Assay Method - Science Direct

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[31] C h o l i n e p h o s p h o t r a n s f e r a s e f r o m M a m m a l i a n S o u r c e s

By ROSEMARY B. CORNELL Introduction Cholinephosphotransferase (CDPcholine: 1,2-diacylglycerol cholinephosphotransferase, EC 2.7.8.2) catalyzes the final reaction in the synthesis of phosphatidylcholine. It is an integral membrane enzyme found CDPcholine + sn-l,2-diacylglycerol--~ CMP + phosphatidylcholine

predominantly in the endoplasmic reticulum but may also be associated with Golgi, mitochondria, or nuclear membranes in some tissuesJ -4 Cholinephosphotransferase (CPT) activity is distinct from ethanolaminephosphotransferase (EPT) activity in several respects, including sensitivity to detergents, heat, CMP, cation selectivity, 5 and trypsin. 6 CPT and EPT activities have been separated chromatographically5 and genetically. 7

Assay Method Principle. A method is described utilizing microsomes and a diacylglycerol suspension produced by cosonicating diacylglycerol, Tween 20, and phospholipids. Preparation of the substrate in this manner yields the highest reported specific activity. 8 CPT can also be assayed in microsomes using endogenously generated membrane-bound diacylglycerol by stimulation of de novo synthesis of diacylglycerol9or by treatment with phospholipase C l° or CMP. ii An assay for CPT using permeabilized HeLa cells has been described. 12In the method described below [methyl-14C]CDPcholine incorporation into phosphatidylcholine is monitored. The water-soluble I C. L. Jelsema and D. J. Morre, J. Biol. Chem. 253, 7960 (1978). 2 M. D. Sikpi and S. K. Das, Biochim. Biophys. Acta 899, 35 (1987). 3 j. E. Vance and D. E. Vance, J. Biol. Chem. 263, 5898 (1988). 4 R. R. Baker and H. Y. Chang, Can. J. Biochem. 60, 724 (1982). 5 K.-M. O and P. C. Choy, Biochem. Cell Biol. 67, 680 (1989). 6 R. Coleman and R. M. Bell, J. Biol. Chem. 252, 3050 (1977). 7 M. Polokoff, D. C. Wing, and C. R. H. Raetz, J. Biol. Chem. 256, 7687 (1981). 8 j. C. Miller and P. A. Weinhold, J. Biol. Chem. 256, 12662 (1981). 9 H. Ide and P. A. Weinhold, J. Biol. Chem. 257, 14926 (1982). l0 M. G. Sarzala and L. M. G. van Golde, Biochim. Biophys. Acta 441, 423 (1976). n H. Kanoh and K. Ohno, Biochim. Biophys. Acta 326, 17 (1973). iz p. Lira, R. B. Cornell, and D. E. Vance, Biochem. Cell Biol. 64, 692 (1986).

METHODS IN ENZYMOLOGY, VOL. 209

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substrate is separated from the insoluble product by a Bligh-Dyer extraction. 13

Reagents /zl (/~g)/assay (50/zl) 1 M Tris-HC1, pH 8.5 0.2 M MgC12 10 mM EGTA

Final concentration

8 mM [methyl-14C]CDPcholine,

2.5/zl 2.5/~1 2.5/zl 2.5/zl

50 mM 10 mM 0.5 mM 0.4 mM

specific radioactivity 0.5 16 mM sn-l,2-Diolein emulsion Microsomes Water

7.5/xl 30/zg protein To 50/zl

2.4 mM

Procedure 1. [methylJ4C]CDPcholine is purchased from New England Nuclear (Boston, MA, 40-60 mCi/mmol) and diluted with unlabeled CDPcholine (Sigma, St. Louis, MO) to the above concentration and specific radioactivity. 2. An emulsion of 1,2-diolein (Sigma), asolectin (soy phospholipids, Associated Concentrates, Woodside, NY), and Tween 20 (Sigma) is prepared as follows: a. Five milligrams (-8/zmol) diolein from a chloroform stock is dried down under nitrogen in a small round-bottomed glass tube or flask. Then 0.25 mg Tween 20 is added from a 25 mg/ml aqueous stock, and the mixture is vortexed vigorously. b. Asolectin (50 mg/ml water) is vortexed vigorously for 30 min into a homogeneous suspension. One-half milliliter asolectin suspension is added to the diolein-Tween mixture. c. The complete mixture is sonicated under nitrogen for about 5 min using a microtip probe sonicator (e.g., Heat Systems, Plainview, NY, 375 W), with the sample immersed in an ice bath. A suspension appearing homogeneous to the eye results. The emulsion can be used for a few days without a noticeable effect on enzyme activity when stored under nitrogen at 4° , but prolonged storage is accompanied by generation of the 1,3-isomer and inhomogeneity of the suspension. 3. The reaction is set up in disposable 13 x 100 mm glass tubes. Tris, MgCI2, and EGTA are added from a cocktail followed by water, 13 E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).

