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 lipid dependency of apocytochrome c binding to model membranes and of the translocation of the precursor protein across these membranes was studied by using large unilamellar, trypsin-containing vesicles. These vesicles were improved with respect to those used in a previous article (Rietveld, A., and de Kruijff, B. (1984) J. Biol. Chem. 259, 6704-6706), in the sense that a lower amount of trypsin was enclosed. In mixed egg phosphatidylcholine/bovine brain phosphatidylserine vesicles, both the Kd of apocytochrome c binding (about 20 microM) and the number of phosphatidylserine molecules interacting with the protein was found to be constant. When the phosphatidylserine fraction in the vesicles is more than 15-30% apocytochrome c addition results in the exposure of (a part of) the protein to the internal, trypsin-containing vesicle medium, which process we conceive as a translocation event. Also the interaction of apocytochrome c with vesicles composed of phosphatidylcholine and another acidic phospholipid in a 1:1 ratio, leads to the translocation of the protein across the model membrane. The affinity of this binding was found to be in the order cardiolipin greater than phosphatidylglycerol greater than phosphatidylinositol greater than phosphatidylserine. By varying the lipid composition of the vesicles, it could be demonstrated that the translocation requires a fluid bilayer. In addition, protein specificity was shown for the translocation process. Although apocytochrome c-lipid interaction causes vesicle aggregation, fusion by lipid mixing could not be detected. Due to the apocytochrome c-lipid interaction also, protein aggregates and oligomers have been formed. These results will be discussed in the light of a model for translocation of a precursor protein across a model membrane. The relevance for the mitochondrial system will also be discussed.
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PMID:Studies on the lipid dependency and mechanism of the translocation of the mitochondrial precursor protein apocytochrome c across model membranes. 300 2

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

A search for maturating peptidases of the precursor protein of the mating hormone (pheromone) alpha-factor of Saccharomyces cerevisiae was performed using short model peptides representing those sequences of the precursor protein, where cleavage is thought to occur in vivo. This search was done in a mutant lacking several of the unspecific vacuolar peptidases. The chromogenic peptide Cbz-Tyr-Lys-Arg-4-nitroanilide led to the detection of a membrane-bound enzyme called proteinase yscF. Cleavage of the synthetic peptide derivative occurs after the basic amino acid pair, a proposed signal for hormone processing. Optimum pH for the reaction is 7.2. The enzyme does not cleave after single basic amino acid residues indicating that it is distinct from trypsin-like proteinases. Proteolytic activity is enhanced by Triton X-100. The enzyme is strongly inhibited by EGTA, EDTA and mercurials but insensitive to phenylmethylsulfonyl fluoride. The enzyme activity is strongly dependent on Ca2+ ions. In a mutant (kex2), which accumulates an over-glycosylated alpha-factor precursor, no proteinase yscF activity can be found. Membrane-bound peptidase activity possibly involved in removal of the arginyl and lysyl residues remaining at the carboxy terminus of the alpha-factor pheromone peptide after the initial cut of the precursor molecule could be identified by using the model peptides Cbz-Tyr-Lys-Arg and Cbz-Tyr-Lys.
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PMID:Hormone processing and membrane-bound proteinases in yeast. 389 3

Maltose binding protein, like most periplasmic proteins, is resistant to a variety of proteinases. Treatment of pre-maltose binding protein with trypsin, chymotrypsin, or proteinase K removes an amino-terminal domain of the same approximate size as the leader sequence without degrading the mature portion of the protein. In addition, pre-maltose binding protein is as active as mature in binding maltose (Ferenci, T., and Randall, L.L. (1979) J. Biol. Chem. 254, 9979-9981). By these criteria, the precursor and mature proteins are in the same conformation except for the exposed leader sequence on the precursor. We have compared the ability of these proteins to interact with amphipaths, such as detergents. The precursor protein binds to Triton X-100, while the mature protein does not. We propose that the leader domain is responsible for detergent binding. Mutations in the leader region of the precursor which block export in vivo prevent detergent binding in vitro. A mutant with a mild export defect can still bind detergent. This correlation between detergent binding by precursors with related leaders and export efficiency of each precursor suggests that hydrophobic partition of the leader may initiate pre-protein transfer across the membrane.
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PMID:The leader region of pre-maltose binding protein binds amphiphiles. A model for self-assembly in protein export. 390 99

Human B-cell growth factor has been described as a trypsin-sensitive protein of Mr 12,000-14,000. Evidence is provided herein that this relatively low molecular weight product may be released from a larger precursor molecule of Mr 60,000-80,000. The precursor protein is confined to the cytosol of freshly isolated T lymphocytes, and only the Mr 12,000-14,000 moiety is released upon lectin stimulation. The precursor protein was subjected to limited tryptic digestion, which demonstrated that the biologically active fraction of the moiety resided in a relatively low molecular weight fragment. The T lymphocyte routinely possessed an intracytoplasmic pool of the precursor protein, the amount of which cyclically varied depending upon its depletion by the secretion process of a lower molecular weight product. Analysis of the mRNA size coding for the majority of B-cell growth factor activity, determined by translation in Xenopus laevis oocytes, suggested that the B-cell growth factor-specific mRNA resided in the greater than or equal to 15S range. This value is consistent with the size of the larger precursor. Therefore, it is proposed that a precursor-product relationship exists for the processing of human B-cell growth factor, analogous to that which has been described for several other cytokines.
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PMID:Evidence for an intracellular precursor for human B-cell growth factor. 608 64

