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)

Location of electron transport chain components in chloroplast membranes of chlamydomonas reinhardi, y-1 was investigated by use of proteolytic digestion with soluble or insolubilized trypsin. Digestion of intact membrane vesicles with soluble trypsin inactivates the water-splitting system, the 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition site of Photosystem II, the electron transport between the two photosystems as well as the ferredoxin NADP reductase. Reduction of NADP with artificial electron donors for Photosystem I could be restored, however, by addition of purified reductase to trypsin-digested membranes. Electron transfer activities of Photosystems I and II reaction centers were resistant to trypsin digestion either from outside or from within the thylakoids when active trypsin was trapped inside the membrane vesicles by sonication and digestion carried out in the presence of trypsin inhibitor added from outside. In the latter case, the water-splitting system was also found to be resistant to digestion. Polyacrylamide-bound insolubilized trypsin inactivated only the ferredoxin NADP reductase. Photosynthetically active membranes obtained at different stages of development showed a basically similar behavior toward trypsin.
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PMID:Trypsin-sensitive photosynthetic activities in chloroplast membranes from Chlamydomonas reinhardi, y-1. 0 Apr

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

The bovine adrenal cortex adrenodoxin gene was inserted into pTZ19 under T7 promoter control. The adrenodoxin mRNA was synthesized with T7 RNA polymerase and then translated in the reticulocyte cell-free translation system. The protein product was identified as the adrenodoxin precursor with molecular weight 22000. The import of the precursor into isolated yeast mitochondria was carried out. The protein was found to be inserted into the trypsin-insensitive compartment of mitochondria via an energy dependent way. This resulted in the processing of the precursor to the 12000-mature form. Thus, the precursor of mammalian adrenodoxin can be normally imported into yeast mitochondria.
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PMID:[Synthesis of a bovine adrenodoxin precursor in vitro and its import into yeast mitochondria]. 150 62

The precursor of bovine adrenodoxin (pAd), a mitochondrial protein, was expressed in Escherichia coli. The cloned cDNA of pAd was ligated to an expression vector pET-3d, and silent mutations were introduced into the N-terminal portion of the cDNA in order to increase the expression. The precursor was highly expressed (approximately 20% of the total cell protein) as the inclusion body, and contained an iron-sulfur center as judged from its optical absorption spectra. The inclusion body was solubilized with 7 M urea and pAd was purified in the presence of urea. The purified pAd was efficiently imported into isolated bovine adrenal cortex mitochondria and processed to the mature form. The import reaction required ATP inside the mitochondria in addition to the inner membrane potential, and was strongly inhibited by trypsin treatment of the mitochondria, as in the case of the in vitro translated precursor. It was, however, not dependent on the unfolding activity of the cytosolic factor with extramitochondrial ATP.
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PMID:Characterization of adrenodoxin precursor expressed in Escherichia coli. 161 34

Bovine, porcine and sheep adrenodoxin, and the trypsin-resistant form of bovine adrenodoxin have been studied by one- and two-dimensional 1H-NMR spectroscopy. Assignment of the resonances for all the aromatic amino acids with resolved aromatic resonances have been made by correlating NMR spectra with the amino acid sequences from various species. Slowly exchanging amide protons and downfield shifted alpha-protons of His10 and Phe11 suggest possible involvement in beta-sheet structure. The effects on the assigned resonances due to the specific spin-label with a nitroxide radical at Cys95 have been analyzed on a two-dimensional 1H-NMR spectrum. The present results provide evidence for a structural similarity with a model for the structure of adrenodoxin based on a sequence alignment with that of Spirulina platensis ferredoxin, for which X-ray crystallographic data is available. epsilon-Methyl groups of Met120 and Met122 have been assigned by comparing 1H-NMR spectra of adrenodoxin with those of the trypsin-resistant form of adrenodoxin which is specifically cleaved at Arg115. epsilon-Methyl groups of Met120 and Met122 have an exceptionally long longitudinal relaxation time compared with those of valyl and leucyl methyl groups, suggesting that the COOH-terminal peptide spanning over 13 amino acids rotates rather freely in the solvent.
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PMID:Proton nuclear magnetic resonance investigation of adrenodoxin. Assignment of aromatic resonances and evidence for a conformational similarity with ferredoxin from Spirulina platensis. 202 4

