0022-3042 78 1001-109510200~0
Josmal o/ Nuemrhu,ritrfn Vol. 31. pp 1095- 1099 Pergamon Press Ltd. 1978. Printed in Great Britain 0 International Society lor Neurochemislry Ltd
isoelectric focusing of proteolipid (Receioed 21 March 1978. Accepted 5 May 1978)
Crude bovine white matter proteolipid was prepared by the emulsion-centrifugation procedure of FOLCHet al. (1959), omitting the final organic solvent extractions. The preparations contained 5&55% lipid, but the protein moiety was equivalent to that of the apoprotein described above. Focusing in gels. Isoelectric focusing was carried out in polyacrylamide tube gels containing a final pH gradient between 6.6 and 9.3. The gel solution (5% T, 5% C) consisted of 1.5% Triton X-100, 6 M-urea, and a 1 : I mixture of pH 3.5-10 and pH 9-1 1 ampholytes at a final ampholyte concentration of 2%. Although the ampholyte mixture has a range of pH3.5-11, the final pH gradient obtained in polyacrylamide gels was considerably narrower than the theoretical range. The gel solution was degassed at 15 mm-Hg for 5 min and potassium persulfate added to give a final concentration of 0.72%. Fifty micrograms of apoprotein in Triton X-100 was combined with 2ml of gel solution and the mixture poured into a 12 x 0.5cm gel tube. A flat gel surface was obtained by carefully overlaying the gel solution with distilled water. Polymerization was completed within 1 h. Electrofocusing was performed for 12h at constant voltage with 2 0 m ~ - N a O Has the catholyte and I O ~ M - H ~ P Oas, the anolyte. For the first hour the current was 0.5 mA per gel tube; for the remaining period, 2 mA per gel tube. At the completion of focusing 1 ml of the catholyte above the surface of each gel MATERIALS AND METHODS was dialyzed against water and analyzed for protein by Chemicals. Triton X-100 was obtained from Sigma the procedure of LOWRYet a/. (1951) as modified by LEES (1972). Gels were fixed and washed exhaustively Chemical Co., St. Louis, MO, Brij 35 from Pierce Chemical & PAXMAN Co., Rockford, IL and ultra pure urea from Schwarz- in 10% trichloroacetic acid to remove amphplyte and Mann, Orangeburg, NY. All ampholytes were from LKB- detergent. Proteins were then stained in 1% Coomassie Producter AB, Sweden. Chymotrypsinogen A was pur- blue in 7% acetic acid for 1 h, and gels were destained chased from Worthington Biochemical Corp., Freehold, by diffusion in acetic acid-methanol-water, 7:30:63 by vol. The pH gradient was determined on 1.5mm slices of a NJ. Proteolipid preparations. Proteolipid apoprotein was pre- blank electrofocused gel with a Sargent-Welch Model NX pared from a concentrated total lipid extract of bovine pH meter. Focusing in .sucrose gradients. Isoelectric focusing in a white matter by dialysis against neutral and acidified chloroform-methanol as described by FOLCH-PI& sucrose gradient was performed by the method of VESTERSTOFFYN (1972). The apoprotein contained c 0.04% phos- BERG (1971) but with the inclusion of Brij 35 at a concenphorus and was stored in 2:l chloroform-methanol at a tration of 1% in both the dense and the light sucrose soluconcentration of approx 5 mg/ml. For isoelectric focusing tions. Focusing was carried out in an electrofocusing in polyacrylamide gels, the apoprotein was converted to column (LKB No. 8102) with a 1% pH 3.510.0 ampholyte. a water-soluble form by the rapid method of SHERMAN Apoprotein (7.6mg) was mixed with the dense sucrose & FOLCH-PI (1970) and diluted with Triton X-100 to give solution. The anolyte consisted of 10% concentrated a final concentration of 1 mg protein/ml in 1.5% Triton H 2 S 0 4 prepared in 50% sucrose; the catholyte consisted X-100. For focusing in sucrose gradients, the apoprotein of 2% aqueous ethylamine (v/v). Electrofocusing was perwas converted in the presence of 0.1% mercaptoethanol. formed for 48 h at a final potential of 400V with tap water as a coolant. After completion of focusing 2.3 ml fractions were collected, and the pH and absorbance (280nm) of Abbreviation used: SDS, sodium dodecyl sulfate. each fraction was measured. ISOELECTRIC focusing is a sensitive method for determining the homogeneity of a protein and provides information on its physicochemical characteristics. The homogeneity of bovine brain white matter proteolipid has been difficult to assess because of its tendency to aggregate. The protein resolves into multiple bands on SDS polyacrylamide gel electrophoresis, but several approaches suggest a chemical similarity among the different bands. Analysis of Ferguson plots of the bands has suggested the existence of an oligometric series (CHAN& LEES,1974), and analysis of the proteins recovered from the gel bands shows essentially the same amino acid compositions, the same amino- and carboxyl-terminal amino acids and the same sequence of at least the first eight amino acids at the amino terminus et al., 1976; NICOTet a/., 1973; CHAN (VACHER-LEPR~TRE & LEES,1978). However, AGRAWAL et a/. (personal communication) have found that antibodies to the major proteolipid band isolated from rat myelin do not cross react with another gel component, namely, the band designated as DM20 (AGRAWALet al., 1972). The present study describes the isoelectric focusing of bovine white matter proteolipids in both polyacrylamide gels and sucrose gradients and presents evidence for a charge homogeneity of the apoprotein.
