Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The complete amino acid sequence of the mangano superoxide dismutase from Escherichia coli B has been deduced through characterization of peptides from cyanogen bromide, bromonitrophenylsulfenyl skatole, citraconylated tryptic, and succinylated tryptic digests of the intact polypeptide chain and through subfragmentation of selected peptides with chymotrypsin, thermolysin, trypsin, and Staphylococcus aureus V8 extracellular protease. No significant homology is detected on comparison with the sequence of the copper- and zinc-containing superoxide dismutase from bovine erythrocytes, indicating that the manganese-iron and the copper-zinc classes of dismutases arose from independent evolutionary ancestors, a proposal previously based solely on enzymological and NH2-terminal sequence data. The amino acid sequence listed below corresponds to a molecular weight of 22,900 and appears to be identical in each subunit polypeptide of the native enzyme dimer. formula: (see text).
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PMID:The amino acid sequence of mangano superoxide dismutase from Escherichia coli B. 36 8

Diferric ovotransferrin was hydrolyzed by thermolysin, a thermostable protease, at elevated temperatures. At 65 degrees C, the amino(N)-terminal lobe was completely digested into small peptides, while the carboxyl(C)-terminal lobe was significantly resistant to the protease. This permitted the isolation of an iron-bound C-terminal half-molecule consisting of a glycosylated single polypeptide in an excellent yield (about 90%). The fragment comprises the residues from 336 to the C-terminus of ovotransferrin. The results for the visible absorption spectrum of the copper-bound fragment, the stability of the iron-bound fragment in high concentration of urea, and the CD spectra of the fragment in the far and near UV regions indicated that it retains the metal binding activity and conformation of the C-terminal lobe of intact ovotransferrin.
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PMID:The carboxyl-terminal half-molecule of ovotransferrin prepared by selective digestion of the amino-terminal lobe with thermolysin. 136 35

The primary structure of p97 (melanotransferrin) has been compared with other members of the transferrin superfamily. A molecular structure of p97 has been modelled based on the crystal structure of diferric rabbit serum transferrin. The most significant amino acid substitutions in p97 are almost exclusively limited to only two regions; the C-lobe iron-binding cleft and the interlobe contact region. The latter includes within the N-terminal lobe a Zn-binding consensus sequence found in metallopeptidases, and in the C-terminal lobe a glutamic acid residue (Glu-394) capable of completing a potential thermolysin-like Zn-binding site. Thus, p97 may have a Zn-binding potential, unique amongst the transferrin superfamily.
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PMID:A molecular model for the tumour-associated antigen, p97, suggests a Zn-binding function. 163 59

We have investigated the effect in solution of synthetic carrier ampholytes on the saturation of human serum transferrin. By spectrophotometric titrations of human serum transferrin with various Fe3+-carrier ampholyte solutions, we demonstrated that under these conditions carrier ampholytes behave as typical chelators, their binding curves being very similar to that obtained with disodium nitrilotriacetate. On performing titration experiments at three different pH values, carrier ampholytes act like nitrilotriacetate at pH 7.5, but the former are more effective iron donors at pH 8.4 and worse iron donors at pH 5.2. Spectrophotometric titrations of isolated C-terminal and N-terminal fragments obtained from human serum transferrin by thermolysin cleavage show no differences between them, and no differences with respect to the whole protein except that they contain half the number of binding sites. In order to determine a site-specificity of iron in the presence of ampholytes, the classical urea/polyacrylamide-gel-electrophoresis technique was adopted. Under saturating conditions carrier ampholyte solutions act mostly on the C-terminal site, whereas desaturating agents remove iron preferentially from the N-terminal site. Our findings support the hypothesis that Ampholine may chelate Fe3+ as well as many other compounds.
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PMID:Effect of synthetic carrier ampholytes on saturation of human serum transferrin. 273 May 92

A single-sited iron-binding fragment of human transferrin has been obtained by thermolysin cleavage of the protein, selectively loaded with iron in the C-terminal binding site, in a urea-containing buffer. The fragment contains carbohydrate, and hence derives from the C-terminal half of transferrin. Its metal-binding site accepts Fe3+ and Cu2+ with bicarbonate as accompanying anion, but only Fe3+ with oxalate as anion. EPR spectroscopic properties of the fragment are similar to those of the corresponding site in the intact protein. However, iron-binding by the fragment is weaker than by the C-terminal site of the intact protein, particularly at low pH, suggesting that overall as well as local protein conformation influences the metal-binding functions of the site.
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PMID:Preparation and properties of a single-sited fragment from the C-terminal domain of human transferrin. 298 30

The aim of the present study was to determine if human N-terminal half-transferrin (N- fragment), prepared by thermolysin cleavage of diferric transferrin, would bind to the rat hepatocyte transferrin receptor and donate iron to the cell. Competition experiments between 125I-labelled N-fragment and diferric transferrin revealed no receptor binding of the half-transferrin. Still, the N-fragment delivered iron to the cells in amounts approximately 30-fold above what could be accounted for by uptake of the fragment itself. The rate of cellular iron uptake from the fragment was comparable to what is seen with the intact transferrin. The uptake of 125I-labelled N-fragment was not inhibited by excess non-radioactive diferric transferrin. By comparison, the uptake of 59Fe from the N-fragment was inhibited 70% by excess nonradioactive diferric transferrin. This suggests that iron derived from diferric transferrin competes with the iron derived from the N-fragment for a common transport pathway. Although some cellular degradation of the N-fragment occurred, the extent of degradation was too low to explain the amount of iron accumulated by the cells. The results show that the hepatocyte has an effective transferrin-receptor-independent mechanism for accumulation of iron from transferrin.
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PMID:Uptake of iron from N-terminal half-transferrin by isolated rat hepatocytes. Evidence of transferrin-receptor-independent iron uptake. 755 41

