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)

The G-200 flow-through fraction of the extract of sea urchin eggs contained a complex form of glutathione reductase (GR) [EC 1.6.4.2]. The complex was unstable and gradually dissociated with ain increase in GR activity. The activation was facilitated by high concentrations of EDTA, KCI or (NH4)2SO4. The rate of activation by salts was apparently dependent on the ionic strength. The complex form was also activated rather quickly by treatment with proteinases such as trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1] or subtilisin [EC 3.4.21.14]. Trypsin caused the complex to release the free form of GR. Even after trypsin treatment, little change was observed in the dependence of the GR activity on GSSG or NADPH concentration. The GR activity of the complex form was not inhibited at all by 0.2 mM N-ethylmaleimide (NEM) in the presence of GSSG, but was reduced to 3% in the presence of NADPH. When excess NEM was sequestered with GSH, the NEM-treated complex form was strikingly activated by trypsin, while no activation was detected with the free form of enzyme pretreated with NEM. These results suggest that the active site of GR in the complex form is largely masked by a polypeptide moiety of theinhbitiory component.
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PMID:Glutathione reductase in the sea urchin egg. III. Activation of the complex form by proteinases. 1 74

The ability of neutrophils to generate free radicals is a crucial component of host defense (Babior, B. M. (1978) N. Engl. J. Med. 298, 659-668, 721-725. Neutrophil oxidants, however, can cause significant host tissue destruction (Weiss, S. J. (1989) N. Engl. J. Med. 320, 365-376), and the regulation of free radical production is not well understood. We have previously shown that recombinant antichymotrypsin (rACT), a serine protease inhibitor, inhibits superoxide production in intact neutrophils (Kilpatrick, L., Johnson, J. L., Nickbarg, E. B., Wang, Z., Clifford, T. F., Banach, M., Cooperman, B. S., Douglas, S. D., and Rubin, H. (1991) J. Immunol. 146, 2388-2393). Using a cell-free NADPH oxidase preparation, we now demonstrate that rACT alone has no effect on superoxide production and that antichymotrypsin-chymotrypsin (rACT.CT) complexes are required to inhibit superoxide, suggesting that neutrophil chymotrypsin-like proteases produce conformational changes in ACT, allowing it to become active in regulating superoxide production. Additionally, we have identified NADPH oxidase itself as the target for rACT.CT and have demonstrated that rACT.CT interferes specifically with activation of the NADPH oxidase without changing the Km for NADPH or the rate constant describing the rate-limiting step in activation. These observations suggest an important role for antichymotrypsin in the regulation of NADPH-oxidase activation, which is a prerequisite for neutrophil superoxide production, and predict possible therapeutic uses for rACT in conditions where unregulated neutrophil-free radical production has been implicated in the mechanism of tissue destruction.
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PMID:Regulation of neutrophil superoxide by antichymotrypsin-chymotrypsin complexes. 131 83

The NADPH-dependent H2O2-generating system in a pig thyroid particulate fraction requires micromolar concentrations of Ca2+ for activity. The H2O2 generator could be Ca(2+)-desensitized (i.e. made fully active in the absence of Ca2+) by limited proteolysis with alpha-chymotrypsin or by treatment with ZnCl2. The Zn2+ effect was temperature- and dose-dependent with an apparent half-maximum concentration of 0.15 mM at 40 degrees C. Ca2+ desensitization was not reversed by adding the Zn2+ chelators, 1,10-phenanthroline and EGTA, but about one-third of the Ca(2+)-sensitivity was recovered after addition of 10 mM-dithiothreitol. The proteolysed enzyme and the Zn(2+)-treated enzyme had different Km values for NADPH. The Zn2+ effect did not seem to involve proteolysis or membrane fusion. These results indicate that Ca2+ regulation occurs via an autoinhibitory domain or inhibitory protein component of the H2O2-generator system. Its inhibitory effect may be removed by proteolysis or conformational changes, making the catalytic site accessible to the substrate NADPH and/or enabling electrons to be transferred from NADPH to O2.
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PMID:Activation of the NADPH-dependent H2O2-generating system in pig thyroid particulate fraction by limited proteolysis and Zn2+ treatment. 131 20

