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)

The signal peptides of pre-aldehyde dehydrogenase (22-mer) and pre-ornithine transcarbamylase (27-mer) were chemically synthesized and their imports into rat liver mitochondria were studied. Both signal peptides were imported rapidly (within 2 min) in the absence of a membrane potential, exogenous ATP, or rabbit reticulocyte lysate. Signal peptides also were imported into mitochondria treated with a low concentration of trypsin which removed the outer membrane proteins. It was concluded that the chemically synthesized signal peptide could be imported differently than the precursor proteins. The imported signal peptide were found to be associated with both outer and inner membranes. Pulse-chase experiments showed that the import was unidirectional and that the signal peptides associated with inner membranes increased during the chase time. The signal peptides inhibited import of precursor proteins to different extents. Association of signal peptides with inner membrane near or at translocator sites might result in inhibition of precursor import.
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PMID:Import of chemically synthesized signal peptides into rat liver mitochondria. 238 52

Human polymorphonuclear leukocyte elastase (PMN elastase) is inhibited by L-659,286 (7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo-1,2,4- triaz-in-3-yl)thio]methyl]-5-thia-1-aza-6R-bicyclo[4.2.O]oct-2-ene -2- pyrrolidine carboxamide-5,-dioxide) with a Ki of 0.4 microM. This inhibition is time-dependent, rapid, and only slowly reversible, with a t1/2 of greater than 3 days at 25 degrees C. L-659,286 is also highly selective for PMN elastase, as it does not inhibit thrombin, trypsin, papain, plasmin, chymotrypsin, or cathepsin G. L-659,286 administered intratracheally inhibits lung damage caused by administration via the same route of human PMN elastase into hamsters. In marmosets, L-659,286 is cleared from blood very rapidly after an intravenous injection but is recovered in bronchoalveolar lavage fluid for several hours after intratracheal administration.
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PMID:Pharmacological profile of the substituted beta-lactam L-659,286: a member of a new class of human PMN elastase inhibitors. 249 9

We have investigated mitochondrial import and processing of the precursor for human ornithine transcarbamylase (OTC; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) in HeLa cells stably transformed with cDNA sequences encoding OTC precursors carrying mutations in their leader peptides. The mutant precursors studied included two with amino acid substitutions in the 32-amino-acid leader peptide (glycine for arginine at position 23, designated gly23; glycines for arginines at positions 15, 23, and 26, designated gly15,23,26) and two with deletions (deletion of residues 8 to 22, designated d8-22; deletion of residues 17 to 32, designated N16). Specific immunoprecipitation with anti-OTC antiserum of extracts of L-[35S]methionine-labeled cells expressing these mutations yielded only precursor species; neither mature nor intermediate-size OTC subunits were observed. Fractionation of radiolabeled cells, however, revealed important differences among the various mutants: the gly23 precursor was associated with mitochondria and was not detected in the cytosol; the d8-22 and N16 precursors were found with both the mitochondrial fraction and the cytosol; only the gly15,23,26 precursor was detected exclusively in the cytosol. A large fraction of each of the mitochondrially associated OTC species was in a trypsin-protected compartment. In particular, the gly23 precursor behaved in trypsin protection and mitochondrial fractionation studies in a manner consistent with its translocation into the mitochondrial matrix. On the other hand, the lack of binding of the gly23 protein to a delta-N-phosphonoacetyl-L-ornithine affinity column, which specifically recognizes active OTC enzyme, indicated that, despite its intramitochondrial location, the mutant protein did not assemble into the normal, active trimer. Further, the gly23 mutant precursor was unstable within the mitochondria and was degraded with a t1/2 of less further than 4 h. Thus, we have shown that, in intact HeLa cells, cleavage of the OTC leader peptide is not required for translocation into mitochondria, but is required for assembly into active enzyme.
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PMID:Mitochondrial import and processing of mutant human ornithine transcarbamylase precursors in cultured cells. 324 50

The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.
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PMID:Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide. 369 95

Most mitochondrial proteins are encoded in the nucleus and synthesized in the cytoplasm as larger precursors containing NH2-terminal 'leader' peptides. To test whether a leader peptide is sufficient to direct mitochondrial import, we fused the cloned nucleotide sequence encoding the leader peptide of the mitochondrial matrix enzyme ornithine transcarbamylase (OTC) with the sequence encoding the cytosolic enzyme dihydrofolate reductase (DHFR). The fused sequence, joined with SV40 regulatory elements, was introduced along with a selectable marker into a mutant CHO cell line devoid of endogenous DHFR. In stable transformants, the predicted 26-K chimeric precursor protein and two additional proteins, 22 K and 20 K, were detected by immunoprecipitation with anti-DHFR antiserum. In the presence of rhodamine 6G, an inhibitor of mitochondrial import, only the chimeric precursor was detected. Immunofluorescent staining of stably transformed cells with anti-DHFR antiserum produced a pattern characteristic of mitochondrial localization of immunoreactive material. When the chimeric precursor was synthesized in a cell-free system and incubated post-translationally with isolated rat liver mitochondria, it was imported and converted to a major product of 20 K that associated with mitochondria and was resistant to proteolytic digestion by externally added trypsin. Thus, both in intact cells and in vitro, a leader sequence is sufficient to direct the post-translational import of a chimeric precursor protein by mitochondria.
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PMID:A leader peptide is sufficient to direct mitochondrial import of a chimeric protein. 389 25

