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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In a recent study [Shoshan-Barmatz, V., Orr, I., Weil, S., Meyer, H., Varsanyi, M. & Heilmeyer, L. M. G. (1996) FEBS Lett. 386, 205-210] we have demonstrated the presence of the voltage-dependent anion channel (VDAC) in skeletal muscle sarcoplasmic reticulum (SR) as supported here by co-localization of VDAC and (Ca2+ + Mg2+)ATPase in the SR using double-immunogold labeling. The interaction of the carboxyl-modifying reagent dicyclohexylcarbodiimide with the SR-VDAC is characterized by labeling with [14C]dicyclohexylcarbodiimide and by dicyclohexylcarbodiimide modification of the reconstituted-purified VDAC channel activity. In both SR and mitochondrial membranes, [14C]dicyclohexylcarbodiimide most specifically labeled a 35-kDa protein, identified as VDAC by specific anti-VDAC Ig. Labeling of the SR-VDAC was about twofold higher than that of the mitochondrial VDAC, which could result either form higher labeling of the SR protein or from relatively higher amounts of VDAC/mg total protein in the SR membranes. [14C]Dicyclohexylcarbodiimide labeling of the SR, but not the mitochondrial VDAC, was biphasic with respect to time and concentration of [14C]dicyclohexylcarbodiimide. Partial digestion of [14C]dicyclohexylcarbodiimide-labeled SR-VDAC with chymotrypsin yielded five proteolytic fragments which were recognized by the anti-VDAC Ig, and the dicyclohexylcarbodiimide-binding site was localized in the 19-kDa fragment. VDAC was purified from SR and mitochondrial membranes by spermine-agarose column. The interaction of dicyclohexylcarbodiimide with functional carboxyl residue(s) in the purified VDAC is demonstrated by recording its channel activity, following its reconstitution into planar lipid bilayer (PLB). Dicyclohexylcarbodiimide inhibited the channel activity in a voltage-dependent manner, requiring incubation with dicyclohexylcarbodiimide at high (negative or positive) potentials. Dicyclohexylcarbodiimide slowed down the transition from the high-conducting to a long-lived low-conducting states of the channel (approximately 20% of its maximal conductance), by stabilizing the intermediate states. Similar results were also obtained with purified-reconstituted mitochondrial VDAC. Hydrophilic carboxyl reagents [[1-ethyl-3-(3-dimethylamino)propyl] carbodiimide, N-ethyl-phenylisoxazolium-3'-sulfonate] neither modified the channel activity nor prevented [14C]dicyclohexylcarbodiimide labeling. These results indicate that dicyclohexylcarbodiimide interacts with a carboxyl group located in a hydrophobic region of the protein which is involved in the channel gating.
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PMID:Dicyclohexylcarbodiimide interaction with the voltage-dependent anion channel from sarcoplasmic reticulum. 965 59

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the traffic ATPase family that includes multiple proteins characterized by (1) ATP binding, (2) conserved transmembrane (TM) motifs and nucleotide binding domains (NBDs), and (3) molecular transport of small molecules across the cell membrane. While CFTR NBD-1 mediates ATP binding and hydrolysis, the membrane topology and function of this domain in living eukaryotic cells remains uncertain. In these studies, we have expressed wild-type CFTR NBD-1 (amino acids 433-586) or NBD-1 containing the DeltaF508 mutation transiently in COS-7 cells and established that the domain is situated across the plasma membrane by four independent assays; namely, extracellular chymotrypsin digestion, surface protein biotinylation, confocal immunofluorescent microscopy, and functional measurements of cell membrane anion permeability. Functional studies indicate that basal halide permeability is enhanced above control conditions following wild-type or DeltaF508 NBD-1 expression in three different epithelial cell lines. Furthermore, when clinically relevant CFTR proteins truncated within NBD-1 (R553X or G542X) are expressed, surface localization and enhanced halide permeability are again established. Together, these findings suggest that isolated CFTR NBD-1 (with or without the DeltaF508 mutation) is capable of targeting the epithelial cell membrane and enhancing cellular halide permeability. Furthermore, CFTR truncated at position 553 or 542 and possessing the majority of NBD-1 demonstrates surface localization and also confers increased halide permeability. These findings indicate that targeting to the plasma membrane and assumption of a transmembrane configuration are innate properties of the CFTR NBD-1. The results also support the notion that components of the halide-selective pore of CFTR reside within NBD-1.
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PMID:Cystic fibrosis transmembrane conductance regulator (CFTR) nucleotide-binding domain 1 (NBD-1) and CFTR truncated within NBD-1 target to the epithelial plasma membrane and increase anion permeability. 979 Jun 86

