Studies on base-exchange reactions of phospholipids in rat brain

ARCHIVES

OF

Studies

BIOCHEMISTRY

AND

BIOPHYSICS

on Base-Exchange

Reactions

Heterogeneity TAKASHI

654-660 (1976)

175,

of Phospholipids

of Base-Exchange

MIURA’

AND

JULIAN

in Rat Brain

Enzymes KANFER’

Eunice Kennedy Shriver Center at the Walter E. Fern&d State School 200 Trapelo Road, Waltham, Massachusetts 02154, and Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts 02114 Received

November

11, 1975

The solubilization of base-exchange enzymes from rat brain was effected with a combination of 0.8% Miranol H2M with 0.5% sodium cholate. The yields of incorporation activities for ethanolamine and serine in the solubilized supernatant fraction were 74 and 65%, respectively. Choline incorporation enzyme was not solubilized by this detergent mixture. The solubilized activities for ethanolamine and serine were concentrated into the fraction precipitated by (NH&SO, between 35 and 55% in the presence of Asolectin dispersion. The separation of base-exchange enzyme specific for each of the three compounds was accomplished with chromatography using Sepharose 4B, Affi-Gel 102 and DEAE-cellulose. Separate fractions were obtained which possessed principally choline or serine or ethanolamine incorporation activities.

A number of investigators have studied, in vitro, a base-exchange enzymic system which catalyzes the Ca2+-stimulated, nonenergy-requiring incorporation of zrserine, ethanolamine, and choline into their respective phospholipids (l-6). The question concerning the commonality of these incorporations has been an important problem. The participation of more than one enzyme for these base-exchange reactions has been suggested from studies on the heat sensitivity (l), the Ca2+ dependency (1, 2), and the kinetic properties (l-3, 7). In order to provide evidence for this possibility and evaluate the individual reactions it is necessary to purify the baseexchange enzymes. The highest activity of the base-exchange reactions was found in the microsomal fraction from brain and liver (3, 8). The first successful solubilization of the enzymes was achieved from rat brain particles using a zwitterionic detergent, Miranol H2M (1,9). Instability of the ’ Present address: Department of Biochemistry, Faculty of Medicine University of Manitoba, 770 Bannatyne Avenue, Winnipeg, Manitoba Canada R3E OW3. Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

enzymes in the detergent-containing solution prevented further purification. Active enzyme protein was obtained only when the detergent was removed from the %olubilized” preparation using gel filtration on Sephadex G-25. The removal of detergent caused the solubilized proteins to reaggregate. In order to purify these membranebound proteins, it was necessary to establish conditions under which the enzymes are stable in the presence of a solubilizing agent. The present paper deals with the solubilization and the separation of the individual base-exchange enzymes in the presence of detergent and an examination of their heterogeneity. MATERIALS

AND

METHODS

Materials DE32 and Affi-Gel 102 (agarose-NH(CH&NH(CH,),NH,) were purchased from Whatman Biochemicals Ltd., Kent, England, and BioRad Laboratories, Richmond, Calif., respectively. The sources of the radioactive materials employed were: [1,2J4C]ethanolamine (sp act, 33.4 &i/mmol) and n-[U-14Clserine (sp act, 35.1 &i/mmol) International Chemical and Nuclear Corp., Irving, Calif.; 654

