A hydrophobic tryptic peptide from bovine white matter proteolipid

Bioehimica et Biophysica Acta, 702 (1982) 117-124

117

Elsevier Biomedical Press BBA 31112

A HYDROPHOBIC TRYPTIC PEPTIDE FROM BOVINE WHITE MATI'ER PROTEOLIPID MARJORIE B. LEES a,b BETTY H. CHAO a, RICHARD A. LAURSEN c and JAMES J. L'ITALIEN c

Biochemistry Department, E.K. Shriver Center, Waltham, MA 02254, h Department of Biological Chemistry, Harvard Medical School, Boston, MA 02115 and e Department of Chemistry, Boston University, Boston, MA 02215 (U.S.A.) (Received August 12th, 1981)

Key words: Proteolipid; Tryptic peptide; Myelin; (Bovine white matter)

A hydrophobic, chloroform-sohihle tryptic peptide with a molecular weight of approximately 4000 has been purified from the bovine white matter proteolipid protein. Its primary structure was obtained by a combination of solid-phase Edman degradation and mass spectrometry. A major part of the tryptic peptide appears to be inaccessible to the action of proteolytic enzymes. The peptide spans the three cyanogen bromide peptides located by Jollts et ai. (Binchem. Biophys. Res. Commun. (1979) 87, 619-626) at the COOH-terminai region of the intact protein. Secondary structure calculations for this region indicate a segregation into discrete domains, with most of the tryptic peptide corresponding to a highly ordered, hydrophobic domain; an equal probability for a-belicai or ~-structure is predicted for this region.

Introduction The major protein of myelin in the central nervous system is the proteolipid protein, a hydrophobic intrinsic membrane protein which can be extracted from brain white matter with chloroform/methanol mixtures [1-3]. The proteolipid apoprotein, devoid of complex lipids, is completely soluble in chloroform/methanol but can be converted to a water-soluble form. Chemical and physical studies have been difficult because of the aggregation of the water-soluble form of the protein and the inability of denaturing solvents to disaggregate it. The large peptides obtained by chemical or enzymatic cleavage also have a strong tendency to aggregate. Consequently, many conventional methods of protein chemistry have been impossible to apply and this has made determination of the primary structure of the protein exceedingly difficult. Nevertheless, the NH2-terminal se-

Abbreviation: SDS, sodium dodecyl sulfate. 0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

quences (15-30 residues) of the proteolipid isolated from several species have been obtained [4-9], as have the compositions and sequences of several relatively small (less than 12 residues) tryptic peptides [9,10]. A major step towards elucidation of the primary structure was the separation and partial sequencing of four cyanogen bromide peptides from the bovine proteolipid [11]. The present paper reports the sequence of a very hydrophobic, chloroform-soluble tryptic peptide derived from the COOH-terminal region of the protein. It includes a portion of the molecule which had not been sequenced previously. A combination of solid-phase sequence methodology and mass spectrometry was used to obtain the sequence. Methods

Preparation of proteolipid Proteolipid was prepared from a washed chloroform/methanol extract of subcortical white matter [12]. Lipids were partially removed by the emulsion-centrifugation procedure of Folch and co-

118

workers [2,13]. Various preparations contained between 36-42% protein and 1.4-1.5% lipid phosphorous. Lipid-containing preparations were used in the present study since their yield of tryptic peptide was significantly higher than from the apoprotein. SDS-polyacrylamide gel electrophoresis of either lipid-containing preparations or the apoprotein showed a major band corresponding to a molecular weight of approximately 25000 and minor bands at approximately 20000 and 12000. The similarities among the different bands and the suitability of this preparation for sequence studies have been documented [3,5].

COOH-terminal amino acids, samples were dried, resuspended in 0.2 M N-ethylmorpholine and digested with carboxypeptidase A and/or B for 4 h at a 1 : 20 enzyme: substrate ratio. After neutralization, free amino acids were determined on a Beckman amino acid analyzer. For CNBr cleavage, the dried peptide was dissolved in 88% formic acid and treated with a 60-fold weight excess of CNBr. After reaction for 24h, the sample was concentrated by rotary evaporation to remove excess reagent and lyophilized.