A human brain E-type ATPase (HB6 ecto-apyrase) was subjected to site-directed mutagenesis to assess the functional significance of two highly conserved tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly expressed ecto-apyrase completely devoid of nucleotidase activity. Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular distribution clearly different from that of the wild-type enzyme, with the majority of the immunoreactivity concentrated in the interior of the cell. Unlike the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron Blue and was sensitive to proteolytic digestion by chymotrypsin. These results suggest alteration of the tertiary structure, causing the enzyme to be improperly folded and retained within the cell. In contrast, mutation of tryptophan 459 to alanine resulted in an ecto-apyrase with enhanced NTPase activity, but diminished NDPase activity. Immunolocalization of this active mutant ecto-apyrase revealed a cellular pattern similar to that of the wild-type enzyme, distributed along the cell periphery and in cell processes. Coupling this active W459A mutation to a previously described mutation (D219E) resulted in an enzyme which preferentially hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In addition, the double mutant is substantially less sensitive to inhibition by azide, a more potent inhibitor of ecto-apyrases than ecto-ATPases. Thus, mutation of only two amino acids of an E-type ATPase essentially converts an ecto-apyrase to an ecto-NTPase.
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PMID:Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase. 1023 36

Based on the following observations we propose that the cytoplasmic loop between trans-membrane segments M6 and M7 (L6/7) of the alpha subunit of Na(+),K(+)-ATPase acts as an entrance port for Na(+) and K(+) ions. 1) In defined conditions chymotrypsin specifically cleaves L6/7 in the M5/M6 fragment of 19-kDa membranes, produced by extensive proteolysis of Na(+),K(+)-ATPase, and in parallel inactivates Rb(+) occlusion. 2) Dissociation of the M5/M6 fragment from 19-kDa membranes is prevented either by occluded cations or by competitive antagonists such as Ca(2+), Mg(2+), La(3+), p-xylylene bisguanidinium and m-xylylene bisguanidinium, or 1-bromo-2,4, 6-tris(methylisothiouronium)benzene and 1,3-dibromo-2,4,6-tris (methylisothiouronium)benzene (Br(2)-TITU(3+)). 3) Ca(2+) ions raise electrophoretic mobility of the M5/M6 fragment but not that of the other fragments of the alpha subunit. It appears that negatively charged residues in L6/7 recognize either Na(+) or K(+) ions or the competitive cation antagonists. Na(+) and K(+) ions are then occluded within trans-membrane segments and can be transported, whereas the cation antagonists are not occluded and block transport at the entrance port. The cytoplasmic segment of the beta subunit appears to be close to or contributes to the entrance port, as inferred from the following observations. 1) Specific chymotryptic cleavage of the 16-kDa fragment of the beta subunit to 15-kDa at 20 degrees C (Shainskaya, A., and Karlish, S. J. D. (1996) J. Biol. Chem. 271, 10309-10316) markedly reduces affinity for Br(2)-TITU(3+) and for Na(+) ions, detected by Na(+) occlusion assays or electrogenic Na(+) binding, whereas Rb(+) occlusion is unchanged. 2) Na(+) ions specifically protect the 16-kDa fragment against this chymotryptic cleavage.
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PMID:Entrance port for Na(+) and K(+) ions on Na(+),K(+)-ATPase in the cytoplasmic loop between trans-membrane segments M6 and M7 of the alpha subunit. Proximity Of the cytoplasmic segment of the beta subunit. 1063 5

Two isoforms of lobster muscle tropomyosin, a fast muscle type, fTm, and a slow muscle type, sTm1, are identical except for 15 residues within the region of amino acids 39-80, which corresponds to exon 2 of the tropomyosin genes of many phyla. Although the difference in the sequence does not include the terminal regions, the two isoforms are extremely different in viscosity, which is a good measure of the head-to-tail interaction strength and should be dependent on the conformation of the terminal 7-9 residues. To determine the influence of amino-acid replacements in the internal region on the overall conformation and the functional properties of the molecule, we compared the physical properties of the two isoforms and their interactions with other proteins, such as actin and myosin subfragment 1 (S1). Limited proteolysis by trypsin and chymotrypsin showed that sTm1 is more susceptible than fTm at the sites outside the region with the replaced residues. Compared with fTm, sTm1 showed higher viscosity, had a higher actin affinity, and inhibited acto-S1 ATPase to a greater extent. Finally, the binding isotherm of S1-ADP to actin-sTm1 is less sigmoidal than that to actin-fTm. These results indicate that the amino-acid replacements in the internal region alter the conformation and the physical properties of the entire molecule as well as its interactions with actin and myosin.
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PMID:Amino-acid replacements in an internal region of tropomyosin alter the properties of the entire molecule. 1090 22

