- Email: [email protected]
[25] Assay for lecithin: Cholesterol acyltransferase
[25]
ASSAY FOR LECITHIN :CHOLESI"EROL
ACYLTRANSFERASE
375
phase and in chloroform-methanol, 2 : 1, and its fluorescence in the two respective solvents (FDtp, Fc.~l) is recorded in the fluorometer available to the investigator. Since lecithin is insoluble in this medium, it is dissolved in chloroform-methanol, 2 : 1, and its fluorescence is recorded and related to the phosphorus content, thus providing the fluorescence per nanomole of substrate (MFs). The quantity of fatty acid released (P) is obtained by recording its fluorescence (Fp) and utilizing the following equation: p=
Fv x Fc.~l MF~ ~ x91
The multiplication factor, 91, is the mole ratio of total to fluorescent lecithin. In this procedure we used two enzyme preparations and the buffer of Verger et al.28 However, other phospholipases can be assayed with the fluorescent lecithin using established procedures. Comparison with radioactively labeled phosphatidylcholine showed that the two respective enzymes utilize the fluorescent and radioactive molecules with very similar rates.
[25]
A s s a y for L e c i t h i n : C h o l e s t e r o l A c y l t r a n s f e r a s e By RoY B. VERDERY III and SmMON GATT
Lecithin + cholesterol ~ cholesteryl ester + lysolecithin Assay Method The enzyme can be assayed by determining the disappearance of unesterified cholesterol, by measuring the conversion of radioactive cholesterol to radioactive cholesteryl ester, or by observing the incorporation of radiolabeled fatty acids from lecithin into cholesteryl esters. The reaction is usually run near pH 7 in the presence of a chelating agent to inhibit heavy metal-catalyzed peroxidation of the lecithin and in the presence of a reducing agent, which is usually necessary for maximum enzyme activity. 1 t j. A. Glomset,
Biochim. Biophys. Acta
METHODS IN ENZYMOLOGY, VOL. 72
6 5 , 128 ( 1 % 2 ) . Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
376
GENERAL ANALYTICAL METHODS
[25]
Method 12,3 Principle. The quantity of unesterified cholesterol is measured by gasliquid chromatography (GLC). Reagents Cholesterol, 0.5/.tmol plus lecithin, 1.0 ftmol dissolved in 200 gl of absolute ethanol Stigmasterol, 100 ftg/ml dissolved in chloroform : methanol, 1 : 2 (v/v) Buffer, 0.154 M NaCI, 1 mM EDTA, pH 7.4, 20 mM 2mercaptoethanol Hexane, chloroform, methanol Procedure. Inject the solution containing lecithin and cholesterol through a fine needle into 4 ml of buffer undergoing rapid stirring to form liposomes. 4 The liposome suspension, 100 ftl, is mixed with I00 ftl of enzyme at 4°. After incubation for 30 min at 37°, the reaction is stopped by addition of 0.65 ml of chloroform : methanol, 1 : 2. Stigmasterol solution, 100 pl, is then added as an internal standard, followed by chloroform, 0.25 ml, and water, 0.25 ml. After a brief centrifugation, the upper phase is discarded, the lower phase is dried under nitrogen or air, and the lipids are dissolved in hexane and analyzed by GLC. For separation of cholesterol and stigmasterol, a 3 mm × 60 cm column of 3% OV-1 on 80/100 Gas Chrom Q (Supelco, Bellefonte, Pennsylvania) temperature-programmed from 200° to 230° at 3°/min is sufficient. Zero time controls are obtained by stopping the reaction before incubation. Standards are prepared by mixing I0 ftg of cholesterol with 100 ftl of the stigmasterol solution, drying under air, dissolving in hexane and analyzing by GLC as described previously. Variation for Assay in Plasma or Serum. To determine LCAT4a under reducing conditions, plasma or serum, 100/xl, is mixed with buffer, 100 ftl, at 4°, and the mixture is incubated for 30 min at 37°. Termination of the reaction and determination of cholesterol is accomplished as described above except that a stigmasterol solution of 1 mg/ml is employed. For determination in the absence of exogenous reducing agents, the plasma, 100 ftl, is incubated first and the buffer, 100 fd, is added afterward. Calculations. The extent of the reaction is the difference between the quantity of unesterified cholesterol before and after incubation, the enzymatic activity in nanomoles per milliliter per hour can be calculated as follows: 2 W. M. Sperry, J. Biol. Chem. 111,467 (1935). 3 y. L. Marcel and C. Vezina, Biochim. Biophys. Acta 306, 497 (1973). 4 S. Batzri a n d E. D. K o r n , Biochim. Biophys. Acta 298, 1015 (1973). 4a The abbreviation L C A T stands for lecithin : cholesterol acyltransferase.