A second thioredoxin, Ch1, distinct from the one recently reported [Decottignies, P., Schmitter, J.M., Jacquot, J. P., Dutka, S., Picaud, A. & Gadal, P. (1990) Arch, Biochem. Biophys. 280, 112-121] has been purified from the green alga, Chlamydomonas reinhardtii, and its functional and structural properties investigated. Its activity in various enzymatic assays has been compared with the activities of different plant thioredoxins (Ch2 from C. reinhardtii and spinach m and f). Ch1 cannot serve as a substrate for Escherichia coli thioredoxin reductase, but can be reduced by spinach ferredoxin-thioredoxin reductase. It is less efficient than its spinach counterpart in the activation of corn leaf NADP-dependent malate dehydrogenase by light or dithiothreitol, and it only activates spinach fructose-1,6-bisphosphatase at very high concentrations. The complete primary structure of C. reinhardtii thioredoxin Ch1 was determined by automated Edman degradation of the intact protein and of peptides derived from trypsin, chymotrypsin and Staphylococcus aureus V8 protease digestions. When needed, peptide masses were verified by plasma desorption mass spectrometry. Ch1 consists of a polypeptide of 111 amino acids (11634 Da) and contains the well-conserved active site sequence Trp-Cys-Gly-Pro-Cys. Compared to thioredoxins from other sources, the algal thioredoxin Ch1 displays few sequence similarities with all the thioredoxins sequenced so far. Preliminary evidence indicates that Ch1 may be an h-type thioredoxin.
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PMID:Characterization and primary structure of a second thioredoxin from the green alga, Chlamydomonas reinhardtii. 204 Mar 9

The complete primary structure of a Streptomyces griseus (ATCC 13273) 7Fe ferredoxin, which can couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450soy for NADPH-dependent substrate oxidation, has been determined by Edman degradation of the whole protein and peptides derived by Staphylococcus aureus V8 proteinase and trypsin digestion. The protein consists of 105 amino acids and has a calculated molecular weight, including seven irons and eight sulfurs, of 12,291. The ferredoxin sequence is highly homologous (73%) to that of the 7Fe ferredoxin from Mycobacterium smegmatis. The N-terminal half of the sequence, which is the Fe-S clusters binding domain, has more than 50% homology with other 7Fe ferredoxins. In particular, the seven cysteines known from the crystal structure of Azotobacter vinelandii ferredoxin I to be involved in binding the two Fe-S clusters are conserved.
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PMID:Primary structure of a 7Fe ferredoxin from Streptomyces griseus. 210 13

The incubation of chloroplast fructose-1,6-bisphosphatase with both dithiothreitol and protein denaturants made sulfhydryl groups available for reaction with [1-14C]iodoacetamide (10-12 mol iodoacetamide incorporated/mol enzyme). Digestion of S-carboxyamidomethylated enzyme with trypsin and polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, yielded two 14C-labeled fragments whose apparent molecular mass were 10 kDa and 16 kDa. In the absence of either dithiothreitol or protein denaturants the incorporation of iodoacetamide to the enzyme was lower than 4 mol. When chloroplast fructose-1,6-bisphosphatase was initially incubated with dithiothreitol (2.5 mM) and (a) high concentrations of both fructose 1,6-bisphosphate (4 mM) and Ca2+ (0.3 mM) or (b) low concentrations of both fructose 1,6-bisphosphate (0.8 mM) and Ca2+ (0.05 mM) in the presence of either 2-propanol (15%, by vol.), trichloroacetate (0.15 M) or chloroplast thioredoxin-f (0.5 microM) and subsequently subjected to proteolysis and electrophoresis, S-carboxyamidomethylated tryptic fragments had similar molecular masses. Thus, conditions that stimulated the specific activity of chloroplast fructose-1,6-bisphosphatase caused conformational changes which favoured both the reduction of disulfide bridges and the exposure of sulfhydryl groups. In this aspect, thioredoxin exerted structural and kinetic effects similar to compounds not involved in redox reactions (organic solvents, chaotropic anions). These results indicated that the modification of hydrophobic (intramolecular) interactions in chloroplast fructose-1,6-bisphosphatase constituted the underlying mechanism in light-activation by the ferredoxin-thioredoxin system.
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PMID:Concerted action of cosolvents, chaotropic anions and thioredoxin on chloroplast fructose-1,6-bisphosphatase. Reactivity to iodoacetamide. 255 90

