes of buffer, dithiothreitol, MgCI2, and diacylglycerol are mixed and 0.4-ml aliquots placed into test tubes. Fiftymicroliter aliquots of enzyme are added and the reaction started by the addition of 50/zl of the CDPcholine. The assay is run for 30 min with shaking in a water bath at 30° and is stopped by the addition of 3.3 ml of the chloroform-methanol-water mixture followed by mixing with a vortex mixer. Extraction Procedure. One milliliter of chloroform and 3 ml of 2 M KCI are added to each tube, followed by vigorous mixing. The tubes are centrifuged at about 2500 rpm in a tabletop centrifuge. Following this the upper aqueous layer is removed and discarded. The lower chloroform layer is washed twice more with 1- to 3-ml aliquots of 1 M KC1, aspirating and discarding the aqueous layer each time. Radioactivity Measurement. The chloroform extract is placed into scintillation vials and dried completely. Radioactivity is measured by standard techniques.
Properties Substrate Requirements. The apparent Km of the castor bean enzyme for CDPcholine is about l0/.~M, 1° and similar results have been obtained with other tissues. 9 Estimates have not been made for diacylglycerol, since it occurs in the membrane; some stimulation of activity may be achieved by including this substrate in the assay mixture, however/° p H and Metal Ion Requirements. The optimal pH generally is from 7.51° to 8.0, 9'10 and a divalent cation is required. M g 2+ is preferred over Mn 2÷ by the castor bean enzyme, 1° but the two give equal activities with spinach. 9 Ca 2÷ inhibits the activity. 11,12 Inhibitors. Dithiothreitol promotes and sulfhydryl reagents inhibit the reaction to varying degrees, depending on the tissue source. 9,1° EC 2.7.8.1 Ethanolaminephosphotransferase (CDPethanolamine : 1,2-Diacylglycerol Ethanolaminephosphotransferase) CDPethanolamine + sn-l,2-diacylglycero!--~ phosphatidylethanolamine + CMP
11 j. M. Lord, Plant Physiol. 57, 218 (1976). 12 A. Oursel, A. Tr6moli~res, and P. Mazliak, Physiol. Veg. 15, 377 (1977).
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NUCLEI, E R , AND PLASMA MEMBRANE
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This enzyme catalyzes a reaction similar to the cholinephosphotransferase and with a similar high level of activity) 3-15 It has not, however, been as thoroughly examined and demonstrated not to be catalyzed by the same enzyme that synthesizes phosphatidylcholine.16
Assay Method Principle. The reaction is conducted in the presence of the radioactive, water-soluble substrate, followed by extraction of the chloroform soluble product and measurement of its radioactivity. 15 Reagents MES, 50 mM, pH 6.5 MgC12, 15 mM Dithiothreitol, 5 mM CDP[1,2:4C]ethanolamine, 75/zM, 3.0 mCi/mmol Chloroform-methanol-H20 (1 : 2 : 0.3, by volume) Chloroform KC1, 1.0 M Assay Procedure. Equal quantities (0.1 ml each) of buffer, MgC12, and dithiothreitol are combined with 0.1 ml of H20 in a test tube. The enzyme is added, the mixture preincubated at 37°, and the reaction started by the addition of 0.05 ml CDPethanolamine. The final volume is 0.5 ml. The tubes are incubated with shaking at 37° for 30 min and the reaction stopped by the addition of 3.3 ml of chloroform-methanol-H20. Extraction and Radioactivity Measurement. The extraction and radioactivity measurements were as described for the cholinephosphotransferase assay above.
Properties Substrate Requirements. The apparent Km of the enzyme from castor bean endosperm for CDPethanolamine is 6.0/zM, while that for spinach is 20/zM. 14.15Maximum velocities are achieved at 50 to 100/xM CDPethanolamine. 14,15 CDPcholine is a strong competitive inhibitor of ethanolamine incorporation) 4,15 Diacylglycerol requirements have not been defined due to its natural occurrence in the membranes. Metal and pH Requirements. Mg2÷ is most effective for the castor 13 M. O. Marshall and M. Kates, Can. J. Biochem. 52, 469 (1974). ~4B. A. Macher and J. B. Mudd, Plant Physiol. 53, 171 (1974). ~5 S. A. Sparace, L. K. Wagner, and T. S. Moore, Plant Physiol. 67, 922 (1981). 16j. M. Lord, Biochem. J. 151, 451 (1975).
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bean enzyme, being effective at 3 mM, 15 but for spinach Mn 2÷ has been found to be best, giving an optimal reaction between 0.6 and 2 m M ) 4 The optimum pH ranges from 6.5 for castor bean to 7.0-8.0 for spinach) 4,15 E C 2.1.1.17 Phosphatidylethanolamine Methyltransferase (S-Adenosyl-L-Methionine :Phosphatidylethanolamine N-Methyltransferase) Phosphatidylethanolamine + 3 S-adenosyl-L-methionine--~ phosphatidylcholine + S-adenosylhomocystcine
In castor bean endoplasmic reticulum this reaction occurs with about 10% of the activity of the cholinephosphotransfcrase, l° It also has been described from spinach leaf~3 and potato tuber.17 It is not certain how many enzymes are involved in this three-step methylation.
