Minor groove hydration of DNA in aqueous solution: sequence-dependent next neighbor effect of the hydration lifetimes in d(TTAA)2 segments measured by NMR spectroscopy.

The hydration in the minor groove of double stranded DNA fragments containing the sequences 5'-dTTAAT, 5'-dTTAAC, 5'-dTTAAA and 5'-dTTAAG was investigated by studying the decanucleotide duplex d(GCATTAATGC)2 and the singly cross-linked decameric duplexes 5'-d(GCATTAACGC)-3'-linker-5'-d(GCGTTAATGC)-3' and 5'-d(GCCTTAAAGC)-3'-linker-5'-d(GCTTTAAGGC)-3' by NMR spectroscopy. The linker employed consisted of six ethyleneglycol units. The hydration water was detected by NOEs between water and DNA protons in NOESY and ROESY spectra. NOE-NOESY and ROE-NOESY experiments were used to filter out intense exchange cross-peaks and to observe water-DNA NOEs with sugar 1' protons. Positive NOESY cross-peaks corresponding to residence times longer than approximately 0.5 ns were observed for 2H resonances of the central adenine residues in the duplex containing the sequences 5'-dTTAAT and 5'-dTTAAC, but not in the duplex containing the sequences 5'-dTTAAA and 5'-dTTAAG. In all nucleotide sequences studied here, the hydration water in the minor groove is significantly more mobile at both ends of the AT-rich inner segments, as indicated by very weak or negative water-A 2H NOESY cross-peaks. No positive NOESY cross-peaks were detected with the G 1'H and C 1'H resonances, indicating that the minor groove hydration water near GC base pairs is kinetically less restrained than for AT-rich DNA segments. Kinetically stabilized minor groove hydration water was manifested by positive NOESY cross-peaks with both A 2H and 1'H signals of the 5'-dTTAA segment in d(GCATTAATGC)2. More rigid hydration water was detected near T4 in d(GCATTAATGC)2 as compared with 5'-d(GCATTAACGC)-3'-linker-5'-d(GCGTTAATGC)-3', although the sequences differ only in a single base pair. This illustrates the high sensitivity of water-DNA NOEs towards small conformational differences.

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 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.

Chemical, enzymological and pharmacological equivalence of urokinases isolated from genetically transformed bacteria and human urine.

Low molecular weight urokinase (LUK), which was prepared from E. coli containing a plasmid coding for human pro-urokinase, has an amino acid sequence identical to that of LUK isolated from human urine (uLUK) but lacks the carbohydrate side chain at Asn 144 of the B chain. This chemical difference results in an altered mobility in SDS polyacrylamide gel electrophoresis and an apparently increased specific activity of the E. coli-derived product (cLUK) in diffusion-limited test systems (fibrin agar plate tests). Comparative enzymological investigations in homogeneous phases reveal that the active centers and the substrate recognition sites of cLUK and uLUK are congruent. No significant difference between the enzymes was detectable in the following parameters: Michaelis constants and maximum velocities with the synthetic substrate S-2444; activation rates of human and porcine plasminogen; specificity for ten chromogenic substrates; inhibition constants for the competitive inhibitor benzamidine; inhibition by placental urokinase inhibitor and polyclonal antibodies. Further, cLUK and uLUK dissolved fibrin clots prepared from human plasma in vitro with essentially identical velocities. Both, cLUK and uLUK efficiently lysed injected emboli in rabbits and prevented renal fibrin deposition and death due to endotoxin infusion in rats. It is concluded that cLUK, despite the lack of the carbohydrate side chain, is functionally identical and pharmacologically equivalent to uLUK.

The amino-acid sequence of bovine glutathione peroxidase.

The amino-acid sequence of the seleno-enzyme glutathione peroxidase from bovine erythrocytes was completely determined. Fragmentation of the carboxymethylated protein comprised cleavages with trypsin, with endoproteinase Lys-C, and with cyanogen bromide in 70% formic acid. The resulting peptides were separated by reversed-phase high-performance chromatography or by gel filtration. For sequence determination automated solid or liquid phase techniques of Edman degradation were used. The proper alignment of fragments was experimentally proven in all but one instance. In this case, consistent indirect evidence was provided. The monomer of glutathione peroxidase was shown to consist of 198 amino acids representing a molecular mass ob about 21 900 Da. The active site selenocysteine was localized at position 45. In addition, four cysteine residues were found at positions 74, 91, 111, and 152. The N-terminal part of the sequence obtained revealed a pronounced homology with a partial sequence of the rat liver enzyme. Moreover, tentative sequence data predicted from X-ray crystallographic analysis of bovine glutathione peroxidase were found to agree in about 80% of the residues with the sequence presented. Differences between the predicted and the experimentally determined sequence are discussed.

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.

The primary structure of Cu-Zn superoxide dismutase from Photobacterium leiognathi: evidence for a separate evolution of Cu-Zn superoxide dismutase in bacteria.

The complete amino-acid sequence of the copper-zinc superoxide dismutase of the Photobacterium leiognathi was determined. The fragmentation strategy employed included cyanogen bromide cleavage at its methionine residues and the only tryptophan residue. The S-carboxymethylated chain was further cleaved by means of trypsin, in order to obtain overlapping fragments. For sequence determination automated solid or liquid-phase techniques of Edman degradation were used. C-Terminal amino acids of the entire chain were determined after treatment with carboxypeptidase A. Comparison of the primary structure of this bacterial Cu-Zn superoxide dismutase with the established amino-acid sequences of the other eukaryotic Cu-Zn superoxide dismutases revealed clear homologies. Correspondingly, the Cu-Zn-binding amino-acid residues of the active centre were localized: His45, His47, His70, His79, His125 and Asp91. The two cysteine residues in position 52 and 147 were homologous to the cysteine residues, modelling the essential intrachain disulfide bridge of the corresponding bovine enzyme. As only 25-30% of aligned sequence positions were found to be identical, the enzyme of P. leiognathi shows only a remote phylogenetic relationship towards eukaryotic Cu-Zn superoxide dismutases. When compared to the established phylogenetic tree of the cytochrome c family, this indicates a separate evolution of Cu-Zn superoxide dismutase in Photobacterium. Therefore, a natural gene transfer from the eukaryotic host (ponyfish) to the prokaryotic photobacterium, which Martin and Fridovich postulated 1981 (J. Biol. Chem. 256, 6080-6089) on the basis of amino-acid compositions, can be excluded.

The primary structure of high molecular mass urokinase from human urine. The complete amino acid sequence of the A chain.

The complete sequence of 157 amino acids of the light (A) chain of high molecular mass urokinase from human urine was determined. The fragmentation strategy included cyanogen bromide cleavage of the S-carboxymethylated A chain at the methionine and/or tryptophan residues and use of the specific endoproteinase Lys-C. For sequence determination automated solid- or liquid-phase techniques of Edman degradation were used. C-terminal amino acids of the A chain were determined by consecutive treatment with carboxypeptidase A and B. The amino acid sequence obtained revealed a significant homology to peptide chains of other serine proteinases. Accordingly, the sequence of the A chain can be divided into three domains: 1) The growth factor domain with homologies to murine epidermal growth factor and a particular sequence of bovine clotting factor X, 2) The "kringle" domain with homologies to "kringle" structures, e.g. in plasminogen, and 3) the connecting peptide domain containing the A1 chain of low molecular mass urokinase. Together with the amino acid sequence of the B chain, which was presented by us in an earlier communication, the sequence data presented complete the primary structure of high molecular mass urokinase from human urine.

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.

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.