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Query: UNIPROT:P06889 (Mol)
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To understand the nature of the ATP-induced structural change in myosin subfragment-1, rabbit and chicken skeletal subfragments-1s were cleaved by various proteolytic enzymes in the absence, and in the presence, of ATP and the exact locations of the cleavage sites that were affected by ATP were determined from the amino end analysis of fragments by the use of a protein sequencer. It was found that subtilisin cleaved a site between Gln27 and Asn28 of rabbit subfragment-1 and between Gln28 and Asn29 of chicken subfragment-1 only in the presence of ATP. Thermolysin cleaved a site between Pro31 and Phe32 of chicken subfragment-1 in the presence of ATP, but the same site of rabbit subfragment-1 was not cleaved. The location of these sites is quite similar to the ATP-induced chymotryptic cleavage site of chicken gizzard heavy meromyosin, between Trp29 and Ser30 as reported by others. It is suggested, therefore, that the structure and the ATP-induced structural change in the regions are similar in these subfragment-1s. ATP also changes the cleavage rate of the 26K-50K junction by many proteases. Exact cleavage sites were determined and the relationship between their location and the suppression or the enhancement by ATP of the cleavage was studied. It was found that the cleavage sites were restricted to a quite narrow region and only the cleavage by thermolysin that attacked the middle of the region was enhanced by ATP. The distribution of the cleavage sites and the effect of ATP suggest that ATP induces drastic structural change at the middle of the 26K-50K junction region. The region attacked easily by many proteases coincided very well with a hydrophilic region indicated by the hydropathy index. The region probably protrudes outside and is, therefore, easily attacked by many proteases.
J Mol Biol 1989 Oct 20
PMID:ATP-induced structural change in myosin subfragment-1 revealed by the location of protease cleavage sites on the primary structure. 258 5

The binding energy and the geometrical arrangements of the complexes formed by the zinc dication with OH-, one, four, five or six water molecules, SH-, H2S, formic acid, the formate anion, imidazole, its anion and formamide are calculated using the MNDO method. The comparison of the results obtained with those of ab initio computations on the same complexes induced us to propose for Zn++ a set of parameters different from the one determined by Dewar for the neutral metal atom. Using the two MNDO parametrizations, similar calculations are carried out for Zn++ interacting with two molecules of 2-aminoethanethiol and with models of the four ligands which are present at the thermolysin active site, in order to evaluate the possibilities and limitations of this semiempirical method for theoretical studies concerning zinc metalloenzymes. In the last case, the results obtained suggest that, in the crystal state, the water molecule could be deprotonated. This finding is discussed in relation with the mechanism of action of the enzyme which has been proposed.
J Comput Aided Mol Des 1989 Mar
PMID:A theoretical study of Zn++ interacting with models of ligands present at the thermolysin active site. 271 93

Sedimentation analysis in the analytical ultracentrifuge has been used to characterize the size and shape of thermolysin and a number of its fragments obtained by chemical or enzymatic cleavage of the protein. Four fragments (121-316, 206-316, 225/226-316 and 255-316) originate from the C-terminal domain, and two (1-155 and 1-205) from the N-terminal domain of the intact molecule. In aqueous solution at neutral pH the hydrodynamic properties of the C-terminal fragments, except 255-316, are consistent with compact homogeneous monomers. Fragment 255-316 is a monomeric species below 0.08 mg/ml concentration and forms a dimer above this concentration. Dimerization does not lead to changes in fragment conformation, as determined by far-ultraviolet circular dichroic measurements, but to an increase of 5.6 degrees C (to 68.2 degrees C at 1.0 mg/ml) in the temperature for thermal unfolding and a corresponding increase of 4.6 kJ/mol in the free energy of unfolding. Fragments derived from the N-terminal domain show a strong tendency to form high-molecular-mass aggregates. Previous experiments utilizing circular dichroic measurements and antibody binding data suggested that the C-terminal fragments listed above are able to refold in aqueous solution at neutral pH into a stable conformation of native-like characteristics [Dalzoppo, D., Vita, C. & Fontana, A. (1985) J. Mol. Biol. 182, 331-340] (and references cited therein). Present data establish that all these C-terminal fragments are globular monomeric species in solution (at concentrations approximately 0.1 mg/ml) and thus represent 'isolated' domains (or subdomains) with intrinsic conformational stability typical of small globular proteins.
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PMID:Folding of thermolysin fragments. Hydrodynamic properties of isolated domains and subdomains. 277 48