Assay Method Principle. S-Adenosyl-L-[methyl.14C]methionine is fed as the aqueous substrate and the radioactive phosphatidylcholine product is extracted into chloroform, followed by measuring its radioactivity.l° Reagents Tris-HC1, 250 mM, pH 9.0 Phosphatidylethanolamine, 1.0 mM
S-Adenosyl-t-[methyl-14C]methionine, 1.0 mM, 1 mCi/mmol HC1, concentrated Chloroform-methanol-H20 (1 : 2 : 0.3, by volume) Chloroform KCI, 1.0 M Assay Procedure. Aliquots of buffer, phosphatidylethanolamine, and S-adenosylmethionine (0.1 ml each) are added to assay tubes along with sufficient water to give a final volume (with enzyme) of 0.5 ml. The tubes are preincubated at 37° for 5 rain, after which the reaction is started by the addition of enzyme and the tubes incubated with shaking in a water bath at 37° for 30 min. The reaction is stopped by the addition of 50/~1 of HCI, followed by 3.3 ml of the chloroform-methanol-H20 mixture, and then incubated at room temperature for 1 hr. Extraction and Radioactivity Measurement. The radioactive product is extracted into chloroform and the radioactivity measured as described for the cholinephosphotransferase assay. 17 W. Tang and P. A. Castelfranco, Plant Physiol. 43, 1232 (1968).
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NUCLEI, E R , AND PLASMA MEMBRANE
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Properties Substrate Requirements. The castor bean endoplasmic reticulum reaction(s) demonstrates an apparent Km for S-adenosylmethionine of 31 /.tM; phosphatidylethanolamine stimulates only about 33% 1° and does not stimulate the spinach activity.~4 The single- and double-methylated lipid intermediates stimulate the reaction in both tissues) °,~4 pH Requirement. The optimal pH for the enzyme is around 8.0 to 9.0 in both castor bean and spinach.~°.~4 Products. The product of the reaction under these conditions is primarily phosphatidylcholine, but the mono- and dimethylated intermediates also may occur) °,14 Phosphatidylethanolamine
: L-Serine Phosphatidyltransferase
Phosphatidylethanolamine + L-serine --> phosphatidyiserine + ethanolamine
Phosphatidylserine generally occurs only in small concentrations in plant tissues, but may play an important role) 8 The reaction described here is an exchange-type reaction (exchanging serine for ethanolamine), but another reaction utilizing CDPdiacylglycerol and t-serine also has been described in plants. ~3
Assay Procedure Principle. The CaE+-stimulated exchange reaction is measured by the incorporation of the water-soluble L-serine into the chloroform-soluble lipid product. 19 Reagents HEPES, 200 mM, pH 7.8 CaCI2, 10 mM L-[3-~4C]Serine, 2.0 mM, 2.5 mCi/mmol Chloroform-methanol-H20 (1 : 2 : 0.3, by volume) Chloroform KC1, 1.0 M Assay Procedure. Aliquots (0.1 ml each) of the buffer, CaClz, and Lserine, plus an appropriate amount of water to give a final volume (including enzyme) of 0.5 ml, are added to assay tubes. The tubes are preincuis N. Murata, N. Sato, and N. Takahashi, in "Structure, Function and Metabolism of Plant Lipids" (P.-A. Siegenthaler and W. Eichenberger, eds.), p. 153. Elsevier, Amsterdam, 1984. 19T. S. Moore, Plant Physiol. 56, 177 (1975).
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bated at 30° for 5 min and the reaction started by adding enzyme. Further incubation at 30°, with shaking in a water bath, is performed for 60 min. The reaction is stopped by the addition of 3.3 ml of chloroform-methanol-H20 followed by incubation on ice for 30 min. Extraction and Radioactivity Determination. The extraction and measurement of radioactivity in the product were as described for the cholinephosphotransferase assay.
Properties Substrate Requirements. The Km for serine is about 20/zM. 19 Ethanolamine was found to compete with L-serine but D-serine and choline did not. 19 Phosphatidylethanolamine stimulated the reaction, but it may be nonspecific since CDPdiacylglycerol did also. 19 Metal and pH Requirements. Calcium is required, with the optimum concentration being 2 mM. 19 The best pH at that Ca 2÷ concentration is 7.8, with a shift to a lower pH at higher Ca 2÷ concentrations. 19 Products. Both phosphatidylserine and phosphatidylethanolamine occurred as products of the reaction, suggesting the presence of a decarboxylase enzyme in the endoplasmic reticulum.19 EC 2.7.7.41 Phosphatidate Cytidylyltransferase (CTP : Phosphatidate Cytidylyltransferase) CTP + phosphatidate --* CDPdiacylglycerol + PP~
This enzyme produces a lipid which serves as a precursor to the synthesis of both phosphatidylglycerol and phosphatidylinositol in the endoplasmic reticulum. CDPdiacylglycerol occurs at only very low concentrations in membranes. 2°
Assay Method Principle. The enzyme utilizes the water-soluble substrate, CTP, labeled in the cytidine ring and produces the chloroform-soluble CDPdiacylglycerol which is extracted and its radioactivity measured. 2° Reagents MES, 50 mM, pH 6.5 MnC12, 37.5 mM Phosphatidate, 12.5 mM, derived from egg lipids [3H]CTP, 0.6 mM, 32.2 mCi/mmol 20 K. F. Kleppinger-Sparace and T. S. Moore, Plant Physiol. 77, 12 (1985).
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NUCLEI, ER, AND PLASMA MEMBRANE
[SS]
Chloroform-methanol-H20 (1 : 2 : 0.3, by volume) Chloroform KCI, 1.0 M Assay Procedure. One-tenth-milliliter fractions of buffer, MnCI2, phosphatidate, and CTP are added to an assay tube, along with sufficient water to make a final volume (after enzyme addition) of 0.5 ml. The reaction is initiated by the addition of enzyme. This mixture is incubated at 37° for 30 min in a shaker waterbath. The reaction is stopped by the addition of 3.3 ml of the chloroform-methanol-water solution. Extraction and Radioactivity Measurement. These are carded out as described for the cholinephosphotransferase assay above.
