1322
BIOCHEMICAL SOCIETY TRANSACTIONS
Ubiquitin: preparative chemical synthesis, purification and characterization 0. OGUNJOBI AND R. RAMAGE Depurlmerit of Chemistty Utiiversity of Erlitihirrgh. West Maitis Roud, Editihirrgh E l i 9 3JJ, U.K . Sirmmury An improved total synthesis of ubiquitin has been achieved by the Fmoc/t-butyl solid-phase methodology using NGPmc protection of the Arg residues [ I ] . Optimization of the purification protocol has offered the material in substantial quantity (90 mg) in an overall yield o f 4.2%0. Characterization of the isolated products has opened the way to the studies of the folding pathway and the preparation of structural analogues.
Ititrodirctioti From an organic chemist's viewpoint, a key step in the study of proteins and the structural elements that control or contribute t o their tertiary structure development is the availability o f a reliable synthetic methodology. To this the Merrifield solid-phase approach has made the most significant contribution [ 2 , 3 ] . To explore further the potentials of this approach, it becomes increasingly important that the carefully assembled protein be isolated in preparative quantities (approx. 100-200 mg in one single run) and in high purity ( > 95%) from which useful data (in our case spectroscopic and biological) could be obtained. Total synthesis can be viewed as a potentially rapid and powerful approach capable of yielding proteins in useful quantities, provided that effective and critical purification protocols are followed and assayed by a combination of spectroscopic and electrophoretic methods. The chemical approach is complementary to the recombinant DNA approach, offering the possibility of the introduction of unnatural residues (both isotopically enriched and isosterically altered), but a more specific advantage would be that the protein will not be altered by proteolytic artefacts. At the present time, few reports exist f o r the successful chemical synthesis of proteins and, of these, very low yields (when recorded) - typically less than 1 0 mg recovery - are reported [ 41 for medium-sized peptides of approx. 70- 100 amino acid rcsiducs. As part of our ongoing efforts towards the efficient chemical synthesis and isolation of proteins of molecular mass approx. 10 kDa, we report the improved chemical synthesis and purification of our primary target molecule, human ubiquitin. This highly conserved protein was chosen because o f its size (76 a-amino acids), single polypeptide chain, no cross-links, and its well-established tertiary structure and important biological role.
Experimetird procediire Chemiculs. Fmoc amino acids were purchased from ABI and Novabiochem ( U K ) Ltd, with the exception of FmocAsn(Mbh)OH, FmocGln(Mbh)OH and FmocArg(Pmc)OH, which werc synthesized in our laboratory. The following side-chain-protected amino acids were used: Lys(Boc), His(Trt), Glu(OBu'), Asp(OBu'), Ser(But), Thr(But), Tyr(But). Peptide synthesis gradc N,N'-dimethylAbbreviations used: DIC, di-isopropylcarbodi-imide;HOBT, I -hydroxy-benzotriazole; TFA, trifluoroacetic acid; DMF, N, N'-
dimethylformamide; i.e.f., isoelectnc focusing; r.p., reverse phase; DTT, dithiothreitol.
formamide (DMF), piperidine (used as supplied) and dioxan were purchased from Rathburn Chemicals Ltd and stored over 4 A molecular sieves for 3 days before use. Di-isopropylcarbodi-imide (DIC) (99%) was purchased from Aldrich and used as supplied; 1-hydroxybcnzotriazole (HOBT ) was purchased from Fluka, recrystallized from ethanol, dried over MgSO, and left in the desiccator until required. Acetic anhydride and pyridine were dried, distilled [S]and stored over 4 A molecular sieves until required. Trifluoroacetic acid (TFA)(Fluorochem Ltd) was refluxed over PzOs, fractionally distilled through an efficient column, collecting fraction b.p. 72°C. and stored under dry N, until required. Scavengers were of the best quality available: thioanisole 99%) Aldrich; anisole 99% Aldrich; ethylmethylsulphide 99% Aldrich; P-mercaptocthanol > 99% Biochemika Fluka.
