What is claimed is:
1. A DNA sequence in substantially pure form which codes an eglin or a modified eglin; and fragments thereof.
2. A DNA sequence according to claim 1, which codes eglin C, and fragments thereof.
3. A DNA sequence according to claim 1, which codes modified eglin, in which the modification consists of a shortening of the primary structure of the eglin, whilst maintaining the eglin activity.
4. A DNA sequence according to claim 1, of the formula
42
Met B
5(x)n ATG D
Pro Glu Val Val Gly Lys Thr Val Asp Gln
CCX GAM GTX GTX GGX AAM ACX GTX GAY CAM
Ala Arg Glu Tyr Phe Thr Leu His Tyr Pro
GCX LGN GAM TAY TTY ACX YTZ CAY TAY CCX
Gln Tyr Asp Val WPhe Leu Pro Glu Gly
CAM TAY GAY GTX YAY TTY YTZ CCX GAM GGX
Ser Pro Val Thr Leu Asp Leu Arg Tyr Asn
QRS CCX GTX ACX YTZ GAY YTZ LGN TAY AAY
Arg Val Arg Val Phe Tyr Asn Pro Gly Thr
LGN GTX LGN GTX TTY TAY AAY CCX GGX ACX
Asn Val Val Asn B NON
AAY GTX GTX AAY D TMK (x)m 3
(I)
in which the nucleotide sequence is shown starting with the 5-end and, for better understanding, the aminoacids coded by each triplet are given, and in which D is a direct bond or a nucleotide sequence which codes N-terminal aminoacids of the eglin, and B is a direct bond or the corresponding N-terminal aminoacids chosen from the group
43
Ser PheLeu Lys Ser PheSer Glu Leu Lys
QRS TTY , YTZ AAM QRS TTY , QRS GAM YTZ AAM
Ser PhePhe Gly Ser GluLeu Lys Ser Phe
QRS TTY , TTY GGX QRS GAMYTZ AAM QRS TTY
and
Thr GluPhe Gly Ser GluLeu Lys Ser Phe
ACX GAMTTY GGX QRS GAMYTZ AAM QRB TTY
and D is a direct bond or a nucleotide sequence which codes C-terminal aminoacids of the eglin, and B is a direct bond or the corresponding C-terminal aminoacids chosen from the group
44
His ValHis Val Pro His
CAY GTX , CAY GTX CCX CAY and
His ValPro His Val Gly
CAY GTXCCX CAY GTX GGX
and in which A is deoxyadenosyl, T is thymidyl, G is deoxyguanosyl, C is deoxycytidyl, X is A, T, C or G, Y is T or C, Z is A, T, C or G, if YC, or Z is A or G, if YT, Q is T or A, R is C and S is A, T, C or G, if QT, or R is G and S is T or C, if QA, M is A or G, L is A or C, N is A or G, if LA, or N is A, T, C or G, if LC, K is A or G, if MA, or K is A, if MG, W is Tyr or His, and (X)n and (X)m are each any nucleotide sequences with n and m greater than 3 and less than 100, in particular greater than 5 and less than 12, which can be recognised and cleaved by a restriction enzyme, and fragments of such a double-stranded DNA of the formula I.
5. An expression vector which contains a DNA sequence which codes an eglin or a modified eglin and which is regulated by an expression control sequence such that polypeptides with eglin activity are expressed in a host transformed with this expression vector.
6. An expression vector according to claim 1, in which the DNA sequence codes eglin C.
7. A host transformed with an expression vector according to any one of claims 5 or 6.
8. A transformed host according to claim 7, in which the host is a microorganism or a human or animal cell.
9. A process for the preparation of an eglin compound of the formula
45
(Met)r-B-ProGluValValGlyLysThrValAspGlnAlaArgGlu
TyrPheThrLeuHisTyrProGlnTyrAspValWPheLeuProGluGlySerProValThrLeuAsp
LeuArgTyrAsnArgValArgValPheTyrAsnProGlyThrAsnValValAsn-B
(XIV),
in which B is a direct bond or a peptide radical comprising 1-10 aminoacid units from the N-terminus of the natural eglins, for example such a radical chosen from the group comprising SerPhe, LeuLysSerPhe, SerGluLeuLysSerPhe, PheGlySerGluLeuLysSerPhe and ThrGluPheGlySerGluLeuLysSerPhe, and B is not a peptide radical or is a peptide radical which comprises 1-6 aminoacid units from the C-terminus of the natural eglins, for example such a radical chosen from the group comprising -HisVal, -HisValProHis or -HisValProHisValGly, W is Tyr or His and r is 0 or 1, and in which, in compounds of the formula XIV in which r is 0, the N-terminal aminoacid is free or N-acetylated, and of a salt of such a compound, which comprises culturing a host transformed with an expression plasmid containing an eglin-coding DNA sequence regulated by an expression control sequence, in a liquid nutrient medium containing assimilatable sources of carbon and nitrogen, releasing the product from the host cells and isolating it, or, for the preparation of compounds of the formula XIV, in which r is 0 and the N-terminal aminoacid is N-acetylated, acetylating a compound of the formula XIV with a free N-terminal amino group and, if desired, converting an eglin compound of the formula XIV, which can be obtained, into another eglin compound of the formula XIV, and, if necessary, separating a mixture, obtainable according to the process, of compounds of the formula XIV into the individual components, andor, if desired, converting a resulting salt into the free polypeptide and converting a resulting polypeptide into a salt thereof.