BASE-EXCHANGE [methyl-‘4Clcholine (sp act, 10.3 pCilmmol1, Amersham/Searle, Chicago, Ill. The specific activities were adjusted by the addition of the appropriate nonradioactive material. Unlabeled ethanolamine, Lserine, and choline chloride were purchased from J. T. Baker Chemical Co. (Phillipsburg, N.J.), Schwarz/Mann (Orangeburg, N.Y.) and Pierce Chemical Co. (Rockford, Ill.), respectively. Hepes buffer2 and sodium cholate were obtained from Sigma Chemical Co. (St. Louis, MO.). Asolectin (soya phospholipids) was obtained from Associated Concentrates, New York, N.Y. Miranol H2M3 and Brij 58 were supplied from Miranol Chemical Co., Irvington, N.J., and Pierce Chemical Co., Rockford, Ill., respectively. All other chemicals were of reagent grade. Methods Fractionation of rut brains. All procedures were performed at 4°C except where noted. Brains were obtained from rats 22 to 29 days of age (1) and homogenized in nine volumes of 0.32 M sucrose containing 2 mM Hepes and 1 mM EDTA, pH 8.0. Homogenization was performed with a Polytron instrument (Kinematica, CMBH, Luzern, Switzerland) at maximum speed for 30 s. The homogenate was centrifuged at 12,OOOgfor 10 min, and the supernatant was centrifuged at 56,000g for 60 min. The pellet thus obtained was suspended in the homogenization medium with a Potter-Elvehjem homogenizer and designated as the microsomal fraction. The yield of microsomal activity for the incorporation of the three bases was 44 to 53% of the homogenate, while that of proteins was 20 to 22%. The specific activities increased 2.3- to 2.8-fold during this centrifugation procedure. Solubilization of the enzyme (see flow diagram). The microsomal fraction was suspended in a solution of detergent in 5 mM Hepes, pH 7.23, containing 20% (v/v) glycerol at about 2.5 mg of protein/ml. Sodium cholate and Miranol H2M, individually or combined, were used as the solubilizing agents at the final concentrations indicated in Table I. The * Abbreviations used: DEAE-, diethyl aminoethyl; N-2-Hydroxyethylpiperazine-N’2-ethaneHepes, sulfonic acid, GHM medium, 5 mM Hepes containing 20% glycerol and 1 mM 2-mercaptoethanol; GHMA medium, GHM medium containing Asolectin dispersion; TCA, trichloroacetic acid; AS-supernatant, ammonium sulfate precipitate after dialysis against GHM medium. 3 The structure of Miranol H2M is:

REACTIONS

655

suspension was allowed to stand for 15 min in an ice bath and then centrifuged at 165,500g for 60 min. In order to measure the enzyme activity present, the solubilized supernatant was precipitated with 60% saturation of (NH&SO, and dissolved in 5 mM Hepes, pH 7.23, containing 20% glycerol, 1 mM 2mercaptoethanol (GHM medium) and Asolectin dispersion (3 pg of P/mg of protein) (GHMA medium). The solution was dialyzed overnight against 100 volumes of GHM medium (AS-supernatant). The dialysand was used to assay for base-exchange activities. Ammonium sulfate fractionation of the solubilized supernatunt. The supernatant obtained after solubilizing with a solution of 0.8% (w/v) Miranol H2M and 0.5% (w/v) sodium cholate in 5 mM Hepes, pH 7.23, containing 20% glycerol, was fractionated with (NH&SO, in the presence of Asolectin dispersion (6 fig of P/ml). The ammonium sulfate fractionation was performed in an ice bath, adjusting the pH to 7.2 to 7.4 with concentrated NH,OH. Precipitated material was recovered 20 min later by centrifugation and dissolved in GHMA medium. The solution was dialyzed overnight against 100 volumes of GHM medium. The dialysands were used to assay base-exchange activities. Separation of the individual base-exchange enzymes. 1. Sepharose 4B column. The fraction which precipitated between 35 and 55% (NH&SO, saturation was dissolved in a solution of 0.4% Miranol H2M and 0.2% sodium cholate in GHM medium (approximately 15 mg of protein/ ml). A 5- to 6-ml aliquot was applied to a Sepharose 4B column (2.6 x 40 cm) equilibrated with a solution of 0.25% Miranol H2M in GHMA medium and eluted with the same solvent at a flow rate of 18 ml/h. Enzyme-containing fractions were precipitated with 60% saturation of (NH&SO, in the presence of Asolectin dispersion (6 pg of P/ml). The precipitate was dissolved in the detergent-containing solution (approximately 10 mg of protein/ml). This solution was dialyzed against 30 volumes of the detergent-containing solution. 2. Affi-Gel 102 column. The dialysand was applied 1 h later to a column (1.8 x 20 cm) of Affi-Gel 102, which had been equilibrated with a solution of 0.5% Brij 58 in GHMA medium. The base-exchange activities were eluted both with this solution and a solution of 0.25% Miranol H2M in GHMA medium. 3. DEAE-cellulose column. Fractions 4 to 6 of the Affi-Gel102 column were combined and L-serine was added to make the final concentration 0.1 rnM and, after 15 min, the sample was applied onto a column of DEAE-cellulose (DE32), which had been equilibrated with a solution of 0.5% Brij 58 in GHMA medium and 0.1 mM L-serine. The column was washed with a small amount of the same medium. Elution was conducted in stepwise manner with the same eluant containing 0.15 M NaCl followed by 0.75