[25]
ASSAY FOR LECITHIN"CHOLESTEROLACYLTRANSFERASE
377
LCAT activity = 20 x (cholesterolt=0 - cholesterolt=3o) Since only a small portion of the cholesterol is esterified in this procedure, 5 or 6 replicate determinations before and after incubation must be m a d e ? Method H 5 Principle. Radioactive cholesterol, equilibrated with endogenous cholesterol in heat-inactivated plasma, is converted to radioactive cholesteryl esters that are isolated by thin-layer chromatography (TLC). Reagents
Plasma containing anticoagulant, heated 1 hr at 60° Bovine serum albumin, fraction V, 0.5 g dissolved in 10 ml of 0.154 M NaCI cholesterol (3H or 14C), 10/xCi dissolved in 0.5 ml of absolute ethanol 2-Mercaptoethanol T L C plates of silica gel G Methanol, chloroform, hexane, diethyl ether Procedure. Inject the solution of radioactive cholesterol, 10/xCi, into the albumin solution through a fine needle under the surface. 6 Blow nitrogen across the top o f the mixture until the smell of alcohol is gone. Mix the dispersion with 200 ml of heat-inactivated plasma at 4 °, stir overnight at 4 °, divide into aliquots and store this plasma substrate at - 2 0 °. For the assay, thaw an aliquot of frozen plasma substrate and add 2-mercaptoethanol (0.15% v/v). Mix 100/xl with 100 /xl of enzyme, at 4°, and then incubate for 30 min at 37°. The reaction is stopped by the addition of 1.3 ml c h l o r o f o r m : m e t h a n o l , 1: 2. After a brief centrifugation, 1.0 ml of the supernatant is transferred to another tube, leaving behind the protein. Chloroform (0.3 ml) and water (0.4 ml) are then added and mixed using a vortex mixer, and the two phases are separated by a brief centrifugation. The supernatant phase is discarded, the infranatant is dried under air, and the lipids are dissolved in 200 /zl of hexane. An aliquot, 100/zl, of the hexane is applied to a plate coated with 0.25 mm silica gel G. After developing in hexane : diethyl ether, 90 : 20 (v/v), the cholesteryl ester spot (R s value about 0.97) is visualized by exposure to iodine, scraped, and counted. Residual LCAT activity in heated plasma varies among preparations; therefore, a blank consisting of plasma substrate and normal saline, or another appropriate buffer without enzyme, must be determined. To minimize this blank, the temperature of the plasma must be maintained at 5 j. A. Glomset and J. L. Wright, Biochim. Biophys. Acta 89, 266 (1964). 6 D. Porte and R. J. Havel, J. LipidRes. 2, 357 (1961).
378
GENERAL ANALYTICAL METHODS
[25]
60° for 1 hr, care must be taken, however, to prevent denaturation of the proteins. Calculations. LCAT activity is calculated by the following formula: LCAT activity =
cholesteryl ester(dpm in sample - dpm in blank) unesterified cholesterol specific activity × dilution factor
The dilution factor for the assay as described is 60, and, if the specific activity is measured in units of disintegrations per minute (dpm) per nanomole, the units of enzyme activity are nanomoles per milliliter per hour. M e t h o d III 7 Principle. Radiolabeled lecithin is incubated with cholesterol, and incorporation of the labeled fatty acids into cholesteryl esters is observed. The products are separated from the labeled substrate by a solvent partition step. The blank is reduced by treating the neutral lipid fraction after the partition with silicic acid; separation of fatty acids from cholesteryl esters is obtained by extraction of the washed neutral lipid fraction with a basic aqueous solution, Reagents
Cholesterol, 0.5/xmol, egg lecithin, 1.0/zmol, [14C]lecithin, 1.0/zCi, dissolved in 200/zl of absolute ethanol Buffer, 0.154 M NaCI, 1 mM EDTA, pH 7.4, 20 mM 2-mercaptoethanol Dole's mixtureS: heptane : isopropyl alcohol: 1N H2SO4, 400: 100:10 Silicic acid (Mallinckrodt, St. Louis, Missouri) activated by heating at 100° NaOH, 0.1 N H2SO4, 0.2 N Heptane, water Procedure. Inject the 200 ~1 of ethanolic solution of lecithin and cholesterol containing [14C]lecithin into 4.0 ml of buffer to form substrate liposomes. 3 Mix the liposomal dispersion (100/xl) with enzyme (100/zl) at 4° and incubate at 37° for 30 rain. The reaction is stopped by adding 2.0 ml of Dole's mixture. Heptane, 1.2 ml is then added and mixed thoroughly, followed by 1.0 ml of water. After thorough mixing, the phases are separ R. B. Verderyand S. Gatt, Clin. Chem. (submittedfor publication). 8 V. P. Dole,J. Clin. Invest. 35, 150 (1956).
[25]
ASSAY FOR L E C I T H I N ." C H O L E S T E R O L A C Y L T R A N S F E R A S E
379
rated by centrifugation, and 1.0 ml of the upper heptane-rich phase is transferred to another tube containing 200-300 mg of silicic acid and 2.0 ml of heptane. After thorough mixing, the silicic acid is sedimented by a brief centrifugation; the heptane is transferred to a scintillation vial, and its radioactivity is determined on a liquid scintillation spectrometer. Blanks are determined by incubating substrate liposomes with 100/zl of buffer without enzyme. Calculations. LCAT activity is calculated using the following formula: LCAT activity = (dpm of sample - dpm of blank) × dilution factor specific activity of fatty acid in/3 (2)-position of lecithin The dilution factor in the above assay as described is 28, and if the specific activity of the 2-position fatty acid is expressed in units of disintegrations per minute per nanomole, the units of LCAT activity are nanomoles per milliliter per hour. Two factors affect the specific activity of the 2-position fatty acid: the proportion of radioactivity in the 2-position fatty acid of the "uniformly labeled" [14C]lecithin and the amount of endogenous lecithin in the reaction mixture. The proportion of radioactivity in the 2-position of lecithin from one batch of uniformly labeled lecithin from algae (Chlorella vulgaris, New England Nuclear, Boston, Massachusetts) was 0.68 as determined by treatment with snake venom 9 and thin-layer chromatography of the product fatty acids and lysolecithin. If the enzyme preparation contains significant amounts of lecithin, this must be determined TM and added to the 25 nmol present in the liposomal dispersion. Variation for Improved Specificity. Some LCAT preparations display substantial phospholipase activity. The earlier-described assay does not distinguish between radioactivity due to fatty acids resulting from phospholipase activity and cholesteryl esters from transferase activity. To differentiate between these activities, 1.5 ml of the silicic acid-treated heptane solution is mixed with 1.0 ml of 0.1 N NaOH. After mixing and centrifuging, 1.0 ml of the upper hexane phase is counted to determine transferase activity. One milliliter of hexane and 1.0 ml of 0.2 N H2SO4 are then added. After mixing and centrifuging, 1.0 ml of the upper hexane phase is counted to determine phospholipase activity. A 1.0-ml aliquot of the silicic acid-treated heptane which has not been extracted with NaOH is counted as well. Transferase and total activities are calculated from these counts, respectively, using the formula given previously except that a dilution factor of 84 is employed. Phospholipase activity is estimated as 9 A. F. Robinsonand W. E. M. Lands,Biochemistry 1,804 (1972). 10j. W. Jones and P. Ways,J. Clin. Invest. 46, 1151 (1967).