Preparation of purified trypticpeptide

Peptides were sequenced by solid-phase Edman degradation [14,15]. The lower phase tryptic peptides were immobilized by adding 1 ml of the peptide solution in chloroform/methanol (80 nmol ml) to p-phenylenediisothiocyanate-activated aminopropyl glass [16]. Triethylamine (100 #1) was added and the mixture was kept at 45°C for 1 h with occasional shaking. Excess isothiocyanate groups were blocked by reaction with 100 ml of ethanolamine for 90 min. The support was then washed three times with 5 ml of methanol and was then dried. Cyanogen bromide peptides containing COOH-terminal homoserine were coupled to /3(aminoethyl)aminopropyl glass according to the method of Bridgen [17]. Chymotryptic peptides were coupled to aminopolystyrene by the carbodiimide method [ 18]. Peptides were sequenced by Edman degradation on a Sequemat Mini-15 solid-phase peptide sequencer using standard reagents and programs. Thiazolinones were converted to phenylthiohydantoins automatically in a Sequemat P-6 Autoconverter using 2 M methanolic HC1. Phenylthiohydantoin amino acids wre identified by HPLC using the methanol/sodium acetate system described by L'Italien and Laursen [18] or by back hydrolysis to free amino acids [19]. All of the common phenylthiohydantoins were resolved and eluted within 22 rain with the exception of phenylthiohydantoin-Thr and phenylthiohydantoin-His, which eluted together. The former was usually detected as the dehydro derivative by monitoring at 313 nm.

A proteolipid sample containing 40 mg of protein was suspended in 20 ml of 0.1 M borate buffer (pH 8.5) containing 0.6% Triton X-100 and was digested with 1 ml of L-(tosylamido-2-phenyl)ethyl chloromethylketone-treated trypsin (Millipore Corp.) (1 mg/ml) for 16h at 37°C with gentle shaking. 4 vol. of chloroform/methanol, 2 : 1 (v/v) were added and, after mixing and centrifugation, the lower phase was collected and concentrated to a small volume in a rotary evaporator. The sample was chromatographed on a Sephadex LH60 colu m n (0.9 X 144 cm) equilibrated with chloroform/methanol 2 : 1 (v/v). Fractions of 1.25 ml were collected and monitored at 280 nm. The appropriate peak was re-chromatographed on the same column, and fractions containing the purified peptide were pooled and stored as a chloroform/methanol solution.

Cleavage procedures The purified peptide, designated tryptic lower phase peptide, was converted to the water-soluble form by the same procedure which had been developed for conversion of the chloroform/methanolsoluble apoprotein to its water-soluble form [13]. Ammonium acetate buffer (0.1 M, pH 8.5) containing 0.6% Triton X-100 was added and the peptide was digested with chymotrypsin or elastase at an enzyme:substrate ratio of 1:40 (w/w) for 4 h. Digestion with pepsin was carried out in 0.01 M HC1/0.6% Triton at an enzyme:substrate ratio of 1 : 20. Chloroform/methanol was added to the digest to form a two-phase system and each of the phases was collected separately. To determine

Solid-phase sequencingprocedures

Gas chromatography-mass spectrometry (GC-MS) Derivatization of the peptide mixture obtained

119

by digestion with pepsin was carried out by the procedures developed by Biemann and collaborators [20]. Briefy, approximately 100 nmol of peptide mixture were methylated with 1 rnl of anhydrous 3 N methanolic HCI for 30 rain at room temperature. Trifluoroacetylation was carried out at a pH of over 8 overnight at room temperature with 0.5 ml of 1 : 1 methyl trifluoroacetate/methanol. The trifluoroacetylated peptide methyl esters were reduced with B22H6 (200/~l/mg protein) in tetrahydrofuran. After reaction at 90°C for 30 rain, excess B22H6 was destroyed with methanol. The resulting boroethers and boroamines were cleaved by methanolysis in 300/tl 1 N anhydrous methanolic HC1 at 90°C for 30 min and extracted three times with methylene chloride from 25% K2CO3 and then from saturated K2CO 3. The combined extracts were trimethylsilylatedwith 80 #1 1 : 1 pyridine/trimethylsilyldiethylamine at 55°C for 30 rain. Approximately 1 #1 of the derivatized peptide solution was chromatographed on a 6 foot × 2 mm GC column packed with 3% OV-17 on 80-100 mesh Supelcoport. The column temperature was programmed to increase from 70 to 335°C at 5°C per min. The GLC was interfaced with a Finnegan 4000 mass spectrometer and spectra were acquired repetitively from rn/z 70 to 750 every 4.6 s. Data were analyzed by manual computation.

