Adaptation of plasminogen activator sequences to known protease structures.

The sequences of urokinase (UK) and tissue-type plasminogen activator (TPA) were aligned with those of chymotrypsin, trypsin, and elastase according to their 'structurally conserved regions'. In spite of its trypsin-like specificity UK was model-built on the basis of the chymotrypsin structure because of a corresponding disulfide pattern. The extra disulfide bond falls to cysteines 50 and 111d. Insertions can easily be accommodated at the surface. As they occur similarly in both, UK and TPA, a role in plasminogen recognition may be possible. Of the functional positions known to be involved in substrate or inhibitor binding, Asp 97, Lys 143 and Arg 217 (Leu in TPA) may contribute to plasminogen activating specificity. PTI binding may in part be impaired by structural differences at the edge of the binding pocket.

Studies on cytochrome c oxidase, IV[1--3]. Primary structure and function of subunit II.

The amino acid sequence of polypeptide II from beef heart cytochrome c oxidase is described. Comparision of this primary structure with those of azurins, plastocyanins and stellacyanins reveals clear homologies among them. Thus subunit II of the oxidase is a member of this copper protein family. The sequence homology indicates a copper binding site consisting of two invariant histidines and two sulfur-containing amino acids. Thus subunit II is like a blue copper protein with type I copper.

Studies on cytochrome c oxidase, II. The chemical constitution of a short polypeptide from the beef heart enzyme.

A low molecular weight (approximately 6000) polypeptide fraction was isolated from beef heart cytochrome c oxidase, consisting of three peptides with the N-terminal end groups isoleucine, phenylalanine and serine. The complete amino acid sequence of the serine component is described. From the chemical constitution, a site-specific cleavage from a precursor protein and a possible function in membrane penetration and complex formation of the oxidase is inferred.

Inhibitory characteristics and oxidant resistance of site specific variants of recombinant human antileukoproteinase (ALP).

Tandem gene plasmids were constructed and used to express inactive proteins equivalent to human antileukoproteinase (ALP) and the variants [Leu73]-ALP and [Leu73, 82, 94, 96]-ALP in E. coli K12. After extraction, refolding, and purification, highly pure and active inhibitors were obtained in good yields. Inhibitory constants for human leukocyte elastase and cathepsin G were found to be similar. The variants in which methionines were exchanged for leucines were shown to be more resistant to inactivation by oxidizing agents than native ALP. As oxidizing conditions exist at sites of inflammation, these ALP variants are promising candidates for therapies involving suppression of elastase-mediated injury.

[Hemoglobins, XXV. Hemoglobin (erythrocruorin) CTT III from Chironomus thummi thummi (Diptera). Primary structure and relationship to other heme proteins (author's transl)]. / Hämoglobine, XXV. Hämoglobin (Erythrocruorin) CTT III aus Chironomus thummi thummi (Diptera). Primärstruktur und Beziehung zu anderen Hämproteinen.

The amino acid sequence analysis of hemoglobin (erythrocruorin) CTT III from Chironomus thummi th. (Diptera) has been checked with automatic methods and completed. The protein chain consists of 136 amino acids and contains a neutral exchange isoleucine/threonine in position 57. The molecular weight of the heme protein (Thr) is 15400. The primary structure gives the chemical basis for the refinement of the X-ray structure and the understanding of the mechanism of the Bohr effect in this monomeric hemoglobin. A homologous alignment to vertebrate globins is reported. The resulting data for the phylogeny of proto-and deuterostomian animals and the function of this hemoglobin are discussed.

A strong thrombin-inhibitory prourokinase derivative with sequence elements from hirudin and the human thrombin receptor.

In order to design plasminogen activators with improved thrombolytic properties, bifunctional proteins with both plasminogen-activating and anticoagulative activity were constructed by fusing a thrombin-inhibitory moiety itself comprises four elements: linker 1, a motif directed to thrombin's active site, linker 2 and a fragment of hirudin which binds to the fibrinogen-recognition site of thrombin. In order to improve further the anticoagulative activity, the thrombin-inhibitory domain was modified by substituting linker 2. Introduction of a linker (FLLRNP) from the human thrombin receptor conferred about a 10-fold increase in anticoagulative activity in protein M37 compared with the parent molecule M23 carrying an aliphatic linker. The increase in anticoagulative activity was also reflected in the shift of the Ki value from 159 +/- 20 nM for M23 to 2.0 +/- 0.5 nM for M37. The increased thrombin-inhibitory activity of M37 may be due to the presence of an arginine in the linker from the thrombin receptor which may interact with one of two glutamic acid residues located at the exit of the thrombin substrate binding pocket. This explanation is supported by the observation that another chimera (M35) carrying a linker sequence with two acidic residues has relatively weak thrombin-inhibitory activity. The thrombin-inhibitory activity of M37 may be strong enough to substitute anticoagulative co-medication during fibrinolytic treatment.