Peptide synthesis The chain assembly o f ubiquitin was carried o u t by stepwise method on the p-alkoxybenzylalcohol resin using the Applied Biosystems 430A automated peptidc synthesizer starting wth 0.25 mmol of FmocGly-alkoxybcnzylcstcr-resin (600 mg: substituted at 0.42 mmol of FmocGly/g of resin). The quality of the FmocGly-resin was controlled as prcviously described [6] and corroborated by Br elcmental analysis of p-bromobenzoylatcd sample o f FmocGly-resin. The residual O H groups were capped by bcnzoylation in the presence of pyridine at 0°C for 20 min before the start o f the chain assembly. Throughout the synthesis, DMF/dioxan ( 1 : 1, v/v) mixture was used as the reaction solvent. The synthetic cycle included the following. 1. Na-Fmoc removal, which involved successive treatments with 20'5: (v/v) piperidine in DMF for periods o f 5 , 3 , 3 and 1 min. Real-time assessment of the progress of the synthesis was afforded by measuring spectrophotometrically the chromophoric Fmoc dcgradation product at A 313 nm. This provided information on the efficiency and indeed the quality of the chain assembly cycles. 2. Coupling was effected by triple coupling throughout by one symmetrical anhydride coupling ( 2 mmol of amino acid) and two HOBT active ester couplings ( 1 mmol) for 30 min giving 1.5 h/residue, employing DIC alone and DIC/HOBT, respectively. the exceptions to this protocol were FmocGlyOH single soupling (symmetrical anhydride, 6 mmol of amino acid) with a coupling time of 45 min; FmocArc(Pmc)OH, FmocAsn(Mbh)OH and FmocGln(Mbh)OH involved only DIC/HOBT in triple coupling (3x 1 mmol) for 30 min each giving 1.5 h/residue. 3. The 'capping' protocol utilized the separatc delivery of acetic anhydride/DMF (0.5 M ) and pyridine/DMF (0.5 M) for 6 min in every step. On completion of the assembly of protected ubiquitin, the final weight of peptide-resin was 2.93 g, which was found t o be -80%1 of the expected weight bascd o n the initial FmocGly-resin (600 mg; 0.25 mmol). Quantitative U.V. analysis of the Fmoc group on methionine gavc a substitution value of 0.04 mmol/g (cf. 0.063 mmol of peptidc/g calculated value) in three separate experiments. (This takes into account that - 20% sample was removed at residuc 1 1 for another purpose.) The fully protected sample was stored at - 20°C until needed and Na-terminal Fmoc was removed as required using 20%, piperidinc/DMF and sonication for 10 min. Cleavage and deprotection of the peptide from the resin support was carried out by splitting the material into three 1990
PEPTIDE A N D PROTEIN GROUP approximately equal batches by treating the peptide resin with thioanisolc ( 1 ml; S%), v/v). ethylmethylsulphide ( 1 ml; S ' L . v/v) and anisole ( 1 ml; S%, v/v) for 20 min followed by the addition of 95% ( v / v ) aqueous TFA (20 ml) at room temperature for 3 h. , After the TFA was removed iri V ~ C I I OP-mercaptoethanol (2%, v/v) in diethyl ether (SO ml) was slowly added to the gently stirred residue t o remove the organic scavengers. The diethyl ether was removed by decantation and the procedure was repeated twice. The white solid obtained was immediately dissolved in N,-saturated urea buffer [SO mMNH,OAc/8 M-urea/lO mwdithiothreitol (DTT), pH 4.51 and left t o equilibrate at 4°C overnight with gentle stirring, and at room temperature for a few hours before it was applied t o a high resolution Sephadex G-SO column (2.6 cm x 136 cm), previously equilibrated with urea buffer. The fractions obtained were sequentially dialysed against decreasing