Other methods Amino acid analyses were carried out by standard procedures on a Beckman model 119C amino acid analyzer after hydrolysis of samples in vacuo for 24h at l l0°C in 6N HCI. For tryptophan determination, hydrolysis was carried out with 4 N methanesulfonic acid [21]. Protein was determined by the method of Lowry et al. [38] as modified by Lees and Paxman [22]. Phosphorus was determined by the method of Bartlett [23]. Dansylation was carried out by a modification of the method of Tamura et al. [24], in which the peptides and reagents were dissolved in 2:1 (v/v) chloroform/methanol. Dansyl amino acids were identified by one-dimensional thin-layer chromatography on polyamide plates using benzene/acetic acid (9: 1, v/v) followed by ethyl acetate/acetic acid/methanol (20:1:1, v/v) as the solvent systems. SDS-polyacrylamide gel electrophoresis was

carried out on 8-22% slab gels as described by Laemmli [25]. Reverse-phase HPLC was carried out on a Whatman ODS -10 column in 20 mM phosphate buffer, pH 2.5, with a gradient of 0-30% acetonitrile using an Altex model 110 pump system and a variable wave length detector. Detection was at 215 rim. Results and Discussion

Purification and characterization of lower-phase tryptic peptide The peptides in the tryptic digest of the proteolipid were initially partitioned in a two-phase chloroform/methanol/water system. Peptide maps of the small water-soluble peptides which distributed into the upper, methanol/water phase were identical to those of the acid-soluble peptides previously reported [10]. The insoluble material at the interface was not characterized but presumably consisted of incompletely digested protein and aggregated peptides. The lower, chloroform phase contained approximately 10% of the amino acids of the starting material along with lipids and detergent. A major, hydrophobic peptide was purified from this fraction by Sephadex LH60 chromatography. The peptide (peak II, Fig. la) was separated from the aggregated material in the void volume (peak I) and from a large lipid and detergent-containing peak (peak III). Rechromatography of peak II resulted in a single, slightly asymmetric peak at a position corresponding to a 2.0 a

15

A280 ID

I

04 05 0.2

I 20

1I

40

m 610

FRACTION NUMBER

8O

20 40 60 FRACTION NUMBER

Fig. I. Purification of tryptic lower phase peptide, a,-Initial separation; b, rechromatography of peak II. Conditions: Sephadex LH 60 c o l u m n 0.9 X 144 cm; eluant, chloroform/methanol, 2: I v/v; flow rate, 6 ml/h; fraction size 1.25 ml~ detection. ,428Onm.

120

molecular weight of approximately 4000 (Fig. lb). Upon SDS-polyacrylamide gel electrophoresis, the peptide migrated to a position consistent with its molecular weight determined by chromatography. Dansylation showed a single NH 2-terminal amino acid which was identified as threonine. Reaction with carboxypeptidaseB released arginine as the only COOH-terminal amino acid, whereas reaction with carboxypeptidaseA was negative, as expected for a tryptic peptide. It was concluded therefore that the peptide consisted of a single chain containing an internal lysine which was inaccessible to trypsin. The amino acid composition of the purified peptide indicated an extremely hydrophobic molecule with the four amino acids, valine, alanine, phenylalanine and leucine, accounting for more than half of the total amino acids. On the basis of integral mol ratios, the peptide contained about 43 amino acid residues and included 2 mol of methionine (Table I). Cysteine, tryptophan and proline were absent.

Solid-phase sequence analysis of tryptic lower-phase peptide The tryptic lower-phase peptide was coupled to the solid support in good yield while still dissolved in chloroform/methanol, thus avoiding the problem of dissolving the peptide in the aqueous solvents usually used [26]. Edman degradation gave the first 23 residues of the peptide, except for the NH2-terminal, which remained attached to the support, and residue 9, for which no phenylthiohydantoin amino acid could be detected (Fig. 2). The NH2-terminal residue was identified as threonine by dansylation and by Edman degradation in the spinning cup sequenator [27]. Only very limited sequence information could be obtained by the latter method because of washout of the peptide into the organic solvents required for extraction of phenylthiohydantoin amino acids. The absence of a detectable residue at position 9 remains unexplained, although one possibility is that it is a site for the attachment of covalently bound fatty acid [28-30].