R67 dihydrofolate reductase (DHFR) is a novel protein that provides clinical resistance to the antibacterial drug trimethoprim. The crystal structure of a dimeric form of R67 DHFR indicates the first 16 amino acids are disordered [Matthews et al. (1986) Biochemistry 25, 4194-4204]. To investigate whether these amino acids are necessary for protein function, the first 16 N-terminal residues have been cleaved off by chymotrypsin. The truncated protein is fully active with kcat = 1.3 s-1, Km(NADPH) = 3.0 microM, and Km(dihydrofolate) = 5.8 microM. This result suggests the functional core of the protein resides in the beta-barrel structure defined by residues 27-78. To study this protein further, synthetic genes coding for full-length and truncated R67 DHFRs were constructed. Surprisingly, the gene coding for truncated R67 DHFR does not produce protein in vivo or confer trimethoprim resistance upon Escherichia coli. Therefore, the relative stabilities of native and truncated R67 DHFR were investigated by equilibrium unfolding studies. Unfolding of dimeric native R67 DHFR is protein concentration dependent and can be described by a two-state model involving native dimer and unfolded monomer. Using absorbance, fluorescence, and circular dichroism techniques, an average delta GH2O of 13.9 kcal mol-1 is found for native R67 DHFR. In contrast, an average delta GH2O of 11.3 kcal mol-1 is observed for truncated R67 DHFR. These results indicate native R67 DHFR is 2.6 kcal mol-1 more stable than truncated protein. This stability difference may be part of the reason why protein from the truncated gene is not found in vivo in E. coli.
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PMID:Construction of a synthetic gene for an R-plasmid-encoded dihydrofolate reductase and studies on the role of the N-terminus in the protein. 193 13

Sarcolemmal vesicles prepared by a new procedure from bovine tracheal smooth muscle were found to have a Na-Ca exchange activity that is significantly higher than that reported for different preparations from other types of smooth muscle. The exchange process system co-purified with 5'-nucleotidase, a plasma membrane marker enzyme, and was significantly enriched (over 100-fold) compared to mitochondria (cytochrome-c oxidase) but only slightly enriched (4-fold) compared to sarcoplasmic reticulum (NADPH-cytochrome-c reductase). The Na+ dependence of Ca2+ transport was demonstrated through both uptake and efflux procedures. The uptake profile with respect to Ca2+ was monotonic with a linear vo VS. vo.S-1 plot. The resultant Km of Ca2+ from the airway sarcolemmal vesicles (20 microM) was similar in magnitude to the Km of cardiac sarcolemmal vesicles (30 microM). Tracheal vesicles demonstrated a Vmax of 0.3-0.5 nmol.mg-1.s-1 which is significantly higher than that reported in preparations from other smooth muscle types. Furthermore, two processes found to stimulate cardiac Na-Ca exchange, pretreatment with either a mixture of dithiothreitol and Fe2+ or with chymotrypsin, were ineffective on the tracheal smooth muscle. Thus, the Na-Ca exchanger identified in tracheal smooth muscle appears to be different from that observed in cardiac muscle, implying that regulation of this activity may also be different.
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PMID:Sodium-calcium exchange in sarcolemmal vesicles from tracheal smooth muscle. 282 16

A procaryotic high-level expression vector for human dihydrofolate reductase has been constructed and the protein characterized as a first step toward structure-function studies of this enzyme. A vector bearing the tac promoter, four synthetic oligodeoxynucleotides, and a restriction fragment from the dihydrofolate reductase cDNA were ligated in a manner which optimized the transcriptional and translational frequency of the enzyme mRNA. The reductase, comprising ca. 17% of the total soluble protein in the host bacteria, was purified to apparent homogeneity as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and characterized by amino acid composition, partial amino acid sequence, and steady-state kinetic analysis. This expression vector has been used as a template for double-stranded plasmid DNA site-specific mutagenesis. Functional studies on a Cys-6----Ser-6 mutant enzyme support the contention that Cys-6 is obligatory for organomercurial activation of human dihydrofolate reductase. The Ser-6 mutant enzyme was not activated to any extent following a 24-h incubation with p-(hydroxymercuri)benzoate and nicotinamide adenine dinucleotide phosphate (reduced) (NADPH), whereas the kcat for Cys-6 reductase increased 2-fold under identical conditions. The specific activities of the Cys-6 and Ser-6 enzymes were virtually identical as determined by methotrexate titration as were the Km values for both dihydrofolate and NADPH. The Ser-6 mutant showed a decreased temperature stability and was more sensitive to inactivation by alpha-chymotrypsin when compared to the wild-type enzyme. These results suggest that the Ser-6 mutant reductase is conformationally altered relative to the Cys-6 native enzyme.
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PMID:Expression and site-directed mutagenesis of human dihydrofolate reductase. 304 47