380
GENERAL ANALYTICAL METHODS
[25]
the difference between total and transferase activity. Because of the high dilution factor, the accuracy of this variation is not as great as the accuracy of the previously described method, which gives total activity alone. Variation for Assay in Plasma or Serum. Serum or plasma containing anticoagulant is obtained, and DTNB is added to a final concentration of 1.4 mM 1~ to inhibit the LCAT. [~4C]Lecithin (10-100 nCi) in 100/.d of absolute ethanol is injected through a fine needle into 2.0 ml of this plasma or serum undergoing rapid mixing. The mixture of lecithin and plasma is then incubated 30 min at 37° to permit equilibration of the radioactive and endogenous lecithin. The reaction is started by mixing 100 /xl of the plasma containing [~4C]lecithin with 100 /zl of buffer containing 2-mercaptoethanol. The 2-mercaptoethanol reverses the inhibition caused by DTNB. After incubation for 30 min at 37°, the reaction is terminated and LCAT activity is determined as described previously. Accurate calculation of activity in terms of nanomoles per milliliter per hour requires independent determination of the lecithin concentration in the plasma. Remarks The three methods described above were chosen because they have unique features. The first method is a modification of the determination that originally led to the discovery of LCAT.12 The major advantage of this method is that LCAT is determined unambiguously in terms of nanomoles of cholesterol per hour, independent of the requirement t h a t a labeled substrate be completely equilibrated with any endogenous substrate. The disadvantages are its lack of sensitivity, since only small changes in cholesterol content occur during the incubation and the requirement that many time-consuming determinations of cholesterol be made for each assay. The second method is an example of the numerous assay methods that have been used which employ radioactive cholesterol as labeled substrate. The advantage of this particular method is that the incubation mixture contains all the plasma lipoproteins as well as the LCAT activator apolipoprotein, A-I, and the cholesteryl ester transferase protein. It therefore contains all the potential cofactors that affect LCAT activity. There are several major disadvantages to this method: TLC separation of unesterified cholesterol from cholesteryl ester is time consuming; the measured activity may depend on the particular plasma used for preparation of the substrate since the state of nutrition 13 and sex 4 of the donor may change LCAT activity; if the enzyme preparation contains a substantial 1~ K. T. Stokke a n d K. R. N o r u m , Scand. J. Clin. Lab. Invest. 27, 21 (1971). 12 j. A. G l o m s e t , F. Parker, M. Tjaden, and R. H. Williams,Biochim. Biophys. Acta 58, 398 (1962). ~3 H. G. Rose a n d J. Juliano, J. Lab. Clin. Med. 89, 524 (1977).
[25]
ASSAY FOR LECITHIN :CHOLESTEROL ACYLTRANSFERASE
381
amount of cholesterol, isotopic dilution will affect the measured amount of LCAT activity. The third method is a variation of the methods that employ liposomal substrates 14and was developed to provide these two advantages: (a) the procedure utilizes a commercially available radioactive substrate and a rapid method to separate a labeled product from a labeled substrate; (b) it can be used conveniently to assay many samples. The major disadvantage is that if lecithin is present in the enzyme preparation, as in the case for assays in plasma, its amount must be determined before activity can be expressed in absolute units. It should be emphasized that with all these methods the assay in plasma is complicated by the simultaneous presence of variable amounts of both enzyme and substrate and the possibility that variable amounts of activating cofactor proteins might also be present. Partial Purification Purification of LCAT from plasma is made difficult by the large amount of albumin present, the binding of LCAT to lipoproteins, 1~ and the short half-life16 displayed by the partially purified enzyme. The procedure to be described was designed to give reproducible high yields of the enzyme but does not give a pure protein preparation.
Step 117 Principle. Albumin, LCAT, and most of the lipoproteins are separated by sequential ultracentrifugation. Reagents KBr Plasma containing anticoagulant Procedure. Add 0.398 g of KBr per milliliter to plasma to a final density of 1.25 g/ml. Centrifuge for 44 hr at 50,000 rpm in a Beckman 60 Ti rotor. Remove the floating lipoproteins containing bound LCAT by tube-slicing 1.5 cm from the top of the tube. Readjust the lipoproteins to 1.21 g/ml using KBr; centrifuge and remove the lipoproteins by tube slicing as before. The LCAT is in the infranatant. The yield is approximately 33%, and the purification from plasma is about 20-fold. The major contaminating protein is albumin. 14 A. V. Nichols and E. L. Gong, Biochim. Biophys. Acta 231, 175 (1971). 13 y. A k a n u m a and J. Glomset, Biochem. Biophys. Res. Commun. 32, 639 (1968). 16 C. J. Fielding and P. E. Fielding, FEBS Lett. 15, 355 (1971). lr K. R. N o r u m , J. A. Glomset, A. V. Nichols, T. Forte, J. J. Albers, W. C. King, C. D. Mitchell, K. R. Applegate, E. L. Gong, V. Cabana, and E. Gjone, Scand. J. Clin. Lab. Invest. 35, Suppl. 142, 31 (1975).
382
GENERAL ANALYTICAL METHODS
[25]
Step 215 Principle. LCAT is bound to HDL3 coupled to Sepharose and is eluted with 0.5 mM taurocholate. Reagents Sepharose 4B (Pharmacia, Piscataway, New Jersey) to which HDLn (4 mg of protein per I ml of gel) has been bound using CNBr '8 Buffer 1: 0.14M NaC1, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 7.4 Sodium taurocholate, 0.5 mM Procedure. The LCAT containing infranatant of step 1 is dialyzed against buffer 1 and applied to the HDL3-Sepharose atfinity column previously equilibrated with the same buffer. After rinsing with 0.5 bed volume of buffer 1, the column is eluted with 0.5 mM sodium taurocholate. The LCAT elutes as a single peak at the sodium taurocholate front and can be monitored by absorbance at 280 nm OD2s0. The yield of this step is approximately 70-80%, and the purification is approximately 10-fold. The major contaminating protein is apolipoprotein A-I.