Properties Substrate Requirements. The apparent Km of the castor bean enzyme for CTP is about 17/zM, z° which is lower than those reported for other plant microsomal activities. 21 The best stimulation is apparent with mixed phosphatidates, but endogenous lipid substrate prevents an estimation of the precise requirements. 2° pH and Metal Ion Requirements. Manganese is strongly preferred over Mg 2÷ and was optimal at 7.5 mM. The best pH is 6.5. 20 Detergent Effects. Both deoxychoclate and Triton X-100 strongly inhibit the enzyme at concentrations as low as 0.01% (w/w). 2° EC 2.7.8.5 CDPdiacylglycerol-Glycerol-3-phosphate 3-Phosphatidyltransferase (CDPdiacylglycerol : sn-Glycerol-3-phosphate 3-Phosphatidyltransferase) EC 3.1.3.27 Phosphatidylglycerophosphatase (Phosphatidylglycerophosphate Phosphohydrolase) sn-l-Glycerol 3-phosphate + CDPdiacylglycerol
3-sn-phosphafidyl-l'-sn-glycerol 3'-phosphate + CMP 3-sn-Phosphatidyl-1 '-sn-glycerol Y-phosphate ~ 3-sn-phosphatidyl-I '-sn-glycerol + Pi
These two enzymes have not been separated from plant tissues, but the reaction is strongly pulled to the production of phosphatidyglycerol and so assays measure the production of that final product. 22 The reac-
21 j. Bahl, T. GuiUot-Salomon, and R. Douce, Physiol. Veg. 8, 55 (1970). zz T. S. Moore, Plant Physiol. 54, 164 (1974).
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tions were first demonstrated in spinach 4 and have since been studied further in spinach 23 as well as in cauliflower 24 and castor bean. 22
Assay Method Principle. Radioactive sn-glycerol 3-phosphate is incorporated into a chloroform-soluble product, which is extracted and the radioactivity measured. 22 Reagents Tris-HCl, 2.0 M, pH 7.3 MnC12, 40 mM Triton X-100, 0.6% (w/w) CDPdiacylglycerol, 0.8 mM sn-[U-14C]Glycerophosphate, 4.0 mM, 1.9 mCi/mmol Chloroform Methanol KCI, 1.0 M Assay Procedure. Twenty-five microliters each of buffer, MnC12, detergent, and the two substrates are added to assay tubes along with sufficient water to give a final volume of 0.2 ml following further additions. The tubes are preincubated for 5 min at 30° in a water bath. Enzyme is added to start the reaction and the tubes are incubated, with shaking, for an additional 1.5 hr. The reaction is stopped by adding 2.0 ml of methanol followed by incubating on ice for 1 hr. Extraction Procedure. Two milliliters of chloroform is added to each assay tube and mixed. The emulsion is broken by centrifugation at 2500 rpm in a tabletop centrifuge. The upper aqueous layer is removed and discarded, and the lower chloroform layer washed twice with 5.0-ml aliquots of the KC1 solution, followed by twice with distilled water. Each time the tubes are mixed, centrifuged, and the upper layer removed. Radioactivity Measurement. This is as described in the section on cholinephosphotransferase.
Properties Substrate Requirements. Calculated Km values for glycerol-P range from 50 to 250 /~M, 22,z3 while that for CDPdiacylglycerol is about 2-3/~M. 22"23 23 M. O. Marshall and M. Kates, Biochirn. Biophys. Acta 260, 558 (1972). 24 R. Douce and J. Dupont, C.R. Hebd. Seances Acad. Sci., Ser. D 268, 1657 (1969).
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NUCLEI; E R , AND PLASMA MEMBRANE
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Metal and pH Requirements. Mn 2÷ stimulates the reaction best at 5 mM, and Mg 2÷ is only partially effective. 22 The pH optimum is 7.3. 22,23 Detergent Effects. Triton X-100 stimulates at concentrations up to 0.075% (W/W). 22 Products. The major product is phosphatidyglycerol, but the phosphorylated intermediate is found in low quantities in some cases. 22,23 EC 2.7.8.11 CDPdiacylglycerol-Inositol 3-Phosphatidyltransferase (CDPdiaeyglycerol : rayo-Inositol 3-Phosphatidyltransferase) CDP-sn-l,2-diacylglycerol + myo-inositol --> phosphatidylinositol + CMP
This enzyme activity has now been described from several plant species I and solubilized from soybean. 25 It was first described from cauliflower by Sumida and Mudd. 26
Assay Method Principle. Radioactive myo-inositol is incorporated into phosphatidylinositol, which can be extracted into chloroform and its radioactivity determined. 17 Reagents Tris-HC1, 250 mM, pH 8.5 MnCI:, 7.5 mM CDPdipalmitoylglycerol, 5.0 mM myo-[2-3H]Inositol, 6.0 mM, 1.79 mCi/mmol Chloroform-methanol-H20 (I : 2 : 0.3, by volume) Chloroform KC1, 1.0 M Assay Procedure. A final volume of 0.5 ml is achieved by the addition of 0.1 ml each of buffer, MnCI2, CDPdipalmitoylglycerol, and radioactive myo-inositol, along with sufficient water to achieve the volume after enzyme addition. The contents of the tubes are mixed, the tubes preincubated 5 min in a water bath at 37°, and the reaction started by addition of enzyme. The tubes are then incubated for 30 min at 37°, following which the reaction is terminated by addition of chloroform-methanol-H20. The radioactive product is extracted and the radioactivity measured as described for the cholinephosphotransferase assay. M. L. Robinson and G. M. Carman, Plant Physiol. 69, 146 (1982). S. Sumida and J. B. Mudd, Plant Physiol. 45, 712 (1970).
