Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

By means of a monospecific antibody, dopamine beta-hydroxylase was monitored immunoelectrophoretically in various extracts of chromaffin granules. Approximately one-third of the dopamine beta-hydroxylase present was located in the membrane fraction and could only be liberated with detergent. The dopamine beta-hydroxylases of the buffer and membrane fractions were antigenically identical, but differed in their amphiphilicity, as demonstrated by the change in precipitation patterns on removal of Triton X-100 from the gel, on charge-shift crossed immunoelectrophoresis and on crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose. Furthermore, immunoelectrophoretic analysis in the presence of Triton X-100 plus the cationic detergent cetyltrimethylammonium bromide indicates additional heterogeneity of the membrane-bound dopamine-beta-hydroxylase. By limited proteolysis with chymotrypsin and thermolysin the amphiphilic form could be convered into its hydrophilic counterpart.
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PMID:Immunochemically identical hydrophilic and amphiphilic forms of the bovine adrenomedullary dopamine beta-hydroxylase. 48 54

The amino terminus of bovine rhodopsin is blocked and has the sequence x-Met-Asn(CHO)-Gly-Thr-Glu-Gly-Pro-Asn-Phe-Tyr-Val-Pro-Phe-Ser-Asn(CHO)-Lys-Thr-Gly-Val-Val-Arg, where CHO represents sites of carbohydrate attachment. The carboxyl-terminal sequence of rhodopsin is Val-Ser-Lys-Thr-Glu-Thr-Ser-Gln-Val-Ala-Pro-Ala. Upon short-term digestion of rod outer segment (ROS) membranes with thermolysin, opsin (similar to 35,000 daltons) is converted to a membrane-bound fragment O' (similar to 30,500 daltons) and 2 peptides containing 12 amino acids are released from the carboxyl terminus of rhodopsin into the supernatant. Upon long-term digestion of ROS with thermolysin, opsin and O' are replaced by the membrane-bound fragments F1 (similar to 25,000 daltons), and F2 (similar 9,500 daltons). When 32P-ROS are digested, F2 carries the 32P. Both O' and F1 contain the amino-terminal glycopeptide.
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PMID:The amino- and carboxyl-terminal sequence of bovine rhodopsin. 59 23

Papain and thermolysin are shown to cleave bovine rhodopsin in native membranes in two temporally distinct steps at room temperature. The final product of the proteolysis consists of two membrane-bound fragments of molecular weights 27 000 (Rh27) and 12 500 (Rh12). The molecular weights are not changed by reduction with dithiothreitol. The two fragments remain closely associated in both the membrane and nondenaturing detergents before and after bleaching and can be selectively cross-linked with carbodiimides. The sulfhydryl chemistry of the cleaved protein in nearly indistinguishable from native rhodopsin, and of the total of six sulfhydryl groups, two are located on Rh12 and four on Rh27. In the membrane-bound protein, two sulfhydryl groups are accessible for modification, one on Rh12 and the other on Rh27. The sulfhydryl on Rh12 is particularly reactive and may be selectively labeled with maleimides. Continuous irradiation with white light induces additional sulfhydryl reactivity on Rh27.
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PMID:Organization of rhodopsin in photoreceptor membranes. 1. Proteolysis of bovine rhodopsin in native membranes and the distribution of sulfhydryl groups in the fragments. 71 46

The folding of the peptide chain of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was investigated by enzymatic and immunochemical approaches, using specific proteases and polyclonal antibodies directed against the whole protein and specific regions of the carrier. The accessibility of the membrane-bound ADP/ATP carrier to proteases was followed by immunodetection of the cleavage products, using mitochondria devoid of outer membrane (mitoplasts) and inside-out submitochondrial particles (SMP) in the presence of either carboxyatractyloside (CATR) or bongkrekic acid (BA), two specific inhibitors which are able to bind to the outer face or the inner face of the carrier, respectively. Four types of particles were investigated, namely, mitoplasts-CATR, mitoplasts-BA, SMP-CATR, and SMP-BA. Only the ADP/ATP carrier in SMP-BA was cleaved by two specific proteases, namely, trypsin and lysine C endoprotease, at low doses for short periods of time. Two initial cleavage sites were found between Lys-42 and Glu-43, and between Lys-244 and Gly-245. After a longer period of incubation, an additional cleavage site between Lys-146 and Gly-147 could be demonstrated. Despite cleavage of the membrane-embedded carrier, the binding capacity and affinity of SMP for BA were not altered. A number of other proteases tested, including V8 protease, proline C endoprotease, thrombin, alpha-chymotrypsin, and thermolysin had virtually no effect. These results are explained by a dynamic model of the arrangement of the peptide chain of the ADP/ATP carrier.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Topography of the membrane-bound ADP/ATP carrier assessed by enzymatic proteolysis. 156 52

