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)

A marked increase in water permeability can be induced in Xenopus oocytes by injection of mRNA from tissues that express water channels, suggesting that the water channel is a protein. In view of this and previous reports which showed that proteinases may interfere with mercurial inhibition of water transport in red blood cells (RBC), we examined the influence of trypsin, chymotrypsin, papain, pronase, subtilisin and thermolysin on water permeability as well as on ATPase activity, H(+)-pump, passive H+ conductance, and Na+/H+ exchange in apical brush-border vesicles (BBMV) and endosomal (EV) vesicles from rat renal cortex. H+ transport was measured by Acridine orange fluorescence quenching and water transport by stopped-flow light scattering. As measured by potential-driven H+ accumulation in BBMV and EV, proteinase treatment had little effect on vesicle integrity. In BBMV, ecto-ATPase activity was inhibited by 15-30%, Na+/H+ exchange by 20-55%, and H+ conductance was unchanged. Osmotic water permeability (Pf) was 570 microns/s and was inhibited 85-90% by 0.6 mM HgCl2; proteinase treatment did not affect Pf or the HgCl2 inhibition. In EV, NEM-sensitive H+ accumulation and ATPase activity were inhibited by greater than 95%. Pf (140 microns/s) and HgCl2 inhibition (75-85%) were not influenced by proteinase treatment. SDS-PAGE showed selective digestion of multiple polypeptides by proteinases. These results confirm the presence of water channels in BBMV and EV and demonstrate selective inhibition of ATPase function and Na+/H+ exchange by proteinase digestion. The lack of effect of proteinases on water transport by mercurials. We conclude that the water channel may be a small integral membrane protein which, unlike the H(+)-ATPase and Na+/H+ exchanger, has no functionally important membrane domains that are sensitive to proteolysis.
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PMID:Proteinases inhibit H(+)-ATPase and Na+/H+ exchange but not water transport in apical and endosomal membranes from rat proximal tubule. 130 58

Three-headed Tetrahymena 22S ciliary dynein was found to consist of three heavy chains (HCs) and decompose into two-headed and single-headed fragments upon chymotrypsin digestion. The three HCs (A alpha, A beta, and A gamma) were immunologically different, and presumed to be located on each of the head regions. The two-headed fragment contained A beta and A gamma HCs, while the A alpha HC originated in the single-headed fragment. Both fragments were associated with ATPase activity (Toyoshima, Y. (1987a) J. Cell Biol. 105, 887-895 and Toyoshima, Y. (1987b) J. Cell Biol. 105, 897-901). Using the two-headed dynein fragment, we attempted to determine the site of ATP hydrolysis in the fragment. After digestion of the fragment with 100 micrograms/ml thermolysin for 45 min, we noted eight thermolysin-digested polypeptides (TH 1, 2, 3, 4, 5 alpha, 5 beta, 6 alpha, and 6 beta). By precisely analyzing the degradation process and the products using peptide mapping, immunoblotting and high pressure liquid chromatography, it appeared that the two-headed fragment is dissociated as two separate fragments, each of which contained A beta or A gamma HC. Thermolysin digests, TH 1, 2, 5 alpha and 6 beta were found to be derived from A beta HC, while TH 3, 4, 5 beta and 6 alpha originated in the A gamma HC. Based on the measurements of ATPase activity of these polypeptides, we concluded that the ATPase site is located in the A beta and A gamma HCs, which may have their origins in each head of the two-headed fragment of Tetrahymena 22S ciliary dynein.
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PMID:ATPase sites in two-headed fragment of Tetrahymena 22S ciliary dynein. 153 22