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Properties Substrate Requirements. Reported Km values for myo-inositol range from 0.045 to 0.3 mM, 26,27 and for CDPdiacylglycerol from 0.027 to 1.35 mM. 26,E7 Excess levels of CDPdiacylglycerol can inhibit the reaction. 27 Metal and pH Requirements. A divalent cation is absolutely required, and Mn 2+ is strongly preferred. E6,27 Detergent Effects. Stimulation of activity can be achieved in some cases by the addition of low concentrations of detergents,27 but inhibition also may result. 27 Phosphatidylinositol : myo-Inositol Phosphatidyltransferase Phosphatidylinositol + myo-inositol* ---> phosphatidylinositol* + myo-inositol
This unusual activity has been reported only twice with plant tissues. E8'29 The role of the enzyme is unknown.
Assay Method Principle. The incorporation of radioactive myo-inositol into phosphatidylinositol, under conditions which minimize the activity of CDPdiacylglycerol : myo-inositol phosphatidyltransferase, is followed, z8 Reagents HEPES, 250 mM, pH 8.0 MnC12, 125 mM myo-[2-aH]Inositol, 2.5 raM, 1.79 mCi/mmol Chloroform-methanol-HEO (1:2:0.3, by volume) Chloroform KC1, 1.0 M Assay Procedure. Buffer, MnCI2, and radioactive inositol are added to each assay tube (0.1 ml of each), along with adequate water to give a final volume with enzyme of 0.5 ml. The tubes are mixed, preincubated at 37° for 5 min, and the reaction started by adding enzyme. The tubes are shaken in a water bath at 37 ° for 1 hr, followed by the addition of 3.3 ml of chloroform-methanol-HEO to stop the reaction. Extraction and Radioactivity Measurement. The radioactive product is extracted into chloroform and the radioactivity measured as described for the cholinephosphotransferase. 27 j. C. Sexton and T. S. Moore, Plant Physiol. 62, 978 (1978). 2s j. C. Sexton and T. S. Moore, Plant Physiol. 68, 18 (1981). 29 D. J. Morr6, B. Gripshover, A. Monroe, and J. T. Moore, J. Biol. Chem. 259, 15364 (1984).
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NUCLEI, ER, AND PLASMA MEMBRANE
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Properties Substrate and Phospholipid Effects. The apparent Km for myo-inositol is 26 /zM. 2s Phosphatidylinositol from yeast and castor bean endosperm stimulates the reaction, but that from soybean does not2S; however some other phospholipids can also stimulate the response, z8 Metal and pH Requirements. A divalent cation is absolutely required, and Mn 2+ is strongly preferred. 2s The pH optimum is 8.0. 2s Detergent Effects. Triton X-100 stimulates the reaction at concentrations up to 0.025% (W/W). 28 Nucleotide Effects. CMP, CDP, and CTP all stimulate the reaction 15-fold at 40/~M, while CDPcholine, CDPethanolamine, and CDPdiacylglycerol all stimulate to a lesser extent. 28 Acknowledgments Much of the work described here was supported by grants from the National Science Foundation, with preparation of this manuscriptbeing partially supported by NSF Grant PCM-8402001.
P H O S P H O G L Y C E R I D E S Y N T H E S I S IN E R
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[55] P h o s p h o g l y c e r i d e S y n t h e s i s in E n d o p l a s m i c R e t i c u l u m By THOMAS S. MOORE, JR.
The endoplasmic reticulum of plants is thought to play a major role in membrane phosphoglyceride synthesis, L2 similar to the situation in animals, 3 although it is known that the Golgi apparatus, mitochondria, and plastids 1,2 also play a role. The production of phospholipids by an in vitro microsomal fraction was first demonstrated by Sastry and Kates in 1966,4 and such fractions, probably consisting of endoplasmic reticulum (ER) plus various other membranes, have been reported to synthesize a variety of phospholipids.l,2 The purity of the organelle fractions in many studies is uncertain, and so the relative roles of the ER, Golgi apparatus, and other membranes cannot be clearly argued for most plant systems. The castor bean endosperm system has the advantage of a reduced contribution from the Golgi apparatus as well as clean separation of the other organelles present on sucrose density gradients, and the clear compartmentation of most of the phospholipid synthesis into the endoplasmic reticulum of that tissue supports a major role for that organelle in synthesis of many organelle membranes. 5 For these reasons, the enzyme assays described below are based primarily on work with castor bean endosperm (see Fig. 1). EC 2.7.8.2 Cholinephosphotransferase (CDPcholine: 1,2-Diacylglycerol Cholinephosphotransferase) CDPcholine + sn-l,2-diacylglycerol~ phosphatidylcholine+ CMP This activity usually is the most active of the phospholipid synthesizing enzymes, 6 which may be a reflection of its product being the most T. S. Moore, Annu. Rev. Plant Physiol. 33, 235 (1982). 2 T. S. Moore, in "Structure, Function and Metabolism of Plant Lipids" (P.-A. Siegenthaler and W. Eichenberger, eds.), p. 83. Elsevier Science, Amsterdam, New York, 1984. 3 R. M. Bell and R. A. Coleman, Annu. Rev. Biochem. 49, 459 (1980). 4 p. S. Sastry and M. Kates, Can. J. Biochem. 44, 459 (1966). 5 T. S. Moore, this series, Vol. 71, p. 596. 6 T. S. Moore and G. D. Troyer, in "Biosynthesis and Function of Plant Lipids" (W. W. Thomson, J. B. Mudd, and M. Gibbs, eds.), p. 16. Am. Soc. Plant Physiol., Rockville, Maryland, 1983.
