Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The sequence of the amino-terminal portion of human parathyroid hormone, particularly the identity of residues 22, 28, and 30 (the subject of discrepancies in recent published reports), has been reexamined by two basic methods of structural analysis. A fresh lot of human parathyroid hormone isolated from pooled adenoma tissue was analyzed by Edman degradation with identification of critical residues by thin-layer chromatography and gas-liquid chromatography. In the second approach, -14C or tritiated amino acids were incorporated during biosynthesis of the human hormone in slices of parathyroid glands in vitro; the appropriate amino acid residues were then determined as the -14C or tritiated phenythiohydantoin derivatives of the amino acid after Edman degradation, or by peptide isolation after appropriate cleavage with endopeptidase, or both. The results confirm our previous findings that residue 22 is glutamic acid, residue 28 is leucine, and residue 30 is aspartic acid.
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PMID:A reinvestigation of the amino-terminal sequence of human parathyroid hormone. 112 1

The cotyledons of 27 day post-germination jojoba seedlings (Simmondsia chinensis) contained five distinct endopeptidase activities separable by DEAE Bio-Gel and CM-cellulose ion exchange chromatography. The endopeptidases were purified 108- to 266-fold and their individuality was confirmed by activity-specific assays in native acrylamide gels along with differences in their Mr and catalytic properties. The five endopeptidases, which showed activity on model substrates and protein, were named EP Ia, EP Ib, EP II, EP III and EP IV. EP Ia was a serine proteinase with a pH optimum of ca 8 and Mr of 58,000. EP Ib, II and III were discrete cysteine proteinases showing pH optima of ca 6.8, 6.0 and 5.4 and Mr of 41,000, 47,000 and 35,000 respectively. EP IV was an aspartic acid proteinase with a ca pH optimum of 3.5 and Mr of 33,000.
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PMID:Multiple forms of endopeptidase activity from jojoba seeds. 136 94

A glutamic acid-specific protease has been purified to homogeneity from Bacillus licheniformis ATCC 14580 utilizing Phe-Leu-D-Glu-OMe-Sepharose affinity chromatography and crystallized. The molecular weight of the protease was estimated to be approximately 25,000 by SDS-polyacrylamide gel electrophoresis. This protease, which we propose to call BLase (glutamic acid-specific protease from B. licheniformis ATCC 14580), was characterized enzymatically. Using human parathyroid hormone (13-34) and p-nitroanilides of peptidyl glutamic acid and aspartic acid, we found a marked difference between BLase and V8 protease, EC 3.4.21.9, although both proteases showed higher reactivity for glutamyl bonds than for aspartyl bonds. Diisopropyl fluorophosphate and benzyloxycarbonyl Leu-Glu chloromethyl ketone completely inhibited BLase, whereas EDTA reversibly inactivated the enzyme. The findings clearly indicate that BLase can be classified as a serine protease. To elucidate the complete primary structure and precursor of BLase, its gene was cloned from the genomic DNA of B. licheniformis ATCC 14580, and the nucleotide sequence was determined. Taking the amino-terminal amino acid sequence of the purified BLase into consideration, the clones encode a mature peptide of 222 amino acids, which follows a prepropeptide of 94 residues. The recombinant BLase was expressed in Bacillus subtilis and purified to homogeneity. Its key physical and chemical characteristics were the same as those of the wild-type enzyme. BLase was confirmed to be a protease specific for glutamic acid, and the primary structure deduced from the cDNA sequence was found to be identical with that of a glutamic acid-specific endopeptidase isolated from Alcalase (Svendsen, I., and Breddam, K. (1992) Eur. J. Biochem. 204, 165-171), being different from V8 protease and the Glu-specific protease of Streptomyces griseus which consist of 268 and 188 amino acids, respectively.
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PMID:Purification, characterization, cloning, and expression of a glutamic acid-specific protease from Bacillus licheniformis ATCC 14580. 142 18

Twenty strains of Staphylococcus aureus from ATCC type cultures and strains found in clinical studies were cultivated, and their endopeptidase activity specific for glutamic acid was surveyed using benzyloxycarbonyl-Phe-Leu-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. The activity was found in two of the strains, ATCC 12600 and ATCC 25923. A glutamic acid-specific proteinase, which we propose to call SPase, was purified from the culture filtrate of S. aureus strain ATCC 12600 by a series of column chromatographies on DEAE-Sepharose twice and on Sephacryl S-200. A single band was observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified SPase. The molecular weight of the proteinase was estimated to be 34000 by SDS-PAGE. When synthetic peptides and oxidized insulin B-chain were used as substrates, SPase showed the same substrate specificity as V8 proteinase, EC 3.4.21.9, which specifically cleaves peptide bonds on the C-terminal side of glutamic acid and aspartic acid. Examination with p-nitroanilides of glutamic acid and aspartic acid as substrates, however, revealed that both proteinases are highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure of SPase, its gene was cloned from genomic DNA of S. aureus ATCC 12600, and the nucleotide sequence was determined. Taking the amino acid sequence of SPase from the NH2-terminus to the 27th residue into consideration, the clones encode a mature peptide of 289 amino acids, which follows a prepropeptide of 68 residues. SPase was confirmed to be a novel endopeptidase specific for glutamic acid, being different from V8 proteinase which consists of 268 amino acids.
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PMID:Purification, characterization and gene cloning of a novel glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600. 159 45

