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)

Alpha-Lactalbumin isolated from human milk was reacted with tetranitromethane in molar excess of 8-32 mol/mol of tyrosine. After gel filtration on Sephadex G-75, followed by chromatographic fractionation using DEAE-Sephadex A-25, three main components were separated, which differed from one another in the extent of nitration. These protein fractions were found to contain, respectively, one and two nitrotyrosine residues, or two nitrotyrosine residues together with one nitrotryptophan. The lactose synthase specifier activity of each of these components was measured and compared with that of unsubstituted alpha-lactalbumin. Comparison of kinetic parameters showed the chemically modified proteins to be only slightly less active when tyrosines were the sole residues modified. In sharp contrast the additional nitration of a single tryptophan residue totally abolished the specifying activity of alpha-lactalbumin. Circular dichroism spectra of the tryptophan derivative revealed some structural alteration when compared with the other two and with the native protein. The conclusion could also be confirmed by using a double-immunodiffusion technique. After hydrolysis of the derivatives with thermolysin, it was possible to localize the substituted residues in the known sequence of human alpha-lactalbumin. Tyrosine-103 was found to be more easily nitrated than tyrosine-18. These two residues seem, therefore, to be on the outer surface of the molecule and more exposed than tyrosine-36 and tyrosine-50. Some precautions are indicated in the use of tetranitromethane as a nitrating agent on the basis of complex products observed in the nitration of the free amino acids tyrosine and tryptophan and their derivatives.
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PMID:Nitration of tyrosyl residues in human alpha-lactalbumin. Effect on lactose synthase specifier activity. 81

The effect of calcium ion on the thermal stability of thermolysin has been investigated. The native protein undergoes an irreversible structural change and autolysis at high temperature. Analysis of the calcium ion dependence of the apparent melting temperature observed spectroscopically gives an apparent deltaH of -x (130 kcal/mol) where x is either 1 or 2. Neither zinc ion, where bound at the active site, nor terbium ion, which binds very tightly to the double calcium binding site, shows a stabilizing effect. These sites are therefore presumably not coupled to the transition which leads to autolysis. Removal of calcium ion from the native enzyme at temperatures below 50 degrees C results in inactivation but not major autolysis. The addition of 1 equiv of terbium before calcium removal results in a protein species which is 40% active and is no longer subject to thermal stabilization by calcium. These results suggest a pathway for the thermal inactivation of the enzyme which involves an irreversible structural change at one or both of the single calcium ion sites. This change propagates to the active site and results in inactivation. The binding of calcium ion to either or both single sites completely inhibits this structural change. The structural change is apparently cooperative and may correspond to a localized denaturation of the native structure.
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PMID:Role of Calcium in the thermal stability of thermolysin. 81 20

The stabilizing effect of calcium ions on thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease has been investigated. Calcium and zinc ions were removed from the proteases by gel filtration over Sephadex G-25 equilibrated with metal chelating agents. Using these enzymes with different metal content, heat inactivation kinetics were studied at various temperatures. Removal of calcium ions caused a sharp decrease in thermostability and diminished the values of the activation enthalpy (deltaH*) and entropy (deltaS*) for heat inactivation. There was little difference in stability between thermolysin containing 0.3 g-atom/mol and B. subtilis neutral protease containing 1.4 g-atoms/mol. Calcium binding isotherms of the proteases were obtained by equilibrium gel chromatography with various concentrations of free calcium ions. Thermolysin had four independent calcium binding sites with an identical intrinsic binding constant (K) of 2.0 X 10(4) M-1. B. subtilis neutral protease had four independent sites. The K value for three sites was 1.1 X 10(5) M-1 and the binding constant for the other site was 1.5 X 10(3) M-1. There was little difference in total free energy change for calcium binding between these proteases. From these results it is concluded that the stabilizing effect of calcium on these enzymes is almost equal, and the extra thermal stability of thermolysin is likely to come from its polypeptide chain structure.
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PMID:Role of calcium ions in the thermostability of thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease. 81 62