Construction and structure-activity relationships of chimeric prourokinase derivatives with intrinsic thrombin-inhibitory potential.

The blood clotting enzyme thrombin plays a central role in the aetiology of occlusive disorders such as stroke and acute myocardial infarction. During fibrinolytic therapy with plasminogen activators, thrombin is neutralized by anticoagulative drugs. In order to combine plasminogen-activating and thrombin-inhibitory activities we constructed chimeric derivatives of recombinant single-chain, urokinase-type plasminogen activator (rscu-PA) which comprise the kringle and protease domain of rscu-PA fused via a linker sequence to a thrombin-inhibitory domain. The inhibitory domain contains a sequence element directed to the active site of thrombin and a sequence taken from either hirudin or the human thrombin receptor both binding to the fibrinogen recognition site of thrombin. Analysing different sets of point mutants showed that the linker between the protease domain and the active site-directed sequence is contributing significantly to the thrombin-inhibitory potential. Kinetic analysis of thrombin inhibition revealed that most of the chimeras tested competitively inhibit the thrombin-mediated cleavage of a peptide substrate in a concentration-dependent manner; however, in two examples the insertion of one glycine residue into the active site directed-sequence abolished the blockade of the active site. This supports the conclusion that the chimeras with high thrombin-inhibitory potential interact with the active site and the fibrinogen recognition site of thrombin.

Studies on cytochrome c oxidase, VIII. The amino acid sequence of polypeptide VII.

The sequence determination of polypeptide VII from beef heart cytochrome c oxidase is described. The amino acid sequence is deduced from overlapping tryptic peptides and peptides obtained after cleavage with Staphylococcus aureus protease. The protein consists of 85 amino acids corresponding to a Mr of 10026, in agreement with a value of 9500 obtained by sodium dodecyl sulfate gel electrophoresis. The amino acid sequence around the only methionine present is very similar to sequences around the invariant heme binding methionine of the cytochrome c family. This similarity suggests that the protein is one of the heme bindings subunits of the oxidase.

Studies on cytochrome c oxidase, V. Polypeptide IV: alignment and amino acid sequences on cyanogen bromide fragments.

The isolation and chemical characterization of polypeptide IV from beef heart cytochrome oxidase is described. The protein is one of the main (stoichiometric) components of the oxidase. It is the largest polypeptide of the enzyme synthezised in the cytoplasm and has, as such, also been identified in enzyme preparations from yeast and Neurospora. A partial sequence, consisting of 105 amino acid residues which give a frame work of the covalent structure of the polypeptide is obtained from N- anc C-terminal sequencing and from the cyanogen bromide fragments of the chain. The isolation and sequencing of the fragments of this membrane protein are discussed.

Studies on cytochrome c oxidase, VI. Polypeptide IV. the complete primary structure.

The complete primary structure of the cytoplasmically synthesized polypeptide IV from beef heart cytochrome oxidase was determined via isolation and sequencing of overlapping methionine, tryptophan, and arginine fragments. The protein consists of 147 amino acids (Mr 17153). It is characterized as a part of a membrane protein complex by a hydrophobic segment consisting of 19 residues. It is suggested that this segment contacts the lipids of the inner mitochondiral membrane. Additional specific contacts may result from pairwise formation of salt bridges between ionic groups of the protein and the phospholipids. The function of this component of the terminal oxidase is yet unknown.

Studies on cytochrome c oxidase, X. Isolation and amino-acid sequence of polypeptide VIIIb.

The isolation and sequence determination of the cytoplasmically synthesized polypeptide VIIIb from beef heart cytochrome c oxidase is described. Several methods for isolating polypeptide VIIIb with gelchromatographic technics are presented. The complete amino-acid sequence is deduced from a N-terminal sequencer run, overlapping tryptic peptides and peptides obtained after tryptophan specific cleavage with cyanogen bromide in heptafluorobutyric acid/formic acid. The small protein consists of 46 amino acids and has a molecular mass of 4 962 Da. The existence of a hydrophobic segment with a length of 20 residues characterizes it as a membrane penetrating protein. The stoichiometry of this polypeptide in the functional monomer of cytochrome c oxidase (complex IV) is 2 and is thus different from all the other polypeptides constituting the respiratory complex IV. The function of this component of the terminal oxidase is as yet unknown.

