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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The microsomal haem oxygenase activity induced by the administration of CoCl2 was found mainly in the smooth-surfaced microsomal fraction, whereas that of the untreated control animals was widely distributed in smooth-surfaced microsomal, rough-surfaced microsomal and Golgi fractions. 2. When microsomal preparation was incubated and the time course of the distribution of biliverdin between the membranes and the medium was followed, most of the biliverdin formed was found first in the medium. This suggests that the active site of haem oxygenase is exposed on the cytoplasmic surface of the membranes. The possible localization of the enzyme at the outer surface of the membranes was also supported by a digestion experiment with trypsin. The haem oxygenase activity was greatly decreased even at low concentration of the proteinase, which did not affected the NADPH-cytochrome c reductase activity. 3. When microsomal preparation was further fractionated by isopycnic centrifugation in the presence of deoxycholate or by partitioning of sonicated microsomal preparation in aqueous-polymer two-phase systems, most of the haem oxygenase activity was found in a fraction different from the main fraction of the NADH- and NADPH-cytochrome c reductase and NADH--ferricyanide reductase activities. This indicates the different distribution of haem oxygenase from the other enzymes mentioned, on the lateral plane of microsomal membranes, and suggests the different localization of the haem oxygenase system from the electron-transport system linked with cytochrome b5 and cytochrome P-450.
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PMID:Topological arrangement in microsomal membranes of hepatic haem oxygenase induced by cobalt chloride. 44 18

1. Chymotrypsin treatment of chloroplast membranes inactivates Photosystem II. The inactivation is higher when the activity is measured under low intensity actinic light, suggesting that primary photochemistry is preferentially inactivated. 2. Membrane stacking induced by Mg2+ protects Photosystem II against chymotrypsin inactivation. When the membranes are irreversible unstacked by brief treatment with trypsin, Mg2+ protection against chymotrypsin inactivation of Photosystem II is abolished. 3. The kinetics of inactivation by chymotrypsin of Photosystem II indicates that membrane stacking slows down, but does not prevent, the access of chymotrypsin to Photosystem II, which is mostly located within the partition zones. 4. It is concluded that a partition gap exists between stacked membranes of about 45 A, the size of the chymotrypsin molecule. 5. The kinetics of inhibition of the chloroplast flavoprotein, ferredoxin-NADP reductase, bt its specific antibody is not affected by membrane stacking. This indicates that this enzyme is located outside the partition zones.
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PMID:Partition zone penetration by chymotrypsin, and the localization of the chloroplast flavoprotein and photosystem II. 44 96

NADPH-cytochrome P-450 reductase was isolated from liver microsomes of phenobarbital-induced rats. The enzyme exhibits an apparent minimal molecular weight of 76,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 molecule each of FMN and FAD. Trypsin treatment of the reductase yields an enzyme with an apparent minimal molecular weight of 69,000 which retains the ability to reduce cytochrome c but has no activity toward cytochrome P-450. Various spectrophotometric titrations were performed to examine the electron-accepting properties of the purified NADPH-cytochrome P-450 reductase and, in particular, to determine the oxidation state of the stable semiquinone form produced by air oxidation of NADPH-reduced enzyme. Titration of the air-stable semiquinone form of the reductase with ferricyanide indicated that 1 mol/2 mol of flavin was required for complete oxidation. Furthermore, a spectrum corresponding to that of the air-stable semiquinone form was produced by the addition of approximately 0.5 mol of reductant/2 mol of flavin when the oxidized enzyme was titrate with NADPH or dithionite under anaerobic conditions. The spectral changes which accompanied the overall reduction of oxidized enzyme to the reduced form with dithionite produced four sets of isosbestic points, and the spectrophotometric titration curve consisted of four approximately equal phases. In the titration with NADPH, no significant further reduction was observed after the addition of approximately 1.5 mol/2 mol of flavin. However, the enzyme was fully reduced by NADPH when an NAPH-generating system was used to prevent the accumulation of NADP. Our results establish that the air-stable semiquinone form is a 1-electron-reduced form, rather than a half-reduced (2-electron-reduced) form as maintained by others and are in agreement with earlier studies (Iyanagi, T., Makino, N., and Mason, H.S. (1974) Biochemistry 13, 1701-1710) with the purified trypsin-solubilized reductase. Accordingly, the air-stable species represents a form of the NADPH-cytochrome P-450 reductase in which one of the two flavins exists in the semiquinone state and the other in the oxidized state.
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PMID:Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states. 63 95

Ehrlich cell plasma membrane vesicles have a ferricyanide reductase activity that shows two phases. These two phases were kinetically characterized. Evidence is presented for a differential effect of trypsin on both phases.
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PMID:Two phases of ferricyanide reductase activity in Ehrlich cell plasma membranes. 128 32

Using purified bacterially expressed herpes simplex virus type 1 ribonucleotide reductase large subunit (R1) and the proteolytic enzymes chymotrypsin and trypsin, we have generated stable N-terminal truncations. Chymotrypsin removes 246 amino acids from the amino terminus to produce a fragment (dN246R1) which retains full enzymic activity and affinity for the small subunit (R2). Treatment of R1 with trypsin produces a 120K protein and a cleavage at amino acid residue 305 to produce a fragment (dN305R1) which remains associated with a 33K N-terminal polypeptide. Although this 33K-dN305R1 complex retains full binding affinity for R2 its reductase activity is reduced by approximately 50%. Increasing the concentration of trypsin removes the 33K N-terminal polypeptide resulting in dN305R1 which, when bound to R2, has full ribonucleotide reductase activity. Like R1, dN246R1 and dN305R1 each exist as dimers showing that the first 305 amino acids of R1 are not necessary for dimer formation. These results indicate that, in structural studies of subunit interaction, dN246R1 or dN305R1 can be considered as suitable replacements for intact R1.
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PMID:The unique N terminus of the herpes simplex virus type 1 large subunit is not required for ribonucleotide reductase activity. 130 56

