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Bioconjugate Chem. 1002, 3, 160- 166
Photochemical Cross-Linking of Guanosine 5’-Triphosphateto Phosphoenolpyruvate Carboxykinase (GTP) Cristina T. Lewis,+Jerome M. Seyer,i and Gerald M. Carlson’ Department of Biochemistry, The University of Tennessee, Memphis, College of Medicine, Memphis, Tennessee 38163. Received November 19, 1991
Mammalian phosphoenolpyruvate carboxykinase (PEPCK) specifically requires a guanosine or inosine nucleotide as a substrate; however, the structural basis for this nucleotide specificity is not yet known. Because affinity labels derived from guanosine have not yielded a stable, modified peptide in quantities sufficient for sequence analysis, we have investigated the utility of direct photochemical cross-linking of GTP to PEPCK in order to identify the nucleotide binding site. UV irradiation at a distance of 2 cm by a Mineralight lamp (330 pW/cm2) results in the attachment of [ d 2 P 1 G T Pto PEPCK via a stable, covalent linkage in a reaction that is dependent upon GTP concentration and duration of irradiation. After 10 min of irradiation, more than 0.2 mol of [cY-~~PIGTP is incorporated per mole of PEPCK; under these conditions the GTP concentration required for half-maximal labeling is 69 pM. The substrates phosphoenolpyruvate, ITP, and GDP provide protection against photolabeling, as do Mn2+ and Mg2+. One major and one minor radioactive peptide derived from proteolytic digests of photolabeled PEPCK have been isolated and identified. The major modified peptide has been provisionally assigned to an acidic region near the C-terminus, and the minor peptide has been identified as Ser462-Lys471.
INTRODUCTION Mammalian phosphoenolpyruvate carboxykinase (EC 4.1.1.32), referred to hereafter as PEPCK,l catalyzes the conversion of oxaloacetate to phosphoenolpyruvate in the first committed step of gluconeogenesis. The enzyme oxaloacetate + MgGTP(1TP) + phosphoenolpyruvate + MgGDP(1DP) + CO, displays a specific substrate requirement for guanosine or inosine nucleotides; adenosine nucleotides are neither substrates nor inhibitors and do not bind to the enzyme in a detectable manner (Miller et al., 1968). Although the gene codingfor the cytosoliccarboxykinase has been cloned and the amino acid sequence of the enzyme has been determined (Beale et al., 19851, the nucleotide binding site of PEPCK has not been directly identified, and very little is known about the structure of the enzyme’s active site. Furthermore, even though PEPCK does contain three consensus sequences that may represent the guanine nucleotide binding region (Cook et al., 1986), the enzyme does not share extensive regions of homology with other GTP-binding proteins. Chemical modification and affinity labeling are traditional techniques that have made significant contributions to the identification of functionally important enzyme residues, and such procedures have been exploited in several efforts to elucidate the mechanism of PEPCK’s binding specificity. To date, however, modification of + Recipient of the Doggett Predoctoral Fellowship from the College of Graduate Health Sciences, The University of Tennessee, Memphis. Present address: Departments of Chemistry and Molecular Biology, The Scripps Research Institute, 10666 North Torrey Pines Road, La Jolla, CA 92037. Primary address: Veterans AdministrationMedical Center, 1030 Jefferson Avenue, Memphis, TN 38104. * To whom correspondence should be addressed. Abbreviations used: PEPCK,phosphoenolpyruvate carboxykinase (GTP);HPLC, high-performance liquid chromatography; PTC, phenylthiocarbamyl; PTH, phenylthiohydantoin.