Structural relationship between human high and low molecular mass urokinase.

Human low molecular mass urokinase was demonstrated to consist of two polypeptides. The peptide chains of about 30000 and of 2427 Da, respectively, were isolated by gel filtration after reductive cleavage of single interchain disulfide bridge. The complete amino acid sequence of the 2427-Da chain consisting of 21 amino acids was determined. Stoichiometric amounts of hexosamines were found in the 2427-Da chain. The isolated 30000-Da chains of both, human low and high molecular mass urokinases were found to be identical in terms of amino acid composition, of hexosamine content, of N- and of C-terminal amino acid sequences. The amino acid sequence of the 2427-Da chain of the low molecular enzyme was found to be different from the N-terminal sequence of the 20000-Da chain of the high molecular enzyme. The transformation of high to the low molecular form is considered a limited proteolytic degradation of the 20000-Da chain of high molecular mass urokinase, exclusively.

Primary structure of Cu-Zn superoxide dismutase of Brassica oleracea proves homology with corresponding enzymes of animals, fungi and prokaryotes.

The complete amino-acid sequence of Cu-Zn superoxide dismutase from white cabbage (Brassica oleracea) is reported. The polypeptide chain consists of 151 amino acids and has a molecular mass of 15,604 Da. The primary structure of the reduced and S-carboxymethylated protein was determined by automated solid phase sequence analysis of tryptic fragments and peptides obtained by digestion with Staphylococcus aureus proteinase V8. The protein shows a free amino terminus as was found for all non-mammalian Cu-Zn enzymes so far sequenced. Comparison of the amino-acid sequence from the plant Cu-Zn enzyme with those from nine eukaryotic enzymes reveals a high degree of homology (50-64%) among these enzymes. As already described for all the eukaryotic Cu-Zn superoxide dismutases also the plant enzyme shows a low homology (about 28%) with the bacteriocuprein of Photobacterium leiognathi. However, the amino-acid residues involved in metal binding, the half-cystine residues forming the intermolecular disulfide bridge, one of the arginine and some glycine and proline residues are conserved in all eleven Cu-Zn superoxide dismutases. Although the precise role of the 23 completely conserved residues is not yet completely understood, they appear to almost define the minimum structural requirements for optimizing the superoxide dismutation at the catalytic site, since functional differences between the eleven enzymes are not detectable.

The amino-acid sequence of rat Cu-Zn superoxide dismutase.

The primary structure of Cu-Zn superoxide dismutase isolated from rat liver was determined. The enzyme was reduced, carboxymethylated and fragmented by treatment with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by gel filtration or high performance liquid chromatography and sequenced by automated Edman degradation. The total sequence of 153 amino-acid residues per subunit was reconstructed from overlapping peptides. Rat Cu-Zn superoxide dismutase proved to be closely related to the corresponding sequences of other mammals in having more than 80% identical amino-acid residues in homologous position and an acetylated N-terminus. Comparison of the rat Cu-Zn superoxide dismutase structure with those of other species suggests a similar phylogenetic distance between rat, man, pig, cattle and horse and a rapid molecular divergence during vertebrate development compared to earlier evolutionary periods.

Studies on cytochrome c oxidase, VII. Isolation and chemical characterization of polypeptide VII.

Polypeptide VII of cytochrome c oxidase was isolated and purified by gel filtration on Bio-Gel P-10 in 10% acetic acid. Automatic Edman degradation of this peptide chain was not successful, because it is blocked at the N-terminus. The amino acid analysis shows a relatively high content of hydrophilic residues (54%). On the basis of this analysis and the apparent molecular weight by sodium dodecyl sulfate gel electrophoresis and gel filtration, a chain length of about 80 residues was calculated. Among the tryptic peptides one blocked heptapeptide was found. Cleavage of this peptide with thermolysin gave two peptide fragments, one of which was not retained on a cation exchange resin. Mass spectrometric sequence determination of this peptide revealed the structure Ac-Ala-Glu-Asp for the N-terminus of polypeptide VII. Treatment with carboxypeptidase A at two different pH values showed that the C-terminal amino acid is isoleucine and the penultimate amino acid is lysine.