10. A process according to claim 9, for the preparation of a compound of the formula XIV, in which B is a peptide radical selected from the group comprising LeuLysSerPhe, SerGluLeuLysSerPhe, PheGlySerGluLeuLysSerPhe and ThrGluPheGlySerGluLeuLysSerPhe, B is the radical -HisValProHisValGly, W is Tyr and r is 0 or 1; and furthermore also a process for the preparation of an eglin B compound of the formula XIV, in which B is the peptide radical ThrGluPheGlySerGluLeuLysSerPhe, B is the peptide radical -HisValProHisValGly, W is His and r is 0 or 1, the N-terminal aminoacid in compounds of the formula XIV in which r is 0 being-free or N-acetylated, and of a salt of such a compound.
11. A process according to claim 9, for the preparation of a compound of the formula XIV, in which B is PheGlySerGluLeuLysSerPhe, ThrGluPheGlySerGluLeuLysSerPhe or N-acetyl-ThrGluPheGlySerGluLeuLysSerPhe B is the HisValProHisValGly radical, W is Tyr and r is 0, and of a salt of such a compound.
12. A process according to claim 9, for the preparation of eglin C.
13. A process according to claim 9, for the preparation of eglin B.
14. A process according to claim 9, for the preparation of N-acetyl-eglin C.
15. A process according to claim 9, for the preparation of N-methionyl-eglin C.
16. A process according to claim 9, for the preparation of DesThr1Glu2-eglin C.
17. A process according to claim 9, wherein the conversion of a compound of the formula XIV, in which r s 0 and the N-terminal amino group is in the free form, into a corresponding compound of the formula XIV, in which the N-terminal aminoacid is N-acetylated, is carried out by an enzymatic route.
18. A process according to claim 9, wherein the methionyl radical in a compound of the formula XIV, which can be obtained, which has an N-terminal methionyl radical is detached with cyanogen bromide.
19. A process according to claim 9, wherein the acetyl radical in a compound of the formula XIV, which can be obtained, which has an N-terminal acetylated amino group is detached enzymatically.
20. An eglin compound of the formula
46
(Met)r-B-ProGluValValGlyLysThrValAspGlnAlaArgGlu
TyrPheThrLeuHisTyrProGlnTyrAspValWPheLeuProGluGlySerProValThrLeuAsp
LeuArgTyrAsnArgValArgValPheTyrAsnProGlyThrAsnValValAsn-B
(XIV)
in which r is 1 and B is a direct bond or a peptide radical comprising 1-10 aminoacid units from the N-terminus of the natural eglins, for example such a radical chosen from the group comprising SerPhe, LeuLysSerPhe, SerGluLeuLysSerPhe, PheGlySerGluLeuLysSerPhe and ThrGluPheGlySerGluLeuLysSerPhe, and B is not a peptide radical or is a peptide radical which comprises 1-6 aminoacid units from the C-terminus of the natural eglins, for example such a radical chosen from the group comprising -HisVal, -HisValProHis or -HisvalproHisvalGly, W is Tyr or His, or in which r is 0, B is PheGlySerGluLeuLysSerPhe or an N-terminally acetylated peptide radical, selected from the group comprising N-acetyl-SerPhe, N-acetyl-LeuLysSerPhe, N-acetyl-SerGluLeuLysSerPhe, N-acetyl-PheGlySerGluLeuLysSerPhe or N-acetyl-ThrGluPheGlySerGluLeuLysSerPhe, B is as defined and W is Tyr or His, and a salt of such a compound.
21. An eglin compound of the formula XIV according to claim 20 in which r is 0, B is the peptide radical PheGlySerGluLeuLysSerPhe or N-acetyl-ThrGluPheGlySerGluLeuLysSerPhe, B is the peptide radical -HisValProHisValGly and W is Tyr, and a salt of such a compound.
22. N-Acetyl-eglin C and a salt thereof, according to claim 20.
23. N-Methionyl-eglin C or a salt thereof, according to claim 20.
24. Des Thr1Glu2-eglin C or a salt thereof, according to claim 20.
25. A pharmaceutical preparation containing a compound of the formula XIV according to claim 20 or a pharmaceutically acceptable salt thereof.
26. A method of treating pulmonary diseases, septic shock or inflammation in a mammal comprising administering to said mammal a therapeutically effective amount of a compound of the formula XIV according to claim 20 or of a pharmaceutically acceptable salt thereof.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
1. A membrane electrode assembly, including:
an electrolyte membrane;
an anode disposed on one face of said electrolyte membrane; and
a cathode disposed on the other face of said electrolyte membrane, wherein:
said cathode contains a catalyst layer such that the ratio of a pore volume in a second micro-pore diameter to a pore volume in a first micro-pore diameter is in a range of 3.8 to 8.3,
the first micro-pore diameter has a range from 0.01 \u03bcm to less than 0.1 \u03bcm, and
the second micro-pore diameter has a range from 0.1 \u03bcm to less than 1 \u03bcm.
2. A membrane electrode assembly according to claim 1, wherein the catalyst layer contains a platinum-alloy-supported catalyst.
3. A membrane electrode assembly according to claim 1, wherein the catalyst layer contains an ion conductor having an ion-exchange group equivalent weight (EW value) which is less than or equal to 800.
4. A membrane electrode assembly according to claim 2, wherein the catalyst layer contains an ion conductor having an ion-exchange group equivalent weight (EW value) which is less than or equal to 800.
5. A fuel cell having a membrane electrode assembly according to claim 1.
6. A fuel cell having a membrane electrode assembly according to claim 2.
7. A fuel cell having a membrane electrode assembly according to claim 3.
8. A fuel cell having a membrane electrode assembly according to any one of claim 4.