urea concentration: N,-saturated SO mMNH,OAc/4 M urea/lO mM-DTT pH 4.5 ( 2 I, 24 h); N,snturated 50 m ~ - N H , o A c / 2M urea/lO mM-DTT pH 4.5 ( 2 1, 24 h); N,-saturated SO mM-NH,OAc/lO mM-DTT pH 4.5 ( 2 I. 24 h); N,-saturated 5 0 mM-NH,OAc pH 4.5 ( 4 x 2 I. 24 h). Thcse fractions were assayed by isoelcctric focusing (i.c.f.) o n Phast gel pl range 3-9 and reverse phase ( r p )h.p.1.c. The desired fractions wcre further purified by cation-exchange chromatography on a CM-Sepharose CL6B (Pharmacia) column ( 1.6 cm X 40 cm) eluting with SO mM-NH,OAc pH 4.5 ( I bed volume) followed by sequential pH and salt gradients in the order: (1) SO mM-NH,OAc pH 4.5-50 mMNH,OAc pH 5.5; ( i i ) 5 0 mM-NH,OAc pH 5.5-0.3 MNH,OAc pH S.S. The desired material obtained (assayed by Phast i.e.f. and r.p. h.p.1.c.) was purified further by anion-exchange chromatography o n DEAE-Sepharose (Pharmacia) column ( 1.6 cm x 40 cm) eluting with SO mM-NH,HCO, pH 9.3 ( I bed volume) followed by salt gradient elution up to 0.3 MNH,HCO, pH Y.3. Two major peaks werc obtained which assayed similarly by Phast i.e.f. and r.p. h.p.1.c. These peaks. designated A and H. were judged t o be > Y 0 " h and > 80% purc. respectively. Semi-preparative r.p. h.p.1.c. purification gave synthctic ubiquitin A (40mg) and ubiquitin B ( 5 0 mg) in 4.2% overall
1323 yield and in >9SnL1 purity. The synthetic material s o obtained exhibited identical h.p.1.c. and i.e.f. behaviour in comparison with authentic bovine ubiquitin. Amino acid analysis figures agree well with thcorctical values. Elcctrospray mass analysis o f this material showed the expected molecular ion 8564.8. The primary sequence of the synthetic ubiquitin was confirmed by automated Edman degradation. Tryptic digestion of oxidized ubiquitins A and B and bovine ubiquitin gave a very interesting picture which led u s t o speculate that peak B corresponds to a material which may represent an intermediate state of folding. The current hypothe5is is that protein folding takes place in a few discreet steps 17, 81. Isolation of materials at intcrmediate stages in the process opens the way to kinetic and thermodynamic investigation of the development o f tertiary structure. The tryptic digest was found to be most variable in the C-terminal region and one could spcculatc that the fitting o f this region into the final tertiary structure occurs at a late stage. Clearly the ncxt step is direct physical investigation of the materials, which we are carrying out by spectroscopic and electrophoretic methods. We thank the S.E.K.C.. Applied l3iosystems Inc. and Merck. Sharp & Dohmc f o r financial support. We are grateful t o B. Whigham and K. Shaw for their invaluable technical support. Brian Green ( V C Instruments) for the mass analysis. Dr L. A . FothergillCilmore and Ms L. Kerr o f the University o f Edinburgh sequencing unit, WELMET. lor the sequencing o f these samples. Green. J. & Kamage. K.( 1987) Tc~irctheclror~ I.c,tr. 28. 2287 Merrifield. K. B. ( 1963)J . Am. C'hcJtn..Yoc,. 85, 2 147 Merrifield. K.13. ( 1985) S c i e n w 232. 341 -347 Hriand. J.-P., Muller. S.. Raboy. 13. & Van Dorsselaer. A . ( I 989,) /",pi. Hes. 2. 38 1-388 5. Armarego, W. L. F., Perrin, D. I). & Perrin, D. K.( 1980) I'uriJicw riot? oj'luhorcrio~y('lic~rniccrh. 2nd cdn.. Pergamon Press. Oxford 6 . Green, J., Ogunjobi, 0. M. & Kamage. K. ( 1 9 8 9 ) Terrrrheclrorr 1,rrr. 3 0 . 2 149-2 I 5 2 7. Anfinsen, C . 13. & Schcraga. H. A . ( 1075) A d i . . I'roi. (‘hem. 29, 205-300 ( I98 I ) Ad,'. I'tW. C'hc,tt/. 34. 167-339