Thermolysin and neutral protease A are neutral metalloendopeptidases having similar specificity, molecular weight, metal content, and amino acid composition. Thermolysin, derived from the thermophilic organism Bacillus thermoproteolyticus, is heat inactivated at about 84 degrees whereas neutral protease A, derived from the mesophilic organism Bacillus subtilis, is inactivated at about 59 degrees. Structural analyses reveal that the two enzymes are homologous. Of the 326 residues of neutral protease A, 171 have been placed in sequence and 49% of these have been found in identical loci in thermolysin. These include many of the residues corresponding to the active site of thermolysin. The sensitivity of both enzymes to thermal inactivation is dependent upon the presence of calcium and neutral protease appears to bind less calcium than thermolysin. Structural data indicate that many of the ligands associated with calcium sites 1 and 2 (double site of thermolysin) are present in neutral protease and that calcium site 4 cannot exist in neutral protease. The structural homology and functional analogy of these two proteins support the concept that they have similar conformations. The known structure of thermolysin is used as a model to discuss structural differences which might be related to thermal stability.
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PMID:Thermal stability of homologous neutral metalloendopeptidases in thermophilic and mesophilic bacteria: structural considerations. 82 May 64

A series N-hydroxysuccinimide esters of acylamino acids previously shown to acylate and thereby increase the activity of thermolysin by several orders of magnitude (Blumberg, S., and Vallee, B. L. (1975), Biochemistry 14, 2410) has been used to modify the related neutral proteases from Bacillus subtilis, Bacillus megaterium, and Aeromonas proteolytica. Each of these enzymes is activated to a level characteristic of the particular protein and the particular acyl group incorpporated when monitored with the substrate furylacryloyl-Gly-Leu-NH2. Thus, for the modification of B. megaterium, B. subtilis, and A. proteolytica proteases with Ac-Trp-ONSu, kcat/Km increases 11-, 2.5-, and 18-fold whereas those of the Ac-Phe(4-DnpNH)-ONSu modified enzymes before and after deacylation with hydroxylamine indicate that from 1 to 2 residues are modified. The rate of removal of the Ac-Phe(4-DnpNH) label by 0.1 M hydroxylamine correlates directly with that of the return of native enzymatic activity, at a rate comparable with the rate of deacylation of O-acyltyrosine models. The competitive inhibitors Zn2+ and beta-phenyl-propionyl-Phe do not prevent activation indicating that modification occurs at a site(s) distinct from that at which inhibitors bind. The degree of activation depends also on the substrate employed, generally being greater for substrates which the native enzymes hydrolyze slowly. These data are interpreted to indicate the modification of a residue near the active site, but which serves as a subsite for substrate interaction.
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PMID:Superactivation of neutral proteases: acylation with N-hydroxysuccinimide esters. 82 65

The complete amino acid sequence of phosphlipase A2 (EC 3.1.1.4) from horse pancreas was determined. The protein controls of a single polypeptide chain of 125 amino acids and has a molecular weight of 13,927. The chain is crosslinked by seven disulfide bridges. The sequence was determined by automated Edman degradation of the intact protein and several of the large peptide fragments. Smaller peptides were analyzed by manual Edman degradation. Fragmentation of the peptide chain was accomplished by enzymatic digestion with trypsin, chymotrypsin, and thermolysin. The final overlap was found by digestion of the polypeptide with a staphylococcal protease specific for glutamoyl bonds. Phospholipase A2 from horse pancreas shows homology to snake venom phospholipases A2 and to the enzyme from porcine pancreas, provided that the published amino acid sequence of the porcine phospholipase A2 is revised to some extent.
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PMID:Amino acid sequence of phospholipase A2 from horse pancreas. 83 12

1. When thermolysin was treated with a 100-fold molar excess of 2,4,6-trinitrobenzene-1-sulfonate at pH 8.0 and 37 degrees for 7 h, all 12 amino groups in the enzyme were almost completely trinitrophenylated. The fully trinitrophenylated enzyme still retained more than 80% of its original activity. The amino groups are therefore not essential for activity. 2. When treated with a 100- to 1,000-fold molar excess of N-acetylimidazole at pH 6.5 and 23 degrees for 2 h, thermolysin lost about 54% of its activity with concomitant acetylation of 21 tyrosine residues out of the total of 28 residues. The reaction did not easily proceed any further. This partially inactivated enzyme regained almost full activity upon treatment with hydroxylamine. These modified tyrosine residues are therefore not directly involved in the active site. 3. Thermolysin was not inactivated by treatment with about 100- to 150-fold molar excess of 2-hydroxy-5-nitrobenzyl bromide or dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide at pH 6.0 and room temperature for 1 h both in the presence and absence of 8 M urea. Thus the three tryptophan residues in the enzyme are not accessible to these reagents. When treated with a 4.3- to 43-fold molar excess of N-bromosuccinimide over tryptophan, the enzyme was inactivated to varying extents, depending on the reaction conditions used. In this case, the tyrosine residues appeared to be the most rapidly modified, but tryptophan and histidine residues were also modified with extensive inactivation at higher concentrations of the reagent. The presence of 8 M urea retarded the inactivation.
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PMID:Studies on thermolysin. I. Effects of chemical modifications on the activity of thermolysin. 84 38