Purified cytochrome P-45011 beta from bovine adrenocortical mitochondria was successfully incorporated into the liposome membranes composed of phosphatidylcholine, phosphatidylethanolamine and cardiolipin at a molar ratio of 2:2:1. The incorporation of P-45011 beta into the liposome membranes was ascertained by the Ficoll density gradient centrifugation and the protein refractoriness to trypsin digestion. The prepared proteoliposomes containing P-45011 beta and phospholipid at a molar ratio of 1:3000 were unilamellar vesicles of about 40 nm in average diameter. The P-45011 beta embedded in the liposome membranes was found to be more stable than the detergent-solubilized form. The reconstituted system containing the P-45011 beta-proteoliposomes, adrenodoxin and NADPH-adrenodoxin reductase showed catalytic activities not only for the hydroxylation of 11-deoxycorticosterone at 11 beta- and 18-positions but also for its conversion into aldosterone with a turnover number of 2.3 nmol/min per nmol of P-45011 beta. A successive reaction without the intermediates leaving from the enzyme was suggested for the P-45011 beta-mediated conversion of 11-deoxycorticosterone to aldosterone following the result that the formation of aldosterone was linear with respect to time without the lag phase; this was confirmed by the result that radioactivity in aldosterone from 3H-labeled 11-deoxycorticosterone was scarcely decreased by the addition of unlabeled intermediates to the reactions system.
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PMID:Adrenal cytochrome P-45011 beta-proteoliposomes catalyzing aldosterone synthesis: preparation and characterization. 276 39

The immunochemical relatedness between human and bovine proteins catalyzing the cholesterol side-chain cleavage reaction was investigated. In dot-immunobinding analysis, antibodies against bovine adrenocortical cytochrome P-450SCC, adrenodoxin, and adrenodoxin reductase recognized the corresponding proteins in a dose-dependent manner in mitochondrial preparations from human placenta. Limited proteolysis with trypsin cleaved bovine P-450SCC into fragments F1 and F2, which represent the NH2- and C-terminal parts of P-450SCC, respectively. Identical trypsin treatment yielded similar-size fragments from human placental P-450SCC. In Western immunoblots, anti-F1 and anti-F2 antibodies recognized the corresponding fragments in both trypsin-digested bovine and human P-450SCC. Antibodies against bovine P-450SCC, fragments F1 and F2, adrenodoxin and adrenodoxin reductase inhibited cholesterol side-chain cleavage activity in bovine adrenocortical mitochondria by 24-51%, but failed to affect the activity in human placental mitochondria. These data indicate that human and bovine P-450SCC share common antigenic determinants located outside the enzyme active site. The immunological similarity between bovine adrenodoxin and human ferredoxin allowed for a simple purification protocol of human placental P-450SCC by adrenodoxin affinity chromatography. The P-450SCC obtained by this method was electrophoretically homogeneous and showed characteristics typical to P-450SCC.
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PMID:Comparison of the immunochemical properties of human placental and bovine adrenal cholesterol side-chain cleavage enzyme complex. 279 61


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