METHODS IN ENZYMOLOGY, VOL. 148
Copyright © 1987by Academic Press, Inc. All rights of reproduction in any form reserved.
586
NUCLEI, ER, AND PLASMA MEMBRANE
[55]
CMP CDPCho ,~ ~PtdCho
DAG~Aqz''''''7
Ptd
"
,
CDP-DAGe G ~ CMPPtdGroP~PtdGro Pi FIG. 1. Outline of the pathways of phosphoglyceride synthesis in plant endoplasmic reticulum.'The enzymes are indicated by their nomenclature numbers, which correlate with the text. The phosphatidate phosphatase (EC 3.1.3.4) has not been adequately described in plant endoplasmic reticulum, and so is not discussed. The two exchange enzymes (producing Ptdlns and PtdSer) have not been assigned a number. Abbreviations: CDP-DAG, CDPdiacylglycerol; DAG, diacylglycerol; Ptd, phosphatidate; PtdCho, phosphatidylcholine; PtdEth, phosphatidylethanolamine; PtdGro, phosphatidylglycerol; PtdGroP, phosphatidylglycerol-P; PtdIns, phosphatidylinositol; PtdSer, phosphatidylinositol.
abundant phospholipid of many membranes.7,8 The activity was first characterized from plants in 1971.9
Assay Method Principle. The chloroform-soluble product radiolabeled from the water-soluble CDPcholine is extracted from the reaction mixture following the assay and its radioactivity determined.~° Reagents Tris-HCl, 50 mM, pH 7.5 Dithiothreitol, 5 mM MgCI2, 100 mM Diacylglycerol, from egg phosphatidylcholine, 2 mM 7 C. Hitchcock and B. W. Nichols, "Plant Lipid Biochemistry." Academic Press, New York, 1971. 8 M. Kates, Adv. Lipid Res. 8, 225 (1970). 9 K. A. Devor and J. B. Mudd, J. Lipid Res. 12, 403. 10T. S. Moore, Plant Physiol. 57, 382 (1976).
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CDP[1,2A4C]choline, 1 mM, 5 mCi/mmol Chloroform-methanol-H20 (1 : 2 : 0.3, by volume) Chloroform KC1, 1.0 M Assay Procedure. Equal volumes of buffer, dithiothreitol, MgCI2, and diacylglycerol are mixed and 0.4-ml aliquots placed into test tubes. Fiftymicroliter aliquots of enzyme are added and the reaction started by the addition of 50/zl of the CDPcholine. The assay is run for 30 min with shaking in a water bath at 30° and is stopped by the addition of 3.3 ml of the chloroform-methanol-water mixture followed by mixing with a vortex mixer. Extraction Procedure. One milliliter of chloroform and 3 ml of 2 M KCI are added to each tube, followed by vigorous mixing. The tubes are centrifuged at about 2500 rpm in a tabletop centrifuge. Following this the upper aqueous layer is removed and discarded. The lower chloroform layer is washed twice more with 1- to 3-ml aliquots of 1 M KC1, aspirating and discarding the aqueous layer each time. Radioactivity Measurement. The chloroform extract is placed into scintillation vials and dried completely. Radioactivity is measured by standard techniques.
Properties Substrate Requirements. The apparent Km of the castor bean enzyme for CDPcholine is about l0/.~M, 1° and similar results have been obtained with other tissues. 9 Estimates have not been made for diacylglycerol, since it occurs in the membrane; some stimulation of activity may be achieved by including this substrate in the assay mixture, however/° p H and Metal Ion Requirements. The optimal pH generally is from 7.51° to 8.0, 9'10 and a divalent cation is required. M g 2+ is preferred over Mn 2÷ by the castor bean enzyme, 1° but the two give equal activities with spinach. 9 Ca 2÷ inhibits the activity. 11,12 Inhibitors. Dithiothreitol promotes and sulfhydryl reagents inhibit the reaction to varying degrees, depending on the tissue source. 9,1° EC 2.7.8.1 Ethanolaminephosphotransferase (CDPethanolamine : 1,2-Diacylglycerol Ethanolaminephosphotransferase) CDPethanolamine + sn-l,2-diacylglycero!--~ phosphatidylethanolamine + CMP
11 j. M. Lord, Plant Physiol. 57, 218 (1976). 12 A. Oursel, A. Tr6moli~res, and P. Mazliak, Physiol. Veg. 15, 377 (1977).
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NUCLEI, E R , AND PLASMA MEMBRANE
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This enzyme catalyzes a reaction similar to the cholinephosphotransferase and with a similar high level of activity) 3-15 It has not, however, been as thoroughly examined and demonstrated not to be catalyzed by the same enzyme that synthesizes phosphatidylcholine.16
Assay Method Principle. The reaction is conducted in the presence of the radioactive, water-soluble substrate, followed by extraction of the chloroform soluble product and measurement of its radioactivity. 15 Reagents MES, 50 mM, pH 6.5 MgC12, 15 mM Dithiothreitol, 5 mM CDP[1,2:4C]ethanolamine, 75/zM, 3.0 mCi/mmol Chloroform-methanol-H20 (1 : 2 : 0.3, by volume) Chloroform KC1, 1.0 M Assay Procedure. Equal quantities (0.1 ml each) of buffer, MgC12, and dithiothreitol are combined with 0.1 ml of H20 in a test tube. The enzyme is added, the mixture preincubated at 37°, and the reaction started by the addition of 0.05 ml CDPethanolamine. The final volume is 0.5 ml. The tubes are incubated with shaking at 37° for 30 min and the reaction stopped by the addition of 3.3 ml of chloroform-methanol-H20. Extraction and Radioactivity Measurement. The extraction and radioactivity measurements were as described for the cholinephosphotransferase assay above.