656

serine activity M sodium chloride. Aliquots of 20 ~1 of each fraction were used to determine base-exchange activities. Preparation of Asolectin dispersion. Microdispersion of the mixed soya bean phospholipids, Asolectin, was prepared essentially according to the method of Fleischer et al. (101. The mixture was dialyzed at 4°C against approximately 100 volumes of 10 mM Hepes and 1 mM EDTA, pH 7.23, and the phosphorus content was assayed prior to use. Enzyme assay of base-exchange reaction. The method of enzyme assay is that described in previous reports (1, 9). The basic constituents of the reaction mixture were: 10 pmol of Hepes, pH 7.23, Asolectin di-persion (25 pg of P), either 7.2 nmol (0.24 &i) of ethanolamine, 20.2 nmol (0.71 @i) of serine, or 64.8 nmol (0.67 &i) of choline, 2 kmol of CaCl, for ethanolamine and choline incorporations or 6 pmol of CaCl, for serine incorporation, 50 fig of bovine serum albumin, and enzyme protein in a total volume of 0.24 ml. The reaction tubes were incubated at 37°C for 15 min with shaking and terminated by the addition of 1 ml of ice-cold 10% trichloracetic acid (TCA). The contents of the tube were filtered onto a nitrocellulose membrane filter (HA 0.45 Frn, 2.5 cm; Millipore Co., Bedford, Mass.) and washed with 20 ml of ice-cold 5% TCA. The filter

was then dissolved in Aquasol (New England Nuclear, Boston, Mass.) and the radioactivity quantitated in a Packard Model 3380 scintillation spectrometer. Analytical methods. Protein was determined by a biuret reaction according to the method modified by Yonetani (111 with bovine serum albumin as standard. The effect of detergents, when present, in the samples was suitably corrected for. Protein was also measured by ultraviolet absorbance (12). Phosphorus content of Asolectin dispersion was determined after digestion with perchloric acid (13). RESULTS

Solubilization

of the Enzymes

The base-exchange system was solubilized from rat brain microsomal membranes by treatment with 1% Miranol H2M and sonic oscillation (1, 9) with an approximate 12% yield. In the present studies several different detergents were examined for their effectiveness in solubilizing both protein and base-exchange activities from the microsomal fraction. The

BASE-EXCHANGE

657

REACTIONS

particles were incubated for 15 min in an ice bath with a detergent-containing solution and centrifuged at 165,500g for 60 min to obtain the “solubilized” activity. The solubilizing ability of Miranol H2M and sodium cholate is shown in Table I. Baseexchange activities were partially solubilized by 0.5% Miranol H2M without sonic oscillation. However, the incorporation activities for ethanolamine, serine, and choline present in the AS-supernatant were 45,44, and 21% of the microsomal fraction, respectively. Sodium cholate was less effective than Miranol H2M for the solubilization of these activities. When the concentration of Miranol H2M was raised to 0.8%, the specific activities of the AS-supernatant was greater than that of the pellet except for choline incorporation. This treatment solubilized 66% of microsoma1 proteins. The incorporation activities for ethanolamine and serine in the solubilized supernatant fraction were 74 and 65%, respectively, of that in the microsoma1 preparation. Concentrations of Miranol H2M above 0.8% caused a loss of activities. Therefore, a mixture of 0.8% Miranol H2M and 0.5% sodium cholate was used as the solubilization reagent, final pH 7.8.