Digestion of rabbit liver microsomal smooth vesicles with Bacillus subtilis protease released proteins and peptide fragments from the vesicles, without solubilizing phospholipids and cholesterol. The proteolysis was, however, limited when about 30% of the protein had been solubilized. The same limitation was observed when the vesicles were treated with trypsin, chymotrypsin, or their combinations with the bacterial protease. The limited proteolysis was accompanied by selective solubilization of cytochrome b(5) and microsomal NADPH-specific flavoprotein, leaving the CO-binding hemoprotein and some other enzymes still attached to the vesicular membranes. Sucrose density gradient centrifugation of protease-treated vesicles indicated that all the vesicles had been attacked by the protease to similar extents. The behavior of intact and digested vesicles in dextran density gradient centrifugation suggested that the vesicles, even after proteolytic digestion, existed in the form of closed sacs which were impermeable to macromolecules such as dextran and proteases. It was concluded that only the outside surface of the vesicles is susceptible to the proteolytic action and that cytochrome b(5) and the NADPH-specific flavoprotein are located in the susceptible area.
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PMID:Proteolytic microdissection of smooth-surfaced vesicles of liver microsomes. 497 13

Our model of the animal fatty acid synthetase describes a head-to-tail arrangement of two identical subunits and predicts the presence of two centers for fatty acid synthesis. Current experiments which support this conclusion were conducted using the following approach. The thioesterase component of chicken liver fatty acid synthetase was either inhibited using phenylmethanesulfonyl fluoride or diisopropyl fluorophosphate, or released from the synthetase by limited proteolysis with alpha-chymotrypsin, thus ensuring that the fatty acyl products remain bound to the enzyme. Employing such preparations, the amount of NADPH oxidized in the initial burst of fatty acid synthesis was determined by stopped flow techniques. Gas-liquid chromatography showed that C20:0 and C22:0 constituted 85% of the fatty acids formed de novo, a result that was confirmed using [14C]acetyl-CoA in the reaction. These data showed that 1.0 mol of fatty acyl-enzyme product was formed per mol of phosphopantetheine; in addition, the measured stoichiometry of NADPH oxidation was sufficient to account for de novo fatty acid synthesis. Altogether, these results indicate that the two sites for fatty acid synthesis are active and function simultaneously. They also indicate that the thioesterase plays a key role in determining the chain specificity of fatty acid synthesis.
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PMID:On the question of half- or full-site reactivity of animal fatty acid synthetase. 670 71

1. Sequence analysis of the NADPH domain (residues 158--293) and of the interface domain (365--478) was based on 12 CNBr fragments, which were isolated using ion-exchange chromatography and paper methods. Fragments with more than 15 residues were digested further with trypsin and chymotrypsin. The isolated peptides were sequenced by automated solid-phase Edman degradation. All sequenced peptides were ordered and overlapped by computerized comparisons with a complete sequence guessed from the electron density map of the protein. In the case of short CNBr fragments, this alignment was confirmed by the sequence analysis of protein fragments resulting from incomplete CNBr cleavage. 2. In the NADPH domain, residue 197, which is involved in an induced-fit mechanism, was identified as a tyrosine. The structure of the NADPH domain is probably homologous with the NAD domain of lipoamide dehydrogenase and with the FAD domain of several proteins, but not with NADPH domains of known chain-fold in other proteins. 3. The paper completes the sequence analysis of glutathione reductase so that the enzyme is now known in atomic detail. The numbering scheme of the chemically determined sequence will be used henceforth in crystallographic studies also. As inferred from the sequence data each of the two identical chains contains 478 amino acid residues, the composition being Cys10, Asp21, Asn17, Thr31, Ser31, Glu29, Gln11, Pro24, Gly43, Ala42, Val44, Met15, Ile29, Leu34, Tyr13, Phe14, Lys34, His16. Arg17, and Trp3. From these data an Mr of 2 x 51 600 was calculated for the FAD-free apoenzyme and an Mr of 2 x 42 400 for the holoenzyme.
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PMID:Glutathione reductase from human erythrocytes. The sequences of the NADPH domain and of the interface domain. 706 May 51

A new isozyme of cytochrome P-450 has been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated chronically with ethanol. Several criteria indicate that the ethanol-inducible cytochrome, which has a minimal molecular weight of 51,000 and is designated form 3a on the basis of its relative electrophoretic mobility, is distinct from the known isozymes of P-450. As judged spectrally, the new isozyme is high spin in the oxidized state, as is form 4, but differs in that the spin state is unperturbed by nonionic detergents. The absolute spectrum of the ferrous carbonyl complex of form 3a is red shifted as compared to that of forms 2, 3b, 3c, 4, and 6 and exhibits a maximum at 452 nm. The amino acid composition of form 3a is different from that of the other isozymes, and both the NH2- and COOH-terminal sequences are distinct; form 3a has an NH2-terminal alanine and a carboxyl-terminal leucine residue. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following treatment with papain, chymotrypsin, or Staphylococcus aureus V8 protease and by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome displays the highest activity of all of the rabbit isozymes in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline when reconstituted with NADPH-cytochrome P-450 reductase and phospholipid in the presence of NADPH and oxygen.
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PMID:Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits. 708 77


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