TABLE I

Cyanogen bromide cleavage of tryptic lower-phase peptide

AMINO ACID COMPOSITION OF TRYPTIC LOWER PHASE PEPTIDE

By CNBr treatment, only 60% of the methionine was converted to homoserine, regardless of the

Amino acid

Mol percent

Mol ratio io 2o Thr - AIa-G lug,Phe-O In-Val -Thr -Phe-I-Leu-, Phe-Ile- Ala- Ala-Fhe-Val -G iy-$1 a- A1a- AI 8

From amino acid composition Asp Thr Ser Giu Gly Ala Val Met Ile Leu Tyr Phe His Lys Arg Unidentified Total

2.6 10.8 3.0 5.0 5.5 19.8 7.2 5.6 4.6 13.6 2.3 12.7 1.9 3.3 2.0

1.1 4.7 1.3 2.2 2.4 8,5 3,0 2.4 2,0 5,9 1,0 5,5 0,8 1.4 0,9 43 ---+1

From sequence

I 5 1 2 2 9 4 2 2 5 1 6 1 1 1 43

tC1

t

/

/

/

/

/

/

/

/

/

/

/

/

/

/

/

I

I

30 Thr.LeU-,Vlfl.-$et..L eur,T~-Phe.Melh-Zle-Ala.Ala-Thr-Tyr~-Asn.Phe-~Ala-V~-Leu, , ~--T-7 j ,r.~ / / / / ILC~ 2 /

/

l

/

40 Lys-l, eurHet-Gly-Arg rC7

i

I

IICB 3

t

Fig. 2. Amino acid sequence of tryptic lower phase peptide from bovine brain white matter proteolipid protein. Peptide designations: C, chymotryptic pepfides; CB, cyanogen bromide peptides; 7-, residue identified by solid-phase Edman degradation. Unlabelled, underlined sequences were identified by mass spectrometry.

121 I00

~z m

~

The two coupled peptides were sequenced simultaneously and, since the sequence of one of the peptides (the NH2-terminal peptide) was known from the studies described above, the partial sequence of the second peptide could be deduced as X-Ala-Ala-Thr-Tyr.

~

60

Additional enzymatic cleavages

z

40 o

20

20

60

FRACTION NUMBER

Fig. 3. Chromatographyof cyanogenbromide peptides from tryptic lower-phasepeptide. Conditions: Bio-Rad P4 column 0.9 × 120 cm; eluant 88% formicacid; detection,fluorescamine after acid hydrolysis.

amount of CNBr used or the time of reaction. A comparable value has also been obtained with the original protein (unpublished data). The lyophilized CNBr peptides could not be redissolved in aqueous buffers or in any of the organic solvents tested. They could be solubilized in 88% formic acid but precipitated upon dilution of the acid. Elution from a Bio-Rad P4 column with 88% formic acid gave one major asymmetrical peak and a minor peak (Fig. 3). The latter (CB 3, Fig. 2) contained only the dipeptide Gly-Arg which derived from the COOH-terminal region of the peptide since it contained no methionine. Amino acid analysis of samples taken from various portions of the major peak were all similar, suggesting a mixture of uncleaved and aggregated material. Further chromatographic separation of the CNBr peptides did not appear practical. Therefore, the peptide mixture was attached, without prior purification, to a solid support by coupling at homoserine lactone [26]. The COOH -terminal Gly-Arg peptide (CB3) does not couple whereas CB 1 and CB2 were covalently attached to fl(aminoethyl)aminopropyl glass [17]. A coupling yield of 25% of the available groups was obtained.

The tryptic lower phase peptide was digested with chymotrypsin, and the cleavage products were partitioned into aqueous and chloroform phases. Using HPLC, two peptides were isolated: C2, Asn -Phe-Ala-Val-Leu; and C3, Lys-Leu-Met-Gly-Arg, which comprise residues 34-43 of the tryptic peptide. The lower phase contained the remainder of the molecule (C1); no small peptides could be isolated from it by chromatography on Sephadex LH 60. Although chymotryptic digestion is expected to cleave at tyrosine, phenylalanine and other apolar residues, the only cleavages observed were at the COOH-terminal side of residues Tyr-33 and Leu-38. Similarly, elastase appeared to cleave only in the COOH-terminal region of the peptide. Thus, under the experimental conditions described, a major portion of the intact peptide appeared to be inaccessible to digestion with chymotrypsin and elastase and these enzymes could not be used to obtain the sequence of residues 24-29. Our previous experiences suggested that protonation might reduce the tendency for the peptide to aggregate. The pH optimum for pepsin is approximately 2.5 and it was reasonable therefore to assess the results of peptic digestion. In contrast to the limited digestion by chymotrypsin and elastase, pepsin cleaved the tryptic lower-phase peptide extensively. After partition of the peptic digest essentially all of the peptides were found in the methanol/water phase. About 15% of the starting material was recovered from the interface; the only amino acid which could be detected in the chloroform phase was serine (from phosphatidylserine). HPLC of the digest separated many peaks (over 40), but amino acid analysis of selected peaks did not give integral mol ratios, suggesting that the peaks each contained more than one peptide. Purification of this complex mixture was considered impractical and no further attempts were made to purify peptic peptides.