Step 319 Principle. LCAT is further purified diethylaminoethyl(DEAE)-Sepharose.
by gradient
elution
from
Reagents DEAE-Sepharose CL-6B (Pharmacia) packed in a 0.9 × 10 cm column Buffer 2:25 mM NaC1, 5 mM EDTA, pH 7.4, 1 mM Tris-HC1, pH 7.4 Buffer 3:200 mM NaC1, 5 mM EDTA, pH 7.4, 1 mM Tris-HC1, pH 7.4 Procedure. The LCAT eluted from the HDLa affinity column is adjusted to about 4 mS (millisiemens) conductivity by dilution with two volumes of distilled water. It is then applied to the DEAE column equilibrated with buffer 2. After rinsing with 20 ml of buffer 2, the LCAT is eluted with a gradient formed by increasing amounts of buffer 3 added to buffer 2. A closed mixing bottle originally containing 50 ml of buffer 2 into which buffer 3 flows, gives a suitable gradient. The LCAT elutes between 8 and 12 mS conductivity after the main protein peak. The yield from this step is
18 S. C. March, I. Parikh, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974). ~9 K. G. Varma and L. A. Soloff, Biochem. J. 115, 583 (1976).
[25]
ASSAY FOR LECITHIN:CHOLESTEROL ACYLTRANSFERASE
383
about 80%, and the purification is about 2-fold. The major contaminating proteins are apolipoprotein D and apolipoprotein A-I in some fractions.
Step 417 Principle. LCAT is concentrated by adsorption onto a small volume of DEAE-Sepharose and elution with a small volume of buffer. Reagents DEAE-Sepharose CL-6B, 1 ml, packed into a small sintered glass funnel Buffer 2:25 mM NaCI, 5 mM EDTA, pH 7.4, 1 mM Tris-HCl, pH 7.4 2-Mercaptoethanol, 10 mM NaCI solution, 0.5 M Procedure. The LCAT eluted from the DEAE column as described in step 3 is diluted with distilled water to a final conductivity of about 4 mS. It is applied to the 1-ml bed of DEAE-Sepharose CL-6B previously equilibrated with buffer 2. The bed is then rinsed with 3 ml of 10 mM 2-mercaptoethanol and rinsed with 10 ml of distilled water. The LCAT is eluted with 3 ml o f 0 . 5 M NaC1. The yield is nearly I00%, and no purification is provided by this step.
Discussion The overall purification achieved as described above is summarized in the table. The result is a partially purified enzyme containing apolipoproteins A-I and D as well as other plasma proteins in lesser amounts. Several procedures have been described that yield preparations with greater purity. '°-2z Apolipoprotein D can be removed by antibody affinity chromatography or hydroxyapatite. However, hydroxyapatite chromatography, which is widely used, gives variable results; recoveries as low as 10% are common, and some batches of hydroxyapatite from a given manufacturer appear to completely inactivate the enzyme. LCAT is a glycoprotein and can be reversibly adsorbed to concanavalin A. Hence, concanavalin A affinity chromatography can yield preparations devoid of albumin and apolipoprotein A-I. This step in conjunction with hydroxyapatite chromatography23 has therefore been used successfully to give a homogeneous protein preparation. 20 j. j. Albers, V. J. Cabana, and Y. D. B. Stahl, Biochemistry 15, 1084 (1976). 21 L. Aron, S. Jones, and C. J. Fielding, J. Biol. Chem. 253, 7220 (1978). 22 j. C h u n g , D. A. Abano, G. M. Fless, and A. M. Scanu, J. Biol. Chem. 254, 7456 (1979). 23 G. Suzue, C. Vezina, and Y. L. Marcel, Fed. Proc., Fed. Am. Soc. Exp. Biol. 38, Abstract 560 (1979).
384
GENERAL
ANALYTICAL
[26]
METHODS
S U M M A R Y O F O V E R A L L P U R I F I C A T I O N O F L C A T FROM P L A S M A
Yield (%) Step 0. 1. 2. 3.
Plasma After ultracentrifugation After affinity c o l u m n After D E A E c o l u m n
Purification (fold)
Step
Overall
Step
Overall
100 33 75 80
I00 33 25 20
1 20 10 2
1 20 200 400
Acknowledgments The procedures described were developed while R. V. was a fellow in the Howard Hughes Medical Institute, Laboratory of John A. Glomset. S. G. was a visiting scientist of the Center for Advanced Studies in the Health Sciences at the University of Washington. This work was supported in part by NIH Training Grant HL07312 to R. V., NIH Grant NS02967 to S. G., and a grant from Reynolds Industries, Inc.
[26] A Novel Assay Method Galactosyl-
for the Biosynthesis
of
and Glucosylceramides
EC 2.4.1.45 U D P g a l a c t o s e : 2-2-hydroxyacylsphingosine galactosyltransferase E C 2.4.1.80 U D P g l u c o s e : N - a c y l s p h i n g o s i n e glucosyltransferase
By
E. COSTANTINO-CECCAR|NI and A. CESTELLI
U D P g a l a c t o s e + a - h y d r o x y fatty acid ceramide galactosylceramide + U D P U D P g l u c o s e + normal fatty acid ceramide ~ glucosylceramide + U D P Principle Phospholipid vesicles can incorporate a - h y d r o x y or normal fatty acid-containing ceramides and can be used as suitable a c c e p t o r s for the assay o f two different m e m b r a n e - b o u n d glycosyltransferases. The vesicles are readily suspended in aqueous salt solutions and e n z y m a t i c activities can be m e a s u r e d in presence of appropriate phospholipids. Addition o f detergents, Celite, or organic solvents to disperse the lipid acceptors is not required. An additional a d v a n t a g e is that the e n z y m e activities can be m e a s u r e d using v e r y small quantities o f proteins (5-10 ~g).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright O 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
ASSAY FOR LECITHIN :CHOLESI"EROL
ACYLTRANSFERASE
375
phase and in chloroform-methanol, 2 : 1, and its fluorescence in the two respective solvents (FDtp, Fc.~l) is recorded in the fluorometer available to the investigator. Since lecithin is insoluble in this medium, it is dissolved in chloroform-methanol, 2 : 1, and its fluorescence is recorded and related to the phosphorus content, thus providing the fluorescence per nanomole of substrate (MFs). The quantity of fatty acid released (P) is obtained by recording its fluorescence (Fp) and utilizing the following equation: p=
Fv x Fc.~l MF~ ~ x91
The multiplication factor, 91, is the mole ratio of total to fluorescent lecithin. In this procedure we used two enzyme preparations and the buffer of Verger et al.28 However, other phospholipases can be assayed with the fluorescent lecithin using established procedures. Comparison with radioactively labeled phosphatidylcholine showed that the two respective enzymes utilize the fluorescent and radioactive molecules with very similar rates.