The photoaffinity reagent 8-[(4-azidophenyl)-methoxy]-1-tritiomethyl-2, 3-dimethylimidazo-[1,2-alpha]pyridinium iodide ([3H]mDAZIP) has been synthesized and used to photoinactivate and label purified hog gastric H+,K(+)-ATPase. The specific (K(+)-sensitive) components of both photoinactivation and labeling showed dependences on inhibitor concentration consistent with covalent modification at an extracytoplasmic site of reversible K(+)-competitive binding in the dark. The maximum amount of specific labeling (1.2 nmol/mg) was similar to the number of phosphorylation sites measured (1.0 +/- 0.14 nmol/mg). Specific labeling was distributed 76% on the alpha chain, 18% on the beta chain, and 6% on undefined peptides. Various digestions with trypsin, protease V8, and thermolysin were employed to fragment the labeled enzyme. Gasphase sequencing of the radioactive peptides identified the major site of specific labeling to be within a region where only two stretches of amino acids (Leu105 to Ile126 and Leu139 to Phe155, designated H1 and H2, respectively) are predicted to span the membrane. This in turn suggested that the labeling site was located within or close to the proposed loop between them (Gln127 to Asn138). A computer-driven energy minimization protocol yielded a loop structure to which SCH 28080 (the parent structure of [3H]mDAZIP) could be docked. Conversely, modeling of the corresponding region of Na+,K(+)-ATPase (a homologous enzyme with much lower affinity for SCH 28080) yielded no apparent binding site. Similarities in the inhibition of H+,K(+)-ATPase by SCH 28080 and of Na+,K(+)-ATPase by ouabain lead to the hypothesis that, in each case, inhibitor binding to E2-P is associated with an increase in the hydrophobicity of the environment of the loop between H1 and H2.
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PMID:Identification of an extracytoplasmic region of H+,K(+)-ATPase labeled by a K(+)-competitive photoaffinity inhibitor. 165 68

Adenosinetriphosphopyridoxal (AP3PL) specifically modifies Lys684 of Ca2(+)-ATPase of sarcoplasmic reticulum (SR-ATPase) in the presence of Ca2+, leading to its inactivation (Yamamoto, H. et al. (1988) J. Biochem. 103, 452-457). We have now investigated the effects of AP3PL on SR-ATPase in the absence of Ca2+. Similarly to its action in the presence of Ca2+, AP3PL inhibited the Ca2(+)-transporting activity in a dose-dependent manner in the absence of Ca2+ as well. ATP and ADP protected SR-ATPase against inactivation by this reagent. One mole of AP3PL was bound per mol of SR-ATPase with concomitant loss of the Ca2(+)-transporting activity. Binding of AP3PL to SR-ATPase was prevented by ATP. AP3PL-labeled SR membranes were digested with thermolysin and labeled thermolytic peptides were purified through C18 reversed-phase HPLC. Two major AP3PL-labeled peptides were obtained in approximately 1:1 ratio; one was an octapeptide corresponding to 679-ValGluProSerHisLys*SerLys-686, and the other, a nonapeptide corresponding to 487-PheSerArgAspSerLys*ArgMetSer-495 (Lys* indicates a labeled Lys residue) of SR-ATPase. Lys684 in the former turned out to be the same as the highly specific target of AP3PL in the presence of Ca2+ which was identified previously. The target site specificity of AP3PL thus changed significantly but not entirely on binding of Ca2+ to SR-ATPase. This indicates that the spatial arrangement around the gamma-phosphoryl group of the bound ATP is affected by Ca2+ ions bound at the transport site. It is also likely that Lys492 and Lys684 are situated close together in the ATP binding site of SR-ATPase.
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PMID:Ca2(+)-dependent conformational change of the ATP-binding site of Ca2(+)-transporting ATPase of sarcoplasmic reticulum as revealed by an alteration of the target-site specificity of adenosine triphosphopyridoxal. 253 25