Proteolipid apoprotein subjected to isoelectric focusing in polyacrylamide tube gels in the presence of urea and Triton X-I00 exhibited the pattern shown in Fig. I . The single, well-resolved band observed near the cathode had an isoelectric point corresponding to 9.2. There was no evidence of protein in any other part of the gel, and analysis of the solution above the gel for protein gave a value of 3 pg of protein (sample size, SOpg). This amount of protein was less than that observed with the same size sample of pure chymotrypsinogen A standard (PI = 9.1). Focusing was achieved only when the sample was polymerized within the gel; when applied at either the cathode or anode, the protein precipitated. Difficulties arise when a protein to be focused has a PI above 9 because of the instability of the high pH gradient in polyacrylamide gels. Focusing for a shorter period of time (8 h or less) improved the gradient but failed to focus the protein. Focusing in a narrow range gradient was unsuccessful and the protein remained spread throughout the gel, probably as a consequence of the long exposure and concentration of the protein in the high pH region. A mixture of pH 3.5-10 and 9-1 1 ampholytes provided a sufficient acidic region to permit the protein to focus. Crude proteolipid preparations, i.e. protein which contained major amounts of lipid, failed to focus under conditions where the apoprotein focused. Isoelectric focusing of the apoprotein was also carried out in a sucrose-Brij 35 medium which allowed for the formation of a higher pH gradient than was obtained in gels. A single protein peak was recovered which had an isoelectric point between 8.9 and 9.2 (Fig. 2). The position of the peak did not change when the sample was mixed with either the light or dense sucrose solutions. Omission of detergent resulted in precipitation of the apoprotein. Recovery of protein in the major peak from the column was 9796 based on E:; = 13.6 at 280nm (FOLCH & STOFFYN, 1972). The U.V.absorbing material which appears in the region between p H 4 and 6 may be derived from a combination of ampholytes, detergents and mercaptoethenol. As was observed in the gels, preparations which still contained lipids did not focus.
FIG.2. Isoelectric focusing of bovine white matter proteolipid apoprotein (6.7 mg) in a sucrose gradient in the presence of Brij 35. The solid line represents absorbance at 280nm. The dotted line indicates the pH gradient measured at 25-C.
was 1401g per ml. These values are one-eightieth and oneseventh, respectively, the concentrations used on SDS gels and should minimize concentration-dependent aggregation. Despite the differences in concentration. the total amount of protein in focusing gels is the same as used for electrophoresis gels where minor bands are detectable. The DM20 should thus be detectable if it focused differently from the major protein species. Since no additional bands are evident, it appears that the DMZO must habe an isoelectric point which is identical or very close to that of the major protein species. The value of 9.2 obtained in the present study by direct measurement of the PI is consistent with titration data (BRAUN& RADIN,1969; FOLCH-PI& STOFFYN,1972) and with obserations on electrophoretic mobility (BRAUN& RADIN,1969; NGLWEN LE et al., 1976). both of which suggest that the apoprotein has a net positive charge at neutral pH. It is also in agreement with the calculated PI value of 9.5 + 0.5 obtained by potentiometric titration DISCUSSION & TER using a mathematical method developed by THOMAS Based on two different methods of isoelectric focusing, MINASSIAN-SARAGA (1976). Although the amino acid coma charge homogeneity of the bovine white matter proteoli- position of the apoprotein shows a higher percentage of pid apoprotein has been demonstrated. The multiple bands dicarboxylic than basic amino acids, no more than half observed on SDS gels may, therefore, correspond to differ- of the acidic residues are titratable in the range between ent physical states of the same protein. These data support pH 3 and 7. Preliminary data on the amide content of the concept of a single molecular species suggested by Fer- the apoprotein suggest the presence of high amounts of guson plots of the different bands (CHAN& LEES, 1974) glutamine and/or asparagine (BRAUN& RADIX, 1969: MARFEY,1973). The electrophoretic behaviour of peptides and identity of their partial sequences (VACHER-LEPR~TRE, 1976). However, the possibility should be considered that obtained upon tryptic digestion of the apoprotein is consisthe detergent does not adequately dissociate individual tent with this view. At least two peptides which contain proteins and that aggregates of several protein species are a higher percentage of acidic than basic amino acids move focusing as a unit. If this were the case, a diffuse band to the cathode, probably as a result of amidation of acidic of heterogeneous aggregates would be expected; however, residues (LEES& CHAN,1975; CHAN& LEES,1978). Application of isoelectric focusing to proteins having the sharp proteolipid apoprotein band (Fig. 1) argues against this possibility. The studies of FEINSTEIN & FELSEN-high isoelectric points is difficult, and only a limited number of such proteins have been characterized by this FELD (1975) suggest that, in dilute solutions of proteolipids, fewer aggregation artifacts occur. Gel isoelectric focusing means. Another membrane protein which has a high isoelectric point is cytochrome c(pI = 9.3) (RADOLA,1973). was achieved only when the sample was polymerized throughout the gel, and under these conditions the initial In myelin both the basic protein and the proteolipid proprotein concentration was only 12.5 p g per ml of gel solu- tein have isoelectric points greater than 9. The existence tion. The initial protein concentration in sucrose gradients of positively charged proteins at physiological pH would
SLICE NUMBER FIG. 1 . lsoelectric focusing of bovine white matter proteolipid apoprotein (25 l i g ) in polyacrylamide gels in the presence of Triton X-100 and 6 M-Urea. Focusing was performed in the region between pH 6.6 and pH 9.3. The dotted line indicates the pH gradient measured at 2 5 T .
Short communication favor association with acidic lipids and may be pertinent for maintaining membrane stability. Acknowl~,dyemenr-This investigation was supported in part by U S . Public Health Service Grant NS 13649. Department qf Biochemistry, Thc Eunice Kennedy Shriwr Center, Walfham, .MA 02154 U.S.A. and Department of Bioloyicul Chemistry, Harcard Medical School, Boston. MA 02115, U.S.A.
M. DRAPER M. B. LEES D. S. CHAN
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