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.
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PMID:The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase. 155 Mar 61

3-Oxoacyl-[ACP] reductase (E.C. 1.1.1.100, alternatively known as beta-ketoacyl-[ACP] reductase), a component of fatty acid synthetase has been purified from seeds of rape by ammonium sulphate fractionation, Procion Red H-E3B chromatography, FPLC gel filtration and high performance hydroxyapatite chromatography. The purified enzyme appears on SDS-PAGE as a number of 20-30 kDa components and has a strong tendency to exist in a dimeric form, particularly when dithiothreitol is not present to reduce disulphide bonds. Cleveland mapping and cross-reactivity with antiserum raised against avocado 3-oxoacyl-[ACP] reductase both indicate that the multiple components have similar primary structures. On gel filtration the enzyme appears to have a molecular mass of 120 kDa suggesting that the native structure is tetrameric. The enzyme has a strong preference for the acetoacetyl ester of acyl carrier protein (Km = 3 microM) over the corresponding esters of the model substrates N-acetyl cysteamine (Km = 35 mM) and CoA (Km = 261 microM). It is inactivated by dilution but this can be partly prevented by the inclusion of NADPH. Using an antiserum prepared against avocado 3-oxoacyl-[ACP] reductase, the enzyme has been visualised inside the plastids of rape embryo and leaf tissues by immunoelectron microscopy. Amino acid sequencing of two peptides prepared by digestion of the purified enzyme with trypsin showed strong similarities with 3-oxoacyl-[ACP] reductase from avocado pear and the Nod G gene product from Rhizobium meliloti.
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PMID:3-Oxoacyl-[ACP] reductase from oilseed rape (Brassica napus). 156 81

Rat hepatic microsomal squalene synthetase (EC 2.5.1.21) was induced 25-fold by feeding rats with diet containing the hydroxymethylglutaryl-coenzyme A reductase inhibitor, fluvastatin, and cholestyramine, a bile acid sequestrant. A soluble squalene synthetase protein with an estimated mass of 32-35 kDa, as determined by gel filtration chromatography on Sephacryl S-200 column, was solubilized out of the microsomes by controlled proteolysis with trypsin. Approximately 25% of the activity was recovered in a soluble form. The enzyme was purified to homogeneity utilizing a series of column chromatography purification steps on DEAE-cellulose, hydroxylapatite, and phenyl-Sepharose sequentially. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Initial kinetic analysis indicated an S0.5 values for trans-farnesyl diphosphate of 1.0 microM and for NADPH of 40 microM. The Vmax with respect to trans-farnesyl diphosphate was calculated at 1.2 mumol/min/mg. NADH also serves as substrate for the reaction with S0.5 value of 800 microM. Western blot analysis utilizing rabbit antisera raised against the purified, trypsin-truncated enzyme showed a single band for the isolated solubilized enzyme at 32-33 kDa and a band for the intact microsomal enzyme at about 45-47 kDa.
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PMID:Solubilization, purification, and characterization of a truncated form of rat hepatic squalene synthetase. 156 7

A tryptic peptide of heme oxygenase obtained after solubilization of rat liver microsomes by mild trypsin treatment was purified. The purified peptide gave only a single protein band with a molecular mass of 28 kDa on SDS/PAGE. The tryptic peptide, like the native heme oxygenase, readily bound with substrate heme forming a hemeprotein transiently. The absorption spectra of the ferric, ferrous, ferrous-CO and ferrous-O2 forms of the resulting complex resembled those of the corresponding forms of the complex of heme and the native enzyme. Ferric heme bound to the tryptic peptide was quantitatively decomposed to biliverdin on incubation with a mixture of ascorbic acid and desferrioxamine, indicating that the tryptic peptide still retained catalytic activity. These observations suggest that heme oxygenase has two domains, a hydrophilic and a hydrophobic domain, and that the two domains are folded almost independently of each other. An NADPH-cytochrome-P-450 reductase system composed of NADPH and detergent-solubilized NADPH-cytochrome-P-450 reductase readily reduced the ferric heme bound to the tryptic peptide, but failed to transfer the second electron required for rapid heme degradation, suggesting that the hydrophobic domain of heme oxygenase is important for receiving the second electron from the reductase.
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PMID:Degradation of heme by a soluble peptide of heme oxygenase obtained from rat liver microsomes by mild trypsinization. 165 Dec 44

The structural organization of the oxysterol receptor, postulated to be involved in the regulation of 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol biosynthesis in mammalian cells, has been explored by limited proteolysis with trypsin, alpha-chymotrypsin, and endoproteinase GluC. Treatment with each of these proteases converts the receptor from a homodimer of approximately 95 kDa subunits to a 44-kDa form, based on hydrodynamic measurements by sucrose density gradient centrifugation and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of photoaffinity-labeled preparations indicates that the oxysterol binding site is on a 28-kDa fragment within the 44-kDa limit form of the receptor. The limit proteolytic form exhibits the high affinity and structural specificity for oxysterols of the native dimeric receptor with an increase in the rate constant of association for 25-hydroxycholesterol. The proteolytic form also shows an increased binding affinity for nonspecific DNA, but no sequence specificity for the oxysterol regulatory element from the reductase gene was detected.
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PMID:A proteolytic fragment of the oxysterol receptor which retains oxysterol binding activity. 189 49


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