Ca2+-Transporting ATPase of rabbit skeletal muscle sarcoplasmic reticulum contains several SH groups which are reactive with N-ethylmaleimide (MalNEt) at pH 7.0. The location of the one which is most reactive with MalNEt (SHN, Kawakita et al. J. Biochem. 87, 609 (1980)) was identified on the amino acid sequence of the ATPase. SHN was labeled by reacting sarcoplasmic reticulum membranes with [14C] MalNEt to a labeling density of 1 mol/mol ATPase. [14C]MalNEt-labeled membranes were digested with thermolysin and 14C-labeled SHN peptides were fractionated by Sephadex LH-20 chromatography to give two major peaks of radioactivity. [14C]-MalNEt-labeled peptides were further purified to homogeneity by C18-reversed phase HPLC. Two radioactive peptides containing modified cysteine (Cys), Leu-Gly-Cys-Thr-Ser and Val-Cys-Lys-Met, were finally obtained in roughly equal amounts and in reasonable recovery. Both of these sequences were found in the amino acid sequence of Ca2+-transporting ATPase (Brandl et al. Cell 44, 597 (1986)), and Cys344 and Cys364 were identified as the targets of MalNEt-modification. Thus, 0.5 mol/mol ATPase of each Cys residue actually reacted rapidly with MalNEt under the conditions leading to SHN-modification. Modification of either one with MalNEt may negatively affect the reactivity of the other. Both of the highly reactive SH groups are located in the neighborhood of Asp351, the phosphorylation site of ATPase.
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PMID:Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. I. Location of a group which is most reactive with N-ethylmaleimide. 295 11

The reactive sulfhydryl group (SHD) (Kawakita et al. (1980) J. Biochem. 87, 609-617) which is essential for the decomposition of the E-P intermediate of Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum has been identified. One sample of sarcoplasmic reticulum membranes was reacted for 3 min with 0.4 mM N-[3H]ethylmaleimide at pH 7.0 at 30 degrees C to a labeling density of 1 mol/mol ATPase without loss of the Ca2+-transporting activity. Another sample of the membranes was treated similarly with non-radioactive N-ethylmaleimide and then labeled with 0.4 mM N-ethyl[14C]maleimide for 17 min. An extensive loss of the Ca2+-transporting activity occurred during the period of this radio-labeling, thus substantiating the 14C-labeling of SHD. The labeled membranes were digested by thermolysin, and the labeled peptides were fractionated by gel filtration and reversed-phase HPLC. Two major radioactive peptides were present in both 3H- and 14C-labeled thermolytic digests, and each of the major components of 14C-labeled peptides had a counterpart in the major components of 3H-labeled peptides which behaved identically on HPLC. The major 14C-labeled peptides were purified and found to be identical with the two SHN peptides, TL-I and TL-II (Saito-Nakatsuka et al. (1987) J. Biochem. 101, 365-376), and 0.5 mol/mol ATPase each of Cys344 and Cys364 was assigned as SHD. It seems that the Ca2+-transport system retains its activity while either of the two Cys residues is unoccupied, but loses it when both of them are modified with N-ethylmaleimide.
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PMID:Reactive sulfhydryl groups of sarcoplasmic reticulum ATPase. III. Identification of cysteine residues whose modification with N-ethylmaleimide leads to loss of the Ca2+-transporting activity. 295 56

Two series of experiments were carried out to characterize (a) peptide fragments of sarcoplasmic reticulum (SR) ATPase, based on proteolysis with different enzymes and distribution of known labels, and (b) specific labeling and functional inactivation patterns, following ATPase derivatization with dicyclohexylcarbodiimide (DCCD) under various conditions. Digestion with trypsin or chymotrypsin results in the initial cleavage of the SR ATPase in two fragments of similar size and then into smaller fragments, while subtilisin and thermolysin immediately yield smaller fragments. Peptide fragments were assigned to segments of the protein primary structure and to functionally relevant domains, such as those containing the 32P at the active site and the fluorescein isothiocyanate at the nucleotide site. ATPase derivatization with [14C]DCCD under mild conditions produced selective inhibition of ATPase hydrolytic catalysis (EP + H2O in equilibrium E + Pi) without significant incorporation of the 14C radioactive label. This effect is attributed to blockage of catalytically active residues by reaction of the initial DCCD adduct with endogenous or exogenous nucleophiles. ATPase derivatization with [14C]DCCD under more drastic conditions produced inhibition of calcium binding, 14C radioactive labeling of tryptic fragments A1 and A2 (but not of B), and extensive cross-linking. Intermolecular and, to some extent, intramolecular cross-linking were prevented by exogenous nucleophiles. The presence of calcium during derivatization prevented functional inactivation, radioactive labeling of fragment A2, and internal cross-linking of fragment A1. It is proposed that both A1 and A2 fragments participate in formation of the calcium binding domain and that the labeled residues of fragment A2 are directly involved in calcium complexation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Patterns of proteolytic cleavage and carbodiimide derivatization in sarcoplasmic reticulum adenosinetriphosphatase. 296 40