Limited proteolysis of flagellin from Salmonella typhimurium SJW1103 by subtilisin, trypsin and thermolysin results in homologous degradation patterns. The terminal regions of flagellin are very sensitive to proteolysis. These parts are degraded into small oligopeptides at the very early stage of a mild digestion that yields a relatively stable fragment with a molecular weight of 40,000. Further proteolytic degradation results in a stable 27,000 Mr fragment. The 40,000 Mr tryptic fragment has been identified as residues 67 to 446 of the flagellin sequence, while the 27,000 Mr fragment involves the 179 to 418 segment. The NH2-terminal sequence positions for the corresponding fragments produced by subtilisin are 60 and 174 for the 40,000 Mr and 27,000 Mr fragments, respectively. The fragments lost their polymerizing ability. Structural properties of flagellin and its 40,000 Mr tryptic fragment were compared by circular dichroism spectroscopy and differential scanning calorimetry. Analysis of the calorimetric melting profiles suggests that terminal parts of flagellin have no significant internal stability and they are in extensive contact with water. However, these regions contain some secondary structure, probably alpha-helices, as revealed by comparison of the circular dichroic spectra in the far-ultraviolet region. Our results indicate that, although the terminal regions of flagellin may contain some alpha-helical secondary structure of marginal stability, they have no compact ordered tertiary structure in solution. On the contrary, the central region of the molecule involves at least two compact structural units.
J Mol Biol 1989 Sep 05
PMID:Terminal regions of flagellin are disordered in solution. 281 Mar 65

The functional and biochemical characteristics of somatostatin (somatotropin release-inhibiting factor) (SRIF) receptor subtypes were examined in the clonal pituitary cell lines AtT-20 and GH3. SRIF inhibits evoked calcium influx into each of these cell lines. The rank order of potencies of structural analogues of SRIF to inhibit calcium influx into GH3 versus AtT-20 cells was different. Inhibitory actions of SRIF on calcium influx desensitized in AtT-20 cells but not GH3 cells. The biochemical properties of the SRIF receptor subtypes in AtT-20 and GH3 cells were assessed by photoaffinity labeling of each receptor with the nonreducible SRIF analogue [125I]CGP 23996 and the photocrosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate. The covalently labeled receptors in both cell lines had the same size, 55 +/- 5 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The covalent binding of [125I]CGP-23996 to GH3 and AtT-20 cell membranes was blocked by 1 microM SRIF, somatostatin 28, Trp8-SRIF and was GTP sensitive. Analysis of the labeled receptors in GH3 and AtT-20 cell membranes by two-dimensional polyacrylamide gel electrophoresis indicated that they were of similar charge (pI = 6-6.5) and that they comigrate when applied together. Proteolysis of the GH3 and AtT-20 cell SRIF receptors with Staphylococcus aureus V-8 and thermolysin revealed similar peptide maps. Pretreatment of AtT-20 cells with different stable SRIF analogues abolished the subsequent equilibrium or covalent labeling of the SRIF receptor with [125I]CGP-23996. Similar treatment of GH3 cells did not reduce the covalent labeling of the SRIF receptor by [125I]CGP 23996. These studies indicate that the functional characteristics of SRIF receptors in GH3 and AtT-20 cells are different. However, clear differences in the biochemical properties of these receptor subtypes were not observed. Subtle variations in the structure of the SRIF receptors may therefore be responsible for the functional differences.
Mol Pharmacol 1988 Apr
PMID:Somatostatin receptor subtypes in the clonal anterior pituitary cell lines AtT-20 and GH3. 289 89