PEPCK with a variety of nucleotide affinity labels (including8-azidoGTP, 5’-[ p -(fluorosulfonyl)benzoyllguanosine, and the 2’,3’-dialdehyde derivative of GTP) has not resulted in the identification of a modified peptide (Lewis et al., 1989a; Jadus et al., 1981; Anthony et al., 1990). For example, previous results from our laboratory established that the photoaffinity label 8-N3GTP specifically modifies PEPCK, ultimately causing formation of a cystine disulfide, but does not yield sufficient quantities of a stable, derivatized peptide for sequence analysis (Lewis et al., 1989a). These results reflect a significant concern regarding attempts to modify PEPCK with nucleotide affinity labels, namely, potential complications caused by the enzyme’s unusual sulfhydryl chemistry. Recently, we reported the identity of a very reactive cysteine residue (Cys288) that is critical for catalytic activity and that lies between two of the consensus sequences for a GTP binding site (Lewis et al., 1989b;Carlson et al., 1978). In addition, chemical modification and photoaffinity labeling studies provide substantial evidence for the existence of at least two cysteine residues within or near the enzyme’s nucleotide-binding site (Lewis et al., 1989a,b). As part of a continuing effort to characterize the active site of PEPCK, we now report the development of a protocol for directly photolabeling the enzyme with its nucleotide substrate. The technique of direct photolabeling offers several potential advantages over the use of photoaffinity or chemically reactive GTP analogues. Several GTP analogues have a chemically reactive moiety that can react with thiols [e.g., 5’-[p-(fluorosulfonyl)benzoyl]guanosine (Jadus et al., 1981) or 5’-(bromoacetamido)-5’-deoxyguanosine (Samant and Sweet, 198311. Consequently, one might predict that a sulfhydryl-directed probe could readily modify the hyperreactive Cysza; if that were the case, it would be difficult to eliminate the possibility that the thiol was modified simply because of its hyperreactivity rather than because of its location within the enzyme’s active site. Because direct photolabeling can lead to the modification of a variety of residues, this potential difficulty may be avoided. In addition, because it is the C-8 position of GTP that typically participates 0 1992 American Chemical Society
Bioconjugate Chem., Vol. 3, No. 2, 1992
Photolabeling of P E E K with [cY-~*P]GTP
in covalent bond formation during photolabeling (Steinmaus et al., 1971), this technique may target different amino acids than affinity labels derivatized at the phosphoryl portion of the nucleotide, such as GMP-pyridoxal phosphate (Ohmi et al., 1988). Finally, direct photolabeling results in the cross-linking of the natural ligand to the enzyme by "freezing" existing contact points, thus increasing the probability that the modificationis specific and occurs within the active site.
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EXPERIMENTAL PROCEDURES
Materials. Radioactive [aJ2P]GTP (10-25 Ci/mmol) and Staphylococcus aureus V8 protease were from ICN Biomedicals, TPCK-treated trypsin was from Sigma Chemical Co., HPLC-grade acetone was from Mallinckrodt, ultrapure urea was from Research Organics, and amino acid standards were from Pierce Chemical Co. All other reagents were of the highest quality commercially available. All glassware used for amino acid analyses was rinsed with constant-boiling HC1. PEPCK was purified to homogeneity and assayed as previously described (Lewis et al., 1989b). StandardConditions for Direct Photolabeling. As previously observed (Sperling,1976;Sperling and Havron, 1976),the addition of acetone as a photosensitizer increased the extent of covalent incorporation of the radioactive nucleotide (Figure 1A). However, with increasing time and concentrations of acetone, the extent of apparent changes in protein structure was also increased (Figure 1B); after extended irradiation in the presence of higher concentrations of acetone, PEPCK migrated as a more broad, diffuse band during polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. We observed that inclusion of dithiothreitol in the photolabeling reaction mixture decreasedthe amount of apparent changes in protein structure and also caused a significant increase in the extent of nucleotide incorporation (data not