I. 2. 3. 4.
Received 1 May I990
Synthesis, monitoring and structure-function studies on some neurokinin A analogues DAVID J. S. GUTHRIE.* AHMED A. ABU SHANAB,* JAMES M. ALLEN.1- G. BRENT IRVINE.* NEIL V. McFERRAN* and BRIAN WALKER* * Ilitisiori of' Hiochernistty. The School of' HioloD arid Hiochernistry, The Qireeri j. Universiy of' Relfast, Belfast R I ' V 7HL. N. Irelrnd, U.K . and t Riornerlical Sciences Kiwrrrch C'entre. Uriiver.sityof' Ulster, Ncwtowriuhhey H7'37 OQB, N . Irchrid, U.K . Neurokinin A (NKA) is one o f three tachykinins found in mammals, the others being substance P (SP)and neurokinin B (NKB) (Table I ) . Thcse peptides arc widely distributed Abbreviations t-hutoxycarbonyl; substance P; Aib. carhoxylic acid; receptor.
Vol. 18
used: Fmoc. fluorenylmethoxycarbonyl; Roc. NKA. neurokinin A; NKH. neurokinin 13; SP. a-aminobutyric acid; Ach. I -aminocyclohexanef.a.h.. fast atom bombardment; NK2, NKA-
throughout the nervous system and show a variety of activities [ 1 I. Three types of receptor with specificitics matching the thrcc tachykinins ( N K I for SP, NK2 for NKA and NK3 for NKB) have been characterized. The relationships between these peptides and their receptors are very complex. Receptor-stimulating activity resides in the very similar C-terminal regions and a degree o f cross-reactivity has been postulated [ 1 I. Some cells express mainly one receptor type, but many cells express more than one. Furthermore, SP and NKA are synthesized together and arc probably co-released from peripheral neurons. We wanted t o investigate the possible rolc o f tachykinins in conditions such as asthma, where it has been suggested that abnormal release of SP or NKA could be involved in bronchoconstriction [ 2, 31. More recently, it has been shown that in the airways NKA is more potent in contracting smooth muscle, while SP is a more potent stimulant o f mucous hypersecretion. vasodilation and resulting oedema
BIOCHEMICAL SOCIETY TRANSACTIONS
Ubiquitin: preparative chemical synthesis, purification and characterization 0. OGUNJOBI AND R. RAMAGE Depurlmerit of Chemistty Utiiversity of Erlitihirrgh. West Maitis Roud, Editihirrgh E l i 9 3JJ, U.K . Sirmmury An improved total synthesis of ubiquitin has been achieved by the Fmoc/t-butyl solid-phase methodology using NGPmc protection of the Arg residues [ I ] . Optimization of the purification protocol has offered the material in substantial quantity (90 mg) in an overall yield o f 4.2%0. Characterization of the isolated products has opened the way to the studies of the folding pathway and the preparation of structural analogues.
Ititrodirctioti From an organic chemist's viewpoint, a key step in the study of proteins and the structural elements that control or contribute t o their tertiary structure development is the availability o f a reliable synthetic methodology. To this the Merrifield solid-phase approach has made the most significant contribution [ 2 , 3 ] . To explore further the potentials of this approach, it becomes increasingly important that the carefully assembled protein be isolated in preparative quantities (approx. 100-200 mg in one single run) and in high purity ( > 95%) from which useful data (in our case spectroscopic and biological) could be obtained. Total synthesis can be viewed as a potentially rapid and powerful approach capable of yielding proteins in useful quantities, provided that effective and critical purification protocols are followed and assayed by a combination of spectroscopic and electrophoretic methods. The chemical approach is complementary to the recombinant DNA approach, offering the possibility of the introduction of unnatural residues (both isotopically enriched and isosterically altered), but a more specific advantage would be that the protein will not be altered by proteolytic artefacts. At the present time, few reports exist f o r the successful chemical synthesis of proteins and, of these, very low yields (when recorded) - typically less than 1 0 mg recovery - are reported [ 41 for medium-sized peptides of approx. 70- 100 amino acid rcsiducs. As part of our ongoing efforts towards the efficient chemical synthesis and isolation of proteins of molecular mass approx. 10 kDa, we report the improved chemical synthesis and purification of our primary target molecule, human ubiquitin. This highly conserved protein was chosen because o f its size (76 a-amino acids), single polypeptide chain, no cross-links, and its well-established tertiary structure and important biological role.