The domain structure of the HSC70-interacting protein (HIP), a 43-kDa cytoplasmic cochaperone involved in the regulation of HSC70 chaperone activity and the maturation of progesterone receptor, has been probed by limited proteolysis and biophysical and biochemical approaches. HIP proteolysis by thrombin and chymotrypsin generates essentially two fragments, an NH2-terminal fragment of 25 kDa (N25) and a COOH-terminal fragment of 18 kDa (C18) that appear to be well folded and stable as indicated by circular dichroism and recombinant expression in Escherichia coli. NH2-terminal amino acid sequencing of the respective fragments indicates that both proteases cleave HIP within a predicted alpha-helix following the tetratricopeptide repeat (TPR) region, despite their different specificities and the presence of several potential cleavage sites scattered throughout the sequence, thus suggesting that this region is particularly accessible and may constitute a linker between two structural domains. After size exclusion chromatography, N25 and C18 elute as two distinct and homogeneous species having a Stokes radius of 49 and 24 A, respectively. Equilibrium sedimentation and sedimentation velocity indicate that N25 is a stable dimer, whereas C18 is monomeric in solution, with sedimentation coefficients of 3.2 and 2.3 S and f/f(o) values of 1.5 and 1.1 for N25 and C18, respectively, indicating that the N25 is elongated whereas C18 is globular in shape. Both domains are able to bind to the ATPase domain of HSC70 and inhibit rhodanese aggregation. Moreover, their effects appear to be additive when used in combination, suggesting a cooperation of these domains in the full-length protein not only for HSC70 binding but also for chaperone activity. Altogether, these results indicate that HIP is made of two structural and functional domains, an NH2-terminal 25-kDa domain, responsible for the dimerization and the overall asymmetry of the molecule, and a COOH-terminal 18-kDa globular domain, both involved in HSC70 and unfolded protein binding.
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PMID:Domain structure of the HSC70 cochaperone, HIP. 1168 74

Despite the fact that mucus and bicarbonate are important macroscopic components of the gastric mucosal barrier, severe acidic and peptic conditions surely exist at the apical membrane of gastric glandular cells, and these membranes must have highly specialized adaptations to oppose external insults. Parietal cells abundantly express the heterodimeric, acid-pumping H-K-ATPase in their apical membranes. Its beta-subunit (HKbeta), a glycoprotein with >70% of its mass and all its oligosaccharides on the extracellular side, may play a protective role. Here, we show that the extracellular domain of HKbeta is highly resistant to trypsin in the native state (much more than that of the structurally related Na-K-ATPase beta-subunit) and requires denaturation to expose tryptic sites. Native HKbeta also resists other proteases, such as chymotrypsin and V8 protease, which hydrolyze at hydrophobic and anionic amino acids, respectively. Removal of terminal alpha-anomeric-linked galactose does not appreciably alter tryptic sensitivity of HKbeta. However, full deglycosylation makes HKbeta much more susceptible to all proteases tested, including pepsin at pH <2.0. We propose that 1) intrinsic folding of HKbeta, 2) bonding forces between subunits, and 3) oligosaccharides on HKbeta provide a luminal protein domain that resists gastric lytic conditions. Protein folding that protects susceptible charged amino acids and is maintained by disulfide bonding and hydrophilic oligosaccharides would provide a stable structure in the face of large pH changes. The H-K-ATPase is an obvious model, but other gastric luminally exposed proteins are likely to possess analogous protective specializations.
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PMID:Gastric H-K-ATPase and acid-resistant surface proteins. 1201 20