Adenosine triphosphopyridoxal (AP3PL) was used as an affinity label directed toward the ATP binding site of the Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum (SR). The reagent inhibited the ATPase activity competitively with ATP, Ki = 20 microM. Incubation of SR membranes with 100 microM AP3PL followed by treatment with NaBH4 resulted in 90% inactivation of the E-P forming activity as well as of the Ca2+-transporting activity. Adenosine di- and tetraphosphopyridoxals had similar but less pronounced effects on the Ca2+-transport system. AP3PL was bound to ATPase in a one-to-one stoichiometry in parallel with the loss of the enzymatic activities. ATP and ADP prevented the binding of AP3PL and thereby protected the enzyme from inactivation. The SR membranes were labeled with [3H]AP3PL and then digested with thermolysin in order to identify the attachment site of the affinity label. A 3H-labeled peptide (Val-Glu-Pro-Ser-His-Lys* 684-Ser-Lys) was purified to homogeneity by Sephadex LH-20 chromatography and C18-reversed phase HPLC (Lys* denotes the binding site of [3H]AP3PL). These results indicate that the SR-ATPase peptide is folded in such a manner that Lys684 and Asp351, the phosphorylation site, are located very close to each other, since the distance between the 4-formyl group reacting with Lys684 and the gamma-phosphoryl group of the ATP moiety of AP3PL is rather small.
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PMID:Affinity labeling of the ATP-binding site of Ca2+-transporting ATPase of sarcoplasmic reticulum by adenosine triphosphopyridoxal: identification of the reactive lysyl residue. 296 78

The soluble peptides from the peptic digest of the reduced S-carboxymethylated 3-carboxypropionylated adenosine triphosphatase protein have been isolated and most of their structures have been determined. About 397 residues of the protein were represented in these peptides. The reduced S-carboxymethylated protein was digested with thermolysin, and peptides containing arginine or carboxymethylcysteine were isolated and characterized. Some peptides isolated from tryptic and staphylococcal-proteinase digests of the protein are described. The information contained within the structures of these peptides has been used to reconstruct long stretches of the sequence of the ATPase protein that constitute most of the protein structure external to the lipid bilayer (Allen, Trinnaman and Green (1980) Biochem. J. 187, 591-616). The details of some of the chromatographic steps used in the isolation of the peptides and the properties of the peptides are contained in Supplementary Publication SUP 50104 (45 pages), which has been deposited with the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.
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PMID:Primary structure of the calcium ion-transporting adenosine triphosphatase from rabbit skeletal sarcoplasmic reticulum. Some peptic, thermolytic, tryptic and staphylococcal-proteinase peptides. 623 80

Proteolytic digestions of myosin subfragment 1 (S-1) with elastase, subtilisin, papain, thermolysin, and Staphylococcus aureus protease reveal that the two trypsin-sensitive regions in S-1 have broad protease susceptibility. The cleavage of S-1 by these enzymes yields products that correspond within 1-2 kilodaltons (kDa) to the 25-, 50-, and 20-kDa fragments produced by trypsin. Papain and thermolysin cut preferentially at the 26-kDa/70-kDa junction, whereas elastase, subtilisin, and S. aureus protease cleave both the 26-kDa/70-kDa and 75-kDa/22-kDa junctions in S-1. Binding of actin to S-1 decreases the rate of all proteolytic reactions in the 95-kDa heavy chain. The protection of the 26-kDa/70-kDa junction by actin is greatest against papain and thermolysin attack. The reaction times of elastase, subtilisin, and S. aureus protease with S-1 increase 2-fold in the presence of actin. However, in contrast to similar reactions with trypsin, they proceed at both junctions and lead to formation of the 50- and 22-kDa fragments. The cleavage of the 22-kDa/50-kDa junction by elastase increases the Km value for the actin-activated ATPase. The presence of the two protease-sensitive regions in S-1 is consistent with a three-domain structure of the myosin head and may have important implications to the mode of intersite communication in this protein.
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PMID:Protease-sensitive regions in myosin subfragment 1. 635 63


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