Experimetird procediire Chemiculs. Fmoc amino acids were purchased from ABI and Novabiochem ( U K ) Ltd, with the exception of FmocAsn(Mbh)OH, FmocGln(Mbh)OH and FmocArg(Pmc)OH, which werc synthesized in our laboratory. The following side-chain-protected amino acids were used: Lys(Boc), His(Trt), Glu(OBu'), Asp(OBu'), Ser(But), Thr(But), Tyr(But). Peptide synthesis gradc N,N'-dimethylAbbreviations used: DIC, di-isopropylcarbodi-imide;HOBT, I -hydroxy-benzotriazole; TFA, trifluoroacetic acid; DMF, N, N'-
dimethylformamide; i.e.f., isoelectnc focusing; r.p., reverse phase; DTT, dithiothreitol.
formamide (DMF), piperidine (used as supplied) and dioxan were purchased from Rathburn Chemicals Ltd and stored over 4 A molecular sieves for 3 days before use. Di-isopropylcarbodi-imide (DIC) (99%) was purchased from Aldrich and used as supplied; 1-hydroxybcnzotriazole (HOBT ) was purchased from Fluka, recrystallized from ethanol, dried over MgSO, and left in the desiccator until required. Acetic anhydride and pyridine were dried, distilled [S]and stored over 4 A molecular sieves until required. Trifluoroacetic acid (TFA)(Fluorochem Ltd) was refluxed over PzOs, fractionally distilled through an efficient column, collecting fraction b.p. 72°C. and stored under dry N, until required. Scavengers were of the best quality available: thioanisole 99%) Aldrich; anisole 99% Aldrich; ethylmethylsulphide 99% Aldrich; P-mercaptocthanol > 99% Biochemika Fluka.
Peptide synthesis The chain assembly o f ubiquitin was carried o u t by stepwise method on the p-alkoxybenzylalcohol resin using the Applied Biosystems 430A automated peptidc synthesizer starting wth 0.25 mmol of FmocGly-alkoxybcnzylcstcr-resin (600 mg: substituted at 0.42 mmol of FmocGly/g of resin). The quality of the FmocGly-resin was controlled as prcviously described [6] and corroborated by Br elcmental analysis of p-bromobenzoylatcd sample o f FmocGly-resin. The residual O H groups were capped by bcnzoylation in the presence of pyridine at 0°C for 20 min before the start o f the chain assembly. Throughout the synthesis, DMF/dioxan ( 1 : 1, v/v) mixture was used as the reaction solvent. The synthetic cycle included the following. 1. Na-Fmoc removal, which involved successive treatments with 20'5: (v/v) piperidine in DMF for periods o f 5 , 3 , 3 and 1 min. Real-time assessment of the progress of the synthesis was afforded by measuring spectrophotometrically the chromophoric Fmoc dcgradation product at A 313 nm. This provided information on the efficiency and indeed the quality of the chain assembly cycles. 2. Coupling was effected by triple coupling throughout by one symmetrical anhydride coupling ( 2 mmol of amino acid) and two HOBT active ester couplings ( 1 mmol) for 30 min giving 1.5 h/residue, employing DIC alone and DIC/HOBT, respectively. the exceptions to this protocol were FmocGlyOH single soupling (symmetrical anhydride, 6 mmol of amino acid) with a coupling time of 45 min; FmocArc(Pmc)OH, FmocAsn(Mbh)OH and FmocGln(Mbh)OH involved only DIC/HOBT in triple coupling (3x 1 mmol) for 30 min each giving 1.5 h/residue. 3. The 'capping' protocol utilized the separatc delivery of acetic anhydride/DMF (0.5 M ) and pyridine/DMF (0.5 M) for 6 min in every step. On completion of the assembly of protected ubiquitin, the final weight of peptide-resin was 2.93 g, which was found t o be -80%1 of the expected weight bascd o n the initial FmocGly-resin (600 mg; 0.25 mmol). Quantitative U.V. analysis of the Fmoc group on methionine gavc a substitution value of 0.04 mmol/g (cf. 0.063 mmol of peptidc/g calculated value) in three separate experiments. (This takes into account that - 20% sample was removed at residuc 1 1 for another purpose.) The fully protected sample was stored at - 20°C until needed and Na-terminal Fmoc was removed as required using 20%, piperidinc/DMF and sonication for 10 min. Cleavage and deprotection of the peptide from the resin support was carried out by splitting the material into three 1990