Polypeptide 3, the major membrane-penetrating protein of the human erythrocyte membrane, was characterized, together with two major fragments derived by specific proteolysis of the native protein in the membrane. One fragment (fragment 3f) was obtained from thermolysin cleavage in the extracellular region of the protein, and the other (fragment T1) was derived from tryptic cleavage in the intracellular region of the protein. The results of N- and C-terminal group analysis suggest that fragment 3f contains the N-terminal region of polypeptide 3 and fragment T1 contains the C-terminal part of the molecule. The carbohydrate contents of the polypeptides suggest that carbohydrates are present in three regions of the molecule, much of this carbohydrate being present in the C-terminal part of the molecule. This region of the protein also contains the receptors for concanavalin and the lectins from Phaseolus vulgaris and Ricinis communis, and our results suggest that there is heterogeneity in the carbohydrate chains present in the C-terminal region of polypeptide 3. These data are related to the folding of polypeptide 3 in the erythrocyte membrane.
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PMID:The structure of the major protein of the human erythrocyte membrane. Characterization of the intact protein and major fragments. 85 16

The alfalfa mosaic virus protein was submitted to the action of cyanogen bromide. Four peptides were isolated. Study of these peptides allowed us to determine the order. Then protein was submitted, after S-carboxymethylation or S-aminoethylation, to the action of different proteolytic enzymes: trypsin, chymotrypsin, thermolysin and papain. The peptides issued from these different hydrolysis were separated on Dowex 50 X4 and Dowex 1 X2, and their amino acid composition was determined. The use of classical methods of sequence determination, of mass spectrometry and for one case the use of a sequencer, lead to the obtention of the primary structure of all the tryptic peptides. The studies of chymotryptic, thermolytic and papainic hydrolysates, and of cyanogen bromide rupture, allowed us to isolate the overlapping peptides which were necessary for the reconstitution of the complete proteic chain.
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PMID:[Determination of the primary structure of alfalfa mosaic virus (strain S) coat protein. II. Complete sequence of the protein (author's transl)]. 88 29

By combinations of selective chemical cleavage (cyanogen bromide), selective enzymatic cleavage (trypsin, thermolysin), and random cleavage (partial acid hydrolysis) a series of disulfide-containing peptides have been isolated from ovine lutropin beta subunit. These peptides suggest six disulfide linkages between half-cystine residues in positions 23-72, 26-110, 93-100, 34-88, 9-90, and 38-57. The latter pair was placed by elimination of other possibilities. The first three pairs are in agreement with a report by Chung, D., Sairam, M. R. and Li, C. H. (1975) Int. J. Peptide Protein Res. 7, 487-493; the pair 93-100 has also been detected by Reeve, J. R., Cheng, K. W. and Pierce, J. G. (1975) Biochem. Biophys. Res. Commun. 67, 149-155, using partial reduction and alkylation. In an attempt to improve the efficiency of enzymatic attack, a preliminary partial reduction as per Reeve et al. [16] was done. In this instance a peptide suggesting an additional disulfide linkage between half-cystines 23-26 was obtained as well as peptides consistent with the 23-72 and 26-110 placements. This was interpreted as an artifactual opening and recombining during partial reduction-reoxidation to produce the 23-26 linkage. The placement of three disulfide bonds (34-88, 9-90, and 38-57) is in disagreement with the pairings Chung et al. [15] suggest for these six half-cystine residues. Six reasons for uncertainty in the placement of disulfide bonds are discussed. It is concluded the definitive placement of the disputed three disulfide bonds in ovine lutropin beta subunit remains an open question.
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PMID:Studies of disulfide bond location in ovine lutropin beta subunit. 88 34


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