The complete amino acid sequence of low molecular mass urokinase from human urine.

The sequence of all 253 amino acids of the heavy (B-) chain of human urinary urokinase was determined. The fragmentation strategy employed included cyanogen bromide cleavage of S-carboxymethylated B-chain at Met and/or Trp residues, cleavage of acid-labile Asp-Pro bonds, and the use of the specific endoproteinases Lys-C and Arg-C for generation of overlapping fragments. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. The amino acid sequence obtained substantiates the serine protease character of the B-chain of urokinase: a considerable homology with other serine proteinases, especially with the B-chain of human plasmin, was proved. The pertinent active site amino acids were localized: His-46, Asp-97, and Ser-198. A carbohydrate side chain, containing at least 4 glucosamine and 2 galactosamine residues, was demonstrated to be fixed at asparagine in position 144. The sequence data presented, together with the sequence of the second (A1-) chain of low molecular mass urokinase which was reported by us in an earlier communication, complete the knowledge of the whole primary structure of an active form of human urinary urokinase.

The amino-acid sequence of rabbit Cu-Zn superoxide dismutase.

The primary structure of Cu-Zn superoxide dismutase from rabbit liver was investigated. The reduced and S-carboxymethylated enzyme was treated with cyanogen bromide, trypsin or Staphylococcus aureus proteinase V8. The resulting peptides were separated by high-performance liquid chromatography and sequenced by automated Edman degradation. With the exception of the N- and C-terminus the complete sequence was established by means of overlapping peptides. The N-terminus is blocked and thus not susceptible to Edman degradation. The amino-acid composition of the tryptic N-terminal peptide corresponds to that of the cytoplasmatic Cu-Zn superoxide dismutases of other mammals investigated. The chromatographic behaviour of these N-terminal peptides on a reversed phase C18 column is also identical, thus suggesting also for the rabbit Cu-Zn superoxide dismutase the N-terminal sequence Ac-Ala-Thr-Lys. The C-terminus was demonstrated to have the sequence -Ile-Ala-Pro by enzymatic degradation with carboxypeptidase Y. The complete amino-acid sequence of the rabbit Cu-Zn superoxide dismutase consists of 152 amino-acids and shows the expected homology to other Cu-Zn enzymes published so far. The aspartate and six histidine residues known to complex the metal ions are conserved at homologous positions. This also applies for the arginine residue near the C-terminus which is supposed to direct the anionic superoxide radical towards the active centre of the enzyme. The amino acid sequence of the rabbit Cu-Zn superoxide dismutase corresponds to those of other mammals in more than 80% of its amino-acid residues. From a total of 152 amino-acid residues the rabbit shares with rat 128, with mouse 130, with horse 127, with pig 126/127, with cattle 130 and with man 131 amino acids in homologous positions. However the Cu-Zn superoxide dismutases of closely related mammals like rats and mice differ in only five amino acid residues of their sequence. A phylogenetic closer relatedness between lagomorphs and rodents than between other orders of mammals, could not be derived from the sequence data given. Rather rodents and lagomorphs are to be considered as two evolutionary independent orders of mammals.

Functional properties of a recombinant chimeric protein with combined thrombin inhibitory and plasminogen-activating potential.