Carbamyl phosphate synthetase I and ornithine transcarbamylase are matrix enzymes synthesized outside the mitochondria in the form of larger precursors and are transported rapidly into mitochondria, in association with post-translational proteolytic processing to the mature enzymes. Treatment of isolated rat hepatocytes with 40 micrograms/ml of rhodamine 123 resulted both in a potent inhibition of the processing of the enzyme precursors and in accumulation of the precursors. In pulse-chase experiments, the labeled precursor disappeared much more slowly in the presence of the dye. Rhodamine 123 strongly inhibited the uptake and processing of the ornithine transcarbamylase precursor by isolated rat liver mitochondria. Other positively charged rhodamines such as rhodamines 6G and 6GX were also strongly inhibitory. On the other hand, rhodamine B which has no net charge was much less inhibitory. These results suggest that the positively charged rhodamines inhibit the binding of the positively charged enzyme precursors to a negatively charged protein(s) or to phospholipids of the mitochondrial outer membrane. Potassium and magnesium ions, and probably a cytosolic protein(s), were required for the maximal uptake and processing of the ornithine transcarbamylase precursor by the isolated mitochondria. The concentrations of potassium and magnesium ions required for the maximal transport and processing were about 120 mM and 0.8-1.6 mM, respectively. Dialyzed postribosomal supernatant of rabbit reticulocyte lysate (36-72 mg protein/ml), in combination with potassium and magnesium ions, stimulated the precursor transport and processing 3- to 4-fold. The stimulatory activity of the dialyzed lysate was inactivated by trypsin treatment or heat treatment. No significant amount of the enzyme precursor was associated with the mitochondria when incubation was performed in the absence of these compounds. All these results indicate that potassium and magnesium ions, and probably a cytosolic protein(s), are required for the binding of the ornithine transcarbamylase precursor to the mitochondria or its transport into the organelle.
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PMID:Synthesis, intracellular transport and processing of mitochondrial urea cycle enzymes. 654 81

Ornithine transcarbamylase (OTC) is very stable in rat liver homogenates, or mitochondria. However, pure OTC from rat or beef liver is inactivated by broken lysosomes. ATP and a number of purine nucleotides, particularly epsilon-ATP, stimulate the inactivation of OTC by broken lysosomes. Inactivation of OTC by trypsin and elastase is also stimulated by epsilon-ATP. Carbamoyl phosphate protects OTC against proteolytic attack by elastase or by broken lysosomes. Thus, environmental factors, e.g., ATP and carbamoyl phosphate, appear to influence OTC stability to proteases.
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PMID:ATP and other purine nucleotides stimulate the inactivation of ornithine transcarbamylase by broken lysosomes. 655 91

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

Fluorescence protease assays were investigated with peptide substrates containing a 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine (Dbo) as a fluorescent amino acid. The special characteristic of the fluorophore Dbo is its exceedingly long fluorescence lifetime (ca. 300 ns in water under air), which allows the use of nanosecond time-resolved fluorescence (Nano-TRF) detection to efficiently suppress shorter-lived background emission. In addition, the natural amino acids tryptophan and tyrosine can be employed as intramolecular fluorescence quenchers, which facilitates substrate design. Fourteen synthetic peptide substrates (composed of 2-19 amino acids) and five enzymes (trypsin, pepsin, carboxypeptidase A, leucine aminopeptidase, and chymotrypsin) were investigated and, in all 28 examined combinations, enzymatic activity was detected by monitoring the increase in steady state fluorescence with time and determining the reaction rates as kcat/Km values, which ranged from 0.2 to 80x10(6) M-1 min-1. The results suggest an excellent compatibility of the very small and hydrophilic fluorescent probe Dbo with solid-phase peptide synthesis and the investigated proteases. For all 14 peptides the fluorescence lifetimes before and after enzymatic cleavage were measured and Nano-TRF measurements were performed in 384-well microplates. The fluorescence lifetimes of the different peptides provide the basis for the rational design of Dbo-based fluorescent substrates for protease assays. Measurements in Nano-TRF mode revealed, in addition to efficient suppression of background fluorescence, an increased differentiation between cleaved and uncleaved substrate. The Dbo-based assays can be adapted for high-throughput screening.
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PMID:Design of peptide substrates for nanosecond time-resolved fluorescence assays of proteases: 2,3-diazabicyclo[2.2.2]oct-2-ene as a noninvasive fluorophore. 1713 73

We report the use of the macrocyclic host cucurbit[7]uril (CB7) as a supramolecular additive in nanosecond time-resolved fluorescence (Nano-TRF) assays for proteases to enhance the discrimination of substrates and products and, thereby, the sensitivity. A peptide substrate was labeled with 2,3-diazabicyclo[2.2.2]oct-2-ene (DBO) as a long-lived (>300 ns) fluorescent probe and 3-nitrotyrosine was established as a non-fluorescent fluorescence resonance energy transfer (FRET) acceptor that acts as a "dark quencher." The substrate was cleaved by the model proteases trypsin and chymotrypsin and the effects of the addition of CB7 to the enzyme assay mixture were investigated in detail using UV/VIS absorption as well as steady-state and time-resolved fluorescence spectroscopy. This also allowed us to identify the DBO and nitrotyrosine residues as preferential binding sites for CB7 and suggested a hairpin conformation of the peptide, in which the guanidinium side chain of an arginine residue is additionally bound to a vacant ureido rim of one of the CB7 hosts.
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PMID:Interaction of Cucurbit[7]uril With Protease Substrates: Application to Nanosecond Time-Resolved Fluorescence Assays. 3313 64


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