122

Mass spectrometry The Edman method requires the isolation and purification of peptides prior to their sequencing. By contrast, sequencing by GC-MS can be carried out on mixtures of small peptides, usually dipeptides to hexapeptides. The large number of peptic peptides was therefore ideally suited for GC-MS protein sequencing. Identification of all of the mass ions obtained was not attempted since analysis of the data was carried out manually. Emphasis was placed on finding overlapping peptides from the unsequenced region in the vicinity of methionine 28. However, the sequence of several other regions of the tryptic peptide was also confirmed (Table II and Fig. 2). From the overlapping peptides, the sequence Ser-Leu-ThrPhe-Met was determined and identified as residues 24 through 28. The peptide-Ala-Ala-Ala-Thr-Leu was found in the mixture, confirming the occurrence of three alanine residues at positions 18-20. The sequence presented in Fig. 2 accounts for all of the residues indicated by the amino acid composition (Table I) with the exception of a histidine and possibly an additional leucine. The data in Fig. 2 are also consistent with sequences in which

TABLE II PEPTIDES IDENTIFIED BY MASS SPECTROMETRY The retention index incrementis defined by Nau and Biemann [3 I]. Observed and calculated values agree within 3.5% of one another. Leu and lie cannot be differentiated by mass spectrometry. Designations are made on the basis of other information. Peptide

Thr-Leu Ala-Val-Leu Lys-Leu Val-Ser-Leu Thr-Ala-Glu Phe-Val-Gly Leu-Val-Ser-Leu Leu-Thr-Phe Ala-Ala-Ala-Thr-Leu Phe-lle-Ala-Ala Thr-Phe-Met Phe-Met-Ile-Ala

Retention index increment Observed

Calculated

1673 1851 1952 2059 2241 2341 2457 2519 2571 2 71 I 2891 3188

1650 1865 1910 2070 2240 2355 2545 2600 2645 2 735 2950 3 225

Leu or His-Leu are inserted between Leu-25 and Thr-26, so our proposed sequence must be regarded as tentative.

Secondary structure of the COOH-terminal region of the protein Joll6s and co-workers [5] have separated and partially sequenced the four CNBr peptides (CN 1 -4) from the intact proteolipid protein. The tryptic lower-phase peptide can be located within the COOH-terminal region spanning CN2, CN3 and CN4 (Fig. 4) and its sequence completes a major part of the primary structure of this region. The molecular weight of CN2 determined by gel filtration was 4500--+ 400 [11]. The sum of the 18 CN2 residues sequenced by Joll~s and co-workers and the 28 CN2 residues which we have sequenced accounts for a calculated molecular weight of approximately 4800, assuming a value of 100 for each of the two unknown residues (see Fig.4). CN2 presumably contains a cysteine residue since oxidation was required for its separation from CN 1. The cysteine could occur at the unidentified residue 14 described by Joll6s and co-workers, or after Leu-18. In addition, a lysine or arginine residue theoretically should precede the N H 2terminal threonine of the tryptic peptide. This evaluation indicates the probability that our data comes close to completing the primary structure of CN2, but overlapping sequences will be required to determine the precise number of missing residues. Regardless of the size of the segment missing from CN2, the COOH-terminal region of the proteolipid (CN2-CN4) appears to consist of several distinct domains. Using the method of Chou and Fasman [32], we predict that the region sequenced by Joll6s and co-workers [5] has no regular ordered structure, although fl-turns may occur at several closely spaced sequences. Similarly, the sequence Gly-Arg-Gly-Thr within CN4 probably corresponds to a fl-turn (Pt = 1.26). By contrast, The first 40 residues which we sequenced (Fig. 2) are predominantly hydrophobic amino acids with only a single strong a-breaker (Gly-17) and a single strong fl-breaker (Glu 3). Analysis of this hydrophobic region by the method of Chou and Fasman indicates a highly ordered structure with no 13turns and an equal probability of a-helical or fl-sheet