Properties Substrate Requirements. The apparent Km of the enzyme from castor bean endosperm for CDPethanolamine is 6.0/zM, while that for spinach is 20/zM. 14.15Maximum velocities are achieved at 50 to 100/xM CDPethanolamine. 14,15 CDPcholine is a strong competitive inhibitor of ethanolamine incorporation) 4,15 Diacylglycerol requirements have not been defined due to its natural occurrence in the membranes. Metal and pH Requirements. Mg2÷ is most effective for the castor 13 M. O. Marshall and M. Kates, Can. J. Biochem. 52, 469 (1974). ~4B. A. Macher and J. B. Mudd, Plant Physiol. 53, 171 (1974). ~5 S. A. Sparace, L. K. Wagner, and T. S. Moore, Plant Physiol. 67, 922 (1981). 16j. M. Lord, Biochem. J. 151, 451 (1975).
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PHOSPHOGLYCERIDE SYNTHESIS IN E R
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bean enzyme, being effective at 3 mM, 15 but for spinach Mn 2÷ has been found to be best, giving an optimal reaction between 0.6 and 2 m M ) 4 The optimum pH ranges from 6.5 for castor bean to 7.0-8.0 for spinach) 4,15 E C 2.1.1.17 Phosphatidylethanolamine Methyltransferase (S-Adenosyl-L-Methionine :Phosphatidylethanolamine N-Methyltransferase) Phosphatidylethanolamine + 3 S-adenosyl-L-methionine--~ phosphatidylcholine + S-adenosylhomocystcine
In castor bean endoplasmic reticulum this reaction occurs with about 10% of the activity of the cholinephosphotransfcrase, l° It also has been described from spinach leaf~3 and potato tuber.17 It is not certain how many enzymes are involved in this three-step methylation.
Assay Method Principle. S-Adenosyl-L-[methyl.14C]methionine is fed as the aqueous substrate and the radioactive phosphatidylcholine product is extracted into chloroform, followed by measuring its radioactivity.l° Reagents Tris-HC1, 250 mM, pH 9.0 Phosphatidylethanolamine, 1.0 mM
S-Adenosyl-t-[methyl-14C]methionine, 1.0 mM, 1 mCi/mmol HC1, concentrated Chloroform-methanol-H20 (1 : 2 : 0.3, by volume) Chloroform KCI, 1.0 M Assay Procedure. Aliquots of buffer, phosphatidylethanolamine, and S-adenosylmethionine (0.1 ml each) are added to assay tubes along with sufficient water to give a final volume (with enzyme) of 0.5 ml. The tubes are preincubated at 37° for 5 rain, after which the reaction is started by the addition of enzyme and the tubes incubated with shaking in a water bath at 37° for 30 min. The reaction is stopped by the addition of 50/~1 of HCI, followed by 3.3 ml of the chloroform-methanol-H20 mixture, and then incubated at room temperature for 1 hr. Extraction and Radioactivity Measurement. The radioactive product is extracted into chloroform and the radioactivity measured as described for the cholinephosphotransferase assay. 17 W. Tang and P. A. Castelfranco, Plant Physiol. 43, 1232 (1968).
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NUCLEI, E R , AND PLASMA MEMBRANE
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Properties Substrate Requirements. The castor bean endoplasmic reticulum reaction(s) demonstrates an apparent Km for S-adenosylmethionine of 31 /.tM; phosphatidylethanolamine stimulates only about 33% 1° and does not stimulate the spinach activity.~4 The single- and double-methylated lipid intermediates stimulate the reaction in both tissues) °,~4 pH Requirement. The optimal pH for the enzyme is around 8.0 to 9.0 in both castor bean and spinach.~°.~4 Products. The product of the reaction under these conditions is primarily phosphatidylcholine, but the mono- and dimethylated intermediates also may occur) °,14 Phosphatidylethanolamine
: L-Serine Phosphatidyltransferase
Phosphatidylethanolamine + L-serine --> phosphatidyiserine + ethanolamine
Phosphatidylserine generally occurs only in small concentrations in plant tissues, but may play an important role) 8 The reaction described here is an exchange-type reaction (exchanging serine for ethanolamine), but another reaction utilizing CDPdiacylglycerol and t-serine also has been described in plants. ~3
Assay Procedure Principle. The CaE+-stimulated exchange reaction is measured by the incorporation of the water-soluble L-serine into the chloroform-soluble lipid product. 19 Reagents HEPES, 200 mM, pH 7.8 CaCI2, 10 mM L-[3-~4C]Serine, 2.0 mM, 2.5 mCi/mmol Chloroform-methanol-H20 (1 : 2 : 0.3, by volume) Chloroform KC1, 1.0 M Assay Procedure. Aliquots (0.1 ml each) of the buffer, CaClz, and Lserine, plus an appropriate amount of water to give a final volume (including enzyme) of 0.5 ml, are added to assay tubes. The tubes are preincuis N. Murata, N. Sato, and N. Takahashi, in "Structure, Function and Metabolism of Plant Lipids" (P.-A. Siegenthaler and W. Eichenberger, eds.), p. 153. Elsevier, Amsterdam, 1984. 19T. S. Moore, Plant Physiol. 56, 177 (1975).
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PHOSPHOGLYCERIDE SYNTHESIS IN E R
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bated at 30° for 5 min and the reaction started by adding enzyme. Further incubation at 30°, with shaking in a water bath, is performed for 60 min. The reaction is stopped by the addition of 3.3 ml of chloroform-methanol-H20 followed by incubation on ice for 30 min. Extraction and Radioactivity Determination. The extraction and measurement of radioactivity in the product were as described for the cholinephosphotransferase assay.