[25]
A s s a y for L e c i t h i n : C h o l e s t e r o l A c y l t r a n s f e r a s e By RoY B. VERDERY III and SmMON GATT
Lecithin + cholesterol ~ cholesteryl ester + lysolecithin Assay Method The enzyme can be assayed by determining the disappearance of unesterified cholesterol, by measuring the conversion of radioactive cholesterol to radioactive cholesteryl ester, or by observing the incorporation of radiolabeled fatty acids from lecithin into cholesteryl esters. The reaction is usually run near pH 7 in the presence of a chelating agent to inhibit heavy metal-catalyzed peroxidation of the lecithin and in the presence of a reducing agent, which is usually necessary for maximum enzyme activity. 1 t j. A. Glomset,
Biochim. Biophys. Acta
METHODS IN ENZYMOLOGY, VOL. 72
6 5 , 128 ( 1 % 2 ) . Copyright © 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8
376
GENERAL ANALYTICAL METHODS
[25]
Method 12,3 Principle. The quantity of unesterified cholesterol is measured by gasliquid chromatography (GLC). Reagents Cholesterol, 0.5/.tmol plus lecithin, 1.0 ftmol dissolved in 200 gl of absolute ethanol Stigmasterol, 100 ftg/ml dissolved in chloroform : methanol, 1 : 2 (v/v) Buffer, 0.154 M NaCI, 1 mM EDTA, pH 7.4, 20 mM 2mercaptoethanol Hexane, chloroform, methanol Procedure. Inject the solution containing lecithin and cholesterol through a fine needle into 4 ml of buffer undergoing rapid stirring to form liposomes. 4 The liposome suspension, 100 ftl, is mixed with I00 ftl of enzyme at 4°. After incubation for 30 min at 37°, the reaction is stopped by addition of 0.65 ml of chloroform : methanol, 1 : 2. Stigmasterol solution, 100 pl, is then added as an internal standard, followed by chloroform, 0.25 ml, and water, 0.25 ml. After a brief centrifugation, the upper phase is discarded, the lower phase is dried under nitrogen or air, and the lipids are dissolved in hexane and analyzed by GLC. For separation of cholesterol and stigmasterol, a 3 mm × 60 cm column of 3% OV-1 on 80/100 Gas Chrom Q (Supelco, Bellefonte, Pennsylvania) temperature-programmed from 200° to 230° at 3°/min is sufficient. Zero time controls are obtained by stopping the reaction before incubation. Standards are prepared by mixing I0 ftg of cholesterol with 100 ftl of the stigmasterol solution, drying under air, dissolving in hexane and analyzing by GLC as described previously. Variation for Assay in Plasma or Serum. To determine LCAT4a under reducing conditions, plasma or serum, 100/xl, is mixed with buffer, 100 ftl, at 4°, and the mixture is incubated for 30 min at 37°. Termination of the reaction and determination of cholesterol is accomplished as described above except that a stigmasterol solution of 1 mg/ml is employed. For determination in the absence of exogenous reducing agents, the plasma, 100 ftl, is incubated first and the buffer, 100 fd, is added afterward. Calculations. The extent of the reaction is the difference between the quantity of unesterified cholesterol before and after incubation, the enzymatic activity in nanomoles per milliliter per hour can be calculated as follows: 2 W. M. Sperry, J. Biol. Chem. 111,467 (1935). 3 y. L. Marcel and C. Vezina, Biochim. Biophys. Acta 306, 497 (1973). 4 S. Batzri a n d E. D. K o r n , Biochim. Biophys. Acta 298, 1015 (1973). 4a The abbreviation L C A T stands for lecithin : cholesterol acyltransferase.
[25]
ASSAY FOR LECITHIN"CHOLESTEROLACYLTRANSFERASE
377
LCAT activity = 20 x (cholesterolt=0 - cholesterolt=3o) Since only a small portion of the cholesterol is esterified in this procedure, 5 or 6 replicate determinations before and after incubation must be m a d e ? Method H 5 Principle. Radioactive cholesterol, equilibrated with endogenous cholesterol in heat-inactivated plasma, is converted to radioactive cholesteryl esters that are isolated by thin-layer chromatography (TLC). Reagents
Plasma containing anticoagulant, heated 1 hr at 60° Bovine serum albumin, fraction V, 0.5 g dissolved in 10 ml of 0.154 M NaCI cholesterol (3H or 14C), 10/xCi dissolved in 0.5 ml of absolute ethanol 2-Mercaptoethanol T L C plates of silica gel G Methanol, chloroform, hexane, diethyl ether Procedure. Inject the solution of radioactive cholesterol, 10/xCi, into the albumin solution through a fine needle under the surface. 6 Blow nitrogen across the top o f the mixture until the smell of alcohol is gone. Mix the dispersion with 200 ml of heat-inactivated plasma at 4 °, stir overnight at 4 °, divide into aliquots and store this plasma substrate at - 2 0 °. For the assay, thaw an aliquot of frozen plasma substrate and add 2-mercaptoethanol (0.15% v/v). Mix 100/xl with 100 /xl of enzyme, at 4°, and then incubate for 30 min at 37°. The reaction is stopped by the addition of 1.3 ml c h l o r o f o r m : m e t h a n o l , 1: 2. After a brief centrifugation, 1.0 ml of the supernatant is transferred to another tube, leaving behind the protein. Chloroform (0.3 ml) and water (0.4 ml) are then added and mixed using a vortex mixer, and the two phases are separated by a brief centrifugation. The supernatant phase is discarded, the infranatant is dried under air, and the lipids are dissolved in 200 /zl of hexane. An aliquot, 100/zl, of the hexane is applied to a plate coated with 0.25 mm silica gel G. After developing in hexane : diethyl ether, 90 : 20 (v/v), the cholesteryl ester spot (R s value about 0.97) is visualized by exposure to iodine, scraped, and counted. Residual LCAT activity in heated plasma varies among preparations; therefore, a blank consisting of plasma substrate and normal saline, or another appropriate buffer without enzyme, must be determined. To minimize this blank, the temperature of the plasma must be maintained at 5 j. A. Glomset and J. L. Wright, Biochim. Biophys. Acta 89, 266 (1964). 6 D. Porte and R. J. Havel, J. LipidRes. 2, 357 (1961).