ties were distributed in all fractions with lower specific activities than the AS-supernatant (Table IIA). When Asolectin dispersion was added prior to the addition of (NHJ2S04, the fractionation pattern was changed (Table IIC). Incorporation activities of ethanolamine and serine were concentrated in the second and third fractions with a yield of 66 and 73%, respectively. The specific incorporation activities for these two bases increased by about 1.7 times by this procedure. Approximately 70% of choline incorporation activity was present in the first and second fractions. 2. Sepharose 4B column. The 35-55% ammonium sulfate precipitate was dissolved in a solution containing Miranol H2M and sodium cholate and applied to a Sepharose 4B column to remove large membrane fragments which also were found to contain the bulk of the choline incorporation activity. The turbid front fractions contained more than 60% of the charged proteins. This was followed by a broad peak (110-190 ml) containing almost all of ethanolamine and serine incorporation activities. The choline incorporation activity showed a completely different distribution. More than two-thirds of this activity was in the turbid front fractions and the remainder was in the broad peak containing the enzymes for ethanolamine and serine incorporation. The presence of Asolectin dispersion in the elution solution was required to protect the enzymes. The eluant contained almost all the activities towards ethanolamine and serine and

Separation of the Base-Exchange Enzymes 1. Ammonium sulfate precipitation. Ammonium sulfate fractionation in the absence of Asolectin dispersion did not concentrate the incorporation activities of the three bases (Table IIB). These activiTABLE

I

SOLUBILIZATION OF BASE-EXCHANGE ACTIVITIES AND PROTEINS FROM MICROSOMAL FRACTION OF RAT BRAIN BY MIRANOL H2M AND SODIUM CHOLATE Treatment

Protein Supernatant

Miranol H2M (0.5%) Sodium cholate (0.5%) Miranol H2M (0.5%) and sodium cholate (0.5%) Miranol H2M (0.8%) and sodium cholate (0.5%)

(o/0)(1

(nmol

Incorporation activity h-’ . mg? of supernatanp

Pellet

Ethanolamine

Serine

48.3 19.1 50.8

21.9 66.8 35.2

16.7 5.8 20.7

9.0 3.6 10.3

2.5 2.0 3.6

66.3

18.4

28.8

16.3

3.0

a Percentage of microsomal proteins used. b Fractions precipitated by 60% saturation of (NH&SO, tant).

from the solubilized

supernatant

Choline

(AS-superna-

658

MIURA

AND

KANFER

TABLE

II

FRACTIONATION OF THE SOLUBILIZED SUPERNATANT WITH AMMONIUM Fraction”

Protein

(%)*

Incorporation Ethanolamine

SULFATE

activity Serine

(nmol . h-l

mg-*) Choline

A (100) 68.4

20.5 30.8

9.1 14.1

2.7 3.4

O-35% 35-45% 45-55% 55-75%

35.4 18.5 14.0 12.8

24.6 10.2 17.8 20.1

10.6 11.3 1.4 14.0

3.2 2.8 0.4 1.1

O-35% 35-45% 45-55% 55-75%

31.2 23.8 17.4 8.4

7.8 32.0 28.2 8.2

4.1 20.9 10.0 3.0

4.8 1.9 1.2 1.4

Supernatani? O-60% B

C

(2A = solubilized supernatant and AS-supernatant, B = ammonium sulfate fractionation in the presence of Asolectin dispersion; C = ammonium sulfate fractionation in the absence of Asolectin dispersion. * Percentage of proteins of the solubilized supernatant. c Activities of the sunernatant were assaved after dialysis for two days against 100 volumes of GHM medium without precipkation with (NH&&

these activities precipitated by (NH&SO, in the presence of Asolectin dispersion. The precipitate was dissolved in a solution containing Miranol H2M and sodium cholate and dialyzed against the same solution. A 29 and 32% yield was obtained of ethanolamine and serine incorporation activities, respectively, as well as 21% of proteins. 3. Affi-Gel 102 column. One hour after dialysis the enzyme-containing solution was adsorbed on an AffXel 102 column, and a typical elution pattern is shown in Fig. 1. With a Brij 58 solution as the eluant, the bulk of charged proteins was not retained by the column but emerged as a slightly turbid solution. Some activity for ethanolamine and serine was found in these fractions (fractions 3-6) with a 26 and 18% yield, respectively. Elution with Miranol H2M resulted in removal of the adsorbed proteins and a small peak of choline and ethanolamine activities. This was followed by a major peak of ethanolamine incorporation activity (fractions 23-30) which was essentially free of activity towards the other two bases. The total ethanolamine and serine incorporation activities recovered from the column were 82