Properties Substrate Requirements. The Km for serine is about 20/zM. 19 Ethanolamine was found to compete with L-serine but D-serine and choline did not. 19 Phosphatidylethanolamine stimulated the reaction, but it may be nonspecific since CDPdiacylglycerol did also. 19 Metal and pH Requirements. Calcium is required, with the optimum concentration being 2 mM. 19 The best pH at that Ca 2÷ concentration is 7.8, with a shift to a lower pH at higher Ca 2÷ concentrations. 19 Products. Both phosphatidylserine and phosphatidylethanolamine occurred as products of the reaction, suggesting the presence of a decarboxylase enzyme in the endoplasmic reticulum.19 EC 2.7.7.41 Phosphatidate Cytidylyltransferase (CTP : Phosphatidate Cytidylyltransferase) CTP + phosphatidate --* CDPdiacylglycerol + PP~
This enzyme produces a lipid which serves as a precursor to the synthesis of both phosphatidylglycerol and phosphatidylinositol in the endoplasmic reticulum. CDPdiacylglycerol occurs at only very low concentrations in membranes. 2°
Assay Method Principle. The enzyme utilizes the water-soluble substrate, CTP, labeled in the cytidine ring and produces the chloroform-soluble CDPdiacylglycerol which is extracted and its radioactivity measured. 2° Reagents MES, 50 mM, pH 6.5 MnC12, 37.5 mM Phosphatidate, 12.5 mM, derived from egg lipids [3H]CTP, 0.6 mM, 32.2 mCi/mmol 20 K. F. Kleppinger-Sparace and T. S. Moore, Plant Physiol. 77, 12 (1985).
592
NUCLEI, ER, AND PLASMA MEMBRANE
[SS]
Chloroform-methanol-H20 (1 : 2 : 0.3, by volume) Chloroform KCI, 1.0 M Assay Procedure. One-tenth-milliliter fractions of buffer, MnCI2, phosphatidate, and CTP are added to an assay tube, along with sufficient water to make a final volume (after enzyme addition) of 0.5 ml. The reaction is initiated by the addition of enzyme. This mixture is incubated at 37° for 30 min in a shaker waterbath. The reaction is stopped by the addition of 3.3 ml of the chloroform-methanol-water solution. Extraction and Radioactivity Measurement. These are carded out as described for the cholinephosphotransferase assay above.
Properties Substrate Requirements. The apparent Km of the castor bean enzyme for CTP is about 17/zM, z° which is lower than those reported for other plant microsomal activities. 21 The best stimulation is apparent with mixed phosphatidates, but endogenous lipid substrate prevents an estimation of the precise requirements. 2° pH and Metal Ion Requirements. Manganese is strongly preferred over Mg 2÷ and was optimal at 7.5 mM. The best pH is 6.5. 20 Detergent Effects. Both deoxychoclate and Triton X-100 strongly inhibit the enzyme at concentrations as low as 0.01% (w/w). 2° EC 2.7.8.5 CDPdiacylglycerol-Glycerol-3-phosphate 3-Phosphatidyltransferase (CDPdiacylglycerol : sn-Glycerol-3-phosphate 3-Phosphatidyltransferase) EC 3.1.3.27 Phosphatidylglycerophosphatase (Phosphatidylglycerophosphate Phosphohydrolase) sn-l-Glycerol 3-phosphate + CDPdiacylglycerol
3-sn-phosphafidyl-l'-sn-glycerol 3'-phosphate + CMP 3-sn-Phosphatidyl-1 '-sn-glycerol Y-phosphate ~ 3-sn-phosphatidyl-I '-sn-glycerol + Pi
These two enzymes have not been separated from plant tissues, but the reaction is strongly pulled to the production of phosphatidyglycerol and so assays measure the production of that final product. 22 The reac-
21 j. Bahl, T. GuiUot-Salomon, and R. Douce, Physiol. Veg. 8, 55 (1970). zz T. S. Moore, Plant Physiol. 54, 164 (1974).
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PHOSPHOGLYCERIDE SYNTHESIS IN E R
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tions were first demonstrated in spinach 4 and have since been studied further in spinach 23 as well as in cauliflower 24 and castor bean. 22
Assay Method Principle. Radioactive sn-glycerol 3-phosphate is incorporated into a chloroform-soluble product, which is extracted and the radioactivity measured. 22 Reagents Tris-HCl, 2.0 M, pH 7.3 MnC12, 40 mM Triton X-100, 0.6% (w/w) CDPdiacylglycerol, 0.8 mM sn-[U-14C]Glycerophosphate, 4.0 mM, 1.9 mCi/mmol Chloroform Methanol KCI, 1.0 M Assay Procedure. Twenty-five microliters each of buffer, MnC12, detergent, and the two substrates are added to assay tubes along with sufficient water to give a final volume of 0.2 ml following further additions. The tubes are preincubated for 5 min at 30° in a water bath. Enzyme is added to start the reaction and the tubes are incubated, with shaking, for an additional 1.5 hr. The reaction is stopped by adding 2.0 ml of methanol followed by incubating on ice for 1 hr. Extraction Procedure. Two milliliters of chloroform is added to each assay tube and mixed. The emulsion is broken by centrifugation at 2500 rpm in a tabletop centrifuge. The upper aqueous layer is removed and discarded, and the lower chloroform layer washed twice with 5.0-ml aliquots of the KC1 solution, followed by twice with distilled water. Each time the tubes are mixed, centrifuged, and the upper layer removed. Radioactivity Measurement. This is as described in the section on cholinephosphotransferase.