Neutral endopeptidase (EC 3.424.11, NEP) is a membrane-bound zinc-metallopeptidase. The substrate specificity and catalytic activity of NEP resemble those of thermolysin, a bacterial zinc-metalloprotease. Comparison of the primary structure of both enzymes suggests that several amino acids present in the active site of thermolysin are also found in NEP. Using site-directed mutagenesis of the cDNA encoding the NEP sequence, we have already shown that His residues 583 and 587 are two of the three zinc ligands. In order to identify the third zinc ligand, we have substituted Val or Asp for Glu616 or Glu646. Val616 NEP showed the same kinetic parameters as the non-mutated NEP. In contrast, the mutant Val646 NEP was almost completely devoid of catalytic activity and unable to bind the tritiated inhibitor [3H]N-[2(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxypropyl]gl ycine, the binding of which is dependent on the presence of the zinc ion. Replacing Glu for Asp at position 646 conserved the negative charge, and the mutant enzyme exhibited the same Km value as the non-mutated enzyme, but kCat was decreased to less than 3% of the value of the non-mutated enzyme. When compared to the non-mutated enzyme Asp646 NEP showed a higher susceptibility to chelating agents, but bound the tritiated inhibitor with the same affinity. Taken together, these observations strongly suggest that Glu646 of NEP is the third zinc-coordinating residue and is equivalent to Glu166 in thermolysin.
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PMID:Identification of glutamic acid 646 as a zinc-coordinating residue in endopeptidase-24.11. 167 40

The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking similarities with respect to structural requirements for substrate binding to the hydrophobic pocket at the S1' subsite of the active site. Both enzymes showed the highest reaction rates with substrates having leucine residues in this position while phenylalanine residues gave the lowest Km. The two enzymes were also inhibited by the same N-carboxymethyl peptide inhibitors. Although the mammalian enzyme was more susceptible to inhibition than its bacterial counterpart, structural variations in the inhibitor molecules affected the inhibitory constants for both enzymes in a similar manner. The two enzymes differed significantly, however, with respect to the effect of structural changes in the P1 and P2' positions of the substrate on the kinetic parameters of the reaction. The mammalian enzyme showed the highest reaction rates and specificity constants with substrates having the sequence -Phe-Gly-Phe- or -Phe-Ala-Phe- in positions P2, P1, and P1', respectively, while the sequence -Ala-Phe-Phe- was the most favored by the bacterial enzyme. The sequence -Gly-Gly-Phe- as found in enkephalins was not favored by either of the enzymes. Of the substrates having an aminobenzoate group in the P2' position, the mammalian enzyme favored those with the carboxyl group in the meta position while the bacterial enzyme favored those with the carboxyl group in the para position.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Substrate and inhibitor studies of thermolysin-like neutral metalloendopeptidase from kidney membrane fractions. Comparison with bacterial thermolysin. 351 18

Escherichia coli lactose permease (also referred to as lactose carrier) is an integral protein of the cytoplasmic membrane. Using lactose permease either radiolabeled biosynthetically in plasmid-bearing E. coli minicells or radioalkylated post-synthetically by chemical modification, we have determined sites on the membrane-bound protein accessible to proteolytic attack and we have characterized several high-molecular-mass products. The most prominent polypeptide obtained from lactose permease radiolabeled biosynthetically is observed after digestion with different proteases. The fragment produced by thermolysin was shown to contain the intact N-terminus and to extend into the region around amino acid residue 140 which, according to secondary structure models, is presumed to be less tightly folded than the rest of the molecule. Evidence is presented that the corresponding fragments obtained after digestion with several other proteases also originate from the N-terminal part of the protein. This N-terminal segment of the lactose carrier is resistant to proteolytic digestion even in the presence of non-ionic detergents and it may represent a tightly folded domain. Additional proteolytic cleavage sites located C-terminal of the Cys148 residue can be inferred.
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PMID:Limited proteolysis of lactose permease from Escherichia coli. 352 59

