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)

A homogeneous cytochrome P-450scc preparation with a specific enzyme content of 18 nmol/1 mg protein has been obtained using affinity chromatography on adrenodoxin-Sepharose under optimal conditions of the protein adsorption onto and desorption from the affinity sorbent. The data on the N-terminal amino acid sequence of the enzyme, along with the results of electrophoretic and spectrophotometric analyses favoured the multistage cholesterol transformation to pregnenolone to be catalyzed by single species of cytochrome P-450scc consisting of one polypeptide chain. Limited proteolysis of cytochrome P-450scc with trypsin resulted, at the initial stages, in the formation (in an equimolar ratio) of two large polypeptide fragments, I and II, with Mr 27000 and 22000, respectively. Prolonged action of trypsin led to the digestion of fragment II and the formation of a stoichiometric amount of fragment III, Mr of about 14000. Cytochrome P-450scc converted by trypsin into equimolar mixtures of fragments I and II or I and III retained the major spectral and functional properties of the native protein. The aspartyl-prolyl linkages, sulphhydryl groups, and surface tyrosine residues are distributed nonuniformly among fragments I and II. These data, as well as a different resistance of the fragments to the action of trypsin, suggest that cytochrome P-450scc consists of two independently folded domains linked with a short loop of the polypeptide chain, the domains being rigidly associated under neutral conditions.
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PMID:The domain structure of the cholesterol side-chain cleavage cytochrome P-450 from bovine adrenocortical mitochondria. 672 73

The mitochondrial proteins involved in adrenocortical steroidogenesis are synthesized as higher molecular weight precursors which require processing by the mitochondria to their mature sizes. The post-translational maturation of two of these proteins has been examined: the cholesterol side chain cleavage cytochrome P-450 (P-450scc) and the iron-sulfur protein, adrenodoxin. Total translation products synthesized in a cell-free system programmed by bovine adrenocortical poly(A+) RNA were incubated with isolated bovine adrenocortical or heart mitochondria followed by immunoisolation of radiolabeled P-450scc or adrenodoxin. In the presence of adrenocortical mitochondria, the precursor form of P-450scc was converted into a trypsin-resistant form that had the same molecular weight as mature P-450scc. Unlike adrenocortical mitochondria, heart mitochondria were unable to process the P-450scc precursor which remained unaltered and trypsin-sensitive. In addition, a matrix fraction of heart mitochondria did not cleave the P-450scc precursor. In contrast, the adrenodoxin precursor did not exhibit similar specificity as it was processed to the mature form by both adrenocortical and heart mitochondria. Also, the adrenocortical mitochondria were not restricted to processing endogenous proteins as they imported and cleaved the precursor to ornithine transcarbamylase. The results indicate that some mitochondrial precursor proteins have tertiary structures which allow them to be recognized by all mitochondria while other mitochondrial precursor proteins have structures recognizable by only specialized mitochondria.
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PMID:Discriminatory processing of the precursor forms of cytochrome P-450scc and adrenodoxin by adrenocortical and heart mitochondria. 673 44

The complete amino acid sequence for a 3Fe:3S ferredoxin from the "archaebacterium" Methanosarcina barkeri (DSM 800) was determined by repetitive Edman degradation on the whole protein and peptides derived from trypsin, thermolysin, and Staphylococcus aureus protease digestion. The protein has 59 residues of which 8 are cysteines. The latter have the same spacing and distribution as found for the clostridial-type 2 x 4Fe:4S ferredoxins. Also, the sequence had evidence of internal homology which is indicative of gene duplication prior to the divergence of the archaebacteria and the eubacteria. This is the first sequence to be reported for a methanogen ferredoxin and only the fourth for a 3Fe:3S ferredoxin from any source.
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PMID:Amino acid sequence of a 3Fe:3S ferredoxin from the "archaebacterium" Methanosarcina barkeri (DSM 800). 675 24

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent Km for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.
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PMID:Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids. 683 5

Protein insertion into mitochondrial outer membrane (OM) vesicles isolated from Neurospora crassa has recently been reported. The N. crassa OM vesicles retained the features of the intact mitochondria concerning the dependency of insertion on the receptor protein [A. Mayer et al. (1993) J. Cell Biol. 121, 1233-1243]. In this study, OM vesicles were purified from bovine adrenal cortex mitochondria, and unilamellar proteoliposomes were reconstituted from OM vesicles using heptyl beta-thioglucoside. Both OM vesicles and the reconstituted outer membrane vesicles (ROM) were able to import porin, but unable to import the precursor of adrenodoxin, which translocates across both the outer and inner membranes of intact mitochondria. Porin insertion into both OM vesicles and ROM was inhibited in the presence of purified recombinant adrenodoxin precursor and also by ATP depletion, and was dependent on the trypsin-sensitive membrane surface factor, suggesting that the purified OM vesicles as well as ROM retained the properties of the intact OM concerning porin insertion. The protein import machinery of OM seems to be functional for the outer membrane protein without the participation of the inner membrane. The successful reconstitution of the protein import activity from solubilized OM will pave the way for further biochemical characterization of the protein import machinery of OM.
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PMID:Reconstitution of import-competent outer membrane vesicles from mammalian mitochondria. 779 73