123 P4 =

1.17 1.28 II I I I v yGVLPWNAFPGKVXGSNL . . . . YRTAEFQVTFX L F I A A F V G A A A T L V S L T F M I CN2

t I

1.20

1.26 ["

/ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Pa = 0.86 P~= 1.02

Pe= 1.13 P/9= 1.18

I

IAATYNFAVLKLMGRGTKF 11 CN3 II CN4 I .I

Pc= 0.87 P~ = 0.96

Fig. 4. Primary structure of COOH-terminai region of bovine white matter proteolipid. CN2-CN4 refers to the CNBr peptides of Jolles et al. [I 1] who reported the sequencesof CN3, CN4 and the first 18 residues of CN2. -- -- --, tryptic lower phase peptide. P,,, P/~and Pt calculated accordingto the method of Chou and Fasman [32].

structure (P~ = 1.13 and P/~ = 1.18). Although it is possible that the Chou and Fasman method is not applicable to hydrophobic proteins, an alternative explanation is that the data may be predicting correctly a potential for conformational flexibility; i.e. the ability to interconvert between a- and B-forms. The solubility properties of the intact proteolipid protein and its ability to exist in either a water-soluble or an organic solvent-soluble form demonstrate the conformational flexibility of this protein. Circular dichroism studies on lipophilin, the comparable proteolipid isolated from human myelin, indicate an ordered structure containing either 0 or 38% B-pleated sheet, depending on the method of preparation of the protein [33]. Similarly, circular dichroism measurements show either 0 or 43% B-sheet conformation, depending on the solvent, for a synthetic peptide representing the precursor-specific region of preproparathyroid hormone [34], whereas conformational analysis utilizing the Chou and Fasman method predicts an equal probability for a- or B-structure (Pa = 1.14 and P/~ = 1.18). Bacteriorhodopsin has several ordered hydrophobic regions, which Unwin and Henderson [35] have proposed to have an a-hehcal structure, buried within the bilayer. However, analysis of several regions of the reported sequence [36] by the method of Chou and Fasman predicts equal probability for either a- or Bstructure. For example, the relatively hydrophobic region 51-69 of bacteriorhodopsin, which occurs between two B-turns and probably spans the membrane [36], has a value of 1.03 for P~ and 1.13 for P/~. In region 143-157, which is entirely hydrophobic, Pa and P/~ are 1.08 a n d 1.26, respectively. Analysis of the conformations of hydrophobic domains in several membrane-associated proteins

by the method of Chou and Fasman [32] thus results in prediction of an equal probability for a-helical or B-sheet structure or a slight preference for B-sheet. On the other hand, electron diffraction data [35] indicate a-helical structures for bacteriorhodopsin. Furthermore, Engelman and Steitz [37] have argued on theoretical grounds that only hehcal structures are likely to be found in lipid bilayers. Thus, either the Chou and Fasman method does not hold for polypeptides in a lipid environment or B-sheet structures may indeed be found in bilayers despite arguments to the contrary. If the latter were the case, changes from an a-hdical to a B-sheet conformation might provide a mechanism for the modulation of dynamic membrane processes. Regardless of the actual conformation for these hydrophobic domains, the proteolipid region we have sequenced seems to fit the general pattern seen for other hydrophobic, membrane-associated proteins.

Acknowledgements This work has been supported in part by U.S. Public Health Service grant NS13649 (M.B.L.) and National Science Foundation grant PCM79-04910 (R.A.L.). We are indebted to Mr. James Evans for the derivatization of the peptides for mass spectrometry and for carrying out the GC-MS determinations.

