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

Enolase in the presence of Mg2+ catalyzes the elimination of H2O from 2-phosphoglyceric acid (PGA) to form phosphoenolpyruvate (PEP) and the reverse reaction, the hydration of PEP to PGA. The structure of the ternary complex yeast enolase-Mg2(+)-PGA/PEP has been determined by X-ray diffraction and refined by crystallographic restrained least-squares to an R = 16.9% for those data with I/sigma (I) greater than or equal to 2 to 2.2-A resolution with a good geometry of the model. The structure indicates the substrate molecule in the active site has its hydroxyl group coordinated to the Mg2+ ion. The carboxylic group interacts with the side chains of His373 and Lys396. The phosphate group is H-bonded to the guanidinium group of Arg374. A water molecule H-bonded to the carboxylic groups of Glu168 and Glu211 is located at a 2.6-A distance from carbon-2 of the substrate in the direction of its proton. We propose that this cluster functions as the base abstracting the proton in the catalytic process. The proton is probably transferred, first to the water molecule, then to Glu168, and further to the substrate hydroxyl to form a water molecule. Some analogy is apparent between the initial stages of the enolase reverse reaction, the hydration of PEP, and the proteolytic mechanism of the metallohydrolases carboxypeptidase A and thermolysin. The substrate/product binding is accompanied by large movements of loops Ser36-His43 and Ser158-Gly162. The role of these conformational changes is not clear at this time.
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PMID:Mechanism of enolase: the crystal structure of enolase-Mg2(+)-2-phosphoglycerate/phosphoenolpyruvate complex at 2.2-A resolution. 200 20

A rat hepatocyte cell line was cultured in Higuchi's medium with fetal calf serum and insulin and labeled with 35SO2/4-. The cells were treated with a number of ligands to displace the heparan 35SO4 proteoglycan (HSPG) from the pericellular matrix. Maximum release was obtained with D-mannose-6-PO4 (50 mM), D-glucose-6-PO4 (50 mM), myo-inositol-2-PO4 (2-5 mM), myo-inositol hexaphosphate (2-5 mM), and DL-myo-inositol-1-PO4 (1-2 mM). D-myo-Inositol-1,3,4-(PO4)3 (1 mM) and L-myo-inositol-1-PO4 (2 mM) were intermediate in their ability to release the cell surface HSPG, whereas heparin (2 mg/ml), yeast phosphomannan (4 mg/ml), D-xylose-1-PO4 (50 mM), D-glucose-6-SO4 (50 mM), and myo-inositol hexasulfate (5 mM) were ineffective. When 35SO2/4- was added to cell cultures, the total cell surface HSPG increased linearly, but the percentage of the total cell surface [35SO4]HSPG that was released by myo-inositol-PO4 increased with time during the labeling period, reaching a maximum of 65% after 5 h. When cells were labeled for 12 h without insulin in the medium, the maximum amount of cell surface HSPG that was released by myo-inositol-PO4 was reduced to 30%. However, when cells labeled in the absence of insulin were treated with phosphatidylinositol-specific phospholipase C and then myo-inositol-PO4, the release of the cell surface [35SO4]HSPG was increased to 73%. When the [35SO4]HSPG that was released from the cell surface by treatment with myo-inositol-PO4 was added to cultures of unlabeled hepatocytes, it was taken up very rapidly and a portion of the internalized HSPG was converted to free heparan SO4 chains which appeared in the nucleus. Uptake was Ca2+- and Mg2+-independent. The amount of [35SO4]HSPG taken up was markedly reduced when the myo-inositol-PO4-releasable [35SO4]HSPG was pretreated with trypsin, thermolysin, alkaline borohydride, or alkaline phosphatase. When the cells were grown in inositol-deficient medium or in the presence of myo-inositol-PO4, the amount of heparan SO4 found in the nucleus was markedly reduced, and the cells no longer exhibited contact inhibition. These effects of myo-inositol deficiency on the growth and nuclear heparan SO4 were accentuated by addition of LiCl to the cultures to prevent phosphatidylinositol synthesis from the endogenous myo-inositol-PO4.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Involvement of phosphatidylinositol and insulin in the coordinate regulation of proteoheparan sulfate metabolism and hepatocyte growth. 295 71

We have studied the binding of fibronectin and its thermolysin fragments to DNA and heparin. Elution of polypeptides bound to DNA-cellulose and heparin-Sepharose affinity chromatography columns was performed by NaCl linear gradients in buffers at different pH and in the presence and absence of calcium ions. The NaCl concentration required to elute fibronectin from both types of column increased as the pH decreased. Fibronectin was not retained on DNA-cellulose or heparin-Sepharose affinity chromatography columns using a buffer containing physiological concentrations of Ca2+, Mg2+ and NaCl, at pH 7.4. On the other hand at pH 6.4 in conditions of physiological ionic strength, fibronectin was retained by both columns, eluting from the DNA-cellulose at 280 mM NaCl and from the heparin-Sepharose column at 210 mM. Furthermore, we have studied the interaction of thermolysin-digested fibronectin both with DNA-cellulose and heparin-Sepharose using the above procedure. The results demonstrate that there are four distinct domains, which interact both with DNA and heparin. We also report here the modulation by pH and Ca2+ ions of the interaction with DNA and heparin of these different domains.
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PMID:DNA-binding domains of human plasma fibronectin. pH and calcium ion modulation of fibronectin binding to DNA and heparin. 394 64

