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:3.4.24.11 (CD10)
9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The function of the flexible loop which is disordered in crystal structure analysis of glutathione synthetase from Escherichia coli B has been investigated by limited proteolysis and kinetic measurements for the wild-type and mutant enzymes. Proteolysis of the intact enzyme using arginyl endopeptidase or trypsin brought about a time-dependent decrease in the enzymatic activity and the production of protein fragments. SDS-polyacrylamide gel electrophoresis and peptide sequence analysis showed that only a peptide bond between arginine 233 and glycine 234 in the loop was cleaved. Further, native polyacrylamide gel electrophoresis revealed that the cleaved enzyme retained almost the same quaternary structure as that of the wild-type enzyme. Upon protease treatment, the presence of substrates, ATP and/or gamma-L-glutamyl-L-cysteine (gamma-Glu-Cys), protected the loop from cleavage, but the presence of glycine was not capable of protecting it. In addition, replacement of arginine 233 in the loop with lysine by site-directed mutagenesis increased the Michaelis constants for gamma-Glu-Cys and glycine by factors of 28 and 213, respectively. The protection against cleavage on a similar protease incubation of this mutant enzyme was also observed in the presence of ATP and/or gamma-Glu-Cys, but the effect in the presence of both substrates was half as large as that for the wild-type enzyme. These results suggest that the loop covers the active site while ATP and gamma-Glu-Cys bind there and that it protects the unstable gamma-Glu-Cys phosphate intermediate from decomposition by bulk water.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mutational and proteolytic studies on a flexible loop in glutathione synthetase from Escherichia coli B: the loop and arginine 233 are critical for the catalytic reaction. 154 May 81

Airway mucosa consists of several types of cells including ciliated cells, mucus secreting cells, basal cells and Clara cells. In this review, fine structures of these epithelial cells and intercellular junctions are demonstrated by scanning and transmission electron microscopy, and the proposed kinetics of cellular maturation and development are discussed. Airway epithelium not only plays a role as a mechanical barrier at the air-surface interface but also possesses a wide variety of functions. Ciliary beating has been recognized to be one of the important determinants for mucociliary transport by clearing inhaled particles and bacteria from the airway. We found that the motility of cilia can be regulated by intracellular second messengers, such as Ca2+, cAMP, and protein kinase C. When ciliated epithelium is encountered by physicochemical stimuli, these signal transduction systems are activated through phosphatidylinositol turnover and/or Ca2+ channel opening, which subsequently modulate the synthesis of ATP, an energy source of ciliary beating. Airway epithelium contains the enzyme neutral endopeptidase which can degrade several peptides into inactive fragments, thus regulating the actions of tachykinins released from sensory C-fibers via axon reflex. Ion transport across airway mucosa is determined by Cl secretion and Na absorption in airway epithelium. To elucidate the mechanism of airway hypersecretion under several conditions of respiratory diseases, the effects of chemical mediators, neuropeptides, and inflammatory mediators on electrical properties of canine cultured tracheal epithelium were studied. We also expanded this idea to human subjects and found that indomethacin inhalation was valuable in reducing the amounts of sputum production by inhibiting Cl and water secretion into the airway lumen. In addition, airway epithelium can modulate contraction of airway smooth muscle by generating epithelium-derived relaxing factor (EpDRF). We have shown that lipopolysaccharide-induced airway hyperreactivity seems attributable to the loss of airway epithelium with EpDRF.
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PMID:[Structure and function of airway epithelial cells]. 207 99