shown). Inasmuch as sulfhydryls are vulnerable to oxidation by ultraviolet radiation (Zaremba et al., 1984; Nath et al., 1985),increased photolabelingin the presence of dithiothreitol may simply be due to prevention of cysteine oxidation. On the basis of these results, standard conditions were established to maximize photoincorporation of [cu-~~P]GTP while photochemical damage to the enzyme was minimized. PEPCK (2-10 p M ) was incubated in the presence of 6 mM TES, 3% (v/v) glycerol, 0.5 mM EDTA, 100 pM dithiothreitol, 1.1%(v/v) acetone, and 200 pM [ c Y - ~ ~ P I G(30-200 TP Ci/mol) in a glass well in a total volume of 100pL at 0 "C. The sample was irradiated at a distance of 2 cm by a UVG-11 Mineralight lamp (330 pW/cm2) for 10 min. After irradiation, the covalent incorporation of [CY-~~PIGTP into PEPCK was routinely measured by precipitation of the labeled enzyme onto filter paper in the presence of 10% trichloroacetic acid (Reimann et al., 1971;King et al., 1982). The filter paper was washed and the radioactivity was determined by liquid scintillation counting. Alternatively, quantification of [a32P]GTP incorporation was performed by liquid scintillation counting and protein assays after gel filtration of the modified enzyme over a column (1.25 X 37 cm) of Sephadex G-50 in the presence of 100 mM ammonium bicarbonate (pH 8.0), 1 mM EDTA, and 2 M urea; unmodified PEPCK in the presence of an equivalent concentration of urea was used as the protein standard. Preparation and Isolation of Labeled Peptides. PEPCK was labeled under standard conditions, solid urea was added to 6 M, and the modified enzyme was separated from free [o!-~~P]GTPby gel filtration over Sephadex G-
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Figure 1. Panel A time-dependence of photoincorporation. PEPCK was labeled under standard conditions for the indicated times in the presence of 200 p M GTP and increasing concentrations of acetone: 0% (v/v), 0;0.2%, 0;0.4%, A; 0.7%,A; 1.1% , B; 1.5%, 0 ; 2.2%, Panel B: assessment of changes in protein structure. PEPCK was photolabeled under standard conditions for the indicated times and in the presence of increasing concentrations of acetone: 0% (v/v), row 1; 0.4%, row 2; 1.1% , row 3; 2.2 % ,row 4. After irradiation, an aliquot of each reaction mixture was subjected to polyacrylamide gel electrophoresis in the presence of sodiumdodecylsulfate. Only the region of interest of the polyacrylamidegel is shown;no other bands were apparent in any other area of the gel.
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50 in the presence of 100 mM ammonium bicarbonate (pH 8.0), 1 mM EDTA, and 2 M urea. Tryptic digests were performed in this buffer in the presence of 2 % (w/w) trypsin for 36 h at 37 "C, with an additional 2% (w/w) trypsin added after the first 12 h. Proteolytic (S. aureus V8 protease) and CNBr digests were also prepared from enzyme labeled under standard conditions. Solid guanidine hydrochloride was added to 4 M, and the modified enzyme was separated from free [ c ~ - ~ ~ P ] GbyTgel P filtration over a column (1.25 X 37 cm) of Sephadex G-50 in the presence of 10 mM ammonium bicarbonate (pH 8.0) and 2 M guanidine hydrochloride. The sample was then digested with 2% V8 protease for 30 h at 37 "C, with an additional 2% V8 protease added after the first 12 h (Drapeau, 1976). Alternatively, the gel-filtered enzyme was lyophilized, solubilized in 70 % formic acid, and digested with CNBr (60-foldmolar excess over total methionine residues) under nitrogen for 24 h at 25 "C. Initial attempts to purify the radiolabeled tryptic peptide(s) were performed by reversed-phase HPLC over a CIScolumn (Delta-Pak, 300 A,3.9 X 300 mm, 15pm; Waters Associates). Tryptic peptides were eluted with 20 mM ammonium acetate (pH 6.0) and acetonitrile, using a triphasic linear gradient of acetonitrile: 0-25% from 10 to 100 min, 25-35% from 100 to 200 min, 35-80% from 200 to 230 min. All subsequent proteolytic digests were
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162 Bloconlugate Chem., Vol. 3, No. 2, 1992