The flavoprotein ferredoxin-NADP reductase (FNR) was isolated from the unicellular green alga, Chlamydomonas reinhardtii. FNR is a monomeric protein containing one FAD and exhibiting ferredoxin-dependent cytochrome c reduction activity. Its complete primary structure was investigated by sequencing overlapping peptides generated by cleavage with trypsin and SV8 protease and confirmed by partial (80%) nucleotidic sequence. C. reinhardtii FNR contains 320 residues, corresponding to a calculated mass of 35,685 and 36,470 including FAD, in agreement with the values measured by laser desorption mass spectrometry. The combination of both amino acid and nucleotidic sequencing, in association with mass spectrometry of peptides, allowed the identification of two N epsilon-trimethyllysines at positions 83 and 89 and one N epsilon-dimethyllysine at position 135. Comparison of the primary structure of C. reinhardtii FNR with the known sequences shows 41-46% identity.
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PMID:Primary structure and post-translational modification of ferredoxin-NADP reductase from Chlamydomonas reinhardtii. 784 Jun 25

Mitochondrial import stimulation factor (MSF) unfolds wheat germ lysate synthesized aggregated mitochondrial precursor proteins and stimulates their mitochondrial import in an ATP dependent manner. Here we analysed the function of MSF mainly by utilizing chemically pure adrenodoxin precursor (pAd). MSF bound to the unfolded pAd and prevented it from losing import competence and also restored the import competence of the aggregated pAd dependent on ATP hydrolysis. The import incompetent aggregated mitochondrial precursors induced the ATPase activity of MSF and the activity was strongly inhibited by isolated mitochondrial outer membrane (OM) but not by trypsin treated outer membrane (tOM). The precursor induced ATPase activity of N-ethylmaleimide (NEM)-treated MSF was not inhibited by OM. In this context, the MSF-precursor complex specifically bound to OM and binding was abolished both by the treatment of OM with trypsin and by the treatment of MSF with NEM. These results show that MSF is a novel cytoplasmic chaperone protein with a mitochondrial precursor-targeting function.
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PMID:MSF, a novel cytoplasmic chaperone which functions in precursor targeting to mitochondria. 795 79

Cys264 of cytochrome P450scc (CYP11A1) was selectively labelled with diiodofluorescein iodacetamide in solution and in proteoliposomes. The labelling affected the interaction of P450scc with adrenodoxin and significantly inhibited the side-chain cleavage activity of the soluble and membrane-bound hemeprotein in the reconstituted system. In proteoliposomes both the labelled and unlabelled hemeproteins were susceptible to trypsin and split into F1 and F2, two fragments corresponding to the two main domains of P450scc. These results suggest that the hinge connecting the two domains in the region Arg250-Asn257 is exposed to the surface of the membrane and involved in the interaction of P450scc with adrenodoxin.
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PMID:Selective chemical modification of Cys264 with diiodofluorescein iodacetamide as a tool to study the membrane topology of cytochrome P450scc (CYP11A1). 811 13

Bovine adrenocortical cytochrome P450scc (CYP11A1) was selectively modified with diiodofluorescein iodoacetamide (DIFIA). Only Cys264 is labeled in the P450 polypeptide chain. The modification significantly affected the cholesterol-hydroxylating activity in the reconstituted system containing NADPH, adrenodoxin reductase, adrenodoxin, and soluble or membrane-bound P450scc. The inhibitory effect correlates with decreased affinity of cytochrome P450scc to intermediate electron carrier, adrenodoxin. Cytochrome P450scc is modified in liposomes and the modified membrane-bound protein is cleaved by trypsin forming two large fragments F1 and F2 corresponding to the N- and C-terminal regions of the molecule. The data indicate that the Cys264-containing region of the cytochrome P450scc molecule is exposed to the surface of protein globule, located outside of the membrane, and can participate in protein-protein interactions.
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PMID:[Selective chemical modification of cytochrome P450scc (CYP11A1) with diiodofluorescein iodoacetamide in the study of the role and topology of interdomain hinge of the hemoprotein molecule]. 915 54

In many physiological studies dehydroascorbate (DHA) reductase is regarded as one of the chloroplast enzymes involved in the protection against oxidative stress. Here, evidence is presented that plant cells do not possess a specific DHA reductase. The DHA reductase activities measured in plant extracts are due to side reactions of proteins containing redox-active dicysteine sites. Native gel electrophoresis combined with specific activity staining revealed three different proteins with DHA reductase activity in leaf and chloroplast extracts. These proteins have been identified as thioredoxins and trypsin inhibitors (Kunitz type) by Western blot analysis. The essential regulatory functions of thioredoxins in chloroplast metabolism are strongly inhibited in the presence of as little as 50 microM DHA. Thus, the intracellular DHA concentration should be kept below 50 microM but not all proteins with DHA reductase activity are effective enough for this purpose. A specific DHA reductase is frequently demanded as part of the enzymatic equipment to avoid oxidative stress. We argue that this is not necessary because in chloroplasts DHA does not accumulate to any significant extent due to the high activities of monodehydroascorbate reductase and of reduced ferredoxin.
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PMID:Dehydroascorbate and dehydroascorbate reductase are phantom indicators of oxidative stress in plants. 932 37


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