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enzyme, and diolein. Tubes are vortexed and placed in a 37° shaking water bath. The reaction is initiated with CDPcholine. Diolein and zero-time "blanks" are included. The reaction is terminated after 15 min by addition of 1.5 ml methanol-chloroform (2 : I, v/v). 4. 14C-Labeled lipids are extracted by addition of 0.3 ml water, 0.5 ml chloroform, and 0.5 ml water in sequence with thorough vortexing after each addition. After centrifugation at 2500 rpm for 5 min, the upper layer is removed and the lower chloroform layer is washed with 2 ml theoretical upper phase (methanol-water-chloroform, 48:47:3, v/v). The washed chloroform layer is transferred to a scintillation vial, the solvent is evaporated under nitrogen, scintillation fluid is added, and the sample is counted. The zero-time blank has typically less than 50 disintegrations/min (dpm). Samples lacking exogenous diolein have significant radioactivity depending on the microsome source.14 More than 95% of the radioactivity in the chloroform phase is associated with phosphatidylcholine (PC). The specific activity of CPT in rat liver microsomes when assayed as described is approximately 30 nmol PC formed/min/mg protein. The Km for CDPcholine is around 200/zM using rat liver microsomes and diacylglycerol prepared as above. The K m for diacylglycerol is approximately 150 /xM. 8 Reactions are linear in the range of 5-30 min and 0 to 50/zg rat liver microsomal protein. Solubilization and Partial Purification Kanoh and Ohno reported the first solubilization procedure for rat liver microsomal CPT. 15 CPT was released from the membrane by sonication in a medium containing 5 mM deoxycholate [approximately the critical micelle concentration (CMC)], and 20% glycerol at pH 8.5. Higher concentrations of deoxycholate led to inactivation. The high pH was required for solubilization. The solubilized preparation was reportedly stable to storage at -20°; however, the lipid content of the final preparation was very high (lipid/protein weight ratio of 1.3). Further treatment with Triton X-100 led to inactivation. O and Choy partially solubilized CPT activity from hamster liver microsomes using Triton QS-15.16 The detergent inactivated CPT,but some activity was regained after dialysis. Attempts to purify the solubilized transferase have met with very limited success. Kanoh and Ohno reported a 4-fold purification, to a 14 H. Kanoh and K. Ohno, this series, Vol. 71, p. 536. 15 H. Kanoh and K. Ohno, Eur. J. Biochem. 66, 201 (1976). 16 K.-M. O and P. C. Choy, Lipids 25, 122 (1990).

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specific activity of 21 nmol/min/mg, by a two-step sonication and centrifugation protocol using 4-5 mM deoxycholate. 15 This is described in detail elsewhere in this series.14 O and Choy reported a 7-fold purification to a specific activity of 3.7 nmol/min/mg after DEAE-Sepharose and Sepharose 6B chromatography.16 Both partially purified preparations appeared to be large lipid-protein aggregates or mixed detergent micelles, judging from the gel filtration elution positions. Further dissolution attempts with higher detergent concentrations led to irreversible inactivation. T M The lack of progress in the purification of mammalian CPT can be attributed to the failure to find conditions suitable for stabilizing the enzyme in the absence of a lipid environment. Reconstitution of Cholinephosphotransferase Activity after Detergent Inactivation Cornell and MacLennan compared the detergent concentration curves for inactivation of CPT and solubilization of sarcoplasmic reticulum membranes containing CPTJ 7 Detergent concentrations required to convert membrane vesicles to detergent-mixed micelles as assessed by loss of turbidity inevitably led to greater than 90% inactivation of CPT. Cholate, deoxycholate, sodium dodecyl sulfate (SDS), Tween 20, octylglucoside, and Triton X-100 were tested. 17 Rat liver CPT is also severely inhibited by 3-[(3-cholamidopropyl)dimethylammonio]-lpropanesulfonate (CHAPS) and Zwittergent solubilization of microsomes. At relatively low detergent concentrations (detergent/protein weight ratio less than 3), even though the solubilized enzyme is inactive, the activity can be reconstituted by addition of a 5-fold excess of crude lipid, such as asolectin, and removal of detergent by dialysis, gel filtration, BioBeads (Bio-Rad, Richmond, CA), or other means effective for the particular detergent. Reconstitution from Triton, octylglucoside, deoxycholate, or cholate solutions has been successful. Solubilization at high detergent/ protein ratios (> 10, w/w) results in irreversible inactivation unless diacylglycerol is present during both the solubilization and reconstitution steps. Glycerol and diacylglycerol also stabilize rat brain EPT activity. Inclusion of these compounds in column chromatography buffers was a key factor in the 37-fold purification of EPT from rat brain. The stabilization by diacylglycerol may be an example of substrate stabilization of the active conformation of the enzyme. An example of a reconstitution protocol using cholate to solubilize microsomes is outlined below. Solubilization. One milliliter (4 mg) microsomal protein in 10 mM Tris17 R. B. Cornell and D. H. MacLennan, Biochim. Biophys. Acta 821, 97 (1985).