Extracts of cell cultures labelled with [3H]leucine were incubated with human alpha 2-macroglobulin (alpha 2M), a plasma proteinase inhibitor. The proteinase-alpha 2M complexes were then precipitated with immobilized monoclonal antibodies to alpha 2M and analysed by SDS/polyacrylamide-gel electrophoresis. Parallel experiments were done with methylamine-inactivated alpha 2M to check for unspecific binding of cell proteins to alpha 2M. Several 3H-labelled cell proteins bound to active, but not to inactivated, alpha 2M. Such proteins are likely to be proteinases. Putative endopeptidases of subunit Mr 112000, 78,000, 53,000, and in some experiments 88,000 and 16,000, were trapped by alpha 2M in supernatant fractions from IMR90 human fibroblasts, EBTr bovine fibroblasts and HeLa human carcinoma cells. No additional proteins were trapped in the presence of ATP. The Mr-78,000 endopeptidase was identified as calpain II by immunoblotting. At pH 5.3 putative endopeptidases of subunit Mr 80,000, 53,000 and 28,000-32,000 were trapped from IMR90-fibroblast extracts. Immunoblotting showed that both cathepsin B and cathepsin D were present in the Mr-28,000-32,000 electrophoretic bands. The use of alpha 2M and immobilized antibody to alpha 2M thus allows a rapid enrichment of endopeptidases from cell extracts. Some potentials and limitations of the method are discussed.
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PMID:Alpha 2-macroglobulin used to isolate intracellular endopeptidases from mammalian cells in culture. 246 15

Ornithine transcarbamoylase (OTC) is inactivated by liver lysosomes. Carbamoyl phosphate prevents the inactivation of OTC by lysosomes, while ATP, ADP, GTP, GDP 1,N6-ethenoadenosine 5'-triphosphate and particularly epsilon-ATP stimulate it. Both stimulation and protection occur at concentrations within the physiological range of ATP and carbamoyl phosphate. Inactivation of OTC is followed by extensive proteolysis. Since the inactivation is prevented by leupeptin, antipain and L-(tosylamido-2-phenyl)ethylchloromethyl ketone, the proteolytic susceptibility of OTC to lysosomes could be due to thiol endopeptidase(s). 1,N6-Ethenoadenosine 5'-triphosphate also markedly increases OTC susceptibility to trypsin and elastase. ATP analogs had no stimulatory effect on OTC inactivation by lysosomes; none of the inhibitors of ATPases tested inhibited the ATP effect. The ATP stimulation does not require Mg2+. These findings indicate a new role for ATP, GTP and related nucleotides in protein breakdown. The ATP, ADP, GTP, GDP stimulation, together with the carbamoyl protection of OTC, agree well with the molecular plasticity hypothesis model.
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PMID:Purine nucleotides stimulate while carbamoyl phosphate protects inactivation of ornithine transcarbamoylase by disrupted lysosomes. 399 99

An endopeptidase catalyzing cleavage of parathyroid hormone to specific C-terminal and N-terminal fragments was identified in a partially purified membrane fraction from rat kidney. Fractionation on sucrose gradient showed that this activity is present primarily in a light membrane fraction rather than in the basal-lateral membranes, or in the classic lysosomal fraction. The endopeptidase can be extracted from the membranes by freezing and thawing, it has an acid pH optimum, and it catalyzes production of specific fragments of PTH. The major C-terminal fragment produced has its N-terminus at residue 39 of the native hormone. This fragment is identical to the primary PTH fragment found in kidney tissue following injection of iodinated PTH into the systemic circulation of rats (D'Amour et al., 1979). Finally, the cleavage of PTH by this acid endopeptidase is activated by physiological concentration of ATP (10(-4) - 10(-3)M). These results suggest that this enzyme may be involved in PTH catabolism by the kidney, that it may be located in a specialized cell fraction and that hormone catabolism may be regulated by the energy status of the cell.
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PMID:Identification of an ATP-activated endopeptidase from rat kidney which catalyzes cleavage of parathyroid hormone to fragments identical to those produced in the rat kidney in vivo. 633 13

A cytosolic enzyme of high molecular weight (about 500 000), which attacks native or denatured proteins (inter alia, casein, globin and hexokinase) was purified about 1000-fold from mixed rat skeletal muscles, including muscles freed of mast cells by prior treatment of the animals with the degranulator, compound 48/80. Peptides of varying size were generated from radioactively labelled globin, but no free amino acids were formed; free tyrosine was also not released from azocasein. The pH optimum was 7.5 and the presence of an essential cysteine group was suggested because dithiothreitol (1 mM) stimulated the activity and N-ethylmaleimide (5 mM) and p-chloromercuriphenylsulphonic acid (1 mM) were inhibitors. The activity was markedly inhibited by Zn2+ but not by leupeptin, chymostatin or pepstatin. The enzyme was stabilized by ATP, at concentrations as low as 0.1 mM, against inactivation at 42 degrees C. The endopeptidase was clearly separated on gel chromatography from another large protease, also sensitive to Zn2+, but with marked aminopeptidase activity and the properties of hydrolase H. The activity levels of the protease, assayed after chromatography on Sepharose 6B of high-speed supernatant fractions, did not vary significantly in skeletal muscle samples which were derived from denervated, starved, diabetic or hyperthyroid animals, in all of which the abnormal physiological states expressed themselves as enhanced rates of tyrosine released by incubated soleus and extensor digitorum longus muscles. Nevertheless, the enzyme described here may be part of an ATP-dependent, multi-component proteolytic system similar to that already known to be present in reticulocytes.
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PMID:A high-molecular-weight cysteine endopeptidase from rat skeletal muscle. 633 37