References 1 Folch, J. and Lees, M. (1951) J. Biol. Chem. 191,807-817 2 Folch-Pi, L and Stoffyn, PJ. (1972) Ann. N.Y. Acad. Sci. 195, 86-107 3 Lees, M.B., Sakura, LD., Sapirstein, V.S. and Curatolo, W. (1979) Biochim.Biophys.Acta 559, 209-230

124 4 Nussbaum, J.L., Rouayrenc, J.F., Mandel, P., Joll~s, J. and Joll~s, P. (1974) Biochem. Biophys. Res. Commun. 57, 1240-1247 5 Vacher-Lepretre, M., Nicot, C., Alfsen, A., Joll~s, J. and Joll/~s, P. (1976) Biochim. Biophys. Acta 420, 323-331 6 Lees, M.B., Chan, D. and Foster, J. (1976) Trans. Am. Soc. Neurochem. 7, 183 7 Nussbaum, J.L., Rouayrenc, J.F., Joll~s, J., Joll~s, P. and Mandel, P. (1974) FEBS Lett. 45, 295-298 8 Boggs, J. and Moscarello, M. (1978) Biochim. Biophys. Acta 515, 1-21 9 Joll6s, J., Nussbaum, J.L., Schoentgen, F., Mandel, P. and Joll6s, P. (1977) FEBS Lett. 74, 190-194 10 Chan, D.S. and Lees, M.B. (1978) J. Neurochem. 30, 983990 11 Joll6s, J., Schoentgen, F., Joll6s, P., Vacher, M., Nicot, C. and Alfsen, A. (1979) Biochem. Biophys. Res. Commun. 87, 619-626 12 Folch, J., Lees, M.B. and Sloane-Stanley, G.H. (1957) J. Biol. Chem. 226, 497-509 13 Lees, M.B. and Sakura, J.D. (1978) in Research Methods in Neurochemistry (Marks, N. and Rodnight, R., eds.), pp. 354-370, Plenum Publishing Corp. New York 14 Laursen, R.A. (1971) Eur. J. Biochem. 20, 89-102 15 Laursen, R.A. and Machleidt, W. (1980) Methods Biochem. Anal. 26, 201-284 16 Wachter, E., Machleidt, W., Hofner, H. and Otto, J. (1973) FEBS Lett. 35, 97-102 17 Bridgen, J. (1975) FEBS Lett. 50, 159-162 18 L'Italien, J.J. and Laursen, R.A. (1981) J. Biol. Chem. 256, 8092-8101 19 Mendez, E. and Lal, C.Y. (1975) Anal. Biochem. 68, 47-53 20 Carr, S.A., Herlihy, W.C. and Biemann, K. (1981) Biomed. Mass. Spectrom. 8, 51-62

21 Simpson, R.J., Neuberger, M.R. and Liu, T.Y. (1976) J. Biol. Chem. 251, 1936-1940 22 Lees, M.B. and Paxman, S.A. (1974) J. Neurochem. 23, 825-933 23 Bartlett, G. (1959) J. Biol. Chem. 234, 466-468 24 Tamura, Z., Nakajima, T., Pisano, J.J. and Udenfriend, S. (1973) Anal. Biochem. 52, 595-606 25 Laemmli, U.K. (1970) Nature 227, 680-685 26 Horn, M.J. and Laursen, R.A. (1973) FEBS Lett. 36, 285288 27 Edman, P. and Begg, G. (1967) Eur. J. Biochem. 1, 80-92 28 Stoffyn, P. and Folch-Pi, J. (1971) Biochem. Biophys. Res. Commun. 44, 157-161 29 Gagnon, J., Finch, P.R., Wood, D.D. and Moscarello, M.A. (1971) Biochemistry 10, 4756-4763 30 Braun, P.E. and Radin, N.S. (1969) Biochemistry 8, 43104318 31 Nan, H. and Biemann, K. (1976) Anal. Biochem. 73, 139153 32 Chou, P.Y. and Fasman, G.D. (1978) Adv. Enzymol. 47, 45-148 33 Anthony, J. and Moscarello, M.A. (1971) FEBS Lett. 15, 335-339 34 Rosenblatt, M., Beaudette, N.V. and Fasman, G.D. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3983-3987 35 Unwin, P.N.T. and Henderson, R. (1975) J. Mol. Biol. 94, 425-440 36 Khorana, H.G., Gerber, G.E., Herlihy, W.C., Gray, C.P., Anderegg, R.J., Nihei, K. and Biemann, K. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5046-5050 37 Engelman, D.M. and Steitz, T.A. (1981) Cell 23, 411-422 38 Lowry, O.H., Rosebrough, N.J., Farr, A. and Randall, R.J. (1981) J. Biol. Chem. 193, 265-275