378
GENERAL ANALYTICAL METHODS
[25]
60° for 1 hr, care must be taken, however, to prevent denaturation of the proteins. Calculations. LCAT activity is calculated by the following formula: LCAT activity =
cholesteryl ester(dpm in sample - dpm in blank) unesterified cholesterol specific activity × dilution factor
The dilution factor for the assay as described is 60, and, if the specific activity is measured in units of disintegrations per minute (dpm) per nanomole, the units of enzyme activity are nanomoles per milliliter per hour. M e t h o d III 7 Principle. Radiolabeled lecithin is incubated with cholesterol, and incorporation of the labeled fatty acids into cholesteryl esters is observed. The products are separated from the labeled substrate by a solvent partition step. The blank is reduced by treating the neutral lipid fraction after the partition with silicic acid; separation of fatty acids from cholesteryl esters is obtained by extraction of the washed neutral lipid fraction with a basic aqueous solution, Reagents
Cholesterol, 0.5/xmol, egg lecithin, 1.0/zmol, [14C]lecithin, 1.0/zCi, dissolved in 200/zl of absolute ethanol Buffer, 0.154 M NaCI, 1 mM EDTA, pH 7.4, 20 mM 2-mercaptoethanol Dole's mixtureS: heptane : isopropyl alcohol: 1N H2SO4, 400: 100:10 Silicic acid (Mallinckrodt, St. Louis, Missouri) activated by heating at 100° NaOH, 0.1 N H2SO4, 0.2 N Heptane, water Procedure. Inject the 200 ~1 of ethanolic solution of lecithin and cholesterol containing [14C]lecithin into 4.0 ml of buffer to form substrate liposomes. 3 Mix the liposomal dispersion (100/xl) with enzyme (100/zl) at 4° and incubate at 37° for 30 rain. The reaction is stopped by adding 2.0 ml of Dole's mixture. Heptane, 1.2 ml is then added and mixed thoroughly, followed by 1.0 ml of water. After thorough mixing, the phases are separ R. B. Verderyand S. Gatt, Clin. Chem. (submittedfor publication). 8 V. P. Dole,J. Clin. Invest. 35, 150 (1956).
[25]
ASSAY FOR L E C I T H I N ." C H O L E S T E R O L A C Y L T R A N S F E R A S E
379
rated by centrifugation, and 1.0 ml of the upper heptane-rich phase is transferred to another tube containing 200-300 mg of silicic acid and 2.0 ml of heptane. After thorough mixing, the silicic acid is sedimented by a brief centrifugation; the heptane is transferred to a scintillation vial, and its radioactivity is determined on a liquid scintillation spectrometer. Blanks are determined by incubating substrate liposomes with 100/zl of buffer without enzyme. Calculations. LCAT activity is calculated using the following formula: LCAT activity = (dpm of sample - dpm of blank) × dilution factor specific activity of fatty acid in/3 (2)-position of lecithin The dilution factor in the above assay as described is 28, and if the specific activity of the 2-position fatty acid is expressed in units of disintegrations per minute per nanomole, the units of LCAT activity are nanomoles per milliliter per hour. Two factors affect the specific activity of the 2-position fatty acid: the proportion of radioactivity in the 2-position fatty acid of the "uniformly labeled" [14C]lecithin and the amount of endogenous lecithin in the reaction mixture. The proportion of radioactivity in the 2-position of lecithin from one batch of uniformly labeled lecithin from algae (Chlorella vulgaris, New England Nuclear, Boston, Massachusetts) was 0.68 as determined by treatment with snake venom 9 and thin-layer chromatography of the product fatty acids and lysolecithin. If the enzyme preparation contains significant amounts of lecithin, this must be determined TM and added to the 25 nmol present in the liposomal dispersion. Variation for Improved Specificity. Some LCAT preparations display substantial phospholipase activity. The earlier-described assay does not distinguish between radioactivity due to fatty acids resulting from phospholipase activity and cholesteryl esters from transferase activity. To differentiate between these activities, 1.5 ml of the silicic acid-treated heptane solution is mixed with 1.0 ml of 0.1 N NaOH. After mixing and centrifuging, 1.0 ml of the upper hexane phase is counted to determine transferase activity. One milliliter of hexane and 1.0 ml of 0.2 N H2SO4 are then added. After mixing and centrifuging, 1.0 ml of the upper hexane phase is counted to determine phospholipase activity. A 1.0-ml aliquot of the silicic acid-treated heptane which has not been extracted with NaOH is counted as well. Transferase and total activities are calculated from these counts, respectively, using the formula given previously except that a dilution factor of 84 is employed. Phospholipase activity is estimated as 9 A. F. Robinsonand W. E. M. Lands,Biochemistry 1,804 (1972). 10j. W. Jones and P. Ways,J. Clin. Invest. 46, 1151 (1967).