Angiotensin-converting enzyme inhibitors promise to make important therapeutic contributions to the control of hypertension and congestive heart failure. The nonapeptide teprotide was the first of these inhibitors to be tested clinically. It was followed by orally active inhibitors, captopril in 1977 and enalapril in 1980. The latter is representative of a new design for the inhibition of metallopeptidases and is the subject of this review. The best of the N-carboxyalkyldipeptide inhibitors inhibits angiotensin-converting enzyme with a Ki of 7.6 X 10(-11) M. This compound is the most potent competitive inhibitor of a metallopeptidase yet to have been reported. The basis of this high potency is beginning to be understood and in part is considered to involve precisely arranged multiple interactions within the enzyme active site. X-ray crystallography of a thermolysin-inhibitor complex has been achieved. Assuming that similar interactions within the active site of angiotensin-converting enzyme are mechanistically probable, the authors hypothesize the binding of enalaprilat to converting enzyme as shown in Figure 24. Such interactions are consistent with kinetic studies (Section V) with the understanding that binding to the enzyme is not sensitive to the inhibitor's state of NH protonation. The reason for this surprising conclusion has not been established. Perhaps counterbalancing factors are involved in the energetics of binding or there may be compensating adjustments made in the enzyme which permit NH protonated and nonprotonated inhibitor to bind equally well. Figure 24 also summarizes present understanding of the conformation of enalaprilat when bound to angiotensin-converting enzyme. From studies on conformationally defined analogs of enalaprilat, it seems likely that the Ala-Pro segment of enalaprilat binds in a conformation that is close to a minimum energy conformer. This situation no doubt contributes to the potency of enalaprilat, since little binding energy would be needed to induce conformational changes in this part-structure of enalaprilat when it is bound to the enzyme. The phenethyl group of enalaprilat is believed to be near the alpha-hydrogen of the L-Ala residue in the enzyme-inhibitor complex. However, the synthesis of conformationally restricted analogs to establish this point has not yet been reached. The N-carboxyalkylpeptide design was developed from Wolfenden's collected product inhibitors of carboxypeptidase-A. Whether or not N-carboxyalkyldipeptides should be classified as collected product or transition state inhibitors is unclear.(ABSTRACT TRUNCATED AT 400 WORDS)
Adv Enzymol Relat Areas Mol Biol 1985
PMID:The design and properties of N-carboxyalkyldipeptide inhibitors of angiotensin-converting enzyme. 299 4

Neutral protease from Bacillus cereus exhibits a 73% amino acid sequence homology to thermolysin, for which an accurate crystal structure exists. The B. cereus enzyme is, however, markedly less thermostable. The neutral protease was crystallized and diffraction data to 3.0 A resolution were recorded by oscillation photography. The crystal structure was solved by molecular replacement methods using thermolysin as a trial molecule. The solution was improved by rigid-body refinement and model rebuilding into electron density omit-maps. The atomic co-ordinates were refined to R = 21.7% at 3.0 A resolution. Comparison of the resultant model with the thermolysin structure shows that the two enzymes are very similar with a root-mean-square deviation between equivalent C alpha-atoms of 0.88 A. The gamma-turn found in thermolysin is transformed into a beta-turn in the neutral protease by the insertion of a glycine residue. There appear to be no contributions to the enhanced thermostability of thermolysin from additional salt bridges, whereas contributions in the form of extra hydrogen bonding interactions could be important. Other factors that may affect thermostability include the two glycine to alanine exchanges and perturbations in the environment of the double calcium site.
J Mol Biol 1988 Feb 05
PMID:Crystal structure of neutral protease from Bacillus cereus refined at 3.0 A resolution and comparison with the homologous but more thermostable enzyme thermolysin. 312 92

The complete amino acid sequence of copper-zinc superoxide dismutase from Neurospora crassa is reported. The subunit consists of 153 amino acids and has a Mr of 15,850. The primary structure was determined by automated and manual sequence analysis of peptides obtained by digestions of the carboxymethylated and aminoethylated enzyme with trypsin and thermolysin. The protein is devoid of tryptophan and methionine and displays a free amino terminus. Comparison of the amino acid sequence with those from human erythrocyte, bovine erythrocyte, horse liver, swordfish liver, and yeast copper-zinc superoxide dismutases reveals a high degree of sequence homology among the six enzymes. Most prominently, the regions containing the amino acid residues participating in the metal-binding and the half-cystine residues forming the intramolecular disulfide bridge are highly conserved. The invariant amino acids Pro 74 and Asp 76 of the four vertebrate and yeast superoxide dismutases were found to be substituted by arginine and alanine, respectively, in the Neurospora enzyme. These radical substitutions occurring in the zinc ligand region, known to form a characteristic loop structure in bovine erythrocyte copper-zinc superoxide dismutase (Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217), however, do not affect the catalytic properties of the Neurospora enzyme.
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PMID:Primary structure of copper-zinc superoxide dismutase from Neurospora crassa. 316 Jun 99