and 84% of that applied. 4. DEAE-cellulose column chromatography. The serine activity passed through the Aft?-Gel 102 column associated with some of ethanolamine activity (Fig. 1). To examine the relationship of these two activities, the eluted fractions from the AffrGel 102 column (fractions 4-6) were applied to a DEAE-cellulose column. Serine incorporation activity was eluted with 0.15 M sodium chloride, completely overlapping the elution of the bulk of proteins when elution was conducted by a Brij 58 solution in the absence of n-serine. When the column chromatographic procedures were conducted in the presence of 0.1 mM Lserine, one-fourth of charged proteins was eluted with 0.15 M sodium chloride (Fig. 2). Serine incorporation activity occurred as a peak well separated from the bulk of proteins with a recovery of 97% of applied activity (fractions 11-14). There was only negligible ethanolamine incorporation activity associated with this peak. DISCUSSION

The procedure for solubilization of baseexchange enzymes was an improvement over that previously employed in two re-

BASE-EXCHANGE

FRACTION NUMBER

FIG. 1. Chromatography of base-exchange enzymes on an Affi-Gel 102 column. The eluted fractions from a Sepharose 4B column, which contained almost all activities for ethanolamine and serine incorporation, were concentrated with 60% saturation of (NH&SO, and dissolved in a solution containing 0.4% Miranol H2M, 0.2% sodium cholate, and GHM medium. This solution was dialyzed for 1 h against 30 volumes of a 0.25% Miranol H2M solution in GHM medium and applied to an Affi-Gel 102 column. The elution was conducted at a flow rate of 40 ml/h with a 0.5% Brij 58 solution in GHMA medium until the point indicated by the arrow and, then, continued with a 0.25% Miranol H2M solution in GHMA medium. Further details of the procedure are described in text. Fractions of 5 ml were collected and aliquots of 20 ~1 of each fraction were used to assay incorporation activities for ethanolamine (O---O), serine (O- . - 0) and choline (X-X 1. The activity is shown as counts per minute of incorporated 14C-labeled base per 20 ~1 of each fraction.

spects. These are the activation by sodium cholate and the solubilization by a lower concentration of Miranol H2M without sonic oscillation treatment. This activation was also effective in the presence of Miranol H2M and, in addition, the presence of sodium cholate decreased the inactivation of the base-exchange enzymes by Miranol H2M. When 0.5% Miranol H2M alone was used, some solubilization of incorporation activities of three bases was achieved. Extraction of the particles with a combination of 0.8% Miranol H2M in the presence of 0.5% sodium cholate resulted in an increased solubilization of ethanolamine and serine incorporation activity with little effect upon choline incorporation activity. The fractionation of the solubilized supernatant with ammonium sulfate was

659

REACTIONS

achieved in the presence of Asolectin dispersion, the role of which is unclear. When the ammonium sulfate fractionation was conducted in the presence of a higher concentration of Asolectin dispersion (12 pg of P/ml), it was necessary to use a higher concentration of (NH&SO, for precipitation of a large part of base-exchange enzymes (unpublished data). Moreover, ASOlectin dispersion reduced the losses of base-exchange enzyme activities during subsequent chromatographic procedures and dialysis against GHM medium. Based upon these observations it seems likely that the enzyme proteins are coprecipitated with phospholipids and the formation of complexes of enzyme-phospholipids is required to retain functional spatial structures of base-exchange enzymes. It is of interest to note that the behavior of choline incorporation enzyme is significantly different from that of the others in all steps of solubilization and fractionation with (NH&SO,. This enzyme was neither activated by sodium cholate nor quantitatively solubilized by Miranol H2M. Moreover, this enzyme was precipitated below 45% saturation of (NH&SO,. These re-

FRACTION

FIG. 2. Chromatography

NUMBER

on a DEAE-cellulose column of the eluant from an Afti-Gel 102 column (fractions 4-6). Fractions 4-6 of an Afi-Gel 102 column were applied to a DEAE-cellulose column. After washing with a 0.5% Brij 58 solution containing GHMA medium and 0.1 mM L-serine the enzymes were eluted at a flow rate of 50 ml/h in stepwise manner with the same eluant containing 0.15 M NaCl followed by 0.75 M NaCl as indicated by the first arrow. The increase in the concentration of sodium chloride is indicated by the second arrow. Fractions of 3 ml were collected and aliquots of 20 ~1 of each fraction were used to assay incorporation activities for ethanolamine (O---O) and serine (O-.-m). The activity is shown as described in the legend of Fig. 1.