Neuropeptides containing the carboxylterminal sequence Arg-Phe-NH2 are found throughout the animal kingdom and are important substances mediating neuronal communication. Here, we have cloned the cDNA coding for the precursor protein of the sea anemone neuropeptide (Antho-RFamide) less than Glu-Gly-Arg-Phe-NH2. This precursor is 334 amino acids in length and contains 19 copies of unprocessed Antho-RFamide (Gln-Gly-Arg-Phe-Gly), which are tandemly arranged in the C-terminal part of the protein. Paired basic residues (Lys-Arg) or single basic residues (Arg) occur at the C-terminal side of each Antho-RFamide sequence. These are likely signals for posttranslational cleavage. The processing signals at the N-terminal side of each Antho-RFamide sequence, however, include acidic residues. Processing at these amino acids must involve either an amino- or an endopeptidase that cleaves C-terminally of aspartic acid or glutamic acid residues. Such processing is, to our knowledge, hitherto unknown for peptidergic neurons. The Antho-RFamide precursor also contains two copies of the putative Antho-RFamide-related peptide Phe-Gln-Gly-Arg-Phe-NH2 and one copy of Tyr-Val-Pro-Gly-Arg-Tyr-NH2. In addition, the precursor protein harbors four other putative neuropeptides that are much less related to Antho-RFamide. This report shows that the biosynthetic machinery for neuropeptides in coelenterates, the lowest animal group having a nervous system, is already very efficient and similar to that of higher invertebrates, such as mollusks and insects, and vertebrates.
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PMID:Primary structure of the precursor for the sea anemone neuropeptide Antho-RFamide (less than Glu-Gly-Arg-Phe-NH2). 170 27

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found in the plasma membrane of many cell types. The cDNA coding for the complete primary structure of neutral endopeptidase has recently been cloned and sequenced (Devault, A. Lazure, C., Nault, C., Le Moual, H., Seidah, N. G., Chretien, M., Kahn, P., Powell, J., Mallet, J., Beaumont, A., Roques, B. P., Crine, P., and Boileau, G. (1987) EMBO J. 6, 1317-1322). Comparison of the sequence of neutral endopeptidase with that of thermolysin, a bacterial Zn-metalloendopeptidase, suggests that Glu-584 in neutral endopeptidase probably corresponds to Glu-143 in thermolysin, which is an essential amino acid involved in catalysis. To test directly the importance of Glu-584 in the catalytic activity of neutral endopeptidase by site-directed metagenesis, we have constructed an expression vector in which the rabbit kidney cDNA encoding the entire neutral endopeptidase sequence is introduced downstream from the SV40 virus early promotor. After transfection in COS-1 monkey kidney cells, this vector was found to promote the expression of a protein with biochemical and catalytic properties identical to kidney neutral endopeptidase. Oligonucleotide-directed mutagenesis of Glu-584 to either valine or aspartic acid completely abolished the enzymatic activity of the recombinant protein without changing its affinity for the substrate-related tritiated inhibitor [3H]N-[(2R,2S)-3-hydroxyamino-carbonyl-2-benzyl-1-oxopropyl]-glycine. This observation clearly identifies Glu-584 as one of the important residues responsible for the catalytic activity of the enzyme.
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PMID:Expression of neutral endopeptidase (enkephalinase) in heterologous COS-1 cells. Characterization of the recombinant enzyme and evidence for a glutamic acid residue at the active site. 289 75

1. The synthetic polypeptides, poly-L-arginine, poly-L-lysine and poly-D-lysine contract guinea-pig isolated trachea in a concentration-dependent, epithelium-independent manner. Indomethacin augmented the contractile response to poly-L-arginine. 2. The contractile response to poly-L-arginine was not significantly inhibited by nicardipine, a selective L-type calcium channel blocker or by the histamine H1-receptor antagonist, mepyramine nor significantly augmented by the neutral endopeptidase inhibitor, phosphoramidon. 3. The contractile response to poly-L-arginine was inhibited in a concentration-dependent manner by prior incubation of guinea-pig tracheal rings with a number of anionic polypeptides including, low molecular weight heparin, poly-L-aspartic acid and bovine serum albumin. 4. In vitro capsaicin-induced desensitization failed to attenuate the contractile response to poly-L-arginine, suggesting little, if any role for sensory neuropeptides in the functional response in the guinea-pig. 5. Synthetic polypeptides induce an epithelium-independent, charge-dependent contraction of guinea-pig isolated trachea.
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PMID:Contractile properties of synthetic cationic polypeptides in guinea-pig isolated trachea. 801 9