fractionated by anion-exchange HPLC after first desalting the lyophilized digests over a column of Bio-Gel P-2 (1.25 X 37 cm) equilibrated in water or in 10 mM ammonium bicarbonate (pH 7.5). Under these conditions the proteolytic peptides (detected by their absorbance at 210 nm) and the radioactivity eluted within the void volume of the P-2 column. The pooled fractions were lyophilized and then relyophilized after solubilization in water. The samples were then dissolved in 10 mM acetic acid (pH 3.5) and were purified by HPLC over an anionexchange column (PL-SAX, 1000 A, 8 Fm, 50 X 4.6 mm, Polymer Laboratories). Peptides were eluted with solvent A (10 mM acetic acid, pH 3.5) and solvent B (500 mh4 acetic acid, pH 3.5) using a linear gradient of 0-60% B (tryptic digest) or 0-100% B (V8 proteolytic digest) from 20 to 110 min. Fractions (2 min) were collected, and the major peak of radioactivity was lyophilized. The radioactive tryptic fragment was further purified by desalting over a column of Sephadex G-10 equilibrated in 10 mM ammonium bicarbonate (pH 7.5), whereas the radioactive V8 proteolytic fragment was rechromatographed over BioGel P-2 in HtO. The pooled fractions were lyophilized prior to amino acid analysis. At each step of the purifications, the yield and quantity of the labeled peptide was estimated on the specific radioactivity of the [a32PlGTP. Sequencing and Amino Acid Analysis of the Labeled Peptides. Independent samples of the purified proteolytic peptides (obtained from different enzyme preparations) were subjected to amino acid sequencing as previously described (Lewis et al., 1989b). Amino acid analyses were performed using standard protocols and according to published procedures (Jarrett et al., 1986). Additional samples of the labeled proteolytic peptides were also submitted to the Harvard Microchem Facility for amino acid analysis or sequencing. Portions of these samples were hydrolyzed in 6 N HCl for 24 h and derivatized with phenyl isothiocyanate using a 420A derivatizer from Applied Biosystems. The resulting PTC amino acids were analyzed via reversed-phase HPLC using an Applied Biosystems 130AHPLC. Aliquota of the samples were also subjected to sequencing using an Applied Biosystems 4776 protein sequencer equipped with a 120A online PTH-amino acid analyzer. RESULTS
Photoincorporation of [cP~~PIGTP. A primary objective of this study was to establish conditions by which radioactive GTP could covalently label PEPCK in yields sufficient for isolation of a modified peptide. Under the established standard conditions described in Experimental Procedures, the extent of photoincorporation is approximately 0.2 mol of [a-32P]GTP/mol of PEPCK (Figure lA), and changes in protein structure, as evidenced by polyacrylamide gel electrophoresis, are minimal (Figure lB, lane 3). The enzyme retains approximately 20% of native activity after irradiation under these standard conditions. Considering the low power of the ultraviolet lamp and the minimal duration of irradiation, the extent of covalent incorporation of the nucleotide is high, and is sufficient for isolation of a labeled peptide. Because it is possible for indiscriminate photochemical reactions to occur during these experiments, it is important to ascertain the specificity of the covalent modification. The data in Table I illustrate that photoincorporation of GTP requires native PEPCK. Enzyme that had been previously denatured by boiling was not labeled, and significant labeling did not occur in the presence of
Table I. Specificity of Photolabeling by [ u - ~ ~ P ] G T P ~ condition % incorporation 2 pM PEPCK 17.8 1.1 2 pM PEPCK + 0.2% sodium dodecyl sulfate 2 pM PEPCK (boiled) 0.9 2 pM albumin 1.2 a Photolabeling was carried out under standard conditions. The data represent the mean for duplicate analyses.
Table 11. Substrate Protection against Photolabeling by [u-~'P]GTP' % of addition control addition none 100 1 mM phosphoenolpyruvate 200 pM ITP 35.0 f 0.1 1 mM oxaloacetate 200 pM GDP 33.7 f 0.3 1.5 mM Mg(CH3COO)z 200 pM AMP 64.1 f 3.3 1.5 mM MnClz
% of control 35.5 f 0.6 89.7 f 0.3 61.4 f 3.7 54.4 f 1.2
Photolabeling was carried out under standard conditions in the presence of 2 pM PEPCK, 200 pM [d2P1GTP,and the indicated concentrations of substrates. Incorporation of [ d P I G T P into PEPCK was measured by precipitation of the modified enzyme onto filter paper as described in Experimental Procedures. The results are expressed as a percentage of the incorporation observed in the absence of substrates (control, set to loo%),and the data represent an average of triplicate experiments (mean f SD).