PEPTIDE A N D PROTEIN GROUP approximately equal batches by treating the peptide resin with thioanisolc ( 1 ml; S%), v/v). ethylmethylsulphide ( 1 ml; S ' L . v/v) and anisole ( 1 ml; S%, v/v) for 20 min followed by the addition of 95% ( v / v ) aqueous TFA (20 ml) at room temperature for 3 h. , After the TFA was removed iri V ~ C I I OP-mercaptoethanol (2%, v/v) in diethyl ether (SO ml) was slowly added to the gently stirred residue t o remove the organic scavengers. The diethyl ether was removed by decantation and the procedure was repeated twice. The white solid obtained was immediately dissolved in N,-saturated urea buffer [SO mMNH,OAc/8 M-urea/lO mwdithiothreitol (DTT), pH 4.51 and left t o equilibrate at 4°C overnight with gentle stirring, and at room temperature for a few hours before it was applied t o a high resolution Sephadex G-SO column (2.6 cm x 136 cm), previously equilibrated with urea buffer. The fractions obtained were sequentially dialysed against decreasing urea concentration: N,-saturated SO mMNH,OAc/4 M urea/lO mM-DTT pH 4.5 ( 2 I, 24 h); N,snturated 50 m ~ - N H , o A c / 2M urea/lO mM-DTT pH 4.5 ( 2 1, 24 h); N,-saturated SO mM-NH,OAc/lO mM-DTT pH 4.5 ( 2 I. 24 h); N,-saturated 5 0 mM-NH,OAc pH 4.5 ( 4 x 2 I. 24 h). Thcse fractions were assayed by isoelcctric focusing (i.c.f.) o n Phast gel pl range 3-9 and reverse phase ( r p )h.p.1.c. The desired fractions wcre further purified by cation-exchange chromatography on a CM-Sepharose CL6B (Pharmacia) column ( 1.6 cm X 40 cm) eluting with SO mM-NH,OAc pH 4.5 ( I bed volume) followed by sequential pH and salt gradients in the order: (1) SO mM-NH,OAc pH 4.5-50 mMNH,OAc pH 5.5; ( i i ) 5 0 mM-NH,OAc pH 5.5-0.3 MNH,OAc pH S.S. The desired material obtained (assayed by Phast i.e.f. and r.p. h.p.1.c.) was purified further by anion-exchange chromatography o n DEAE-Sepharose (Pharmacia) column ( 1.6 cm x 40 cm) eluting with SO mM-NH,HCO, pH 9.3 ( I bed volume) followed by salt gradient elution up to 0.3 MNH,HCO, pH Y.3. Two major peaks werc obtained which assayed similarly by Phast i.e.f. and r.p. h.p.1.c. These peaks. designated A and H. were judged t o be > Y 0 " h and > 80% purc. respectively. Semi-preparative r.p. h.p.1.c. purification gave synthctic ubiquitin A (40mg) and ubiquitin B ( 5 0 mg) in 4.2% overall
1323 yield and in >9SnL1 purity. The synthetic material s o obtained exhibited identical h.p.1.c. and i.e.f. behaviour in comparison with authentic bovine ubiquitin. Amino acid analysis figures agree well with thcorctical values. Elcctrospray mass analysis o f this material showed the expected molecular ion 8564.8. The primary sequence of the synthetic ubiquitin was confirmed by automated Edman degradation. Tryptic digestion of oxidized ubiquitins A and B and bovine ubiquitin gave a very interesting picture which led u s t o speculate that peak B corresponds to a material which may represent an intermediate state of folding. The current hypothe5is is that protein