Flavocytochrome b2 (L-lactate dehydrogenase) from baker's yeast is composed of two structural and functional domains. Its first 100 residues constitute the heme-binding core, which is homologous to cytochrome b5 [B. Guiard, O. Groudinsky & F. Lederer (1974) Proc. Natl Acad. Sci. USA 71, 2539-2543]. We report here the amino acid sequence of the heme-binding domain isolated by tryptic proteolysis of Hansenula anomala flavocytochrome b2. The sequence was established by automated degradation of the whole fragment and of peptides obtained by CNBr cleavage at the unique tryptophan and by proteolysis with thermolysin and endoproteinase Lys C. As isolated, the domain consists of 84 residues without any sulfur amino acids. It shows 49 identities with the heme-binding domain from Saccharomyces cerevisiae and 28 with beef microsomal cytochrome b5. Using the recently published three-dimensional structure of S. cerevisiae flavocytochrome b2 [Z-x. Xia, N. Shamala, P. H. Bethge, L. W. Lim, H. D. Bellamy, N. H. Xuong, F. Lederer and F. S. Mathews (1987) Proc. Natl Acad. Sci. USA 84, 2629-2633], it can be seen that there are only positively charged side chains close to the accessible heme edge, the only negative charges in that area being those of the heme propionates. The implications of this result are discussed in the light of Salemme's model for the cytochrome b5/cytochrome c complex [F. R. Salemme (1976) J. Mol. Biol. 102, 563-568].
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PMID:Amino-acid sequence of the cytochrome-b5-like heme-binding domain from Hansenula anomala flavocytochrome b2. 331 13

Three monoclonal antibodies (mAbs), termed SN2, SN2a and SN2b, were used in the present work to study a human T-cell leukemia-associated cell surface glycoprotein, GP37. Strong specificity of mAbs SN2, SN2a and SN2b for T leukemia cells was demonstrated by radioimmunoassay and fluorescence-activated cell sorter (FACS) analysis. GP37 was not detected on normal human peripheral blood lymphocytes, purified normal T-cells, normal thymocytes nor normal bone marrow cells. Furthermore, GP37 was barely detectable on phytohemagglutinin (PHA)- and Concanavalin A (Con A)-activated T-cells. The results indicate clinical utility of these mAbs. Competitive binding experiments show that the epitopes recognized by SN2 and SN2a are sufficiently close to each other to allow complete reciprocal inhibition of binding whereas the epitopes recognized by SN2 and SN2b are less close to allow only partial reciprocal binding inhibition. The biochemical nature of antigenic determinants defined by these mAbs was studied by treating T leukemia cells with trypsin, chymotrypsin, thermolysin, neuraminidase and mixed glycosidases. The results suggest that the antigenic determinants defined by these mAbs all consist of the protein moiety of the glycoprotein GP37. No significant antigenic modulation was observed when T leukemia cells were reacted with SN2. In a sequential immunoprecipitation experiment, a 125I-labeled leukemia antigen preparation was first treated with a rabbit anti-T leukemia antiserum. The latter had been prepared by immunizing a rabbit with a partially purified human T leukemia antigen preparation and showed a good specificity for T leukemia cells. Subsequent treatment of the labeled antigen preparation with SN2 showed that SN2 antigen had been precleared. Thus, both mouse mAb SN2 and the rabbit anti-T leukemia antiserum react with the same GP37 molecule.
Mol Immunol 1986 Jun
PMID:Human T-cell leukemia-associated cell surface glycoprotein GP37: studies with three monoclonal antibodies and a rabbit antiserum. 348 64


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