A chimeric protein (rscu-PA-40-kDa/Hir), consisting of the C-terminal amino acids 53-65 of hirudin (Hir), fused via a 14-amino-acid linker sequence to the C-terminal of a 40-kDa fragment (Ser47-Leu411) of recombinant (r) single-chain (sc) urokinase-type plasminogen activator (rscu-PA), was produced by expression of the corresponding chimeric cDNA in Escherichia coli cells. The thrombin inhibitory potential of purified rscu-PA-40-kDa/Hir was confirmed by complete inhibition of the coagulant activity of thrombin at 20-30-fold molar excess of the chimera, and by the resistance of rscu-PA-40-kDa/Hir to proteolytic cleavage by thrombin, rscu-PA-40-kDa/Hir prolonged the thrombin time of normal human plasma in a dose-dependent way (reduction of the apparent thrombin concentration to 50% with 95 nM chimeric protein as compared to 4.7 nM hirudin), and inhibited thrombin-mediated platelet aggregation (reduction of the apparent thrombin concentration to 50% with 40 nM chimeric protein). The chimera had a specific activity on fibrin films of 57,000 IU/mg as compared to 95,000 IU/mg for rscu-PA. The urokinase-like amidolytic activity of the single-chain protein was only 220 IU/mg but increased to 169,000 IU/mg after treatment with plasmin, which resulted in quantitative conversion to a two-chain (tc) derivative (rtcu-PA-40-kDa/Hir). Corresponding values for rscu-PA were 270 and 226,000 IU/mg. The catalytic efficiencies for plasmin-mediated conversion to two-chain molecules were comparable for rscu-PA-40-kDa/Hir and rscu-PA (0.63 and 0.65 microM-1.s-1, respectively). The plasminogen-activating potential of the single-chain chimera was comparable to that of rscu-PA; the catalytic efficiencies for plasminogen activation by their two-chain counterparts were also similar (0.55 and 0.73 microM-1.s-1, respectively). In 2 h, 50% lysis of 125I-fibrin-labeled clots prepared from platelet-poor human plasma and immersed in normal plasma was obtained with 1.3 micrograms/ml rscu-PA-40-kDa/Hir and with 0.67 micrograms/ml rscu-PA, with corresponding residual fibrinogen levels of 74% and 87%, respectively. In the absence of fibrin, 50% fibrinogenolysis in 2 h in normal human plasma required 2.1 micrograms/ml rscu-PA, but 7.9 micrograms/ml rscu-PA-40-kDa/Hir. Thus, the chimera rscu-PA-40-kDa/Hir has maintained the specific fibrinolytic and plasminogen activating activity of rscu-PA as well as its fibrinolytic potency in plasma, whereas it displayed a similar or somewhat better fibrin specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibacterial activity of antileukoprotease.

Antileukoprotease (ALP), or secretory leukocyte proteinase inhibitor, is an endogenous inhibitor of serine proteinases that is present in various external secretions. ALP, one of the major inhibitors of serine proteinases present in the human lung, is a potent reversible inhibitor of elastase and, to a lesser extent, of cathepsin G. In equine neutrophils, an antimicrobial polypeptide that has some of the characteristics of ALP has been identified (M. A. Couto, S. S. L. Harwig, J. S. Cullor, J. P. Hughes, and R. I. Lehrer, Infect. Immun. 60:5042-5047, 1992). This report, together with the cationic nature of ALP, led us to investigate the antimicrobial activity of ALP. ALP was shown to display marked in vitro antibacterial activity against Escherichia coli and Staphylococcus aureus. On a molar basis, the activity of ALP was lower than that of two other cationic antimicrobial polypeptides, lysozyme and defensin. ALP comprises two homologous domains: its proteinase-inhibitory activities are known to be located in the second COOH-terminal domain, and the function of its first NH2-terminal domain is largely unknown. Incubation of intact ALP or its isolated first domain with E. coli or S. aureus resulted in killing of these bacteria, whereas its second domain displayed very little antibacterial activity. Together these data suggest a putative antimicrobial role for the first domain of ALP and indicate that its antimicrobial activity may equip ALP to contribute to host defense against infection.

The primary structure of porcine Cu-Zn superoxide dismutase. Evidence for allotypes of superoxide dismutase in pigs.

Cu-Zn superoxide dismutase was purified to homogeneity from mixed pig blood and from a single pig. The isolated product had an absorption ratio 280/260 nm of 0.91, a specific activity of 3 000 +/- 200 units (cytochrome c reduction test), and an isoelectric point of 7.5 (chromatofocusing) or 7.25 (isoelectric focusing), respectively. Sequence determination was performed by automated solid-phase Edman degradation of fragments of the reduced S-carboxymethylated proteins obtained by digestion with trypsin or Staphylococcus aureus proteinase V8 or treatment with cyanogen bromide. Acetylation of the N-terminus was confirmed by comparing high performance liquid chromatography retention times of N-terminal peptides with those of authentic samples. Sequencing of the superoxide dismutase of mixed porcine blood revealed heterogeneity (70% Leu; 30% Val at position 29), whereas the sample derived from a single French pig proved to be homogeneous (100% Leu at position 29). The complete sequence of pig superoxide dismutase comprised 152 amino-acid residues, which corresponds to a theoretical molecular mass of 15 800 Da per subunit, and exhibited the expected high homology with those of other mammals. The aspartate and all 7 histidine residues known to complex the metal ions in bovine superoxide dismutase are conserved in the porcine sequence at the homologous positions Asp82 and His45, His47, His62, His70, His79, His119, respectively.