660

MIURA

AND

KANFER

sults suggest that the choline incorpora- were separated from one another by tion enzyme may still mainly consist of DEAE-cellulose column chromatography membranous fragments even after the in the presence of L-serine. A single serine solubilization treatment employed. This incorporating activity was obtained which assumption was also supported by its be- was devoid of either choline or ethanolhavior on Sepharose 4B column chroma- amine activity. tography. When the fraction precipitated These results provide strong evidence by ammonium sulfate from the superna- for the existence of separate base-extant was applied to a Sepharose 4B column change enzymes specific for each base. The and eluted by Miranol H2M, more than properties of these separated enzymes is a two-thirds of choline incorporation activity subject of present and future studies. was present in the turbid void volume. Therefore, it appears that choline incorpoACKNOWLEDGMENTS ration enzyme is not solubilized under This work was supported by USPHS grants, these conditions and remains associated HD05515 and NS 10330. with the microsomal membrane fragments REFERENCES even after removal of two-thirds of membranous proteins. It could be speculated 1. SAITO, M., BOURQUE, E., AND KANFER, J. (1975) that the choline incorporation enzyme is Arch. Biochem. Biophys. 169, 304. 2. GAITI, A., DEMEDIO, G. E., BRUNETTI, M., tightly bound to the basic membranous AMADUCCI, L., AND PORCELLATI, G. (1974) J. structure or is a component of integral Neurochem. 23, 1153. membrane proteins (14). 3. BJERVE, K. S. (1973) Biochim. Biophys. Acta The clear separation of choline incorpo296, 549. ration activity from the other two activi4. H~~BSCHER, G., DILS, R. R., AND POVER, W. F. R. ties was confirmed by the Am-Gel 102 col(1959) Biochim. Biophys. Actu 36, 518. umn chromatography. Choline incorpora5. BORKENHAGEN, L. F., KENNEDY, E. P., AND tion activity gave two peaks, one eluted by FIELDING, L. (1961) J. Bid. Chem. 236, PC28. Brij 58 just after the unadsorbed proteins 6. DILS, R. R., AND H~BSCHER, G. (1961) Biochim. and the other by replacement of detergent Biophys. Acta 46, 505. with Miranol H2M. Both of these peaks 7. KANFER, J. N. (1972) J. Lipid Res. 13, 468. 8. PORCELLATI, G., ARIENTI, G., PIROTTA, M., AND contain only a small amount of ethanolGIORGINI, D. (1971) J. Neurochem. 18, 1395. amine incorporation activity. It is, there9. SAITO, M., AND KANFER, J. N. (1973) Biochem. fore, concluded that the choline incorporaBiophys. Res. Commun. 53, 391. tion enzyme(s) is different from those for 10. FLEISCHER, S., AND FLEISCHER, B. (1967) in the other two bases. This finding is in Methods in Enzymology (Estabrook, R. W., accord with different characteristics reand Pullman, M. E., eds.), Vol. 10, p. 419, ported for the choline incorporation activAcademic Press, New York and London. ity and the other two activities (1, 15). 11. YONETANI, T. (1961) J. Biol. Chem. 236,168O. The ethanolamine incorporation activ- 12. LAYNE, E. (1957) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), ity was adsorbed to the At&Gel 102 colVol. 3, p. 451, Academic Press, New York and umn and was eluted with Miranol H2M as London. a sharp peak which was free from the activities for incorporation of serine and cho- 13. BARTLETT, G. R. (1959) J. Biol. Chem. 234, 466. line. This indicates that there is at least 14. GULIK-KRZYWICKI, T. (1975) Biochim. Biophys. Acta 415, 1. one enzyme which has only ethanolamine 15. PORCELLATI, G., AND DI JESO, F. (1971) in Memincorporation activity. The serine incorpobrane-Bound Enzymes, Proceedings of an Inration activity was not retained by this ternational Symposium (Porcellati, G., and column and was associated with some ethDi Jeso, F., eds.), p. 111, Plenum Press, New anolamine activity. These two activities York.