Primary structure of carboxypeptidase II from malted barley

0 1. I N T R O D U C T I O N According to MIKOLA(14), germinating bar- ley contains five serine carboxypeptidases of complementary specificities and three of these, carboxypeptidase I, II and III have been isolated and characterised in this laboratory (4, 5, 6). These enzymes in combination with endo- peptidases, very effectively produce free amino acids during germination by cleavage of reserve proteins in the endosperm. We have previously reported the sequence of malt carboxypeptidase 1 Department of Chemistry, Carlsberg Laboratory , Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby - Springer-Verlag is demonstrated that the sequence of this enzyme is almost identical with that of wheat carboxypeptidase II and homologous with that of malt carboxypeptidase I and carboxypeptidase Y. 2. MATERIALS AND M E T H O D S 2.1. Materials Malt carboxypeptidase II was prepared as previously described (6) from malted Gula barley (obtained from the Carlsberg Breweries, Denmark) by affinity chromatography on CABS-Sepharose. Bio-Gels P-200, P-60, P-30 and P-6 were from Bio-Rad, USA. DPCC-treated trypsin and dithiothreitol were from Sigma, USA. S. aureus V8 protease was from Miles, USA. Carboxypeptidase Y was a product ofCarlsberg Biotechnology, Denmark. 2-vinylpyridine was from Janssen Chimica, Belgium. All other chemicals were analytical or HPLC-grade from Merck, W. Germany. 2.2. Methods 2.2.1. Separation of peptide chains After reduction and alkylation with 2vinylpyridine of malt carboxypeptidase II as previously described (5), the A-chain (tool. weight 34,000) was separated from the B-chains (mol.weight 24,000-27,000) by gel filtration on Bio-Gel P-200 (5.0 cm) equilibrated with 5% (v/v) acetic acid. 2.2.2. Cleavage of peptide bonds Chemical cleavage by cyanogen bromide and hydroxylamine and enzymatic digestion with trypsin of the reduced and alkylated chains was performed as previously described (18). Cleavage at the carboxyl group of tryptophan residues in the A-chain was performed according to the procedure described by FONTANAet al. (8). The reaction was carried out in the dark at room temperature overnight. Tyramine hydrochloride (5 rag. ml~) was included as a scavenger in the reagent which consisted of o-iodosobenzoic acid (20 mg. mlz) in 4 M-guanidine hydrochloride and 80% acetic acid. Digestion with S. aureus V8 protease (11) was performed at 25 ~ in 0.1 M-ammonium bicarbonate and 2 M-urea at pH 7.8. The reduced and alkylated peptide chains were dissolved in 6 M-urea and diluted with 2 volumes 0.15 M-ammonium bicarbonate pH 7.8. Then enzyme was added and after 2-3 hours digestion the reaction was stopped by addition of TFA. 2.2.3. C-terminal sequence determination C-terminal sequences of the entire A-chain and the CB4 fragment of the B-chain were determined by digestion with carboxypeptidase Y. The following conditions were used: 0.23 mM A-chain, 0.1 M-Mes, 1 mM-EDTA, 0.1% SDS, pH 7.0, 2.5 tXM carboxypeptidase Y and 0.27 mM-CB4, 0.05 M-sodium acetate, pH 5.0, 7.7 ~tM carboxypeptidase Y. Aliquots were withdrawn during the reaction, pH was adjusted to 2.0 by addition of 0.5 M-HCI and the aliquots were applied directly to a Durrum D500 amino acid analyzer. 2.2.4. Separation of peptide fragments Separation and purification of large peptide fragments were performed by gel filtration through Bio-Gel P-60 or P-30 in 10-30 % (v/v) acetic acid. Purification of small peptides was performed by reverse phase HPLC using equipment from Waters and wide pore C~8 columns i.d. 4.6 m m and length 25 cm (J.T. Baker no. RP 7104-0 or Vydac no. 218TPb). Gradient elution was performed with linear gradients of acetonitrile in 0.1% (w/w) trifluoroacetic acid at a flow rate of 1.0 ml. min -~.The eluate was monitored at 220 nm supplemented with fluorescence detection at 375 nm after excitation at 280 nm, to detect tryptophan containing peptides. Inhomogeneous fractions were rechromatographed in the same system but employing a more flat and narrow gradient of acetonitrile. 2.2.5. Amino acid sequence determinations Peptides were sequenced either on a Beckman model 890C liquid phase sequencer as previously described (12) or on an Applied Biosystems model 470A gas-phase sequencer, using the E L U A T E (ML) program provided by the company. The phenylthiohydantoin amino acid derivatives from the gas-phase sequencer were identified on-line by reverse phase HPLC using equipment from Applied Biosystems, while those from the liquid phase sequencer were identified with a Hewlett-Packard HPLC system using a linear gradient from 10 to 45% methanol, (16). Amino acid compositions were determined as previously described (5). 2.2.6. Peptide nomenclature Peptide fragments obtained by cleavage with cyanogen bromide, hydroxylamine, iodosobenzoic acid, trypsin or S. aureus V8 protease are designated CB, NG, W, T and E, respectively, followed by a number indicating their positions in the polypeptide chain from the N-terminus of the protein. 3. RESULTS 3.1. N-terminal sequences of A- and B-chains The A- and B-chains, as obtained by gel chromatography of the reduced and alkylated enzyme, were subjected to N-terminal sequence determination. The A-chain was sequenced 37 cycles, while the B-chain was found to be N-terminally blocked. The blocking group was both acid and base labile. After its removal, the B-chain could be sequenced 25 cycles. 3.2. Cyanogen bromide fragments According to the amino acid analysis, the A-chain of malt carboxypeptidase II contains four methionyl residues (6) corresponding to five cyanogen bromide fragments. Chromatography of the cyanogen bromide treated A-chain on Bio-Gel P-60 (Figure 1) produced six peaks. Pool 1 contained two peptides which later was demonstrated to originate from incomplete cleavage after two methionines. Pool 2 contained one peptide (CB3, 103 res.) which was sequenced 48 cycles. Pool 3 contained one peptide (CB4+5, 77 res.) originating from incomplete cleavage at Met 24s. Pool 4 contained CB4 (65 res.), which was sequenced 53 cycles. Pool 5 contained CB2 (58 res.) which was sequenced 25 cycles and pool 6 was found to be a mixture of the N-terminal cyanogen bromide fragment (CB1, 22 res.) and the C-terminal cyanogen bromide fragment (CB5, 12 res.). After rechromatography by HPLC the complete sequence of CB5 could be determined. Chromatography of the cyanogen bromide fragments of the B-chain on Bio-Gel P-60 yielded three peaks (Figure 2). Pool 1 contained a single peptide (CB3, 98 res.) which was seFigure2. Separationofcyanogenbromide fragmentsof the B-chain (7 rag) on Bio-Gcl P-60. The column (1.5x88 cm) was eluted with 30% acetic acid at a flow rate of 4 ml. hL Fractions (1.3 ml) were pooled as indicated. < w o z < toO.1 ~e o Figure 3. Separation of hydroxylamine fragments of the A-chain (5.0 rag) on Bio-Gel P-60. The column ( 1.5x88 cm) was eluted with 10%acetic acid at a flow rate of 7 ml. h~. Fractions (1.5 ml) were pooled as indicated. quenced 2 (...truncated)