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.
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PMID:The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro. 645 76

The platelet protein thrombospondin (TSP) which is secreted from alpha-granules upon platelet activation agglutinates trypsinized, glutaraldehyde-fixed human erythrocytes. Optimal conditions for the hemagglutinating activity require that both Ca2+ and Mg2+ be present in final concentrations of 2 mM. In the presence of dithiothreitol (i.e., reduction of disulfide bonds), the lectin-like activity decreases in a manner proportional to the extent of reduction of the molecule from its native trimeric configuration into its Mr 180 000 subunits. Proteolysis of purified TSP with thermolysin, which produces discrete domains with the capacity to bind fibrinogen and heparin, also diminishes, but does not abolish, the hemagglutinating activity. Fibrinogen was without effect on hemagglutinating activity while heparin was found to be a potent inhibitor. Other proteoglycans such as hyaluronic acid, chondroitin sulfate, keratan sulfate, dermatan sulfate, and heparan sulfate had no effect. That portion of the TSP molecule apparently responsible for the hemagglutinating activity was identified by incubating a thermolytic digest of TSP with red blood cells and then determining which fragment was bound to the cell surface. The binding site resides within a peptide fragment of 140 000 daltons but is absent from an Mr 120 000 fragment derived from the Mr 140 000 fragment. Under the conditions for optimal expression of hemagglutinating activity (i.e., 2 mM MgCl2 and 2 mM CaCl2), this Mr 140 000 fragment was also shown to have heparin binding activity.
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PMID:Localization of the hemagglutinating activity of platelet thrombospondin to a 140 000-dalton thermolytic fragment. 650 39

In the previous paper (Block, M. A., Dorne, A.-J., Joyard, J., and Douce, R. (1983) J. Biol. Chem. 258, 13273-13280), we have described a method for the separation of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. The two envelope membranes have a different weight ratio of acyl lipid to protein (2.5-3 for the outer envelope membrane and 0.8-1 for the inner envelope membrane). The two membranes also differ in their polar lipid composition. However, in order to prevent the functioning of the galactolipid:galactolipid galactosyltransferase during the course of envelope membrane separation, we have analyzed the polar lipid composition of each envelope membrane after thermolysin treatment of the intact chloroplasts. The outer envelope membrane is characterized by the presence of high amounts of phosphatidylcholine and digalactosyldiacylglycerol whereas the inner envelope membrane has a polar lipid composition almost identical with that of the thykaloids. No phosphatidylethanolamine or cardiolipin could be detected in either envelope membranes, thus demonstrating that the envelope membranes, and especially the outer membrane, do not resemble extrachloroplastic membranes. No striking differences were found in the fatty acid composition of the polar lipids from either the outer or the inner envelope membrane. The two envelope membranes also differ in their carotenoid composition. Among the different enzymatic activities associated with the chloroplast envelope, we have shown that the Mg2+-dependent ATPase, the UDP-Gal:diacylglycerol galactosyltransferase, the phosphatidic acid phosphatase, and the acyl-CoA thioesterase are associated with the inner envelope from spinach chloroplasts whereas the acyl-CoA synthetase is located on the outer envelope membrane.
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PMID:Preparation and characterization of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts. II. Biochemical characterization. 663 Feb 30

cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) binds tightly to a Zn(2+)-chelate column (Francis, S. H., and Corbin, J. D. (1988) Methods Enzymol. 159, 722-729). Using three different approaches, Zn2+ is now shown to bind to cG-BPDE, and the Kd is determined to be approximately 0.5 microM, with a binding stoichiometry of approximately 3 mol of Zn2+/mol of monomer. A similar concentration range of Zn2+ (0.05-1 microM Zn2+) also supports phosphodiesterase (PDE) catalytic activity. The Zn2+ binding to cG-BPDE is not diminished by, nor is catalysis supported by, relatively high concentrations of Cu2+, Cd2+, Ca2+, or Fe2+. Neither cGMP nor 3-isobutyl-1-methylxanthine affects Zn2+ binding under the conditions used. Mn2+, Co2+, or Mg2+ supports catalysis, but only at significantly higher concentrations (4-, 15-, and 250-fold, respectively) than that required for Zn2+. Two tandem amino acid sequences, which are conserved in the catalytic domains of all characterized mammalian PDEs, resemble the single sequence motif that has been shown to coordinate Zn2+ in the catalytic sites of Zn2+ hydrolases such as thermolysin.
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PMID:Zinc interactions and conserved motifs of the cGMP-binding cGMP-specific phosphodiesterase suggest that it is a zinc hydrolase. 807 92