With the aim of confirming our previous spectrophotometric binding studies ((1978) Eur. J. Biochem. 85, 345-350 and (1980) Eur. J. Biochem. 104, 249-254) and of ascertaining the full physiological significance of ion binding, we investigated the effects of ions and thiol reagents on the proteolysis of yeast phosphoglycerate kinase (ATP: 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3). The single non-essential thiol of the enzyme was modified with 5,5'-dithiobis(2-nitrobenzoic acid) or 2-chloromercuri-4-nitrophenol. Both modifications greatly increased the susceptibility of the kinase to inactivation by trypsin or yeast proteinase A, when compared with that of the native kinase. Electrophoresis in sodium dodecyl sulfate (SDS) revealed that limited proteolysis had occurred. The time courses for the proteolysis and loss of catalytic activity were followed and the active and inactive fragments identified. The molecular masses of the major proteolytic fragments differed with the two endopeptidases. Substrate and non-substrate anions in a concentration-dependent fashion, protected the native and mercurial-labelled kinase from inactivation by trypsin or yeast proteinase A. However, Zn2+, in a concentration-dependent fashion, increased the susceptibility of the native kinase to inactivation by each endopeptidase. The time courses for the inactivation and for the proteolysis allowed the active and inactive fragments to be identified. Zn2+ decreased the rate of inactivation of the mercurial-labelled kinase by proteinase A. The effects of these ions were detected at concentrations compatible with occupancy of an anion binding site and a low affinity Zn2+ binding site, both of which have been indicated from our previous binding studies.
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PMID:The effects of anions, substrates, metal ions and sulfhydryl reagents on the proteolytic susceptibility of yeast phosphoglycerate kinase. 703

1. This study examined the activity and mechanisms of action of urodilatin in bovine bronchi. For comparison, the ability of urodilatin to evoke bronchodilatation or protect against subsequent challenge was compared to that of the closely related peptide alpha-human atrial natriuretic peptide (ANP). 2. Urodilatin reversed methacholine-evoked contraction in a concentration-dependent manner in bovine bronchi. In the absence of any attempt to prevent degradation by neutral endopeptidases, urodilatin was more potent than ANP in this tissue. 3. The bronchodilator properties of urodilatin were significantly augmented by the neutral endopeptidase inhibitor, phosphoramidon (3.68 x 10(-5) M). This provides evidence for at least partial degradation of urodilatin by neutral endopeptidases. With phosphoramidon present, urodilatin and ANP were equipotent. 4. In the presence of phosphoramidon (3.68 x 10(-5) M), pre-incubation with urodilatin (10(-6) M) had a protective effect against subsequent methacholine-induced contraction. This action of urodilatin was quantitatively similar to that of ANP in the presence of this endopeptidase inhibitor. 5. The actions of urodilatin appear to involve ATP-sensitive K+ channels since tolbutamide (10(-6) - 10(-5) M) significantly attenuated the relaxations induced by this peptide. 6. Small conductance Ca(2+)-activated K+ channels seem likewise to be implicated in the actions of urodilatin since blockade of these channels with apamin (10(-7) - 10(-6) M) resulted in a marked attenuation of urodilatin-evoked responses. 7. The presence of charybdotoxin (10-9 M-10-M) had no significant effect on subsequent responses tourodilatin suggesting that large conductance Ca2+-activated K+ channels are not involved in the relaxations evoked by this peptide.8. In the presence of phosphoramidon (3.68 x 10-5 M), urodilatin (10-6 M) evoked elevation of cyclic GMP levels within bovine bronchial tissue. Levels of cyclic GMP increased significantly within 5-10 s in response to this peptide and preceded the initiation of relaxant responses. Maximum increases in cyclic GMP levels were reached within 5 min; the time required for maximal relaxation evoked by this peptide.9. In conclusion, urodilatin, like ANP reversed and protected against, subsequent methacholine-induced bronchoconstriction; an action enhanced by the presence of phosphoramidon (3.68 x 1O-5 M).Associated with these actions of urodilatin was a rise in cyclic GMP levels as well as the opening of ATP-sensitive K+ and small conductance Ca2+-activated K+ channels.
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PMID:Bronchodilator and pre-protective effects of urodilatin in bovine bronchi in vitro: comparison with atrial natriuretic peptide. 760 43