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HCI, pH 8, 0. I mM phenylmethylsulfonyl fluoride (PMSF), 20% glycerol (T/G buffer) is added to 1 ml of a suspension containing 40 mg/ml (~100 mM) cholate, 40% glycerol, 30 mg/ml asolectin, and 10 mg/ml diacylglycerol. The mixture is vortexed and incubated 30 min at 00-4 ° followed by centrifugation at 4° for 30 min at 200,000 g. Pellets are minute or undetectable. Reconstitution. All steps are performed at 4°. Twenty milligrams of additional asolectin is added to the supernatant. The detergent is removed in two steps. (1) The sample is applied to a 20 ml Sephadex G-25 column equilibrated and eluted with T/G buffer. The turbid void volume is collected. (2) The turbid fractions from the Sephadex column are dialyzed against 100 volumes of T/G buffer for at least 24 hr. Bio-Beads SM-2 (Bio-Rad) can be added to the dialysis buffer to absorb detergent and accelerate its removal from the sample. Centrifugation of the samples as above results in sedimentation of the CPT activity. The pellets can be resuspended in a small volume of T/G buffer using a glass-Teflon type tissue grinder and stored at - 7 0 °. Properties

Detergent Sensitioity. According to several reports low, subsolubilizing concentrations of detergents such as Tween 20,18 Triton, 17'18lysolecithin, 19and deoxycholate 15stimulate CPT activity. The stimulation is probably related to their ability to disperse diacylglycerol, and it would depend on the manner of presentation of the diacylglycerol. Membrane-solubilizing concentrations of detergent (i.e., above the CMC or above a detergent/ lipid weight ratio of 2) lead to nearly complete inactivation): Several types of anionic, zwitterionic, and nonionic class A and B detergents have been tried. The effects of cationic detergents, however, have not been reported. CPT may be one of the most detergent-sensitive enzymes to confront lipid enzymologists. Its acute detergent sensitivity suggests that sites critical to its activity are specifically dependent on a phospholipid environment. Metal Ion Requirement. The CPT reaction is selective for Mg2÷ in most animal tissues, although Mn 2÷ can substitute at least partly in the reaction catalyzed by partially purified CPT from hamster liver~ and platelet microsomal CPT. 2° M g 2+ o r M n 2+ in the range of 5-10 mM is optimal. 18G. Arthur, S. Tam, and P. C. Choy, Can. J. Biochem. Cell Biol. 62, 1059 (1984). 19 S. Parthasarathy and W. J. Baumann, Biochem. Biophys. Res. Commun. 91, 637 (1979). 2o S. Taniguchi, S. Morikawa, H. Hayashi, K. Fujii, H. Mori, M. Fujiwara, and M. Fugiwara, J. Biochem. (Tokyo) 100, 485 (1986).

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Ca 2+ is inhibitory in the standard assay in the 10-100/zM range, and the inhibition is competitive with Mg 2÷ or Mn2÷. 2° Topography on Microsomal Membrane. The resistance to extraction from membranes with high salt, EDTA, or low detergent concentrations and the inactivation that coincides with detergent-mediated solubilization of membrane bilayers suggest that CPT is an integral membrane protein. 17 Since CDPcholine is impermeable to intact microsomal membranes, one would anticipate that the active site of CPT faces the cytosol. Evidence supporting this notion includes inactivation by membrane-impermeant proteases or mercury-dextran. 21-23 Reversibility. Incubation of microsomes with CMP inhibits the incorporation of [14C]CDPcholine into PC by stimulation of the back-reaction of CPT. 24 CMP at 0.5 mM inhibits rat liver microsomal CPT 50% when assayed by the method described above. The back-reaction can be used to generate endogenous diacylglycerol. The Km for CMP in the backreaction ranges from 0.18 to 0.35 mM depending on the source. 11,25Pitfalls associated with this procedure have been described in an earlier volume of this series./4 21 R. Coleman and R. M. Bell, J. Cell Biol. 76, 245 (1978). 22 D. E. Vance, P. C. Choy, S. B. Farren, P. Lira, and W. J. Schneider, Nature (London) 270, 268 (1977). 23 L. M. Ballas and R. M. Bell, Biochim. Biophys. Acta 602, 578 (1980). 24 G. Goracci, P. Gresele, G. Arienti, P. Porrovecchio, G. Nenci, and G. Porcellati, Lipids 18, 179 (1983). 2~ G. Goracci, E. Francescangeli, L. Horrocks, and G. Porcellati, Biochim. Biophys. Acta 664, 373 (1981).

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Saccharomyces cerevisiae By RUSSELL H. HJELMSTAD and ROBERT M. BELL Introduction Phosphatidylcholine (PC) and phosphatidylethanolamine (PE), the principal phospholipids of eukaryotic membranes, are synthesized from the common precursor sn-1,2-diacylglycerol and CDPcholine or CDPethanolamine, respectively, in reactions catalyzed by membrane-bound amino alcohol phosphotransferases. 1-3 Comparative enzymological studies in l E. P. Kennedy and S. B. Weiss, J. Biol. Chem. 222, 193 (1956).

METHODSIN ENZYMOLOGY,VOL. 209

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