380
GENERAL ANALYTICAL METHODS
[25]
the difference between total and transferase activity. Because of the high dilution factor, the accuracy of this variation is not as great as the accuracy of the previously described method, which gives total activity alone. Variation for Assay in Plasma or Serum. Serum or plasma containing anticoagulant is obtained, and DTNB is added to a final concentration of 1.4 mM 1~ to inhibit the LCAT. [~4C]Lecithin (10-100 nCi) in 100/.d of absolute ethanol is injected through a fine needle into 2.0 ml of this plasma or serum undergoing rapid mixing. The mixture of lecithin and plasma is then incubated 30 min at 37° to permit equilibration of the radioactive and endogenous lecithin. The reaction is started by mixing 100 /xl of the plasma containing [~4C]lecithin with 100 /zl of buffer containing 2-mercaptoethanol. The 2-mercaptoethanol reverses the inhibition caused by DTNB. After incubation for 30 min at 37°, the reaction is terminated and LCAT activity is determined as described previously. Accurate calculation of activity in terms of nanomoles per milliliter per hour requires independent determination of the lecithin concentration in the plasma. Remarks The three methods described above were chosen because they have unique features. The first method is a modification of the determination that originally led to the discovery of LCAT.12 The major advantage of this method is that LCAT is determined unambiguously in terms of nanomoles of cholesterol per hour, independent of the requirement t h a t a labeled substrate be completely equilibrated with any endogenous substrate. The disadvantages are its lack of sensitivity, since only small changes in cholesterol content occur during the incubation and the requirement that many time-consuming determinations of cholesterol be made for each assay. The second method is an example of the numerous assay methods that have been used which employ radioactive cholesterol as labeled substrate. The advantage of this particular method is that the incubation mixture contains all the plasma lipoproteins as well as the LCAT activator apolipoprotein, A-I, and the cholesteryl ester transferase protein. It therefore contains all the potential cofactors that affect LCAT activity. There are several major disadvantages to this method: TLC separation of unesterified cholesterol from cholesteryl ester is time consuming; the measured activity may depend on the particular plasma used for preparation of the substrate since the state of nutrition 13 and sex 4 of the donor may change LCAT activity; if the enzyme preparation contains a substantial 1~ K. T. Stokke a n d K. R. N o r u m , Scand. J. Clin. Lab. Invest. 27, 21 (1971). 12 j. A. G l o m s e t , F. Parker, M. Tjaden, and R. H. Williams,Biochim. Biophys. Acta 58, 398 (1962). ~3 H. G. Rose a n d J. Juliano, J. Lab. Clin. Med. 89, 524 (1977).
[25]
ASSAY FOR LECITHIN :CHOLESTEROL ACYLTRANSFERASE
381
amount of cholesterol, isotopic dilution will affect the measured amount of LCAT activity. The third method is a variation of the methods that employ liposomal substrates 14and was developed to provide these two advantages: (a) the procedure utilizes a commercially available radioactive substrate and a rapid method to separate a labeled product from a labeled substrate; (b) it can be used conveniently to assay many samples. The major disadvantage is that if lecithin is present in the enzyme preparation, as in the case for assays in plasma, its amount must be determined before activity can be expressed in absolute units. It should be emphasized that with all these methods the assay in plasma is complicated by the simultaneous presence of variable amounts of both enzyme and substrate and the possibility that variable amounts of activating cofactor proteins might also be present. Partial Purification Purification of LCAT from plasma is made difficult by the large amount of albumin present, the binding of LCAT to lipoproteins, 1~ and the short half-life16 displayed by the partially purified enzyme. The procedure to be described was designed to give reproducible high yields of the enzyme but does not give a pure protein preparation.
Step 117 Principle. Albumin, LCAT, and most of the lipoproteins are separated by sequential ultracentrifugation. Reagents KBr Plasma containing anticoagulant Procedure. Add 0.398 g of KBr per milliliter to plasma to a final density of 1.25 g/ml. Centrifuge for 44 hr at 50,000 rpm in a Beckman 60 Ti rotor. Remove the floating lipoproteins containing bound LCAT by tube-slicing 1.5 cm from the top of the tube. Readjust the lipoproteins to 1.21 g/ml using KBr; centrifuge and remove the lipoproteins by tube slicing as before. The LCAT is in the infranatant. The yield is approximately 33%, and the purification from plasma is about 20-fold. The major contaminating protein is albumin. 14 A. V. Nichols and E. L. Gong, Biochim. Biophys. Acta 231, 175 (1971). 13 y. A k a n u m a and J. Glomset, Biochem. Biophys. Res. Commun. 32, 639 (1968). 16 C. J. Fielding and P. E. Fielding, FEBS Lett. 15, 355 (1971). lr K. R. N o r u m , J. A. Glomset, A. V. Nichols, T. Forte, J. J. Albers, W. C. King, C. D. Mitchell, K. R. Applegate, E. L. Gong, V. Cabana, and E. Gjone, Scand. J. Clin. Lab. Invest. 35, Suppl. 142, 31 (1975).
382
GENERAL ANALYTICAL METHODS
[25]
Step 215 Principle. LCAT is bound to HDL3 coupled to Sepharose and is eluted with 0.5 mM taurocholate. Reagents Sepharose 4B (Pharmacia, Piscataway, New Jersey) to which HDLn (4 mg of protein per I ml of gel) has been bound using CNBr '8 Buffer 1: 0.14M NaC1, 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, pH 7.4 Sodium taurocholate, 0.5 mM Procedure. The LCAT containing infranatant of step 1 is dialyzed against buffer 1 and applied to the HDL3-Sepharose atfinity column previously equilibrated with the same buffer. After rinsing with 0.5 bed volume of buffer 1, the column is eluted with 0.5 mM sodium taurocholate. The LCAT elutes as a single peak at the sodium taurocholate front and can be monitored by absorbance at 280 nm OD2s0. The yield of this step is approximately 70-80%, and the purification is approximately 10-fold. The major contaminating protein is apolipoprotein A-I.