Gizzard alpha-actinin binds 45Ca2+ as shown by the calcium overlay method. Flow dialysis measurements in 20 mM Hepes (pH 7.5) reveal 3.5 +/- 1.8 (S.D.) high affinity calcium binding sites per dimer, with Kd1 = 6.36 +/- 0.34 x 10(-6) M, and 87.3 +/- 7.2 sites with Kd2 = 1.66 +/- 0.44 x 10(-4) M. Chymotrypsin and thermolysin digestion yielded peptides of gizzard alpha-actinin which, if they included the putative EF-hands, bound 45Ca2+ in 10 mM imidazole-HCl (pH 7.4) or 60 mM KCl, 10 mM imidazole-HCl (pH 7.4). In addition, peptides which include a region of the molecule more than 27 kDa from the N-terminal also bind calcium. In contrast, when KCl in the binding buffer was increased to 120 mM, calcium binding was eliminated. Flow dialysis data revealed no high-affinity binding and 82.5 +/- 3.3 calcium binding sites with calculated affinities in the millimolar range. These are divalent cation binding sites, not Ca(2+)-specific sites, because they are eliminated by the addition of up to 5 mM Mg2+. Structural changes produced upon cation binding to alpha-actinin measured by circular dichroism, proteolysis and bisANS fluorescence are substantial when binding K+ with only small changes upon binding of Ca2+ or Mg2+ in the presence of 120 mM KCl. These results suggest that monovalent and divalent cations have different effects on different parts of the molecule with a complete elimination of 45Ca2+ binding to the EF-hands being produced by 120 mM KCl.
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PMID:Cation binding to chicken gizzard alpha-actinin. 815 14

The pattern and kinetics of partial proteolysis of Arthrobacter D-xylose isomerase tetramer was studied in order to determine the flexibility of surface loops that may control its stability. It was completely resistant to trypsin, chymotrypsin and elastase at 37 degrees C, but thermolysin cleaved specifically and quantitatively at Thr-347-Leu-348 between helices 10 and 11 to remove 47 residues from the C-terminus of each 43.3 kDa subunit. At high temperatures, helices 9 and 10 were removed from each 38 kDa subunit to give a 36 kDa tetramer. The kinetics of nicking by thermolysin indicated that the Thr-347-Leu-348 loop is locked at low temperatures, but 'melts' at 25 degrees C and is fully flexible above 34 degrees C. The flexibility appears to be associated with binding of Ca2+ ions at the active site, since Co2+, Mg2+ and xylitol protect in proportion to their ability to displace Ca2+. The missing C-terminal helices make many intersubunit contacts that appear in the structure to stabilize the tetramer, but the properties of the purified nicked proteins are almost indistinguishable from the native enzyme. Both the 38 kDa tetramer and the 36 kDa tetramer are identically active and dissociate similarly in urea or SDS to fully active dimers, but the nicked dimers are slightly less stable to urea at 62 degrees C. In the Mg2+ form the thermostability of the 38 kDa tetramer is identical with that of the native enzyme, but the 36 kDa tetramer has a slightly lower 'melting point' (70 degrees C versus 80 degrees C), which may be due to unravelling from the end of helix 8. Since elimination of all the C-terminal helices and many intersubunit contacts has so little effect, one can conclude that the 'weak point' that controls the protein's thermostability lies within the N-terminal beta-barrel domain.
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PMID:Arthrobacter D-xylose isomerase: partial proteolysis with thermolysin. 842 59

The activity of bovine DNase, but not that of porcine DNase, is inhibited by antisera against bovine DNase, and vice versa. Inhibition of DNase is found in the immunoglobulin G-containing fractions, as shown by ion exchange chromatography. Inactive DNase, carboxymethylated specifically at the active site His134, competes with active DNase and reverses the antisera inhibition of DNase, suggesting that the epitode responsible for inhibition does not contain the active site His134. Alignment of the sequences of DNase of these two species shows that the greatest variation occurs between residues 153 and 163, within which are three consecutive peptide bonds, Lys-Trp-His-Leu, that are readily cleaved by trypsin, chymotrypsin, or thermolysin. The 8-hr digest of DNase by each of these three proteases has lost the ability to reverse antisera inhibition. The degree of antisera inhibition varies with the metal ion used as the activator for DNase-catalyzed reactions. When Mn2+, Co2+, or Mg2+ plus Ca2+ are used as activators, inhibition is approximately 50%. When pBR322 plasmid is used as substrate, gel electrophoresis shows that the DNase-catalyzed DNA hydrolysis produces a significant amount of double-strand cuts with Mn2+, Co2+, or Mg2+ plus Ca2+ as activators and antisera inhibit DNase action only on double-strand cuts. With only Mg2+ as the activator no double-strand cuts are observed, either in the presence or absence of antisera, and the DNase activity is not significantly inhibited. We conclude that antisera inhibition is due to antibody binding of the DNase polypeptide chain within residues 153 and 163. These residues are not crucial for catalysis, but are required for DNA binding, which results in double-strand cuts.
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PMID:Mechanism for inhibition of deoxyribonuclease activity by antisera. 911 1


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