denaturant. The inability of [ Q - ~ ~ P I G T toPcovalently label albumin, a protein of similar size but which does not bind the nucleotide, provides further evidence that the photolabeling of PEPCK is not merely due to nonspecific photochemical cross-linking. If the covalent incorporation of [cx-~~PIGTP does occur through a specific reaction at the nucleotide-binding site of PEPCK, then the addition of other substrates should affect the extent of labeling. Table I1 shows that this is indeed the case; alternative substrates such as ITP and GDP provide substantial protection against photoincorporation of [cY-~~PIGTP. AMP, which is not a substrate for the enzyme, is much less effective than ITP or GDP. The protection that is afforded by AMP may be due to the ability of the nucleotide to absorb ultraviolet light at 254 nm and to thus decrease the efficiency of photolabeling. Divalent cations also decrease the extent of photolabeling. Phosphoenolpyruvate affords nearly as much protection against photolabeling as ITP, whereas oxaloacetate has no significant effect. The specificity of the photolabeling reaction was also assessed by measuring the dependence of photoincorporation on [a-32P]GTP concentration. As shown in Figure 2, the extent of labeling displayed saturation at higher nucleotide concentrations, indicating that the covalent modification step is preceded by reversible binding of [a32P]GTPto PEPCK. As determined from the doublereciprocal replot (inset), the concentration of [cY-~~PIGTP required for half-maximal labeling was 69 f 6 pM. Isolation and Characterization of the Modified Tryptic Peptides. Fractionation of a tryptic digest of modified PEPCK by reversed-phase HPLC in 20 mM ammonium acetate (pH 6.0) revealed two peaks of radioactivity (Figure 3). The first peak (designated as peak I) was not retained by the CIScolumn and accounted for 95 % of the total eluted radioactivity. The second peak (peak 11)was only slightly retarded by the CIScolumn and accounted for 5 % of the total eluted radioactivity. Peak I1 was isolated and lyophilized; sequence analysis of the purified peptide yielded exclusivelythe followingsequence: Ser-Glu-Ala-Thr-Ala-Ala-Ala-Glu-X-Lys No PTH-amino acid could be identified for the ninth cycle of sequencing and no significant PTH-derivatives were
Photolabeling of P E E K wRh [cY-~*P]GTP
Bloconjugate Chem., Vol. 3, NO. 2, 1992 109
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[ [ a 3 2 ~ ~(M~ ~ ~ 1 Figure 2. Concentration dependence of photolabeling. PEPCK was labeled under standard conditions in the presence of the indicated concentrations of [(U-~*P]GTP. I
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detected after cycle 10. This sequence matches that of the tryptic peptide Ser42-Lys471of PEPCK, which contains a histidine residue at position 470 (Beale et al., 1985). It should be emphasized that the modified enzyme had been purified from free [ d 2 P 1 G T P prior to tryptic digestion, suggesting that peak I, bearing the majority of the radioactive label, must represent a hydrophilic labeled peptide that was simply not retained by the reversedphase column. Two-dimensionalpeptide maps of tryptic
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Figure 4. Ion-exchange HPLC of the proteolytic digests of modified PEPCK. The tryptic (A) or S. aureus V8 proteolytic (B)digest of PEPCK was prepared and subjected to anionexchange HPLC in 10 mM acetic acid (pH 3.6) as described in Experimental Procedures. Detection of a significant corresponding peak in absorbance at 220 nm was difficult due to a large background subtraction from the acetic acid gradient.
digests of the modified enzyme indicated that the radioactivity was associated with a peptide and did not represent either undigested enzyme, [d2P1GTP,or a fragment of [cz-~~PIGTP. The major radioactive spot derived from the tryptic digest migrated closer to the anode than did [ w ~ ~ P I G Tsuggesting P, the presence of one or more acidic residues (data not shown). Therefore, a purification strategy was devised to take advantage of the highly anionic nature of a tryptic peptide labeled with guanosine triphosphate. After gel filtration, a tryptic digest of modified PEPCK was passed over a strong anion-exchangeHPLC column a t pH 3.6. Under these conditions, most of the tryptic peptides were not retained. The column was then eluted with acetic acid (Figure 4A). The peak eluting between fractions 35 and 45 contained at least 75 % of the total eluted radioactivity; this peak was pooled and subjected to sequencing and amino acid analysis. Under these conditions, this major peak did not have the same retention time as free [aJ2P1GTP. The two smaller radioactive peaks were not analyzed. Three independently derived samplesof the major peak shown in Figure 4A were submitted for sequence analysis; in each case a sequence could not be determined. The yield of PTH derivatives that could be detected at each cycle was very low and accounted for less than 5 % of the applied sample. These results suggested that the amino terminus of this labeled tryptic peptide was blocked; the remaining sampleswere tnerefore characterized by amino acid analysis. The amino acid compositions of each of the three independent samples were very similar, and each contained relatively high amounts of Asx and Glx (data not shown), which is consistent with this tryptic peptide being highly acidic. However, the composition could not be unambiguously assigned to any single modified tryptic peptide predicted from the known sequence of PEPCK (Beale et al., 1985). The quantity of the peptides subjected to amino acid analyses was estimated from the specific radioactivity, assuming one molecule of [CY-~~P] GTP was incorporated per peptide. It should be emphasized that
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Lewls et al.