The proteasome or multicatalytic endopeptidase from eukaryotic cells consists of at least 14 subunits that fall into two families, alpha and beta. Subunit-specific monoclonal antibodies against ten different subunits of human proteasomes have been produced, together with an antibody that reacts with a motif (prosbox 1), common to alpha-type subunits. Four of the subunit-specific antibodies were able to precipitate proteasomes. The subunit composition of HeLa-cell proteasomes precipitated with these four different antibodies were identical, as judged from two-dimensional electrophoresis. One of the four antibodies was used to obtain proteasomes from cell lines (HeLa, Daudi, IMR90 and BSC-1) and human tissues (placenta, kidney, and liver). Electrophoretic analysis of these proteasomes, combined with peptide mapping of some subunits, suggests that they all contain 14 types of subunits as their major constituents. However, one subunit was present in two isoelectric isoforms in all cells examined. Two other subunits occurred in two or three isoelectric isoforms in placenta, liver and kidney, but not in the cell cultures. Extracts of human cells (HeLa, IMR90, Daudi and erythrocytes) were analysed by non-denaturing electrophoresis and immunoblotting. All of the 11 subunits detected by antibodies were present in a pair of ATP-stabilized protein complexes, presumed to be the 26 S proteinase, and in a doublet of complexes which migrated more slowly than purified proteasomes. Besides being present in proteasomes, one subunit was also found to occur in the free state in cell extracts.
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PMID:Human proteasomes analysed with monoclonal antibodies. 782 36

In an attempt to identify putative peroxisomal import receptors, we investigated the cross-linking of a radioiodinated peptide consisting of the 13 last amino acids of acyl-CoA oxidase and comprising the carboxy-terminal SKL-peroxisomal targeting motive, to proteins present in different subcellular fractions from rat liver. The radiolabeled peptide could be cross-linked to an 80 kDa protein present in the cytosol but not to proteins present in other subcellular fractions including highly purified peroxisomes. Binding was reversible, saturable and dependent on the presence of Mg2+ and ATP or GTP but hydrolysis of the nucleotides was not required. Binding was abolished by pretreatment of the cytosol--but not of the peptide--with N-ethylmaleimide. Binding was not specific for peptides containing the carboxy-terminal SKL-motive, since binding was competed for by the SKL-peptide from which the SKL-motive had been deleted, by the SKL-peptide with reversed sequence and by the SV40 T-antigen nuclear localisation signal peptide, but not by other peptides tested. The 80 kDa binding protein cross-reacted with a monoclonal antibody against hsp90. Purification and internal peptide sequencing of the binding protein revealed its identity as prolyl-endopeptidase. In retrospect, we realized that the SKL-peptide and all competing peptides contained a proline residue, which was not present in the non-competing peptides. In recent experiments in yeast McNew et al. (McNew, J.A., Sykes, K. and Goodman, J.M. (1993) Mol. Biol. Cell 4, 223-232) cross-linked a peroxisomal targeting peptide to a 20 kDa cytosolic protein that was identified as proline isomerase despite the fact that the peptide did not contain proline. The experiments by McNew et al. in yeast and our experiments in the rat suggest that the (peroxisomal) targeting sequence cross-linking approach may not be suited for the identification of (peroxisomal) import receptors.
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PMID:The 80 kDa cytosolic protein that binds the C-terminal part of rat acyl-CoA oxidase is not a peroxisomal import receptor but a prolyl-endopeptidase. 794 27


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