folding takes place in a few discreet steps 17, 81. Isolation of materials at intcrmediate stages in the process opens the way to kinetic and thermodynamic investigation of the development o f tertiary structure. The tryptic digest was found to be most variable in the C-terminal region and one could spcculatc that the fitting o f this region into the final tertiary structure occurs at a late stage. Clearly the ncxt step is direct physical investigation of the materials, which we are carrying out by spectroscopic and electrophoretic methods. We thank the S.E.K.C.. Applied l3iosystems Inc. and Merck. Sharp & Dohmc f o r financial support. We are grateful t o B. Whigham and K. Shaw for their invaluable technical support. Brian Green ( V C Instruments) for the mass analysis. Dr L. A . FothergillCilmore and Ms L. Kerr o f the University o f Edinburgh sequencing unit, WELMET. lor the sequencing o f these samples. Green. J. & Kamage. K.( 1987) Tc~irctheclror~ I.c,tr. 28. 2287 Merrifield. K. B. ( 1963)J . Am. C'hcJtn..Yoc,. 85, 2 147 Merrifield. K.13. ( 1985) S c i e n w 232. 341 -347 Hriand. J.-P., Muller. S.. Raboy. 13. & Van Dorsselaer. A . ( I 989,) /",pi. Hes. 2. 38 1-388 5. Armarego, W. L. F., Perrin, D. I). & Perrin, D. K.( 1980) I'uriJicw riot? oj'luhorcrio~y('lic~rniccrh. 2nd cdn.. Pergamon Press. Oxford 6 . Green, J., Ogunjobi, 0. M. & Kamage. K. ( 1 9 8 9 ) Terrrrheclrorr 1,rrr. 3 0 . 2 149-2 I 5 2 7. Anfinsen, C . 13. & Schcraga. H. A . ( 1075) A d i . . I'roi. (‘hem. 29, 205-300 ( I98 I ) Ad,'. I'tW. C'hc,tt/. 34. 167-339
I. 2. 3. 4.
Received 1 May I990
Synthesis, monitoring and structure-function studies on some neurokinin A analogues DAVID J. S. GUTHRIE.* AHMED A. ABU SHANAB,* JAMES M. ALLEN.1- G. BRENT IRVINE.* NEIL V. McFERRAN* and BRIAN WALKER* * Ilitisiori of' Hiochernistty. The School of' HioloD arid Hiochernistry, The Qireeri j. Universiy of' Relfast, Belfast R I ' V 7HL. N. Irelrnd, U.K . and t Riornerlical Sciences Kiwrrrch C'entre. Uriiver.sityof' Ulster, Ncwtowriuhhey H7'37 OQB, N . Irchrid, U.K . Neurokinin A (NKA) is one o f three tachykinins found in mammals, the others being substance P (SP)and neurokinin B (NKB) (Table I ) . Thcse peptides arc widely distributed Abbreviations t-hutoxycarbonyl; substance P; Aib. carhoxylic acid; receptor.
Vol. 18
used: Fmoc. fluorenylmethoxycarbonyl; Roc. NKA. neurokinin A; NKH. neurokinin 13; SP. a-aminobutyric acid; Ach. I -aminocyclohexanef.a.h.. fast atom bombardment; NK2, NKA-
throughout the nervous system and show a variety of activities [ 1 I. Three types of receptor with specificitics matching the thrcc tachykinins ( N K I for SP, NK2 for NKA and NK3 for NKB) have been characterized. The relationships between these peptides and their receptors are very complex. Receptor-stimulating activity resides in the very similar C-terminal regions and a degree o f cross-reactivity has been postulated [ 1 I. Some cells express mainly one receptor type, but many cells express more than one. Furthermore, SP and NKA are synthesized together and arc probably co-released from peripheral neurons. We wanted t o investigate the possible rolc o f tachykinins in conditions such as asthma, where it has been suggested that abnormal release of SP or NKA could be involved in bronchoconstriction [ 2, 31. More recently, it has been shown that in the airways NKA is more potent in contracting smooth muscle, while SP is a more potent stimulant o f mucous hypersecretion. vasodilation and resulting oedema