Properties Substrate Requirements. Calculated Km values for glycerol-P range from 50 to 250 /~M, 22,z3 while that for CDPdiacylglycerol is about 2-3/~M. 22"23 23 M. O. Marshall and M. Kates, Biochirn. Biophys. Acta 260, 558 (1972). 24 R. Douce and J. Dupont, C.R. Hebd. Seances Acad. Sci., Ser. D 268, 1657 (1969).
594
NUCLEI; E R , AND PLASMA MEMBRANE
[55]
Metal and pH Requirements. Mn 2÷ stimulates the reaction best at 5 mM, and Mg 2÷ is only partially effective. 22 The pH optimum is 7.3. 22,23 Detergent Effects. Triton X-100 stimulates at concentrations up to 0.075% (W/W). 22 Products. The major product is phosphatidyglycerol, but the phosphorylated intermediate is found in low quantities in some cases. 22,23 EC 2.7.8.11 CDPdiacylglycerol-Inositol 3-Phosphatidyltransferase (CDPdiaeyglycerol : rayo-Inositol 3-Phosphatidyltransferase) CDP-sn-l,2-diacylglycerol + myo-inositol --> phosphatidylinositol + CMP
This enzyme activity has now been described from several plant species I and solubilized from soybean. 25 It was first described from cauliflower by Sumida and Mudd. 26
Assay Method Principle. Radioactive myo-inositol is incorporated into phosphatidylinositol, which can be extracted into chloroform and its radioactivity determined. 17 Reagents Tris-HC1, 250 mM, pH 8.5 MnCI:, 7.5 mM CDPdipalmitoylglycerol, 5.0 mM myo-[2-3H]Inositol, 6.0 mM, 1.79 mCi/mmol Chloroform-methanol-H20 (I : 2 : 0.3, by volume) Chloroform KC1, 1.0 M Assay Procedure. A final volume of 0.5 ml is achieved by the addition of 0.1 ml each of buffer, MnCI2, CDPdipalmitoylglycerol, and radioactive myo-inositol, along with sufficient water to achieve the volume after enzyme addition. The contents of the tubes are mixed, the tubes preincubated 5 min in a water bath at 37°, and the reaction started by addition of enzyme. The tubes are then incubated for 30 min at 37°, following which the reaction is terminated by addition of chloroform-methanol-H20. The radioactive product is extracted and the radioactivity measured as described for the cholinephosphotransferase assay. M. L. Robinson and G. M. Carman, Plant Physiol. 69, 146 (1982). S. Sumida and J. B. Mudd, Plant Physiol. 45, 712 (1970).
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PHOSPHOGLYCERIDE SYNTHESIS IN E R
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Properties Substrate Requirements. Reported Km values for myo-inositol range from 0.045 to 0.3 mM, 26,27 and for CDPdiacylglycerol from 0.027 to 1.35 mM. 26,E7 Excess levels of CDPdiacylglycerol can inhibit the reaction. 27 Metal and pH Requirements. A divalent cation is absolutely required, and Mn 2+ is strongly preferred. E6,27 Detergent Effects. Stimulation of activity can be achieved in some cases by the addition of low concentrations of detergents,27 but inhibition also may result. 27 Phosphatidylinositol : myo-Inositol Phosphatidyltransferase Phosphatidylinositol + myo-inositol* ---> phosphatidylinositol* + myo-inositol
This unusual activity has been reported only twice with plant tissues. E8'29 The role of the enzyme is unknown.
Assay Method Principle. The incorporation of radioactive myo-inositol into phosphatidylinositol, under conditions which minimize the activity of CDPdiacylglycerol : myo-inositol phosphatidyltransferase, is followed, z8 Reagents HEPES, 250 mM, pH 8.0 MnC12, 125 mM myo-[2-aH]Inositol, 2.5 raM, 1.79 mCi/mmol Chloroform-methanol-HEO (1:2:0.3, by volume) Chloroform KC1, 1.0 M Assay Procedure. Buffer, MnCI2, and radioactive inositol are added to each assay tube (0.1 ml of each), along with adequate water to give a final volume with enzyme of 0.5 ml. The tubes are mixed, preincubated at 37° for 5 min, and the reaction started by adding enzyme. The tubes are shaken in a water bath at 37 ° for 1 hr, followed by the addition of 3.3 ml of chloroform-methanol-HEO to stop the reaction. Extraction and Radioactivity Measurement. The radioactive product is extracted into chloroform and the radioactivity measured as described for the cholinephosphotransferase. 27 j. C. Sexton and T. S. Moore, Plant Physiol. 62, 978 (1978). 2s j. C. Sexton and T. S. Moore, Plant Physiol. 68, 18 (1981). 29 D. J. Morr6, B. Gripshover, A. Monroe, and J. T. Moore, J. Biol. Chem. 259, 15364 (1984).
596
NUCLEI, ER, AND PLASMA MEMBRANE
[55]
Properties Substrate and Phospholipid Effects. The apparent Km for myo-inositol is 26 /zM. 2s Phosphatidylinositol from yeast and castor bean endosperm stimulates the reaction, but that from soybean does not2S; however some other phospholipids can also stimulate the response, z8 Metal and pH Requirements. A divalent cation is absolutely required, and Mn 2+ is strongly preferred. 2s The pH optimum is 8.0. 2s Detergent Effects. Triton X-100 stimulates the reaction at concentrations up to 0.025% (W/W). 28 Nucleotide Effects. CMP, CDP, and CTP all stimulate the reaction 15-fold at 40/~M, while CDPcholine, CDPethanolamine, and CDPdiacylglycerol all stimulate to a lesser extent. 28 Acknowledgments Much of the work described here was supported by grants from the National Science Foundation, with preparation of this manuscriptbeing partially supported by NSF Grant PCM-8402001.