Neutral endopeptidase (EC 3.4.24.11; NEP) is a membrane-bound zinc-metallopeptidase. The catalytic zinc ion is coordinated to three amino acid residues (His538, His587 and Glu646) and a water molecule. Here, we have systematically substituted potential metal-coordinating amino acid residues (His, Glu, Asp, Cys, Tyr, Ser) for each of the three zinc ligands of NEP using a recombinant polymerase chain reaction procedure. NEP mutants at positions 583 and 587 were devoid of catalytic activity. However, Glu587 NEP and Cys583 NEP were able to bind partially a tritiated inhibitor, the binding of which is dependent on the presence of the zinc atom. At position 646, the aspartate and cysteine mutants exhibited activity. For both mutants Km values were unaltered but kcat values were decreased by about 20-fold. Both mutants bound the tritiated inhibitor with Kd values similar to that of the wild-type enzyme. Our data suggest that neither histidine-583 nor -587 can be replaced by any other ligands. On the other hand, the glutamic acid at position 646 can be converted to an aspartic acid or a cysteine indicating the importance of a negative charge at this position.
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PMID:Substitution of potential metal-coordinating amino acid residues in the zinc-binding site of endopeptidase-24.11. 809 56

The design, synthesis, and biochemical profile of meta-substituted benzofused macrocyclic lactams are described. The meta-substituted benzofused macrocyclic lactams were designed to have a degree of flexibility allowing the amide bond to occupy two completely different conformations while maintaining sufficient rigidity to allow for strong interaction between enzyme and inhibitor. Using TFIT, a novel molecular superimposition program, it was shown that the meta analogs could be readily superimposed onto our ACE inhibitor template whereas no low-energy superimpositions of the ortho-substituted macrocycles could be found. The macrocycles were prepared by tethering aldehyde 1 derived from S-glutamic acid or S-aspartic acid to a meta-substituted phosphonium bromide 2. Homologation to a monocarboxylic acid methyl ester malonate followed by deprotection and cyclization gave the macrocyclic frame. Further manipulation gave the desired compounds. Unlike the ortho-substituted benzofused macrocyclic lactams described in the previous paper which are selective NEP inhibitors, the meta-substituted compounds are dual inhibitors of both NEP and ACE. The most potent member of this new series, compound 16a, inhibited both enzymes with an IC50 = 8 nM in NEP and 4 nM in ACE.
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PMID:Meta-substituted benzofused macrocyclic lactams as zinc metalloprotease inhibitors. 904 41

Success in controlling hyperglycemia in type I diabetics will require a restoration of basal insulin. To this end, three plasmid DNAs (pDNA) encoding preproinsulin were compared for constitutive expression and processing to insulin in nonendocrine cells in vitro. The pDNAs were designed to express rat proinsulin I (VR-3501), rat proinsulin I with the B10 aspartic acid point mutation (VR-3502), and a derivative of VR-3502 with a furin cleavage site added at the B-chain and C-peptide junction (VR-3503). Cells transfected with VR-3501 or VR-3502 were able to secrete only proinsulin, whereas transfection with VR-3503 yielded 30-70% mature insulin, which could be increased to >99% by cotransfection with a furin expression plasmid (VR-3505). The insulin produced was biologically active. The bilateral injection of 100 microg of VR-3502 plasmid into the tibialis anterior muscles of mice on two consecutive days yielded, on average, several hundred picograms of heterologous proinsulin per milliliter of serum. In BALB/c mice, serum proinsulin peaked 7-14 days postinjection and declined to preinjection levels by days 21-28. In athymic nude mice, serum proinsulin was sustained for at least 6 weeks. The therapeutic efficacy of delivering insulin via muscle injection of pDNA was evaluated in athymic nude mice made diabetic with the beta cell toxin streptozotocin (STZ). All animals given control DNA died within 1 week of receiving STZ while 40% of the mice coinjected with plasmids VR-3503 and VR-3505 lived through the duration of the 4-week experiment. Muscles of the surviving animals contained 17-100 ng of immune-reactive insulin (IRI), 86-94% of which was mature insulin. The results suggest that heterologous insulin made in muscle increased the survival rate. We propose that insulin plasmid expression in skeletal muscle may be a valid approach to basal insulin delivery. The feasibility of plasmid DNA-based delivery of basal insulin was investigated. An expression system consisting of pDNAs encoding a selectively mutated rat preproinsulin and mouse furin was developed and characterized in vitro and in vivo. When injected with preproinsulin pDNA, the mouse tibialis anterior muscle expressed and released proinsulin into serum at levels comparable to normal basal insulin in rodents. These heterologous proinsulin levels were sustained for several weeks in immune-compromised nondiabetic mice. Mouse muscle coinjected with a pDNA encoding the endopeptidase furin and a pDNA encoding a pre-proinsulin modified to contain two furin cleavage sites produced fully processed insulin. This muscle-made insulin appears to have contributed to the survival of mice treated with a highly diabetogenic dose of streptozotocin, a beta cell toxin. The results demonstrate that skeletal muscle is able to express and deliver therapeutic insulin from plasmid DNA.
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PMID:Insulin delivery with plasmid DNA. 1056 91


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