Step 319 Principle. LCAT is further purified diethylaminoethyl(DEAE)-Sepharose.
by gradient
elution
from
Reagents DEAE-Sepharose CL-6B (Pharmacia) packed in a 0.9 × 10 cm column Buffer 2:25 mM NaC1, 5 mM EDTA, pH 7.4, 1 mM Tris-HC1, pH 7.4 Buffer 3:200 mM NaC1, 5 mM EDTA, pH 7.4, 1 mM Tris-HC1, pH 7.4 Procedure. The LCAT eluted from the HDLa affinity column is adjusted to about 4 mS (millisiemens) conductivity by dilution with two volumes of distilled water. It is then applied to the DEAE column equilibrated with buffer 2. After rinsing with 20 ml of buffer 2, the LCAT is eluted with a gradient formed by increasing amounts of buffer 3 added to buffer 2. A closed mixing bottle originally containing 50 ml of buffer 2 into which buffer 3 flows, gives a suitable gradient. The LCAT elutes between 8 and 12 mS conductivity after the main protein peak. The yield from this step is
18 S. C. March, I. Parikh, and P. Cuatrecasas, Anal. Biochem. 60, 149 (1974). ~9 K. G. Varma and L. A. Soloff, Biochem. J. 115, 583 (1976).
[25]
ASSAY FOR LECITHIN:CHOLESTEROL ACYLTRANSFERASE
383
about 80%, and the purification is about 2-fold. The major contaminating proteins are apolipoprotein D and apolipoprotein A-I in some fractions.
Step 417 Principle. LCAT is concentrated by adsorption onto a small volume of DEAE-Sepharose and elution with a small volume of buffer. Reagents DEAE-Sepharose CL-6B, 1 ml, packed into a small sintered glass funnel Buffer 2:25 mM NaCI, 5 mM EDTA, pH 7.4, 1 mM Tris-HCl, pH 7.4 2-Mercaptoethanol, 10 mM NaCI solution, 0.5 M Procedure. The LCAT eluted from the DEAE column as described in step 3 is diluted with distilled water to a final conductivity of about 4 mS. It is applied to the 1-ml bed of DEAE-Sepharose CL-6B previously equilibrated with buffer 2. The bed is then rinsed with 3 ml of 10 mM 2-mercaptoethanol and rinsed with 10 ml of distilled water. The LCAT is eluted with 3 ml o f 0 . 5 M NaC1. The yield is nearly I00%, and no purification is provided by this step.
Discussion The overall purification achieved as described above is summarized in the table. The result is a partially purified enzyme containing apolipoproteins A-I and D as well as other plasma proteins in lesser amounts. Several procedures have been described that yield preparations with greater purity. '°-2z Apolipoprotein D can be removed by antibody affinity chromatography or hydroxyapatite. However, hydroxyapatite chromatography, which is widely used, gives variable results; recoveries as low as 10% are common, and some batches of hydroxyapatite from a given manufacturer appear to completely inactivate the enzyme. LCAT is a glycoprotein and can be reversibly adsorbed to concanavalin A. Hence, concanavalin A affinity chromatography can yield preparations devoid of albumin and apolipoprotein A-I. This step in conjunction with hydroxyapatite chromatography23 has therefore been used successfully to give a homogeneous protein preparation. 20 j. j. Albers, V. J. Cabana, and Y. D. B. Stahl, Biochemistry 15, 1084 (1976). 21 L. Aron, S. Jones, and C. J. Fielding, J. Biol. Chem. 253, 7220 (1978). 22 j. C h u n g , D. A. Abano, G. M. Fless, and A. M. Scanu, J. Biol. Chem. 254, 7456 (1979). 23 G. Suzue, C. Vezina, and Y. L. Marcel, Fed. Proc., Fed. Am. Soc. Exp. Biol. 38, Abstract 560 (1979).
384
GENERAL
ANALYTICAL
[26]
METHODS
S U M M A R Y O F O V E R A L L P U R I F I C A T I O N O F L C A T FROM P L A S M A
Yield (%) Step 0. 1. 2. 3.
Plasma After ultracentrifugation After affinity c o l u m n After D E A E c o l u m n
Purification (fold)
Step
Overall
Step
Overall
100 33 75 80
I00 33 25 20
1 20 10 2
1 20 200 400
Acknowledgments The procedures described were developed while R. V. was a fellow in the Howard Hughes Medical Institute, Laboratory of John A. Glomset. S. G. was a visiting scientist of the Center for Advanced Studies in the Health Sciences at the University of Washington. This work was supported in part by NIH Training Grant HL07312 to R. V., NIH Grant NS02967 to S. G., and a grant from Reynolds Industries, Inc.
[26] A Novel Assay Method Galactosyl-
for the Biosynthesis
of
and Glucosylceramides
EC 2.4.1.45 U D P g a l a c t o s e : 2-2-hydroxyacylsphingosine galactosyltransferase E C 2.4.1.80 U D P g l u c o s e : N - a c y l s p h i n g o s i n e glucosyltransferase
By
E. COSTANTINO-CECCAR|NI and A. CESTELLI
U D P g a l a c t o s e + a - h y d r o x y fatty acid ceramide galactosylceramide + U D P U D P g l u c o s e + normal fatty acid ceramide ~ glucosylceramide + U D P Principle Phospholipid vesicles can incorporate a - h y d r o x y or normal fatty acid-containing ceramides and can be used as suitable a c c e p t o r s for the assay o f two different m e m b r a n e - b o u n d glycosyltransferases. The vesicles are readily suspended in aqueous salt solutions and e n z y m a t i c activities can be m e a s u r e d in presence of appropriate phospholipids. Addition o f detergents, Celite, or organic solvents to disperse the lipid acceptors is not required. An additional a d v a n t a g e is that the e n z y m e activities can be m e a s u r e d using v e r y small quantities o f proteins (5-10 ~g).
METHODS IN ENZYMOLOGY, VOL. 72
Copyright O 1981 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181972-8