centration of acetone and the duration of irradiation, we the quantity of amino acids detected was within 10% of established standard conditions for photolabeling that this estimate. cause minimal damage to the enzyme. Under these Characterization of the Labeled CNBr Fragment. conditions, the incorporation of the nucleotide is approxIt was anticipated that a cyanogen bromide fragment of imately 0.2 mol/mol of enzyme, as measured by precipthe labeled enzyme might permit the unequivocal idenitation of the labeled enzyme onto filter paper or by gel tification of a modified peptide. Following lyophilizafiltration in the presence of a denaturant. tion, a CNBr digest was solubilized in aqueous 0.1% trifluoroacetic acid and was applied to a CM reversedSeveral lines of evidence indicate that [CY-~~PIGTP is phase HPLC column equilibrated in the same buffer. specifically incorporated into the active site of native Under these conditions, >75 % of the applied radioactivity PEPCK. First, there was no significant photolabeling of was not retained by the column. Because the radioactive denatured enzyme or albumin under conditions in which CNBr fragment(s) eluted from a Bio-Gel P-2 column as incorporation into native PEPCK was approximately 20 % . a broad peak with a greater retention time than [(U-~~PI-Second, the pattern of substrate protection was consistent GTP, we reasoned that the CNBr fragment might be with specific modification of the nucleotide binding site. smaller than the nucleotide itself. This supposition was Substrates that would be expected to compete with [asupported by two-dimensional peptide maps of the CNBr 32P]GTPfor the active site (ITP, GDP, and phosphoedigest, which showed that the radioactive CNBr fragmentnolpyruvate) provided substantial protection against (s) coelectrophoresed with [a-32PlGTP but migrated photolabeling, whereas AMP and oxaloacetate had little somewhat faster than GTP in the second chromatograeffect. It is not clear from these studies whether the diphy dimension (data not shown). These data suggest that valent metal ions decreased the extent of photolabeling the derivatized peptide was not stable under the highly by chelation of the nucleotide or through a general charge acidic conditions used for CNBr cleavage, a result that effect. Third, the extent of labeling displayed saturation has been reported by several other laboratories (Abraham with increasing nucleotide concentration, indicating that et al., 1983; Peter et al., 1988; Hoppe et al., 1983). covalent modification was preceded by reversible binding. Fractionation of a peptic digest by reversed-phase HPLC These data indicate that direct photolabeling by [(u-~~PIalso indicated that most of the radioactivity was not GTP under standard conditions results in the specific retained by the c18 column. We therefore sought yet modification of the enzyme’s active site; thus, the peptide another cleavage method for isolation of the modified pepthat bears the majority of the radioactive label should tide. define a portion of the GTP binding site that interacts Isolation and Characterization of the Modified S. with the purine ring. aureus V8 Proteolytic Peptide. The radiolabeled Isolation of the major radioactive fragment generated fragment derived from a V8 proteolytic digestion of from a V8 proteolytic digest of the modified enzyme yielded modified PEPCKeluted as a single radioactive peak during exclusively the peptide Asp598-Pro,~1~, which lies near the anion-exchange HPLC (Figure 4B). Sequence analysis of carboxyl terminus of this 621 amino acid protein (Beale the purified peptide yielded exclusively the following et al., 1985). Although the sequence of the modified trypsequence: tic peptide derived from peak I1of Figure 3 (Ser~2-Lys471) revealed a distinct blank in one sequencing cycle (usually Asp-Gln-Val-Asn-Ala-Asp-Leu-Pro indicative of a modified residue), the sequence analysis of Although serine and glycine appeared as contaminating the V8 peptide did not display such a pattern. We PTH derivatives in the first cycle (