The present study was initiated to identify the region(s) of ovotransferrin involved in binding to the bacterial transferrin receptors from Haemophilus paragallinarum and Haemophilus avium. Ovotransferrin was digested with either trypsin or thermolysin to obtain its N-lobe and C-lobe fragments. The individual fragments were then purified by a combination of gel exclusion and ion-exchange chromatography. Solid phase binding experiments with the individual fragments demonstrated that the C-lobe fragments blocked the binding of horse radish peroxidase-conjugated ovotransferrin to the transferrin receptors and that much higher concentrations of the N-lobe fragment were required for any detectable blocking. Affinity isolation of the bacterial transferrin receptor from the two Haemophilus species revealed that both native ovotransferrin and its C-lobe fragment were capable of isolating two iron repressible outer membrane proteins. These 95 and 60 kDa outer membrane proteins correspond to Tbp1 and Tpb2, respectively. In contrast, the N-lobe fragment was capable of isolating Tbp2 of H. paragallinarum but not that of H. avium. The inability of the N-lobe and C-lobe fragments from ovotransferrin and human transferrin to support the growth of iron-limited cultures of H. paragallinarum and Neisseria meningitidis, respectively, suggested that interaction with both lobes is necessary for efficient iron acquisition.
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PMID:Transferrin binding protein two interacts with both the N-lobe and C-lobe of ovotransferrin. 872 96

The ability of the partial molecule of transferrin, truncated transferrin (t-Tf), to act as an excretable biologic iron chelator was examined. We confirmed the observations of Zak and Aisen (Zak O, Aisen P. Biochem Biophys Acta 1985;1952:24-8) that thermolysin treatment of human transferrin produces half molecules that retain iron-binding capacity. These molecules are poorly recognized by surface receptors on either human or murine cells. Although the plasma half-life of human transferrin in mice is moderately long (40 hours), injection of t-Tf into mice results in its rapid clearance (half-life = 10 minutes). Injection of iron 59-labeled transferrin results in the deposition of iron in the major hematopoetic organs of mice such as the spleen, bone marrow, and liver. Injection of 59Fe-labeled t-Tf results in the quantitative recovery of iron in the kidneys: 59Fe is retained in the kidney for substantial periods of time with little evidence of its excretion into urine. Injection of iodine 125-labeled t-Tf also results in the deposition of radioactivity in the kidneys, but 125I is rapidly excreted into the urine, where it is detected as free iodine. These results indicate that although t-Tf is directed to the kidney and filtered by the glomerulus, the molecule is reabsorbed and degraded, and iron is retained. These results have implications in the design of iron chelators.
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PMID:Alteration in the organ distribution of iron by truncated transferrin: implications for iron chelation therapy. 934 81

Nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the alpha subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu - Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128. The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from alphaIle107 to alphaTrp117. Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the alpha subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.
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PMID:Structure of the photoreactive iron center of the nitrile hydratase from Rhodococcus sp. N-771. Evidence of a novel post-translational modification in the cysteine ligand. 936 4

Microcin J25 (MccJ25) is a cyclic antibacterial peptide secreted by a fecal isolate of Escherichia coli. It exerts highly potent activity on Salmonella and Escherichia species. The microcin is recognized at the outer membrane of sensitive strains by the FhuA multifunctional protein, which belongs to the iron/siderophore receptor family, and inhibits bacterial transcription through binding to the RNA-polymerase beta' subunit. The mcjABCD genetic system carried by the wild type 50-kb pTUC100 plasmid contains four genes involved in MccJ25 production and immunity. MccJ25 results from the proteolytic cleavage of a 58-residue precursor at a specific Lys-Gly bond. The resulting mature peptide consists of 21 unmodified amino acids, mostly hydrophobic and includes a single dehydration. The initially described macrocyclic structure of MccJ25, which mostly relied on manual Edman sequencing of the thermolysin-cleaved form (t-MccJ25), involved a head-to-tail cyclisation of the 21-residue precursor. This structure did not prove to be consistent with recent IT-MS CID experiments conducted either on the native microcin or on peptides resulting from acidic or enzymatic cleavages, which are in favour of an 8-residue ring followed by a 13-residue tail. Cyclisation thus occurs between the N-terminus (Gly1) and the Glu8 side chain carboxyl group. The solution three-dimensional structure shows threading of the tail into the ring, thus forming a highly stable lasso type structure. Such a structure was described previously for enzyme inhibitors from Actinobacteria and is consistent with the ability of MccJ25 to inhibit RNA polymerase. The lasso structure is discussed in terms of phylogenetical and biotechnological perspectives.
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PMID:Microcin J25, from the macrocyclic to the lasso structure: implications for biosynthetic, evolutionary and biotechnological perspectives. 1554 33


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