Chemotherapy has cachectic effects, but it is unknown whether cytostatic agents alter skeletal muscle proteolysis. We hypothesized that chemotherapy-induced alterations in protein synthesis should result in the increased incidence of abnormal proteins, which in turn should stimulate ubiquitin-proteasome-dependent proteolysis. The effects of the nitrosourea cystemustine were investigated in skeletal muscles from both healthy and colon 26 adenocarcinoma-bearing mice, an appropriate model for testing the impact of cytostatic agents. Muscle wasting was seen in both groups of mice 4 days after a single cystemustine injection, and the drug further increased the loss of muscle proteins already apparent in tumor-bearing animals. Cystemustine cured the tumor-bearing mice with 100% efficacy. Surprisingly, within 11 days of treatment, rates of muscle proteolysis progressively decreased below basal levels observed in healthy control mice and contributed to the cessation of muscle wasting. Proteasome-dependent proteolysis was inhibited by mechanisms that include reduced mRNA levels for 20S and 26S proteasome subunits, decreased protein levels of 20S proteasome subunits and the S14 non-ATPase subunit of the 26S proteasome, and impaired chymotrypsin- and trypsin-like activities of the enzyme. A combination of cisplatin and ifosfamide, two drugs that are widely used in the treatment of cancer patients, also depressed the expression of proteasomal subunits in muscles from rats bearing the MatB adenocarcinoma below basal levels. Thus, a down-regulation of ubiquitin-proteasome-dependent proteolysis is observed with various cytostatic agents and contributes to reverse the chemotherapy-induced muscle wasting.
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PMID:Chemotherapy inhibits skeletal muscle ubiquitin-proteasome-dependent proteolysis. 1201 53

P-glycoprotein (Pgp), an anticancer drug-translocating ATPase, is responsible for multidrug resistance in cancer. We have previously shown (Nuti, S. L., Mehdi, A., and Rao, U. S. (2000) Biochemistry 39, 3424-3432) that tryptic cleavage of Pgp results in the activation of basal and drug-stimulated ATPase functions of Pgp. To understand this phenomenon, we determined the sites cleaved by trypsin and further examined whether the modulation of Pgp function is trypsin-specific or the result of proteolysis in general. The effects of chymotrypsin and proteinase K on Pgp ATPase function were studied. The results show that proteolysis of Pgp irrespective of the protease employed resulted in the activation of basal ATPase activity. However, drug-stimulated ATPase activities were differentially modulated. Immunoblot analysis of proteolytic digests indicated that, irrespective of the protease employed, Pgp was predominantly cleaved in the middle of the molecule. N-terminal amino acid sequencing of Pgp tryptic and chymotryptic peptides indicated Arg(680) and Leu(682) as the sites of cleavage, respectively. These two cleavage sites are part of the predicted linker region that joins the two halves of Pgp. Together, these results suggest that the linker region in Pgp is primarily accessible to protease action and that cleavage of this region modulates Pgp ATPase function.
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PMID:Proteolytic Cleavage of the Linker Region of the Human P-glycoprotein Modulates Its ATPase Function. 1205 98

Insect resistance to the Cry toxins of Bacillus thuringiensis (Bt) has been examined previously using a number of traditional biochemical and molecular techniques. In this study, we utilized a proteomic approach involving two-dimensional differential gel electrophoresis, mass spectrometry, and function-based activity profiling to examine changes in the gut proteins from the larvae of an Indianmeal moth (IMM, Plodia interpunctella) colony exhibiting resistance to Bt. We found a number of changes in the levels of certain specific midgut proteins that indicate increased glutathione utilization, elevation in oxidative metabolism, and differential maintenance of energy balance within the midgut epithelial cells of the Bt-resistant IMM larva. Additionally, the electrophoretic migration pattern of a low molecular mass acidic protein, which apparently is an ortholog of F(1)F(0)-ATPase, was considerably altered in the Bt-resistant insect indicating that variations in amino acid content or modifications of certain proteins also are important components of the resistance phenomenon in the IMM. Furthermore, there was a dramatic decrease in the level of chymotrypsin-like proteinase in the midgut of the Bt-resistant larva, signifying that reduction of chymotrypsin activity, and subsequently decreased activation of Cry toxin in the insect midgut, is an important factor in the resistant state of the IMM. The proteomic analysis of larval gut proteins utilized in this study provides a useful approach for consolidating protein changes and physiological events associated with insect resistance to Bt. Our results support the hypothesis that physiological adaptation of insects and resistance to Bt is multifaceted, including protein modification and changes in the synthesis of specific larval gut proteins. We believe that increased oxidative metabolism may be an adaptive response of insects that undergo survival challenge and that it could mediate detoxification as well as higher rates of generalized and localized mutations that enhance their resistance and provide survival advantage.
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PMID:Insect resistance to Bacillus thuringiensis: alterations in the indianmeal moth larval gut proteome. 1260 Oct 79


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