Neurotensin was inactivated by membrane-bound and soluble degrading activities present in purified preparations of rat brain synaptic membranes. Degradation products were identified by HPLC and amino acid analysis. The major points of cleavage of neurotensin were the Arg8-Arg9, Pro10-Tyr11, and Tyr11-Ile12 peptide bonds with the membrane-bound activity and the Arg8-Arg9 and Pro10-Tyr11 bonds with the soluble activity. Several lines of evidence indicated that the cleavage of the Arg8-Arg9 bond by the membrane-bound activity resulted mainly from the conversion of neurotensin1-10 to neurotensin1-8 by a dipeptidyl carboxypeptidase. In particular, captopril inhibited this cleavage with an IC50 (5.7 nM) close to its K1 (7 nM) for angiotensin-converting enzyme. Thiorphan inhibited the cleavage at the Tyr11-Ile12 bond by the membrane-bound activity with an IC50 (17 nM) similar to its K1 (4.7 nM) for enkephalinase. Both cleavages were inhibited by 1,10-phenanthroline. These and other data suggested that angiotensin-converting enzyme and a thermolysin-like metalloendopeptidase (enkephalinase) were the membrane-bound peptidases responsible for cleavages at the Arg8-Arg9 and Tyr11-Ile12 bonds, respectively. In contrast, captopril had no effect on the cleavage at the Arg8-Arg9 bond by the soluble activity, indicating that the enzyme responsible for this cleavage was different from angiotensin-converting enzyme. The cleavage at the Pro10-Tyr11 bond by both the membrane-bound and the soluble activities appeared to be catalyzed by an endopeptidase different from known brain proline endopeptidases. The possibility is discussed that the enzymes described here participate in physiological mechanisms of neurotensin inactivation at the synaptic level.
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PMID:Degradation of neurotensin by rat brain synaptic membranes: involvement of a thermolysin-like metalloendopeptidase (enkephalinase), angiotensin-converting enzyme, and other unidentified peptidases. 630 59

A thermolysin-like metalloendopeptidase, optimally active at a neutral pH, was identified in human serum. The enzyme cleaves the synthetic substrate glutaryl-Ala-Ala-Phe-2-naphthylamide at the Ala-Phe bond. Activity was determined by measuring the rate of formation of Phe-2-naphthylamide in a coupled enzyme assay in the presence of excess aminopeptidase M. 2-Naphthylamine released during the reaction was determined by a diazotization procedure. Enzyme activity is not affected by inhibitors of serine, thiol, or carboxyl proteases, but is sensitive to inhibition by metal chelators such as EDTA and o-phenanthroline. Dialysis against EDTA leads to loss of activity, which can be fully restored by zinc and cobalt ions. The serum enzyme closely resembles a membrane-bound metalloendopeptidase (EC 3.4.24.11) abundant in lung, spleen, and kidney in that both enzymes are inhibited by the same active-site-directed inhibitors. In addition, an antiserum obtained against the metalloendopeptidase from rabbit kidney shows strong cross-reactivity with the serum enzyme. Metalloendopeptidase activity was measured in 150 controls and in 95 patients with sarcoidosis; the two groups had significantly different enzyme activities (p less than 0.001). The mean enzyme activity in the sarcoidosis group was more than threefold higher than that of the control group. The mean enzyme activity for patients with active disease was more than double that of patients with inactive disease and more than four times that of controls (p less than 0.001). This is noteworthy because angiotensin converting enzyme, a zinc-dipeptidyl carboxypeptidase with a mechanism of action similar to that of the metalloendopeptidase, has also been reported to be increased in the serum of patients with active sarcoidosis. Enzyme activity in patients with active tuberculosis, primary pulmonary neoplasms, and idiopathic interstitial pulmonary fibrosis did not differ significantly from that of controls.
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PMID:Identification of a thermolysin-like metalloendopeptidase in serum: activity in normal subjects and in patients with sarcoidosis. 636 93

Human brain and liver mitochondria contain membrane-bound monoamine oxidase of both A and B types. Monamine oxidase-A (MAO-A), either membrane-bound or in detergent-solubilized extracts from these tissues, was selectively inhibited during incubations with trypsin, chymotrypsin, thermolysin, or papain. MAO-A in solubilized, but not in membrane-bound, preparations was also very sensitive to the action of phospholipase A2, while MAO-B was unaffected. Membrane-bound MAO-A of rat brain mitochondria was more sensitive to phospholipases and less sensitive to proteases than was human brain enzyme, indicating that these agents may reveal species differences in MAO properties. Human brain and liver MAO-A, either solubilized or bound in mitochondrial membranes, apparently contains basic and aromatic peptide moieties that are available to proteases. Hydrolysis of these peptide bonds leads to rapid denaturation unless substrate molecules stabilize the active site. Phospholipase A2 may disrupt the phospholipid microenvironment of MAO-A, the integrity of which is essential for MAO-A activity, but not for MAO-B. No interconversion of the two activities was observed. After phospholipase A2 treatment, remaining MAO-A activity was recovered in low-molecular-weight regions of a gel filtration gradient, suggesting that MAO-A subunits were released. Although these experiments argue against the proposal that phospholipids may regulate the ratio of A/B activities of a single enzyme molecule, it is conceivable that endogenous phospholipases or proteases in mitochondrial membranes may influence MAO-A activity independently of MAO-B activity.
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PMID:Selective effects of proteases and phospholipase A2 on monoamine oxidases A and B of human brain and liver. 637 37


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