Plasma prekallikrein, a precursor protein of kallikrein [EC 3.4.21.8], was highly purified from porcine plasma by chromatography on a DEAE-Sephadex A-50 column, followed by rechromatography on a DEAE-Sephadex A-50 column, chromatography on a CM-Sephadex C-50 column and affinity chromatography on a p-aminobenzamidine-epsilon-aminocaproic acid-Sepharose 4B column. By this procedure, 3.3 mg of purified material was obtained from 1.6 liters of porcine plasma and about 240-fold purification was achieved from the first DEAE-Sephadex A-50 chromatography. The purified protein was found to give a single band on sodium dodecyl sulfate (SDS)-polyacrylamide gel disc electrophoresis. This preparation did not contain kallikrein, Factor XII (Hageman factor) of the blood coagulation system, high molecular weight (HMW) kininogen or plasma kininase. Thus, the material is presumed to be functionally pure. The molecular weight of prekallikrein was estimated to be about 88,000 by SDS-polyacrylamide gel electrophoresis, and prekallikrein consists of a single polypeptide chain. Activation of prekallikrein by trypsin [EC 3.4.21.4] was found to involve the cleavage of a single peptide bond on the disulfide-bridged polypeptide chain, and no change of molecular weight was observed during the activation. This trypsin-activated kallikrein released kinin rapidly from bovine HMW kininogen. However, liberation of kinin was extremely slow from bovine low molecular weight (LMW) kininogen. The kallikrein activity was inhibited by soybean trypsin inhibitor (SBTI) and Trasylol, but not by Polybrene or egg-white trypsin inhibitor (EWTI).
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PMID:Purification of prekallikrein from porcine plasma and its conversion to active kallikrein. 655 87

The content of met-enkephalin (met-EK)-like peptides in a crude extract from intact pulp of the rat markedly increased with tryptic digestion but not with carboxypeptidase B(CPase B) digestion, while the content of leu-enkephalin (leu-EK)-like peptides more markedly with CPase B- than with tryptic digestion. On the other hand, results by High Performance Liquid Chromatography (HPLC) showed that the contents of both met-EK-Arg-Phe and leu-EK in cavity formed pulp increased 6 times as much as those contents in intact pulp, while the met-EK content in cavity formed pulp increased 2 times as much as that in intact pulp. Furthermore, results by gel filtration of crude extract from intact pulp showed that one peak of met-EK-like immunoreactivity (met-EK-IR) appeared at position of approximately 30,000 of molecular weight and a broad peak of leu-EK-IR appeared at position of approximately 60,000 of molecular weight following tryptic digestion. From these results, it was suggested that noxious stimuli might cause activation of trypsin-like enzymes followed by processing from one precursor protein to met-EK-like peptides as well as from another to leu-EK-like peptides in the rat incisor pulps.
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PMID:A possible relationship between processing from precursor proteins to opioid peptides and noxious stimulation in the rat incisor pulp. 666 44

The de novo biosynthesis of the heterotetrameric (alpha 2 beta 2) serum glycoprotein, haptoglobin, was studied using a rabbit reticulocyte cell-free translation system and mRNA preparations from the livers of turpentine-treated rats. Analysis of the translation mixtures with either antisera specific for the alpha-subunit (anti-alpha-IgG) or the beta-subunit (anti-beta-IgG) or the native haptoglobin tetramer (anti-alpha 2 beta 2-IgG) resulted in each instance in the recovery of a single protein exhibiting a Mr = approximately 38,000. Cleavage of the translation product with cyanogen bromide or trypsin resulted in the generation of small peptide fragments that were specifically immunoadsorbed with either anti-alpha-IgG or anti-beta-IgG. These results indicated that the primary translation product of haptoglobin mRNA is a single polypeptide that contains the elements of both the alpha-subunit and the beta-subunit of the protein. Presumably haptoglobin is synthesized in vitro as a single precursor protein that is proteolytically processed post-translationally to form the dissimilar alpha- and beta-subunits of the native protein.
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PMID:Haptoglobin. A novel mode of biosynthesis of a liver secretory glycoprotein. 745 86

We have investigated the cleavages at the flavivirus capsid-prM protein junction in vitro. When expressed in the absence of the flavivirus proteinase, capsid and prM, which are separated by an internal signal sequence, exist as a membrane-spanning precursor protein. Here we show the induction of posttranslational signal peptidase cleavage of prM by trypsin cleavage of a cytoplasmic region of this precursor protein.
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PMID:Posttranslational signal peptidase cleavage at the flavivirus C-prM junction in vitro. 749 34

The three-dimensional structures of a series of 6-kDa trypsin inhibitors isolated from the stigma of the ornamental tobacco Nicotiana alata have been determined by 1H NMR spectroscopy combined with simulated annealing calculations. The proteins, T1-T4, are proteolytically cleaved from a 40.3-kDa precursor protein, NA-proPI, together with a chymotrypsin inhibitor, C1, the structure of which was reported recently [Nielsen, K.J., Heath, R.L., Anderson, M.A., & Craik, D.J. (1994) J. Mol. Biol. 242, 231-243]. Each of the proteinase inhibitors comprises 53 amino acids, including 8 cysteine residues which are linked to form 4 disulfide bridges. The proteins have a high degree of sequence identity and differ mainly in residues around the putative reactive sites. The structure of T1 was determined using a set of 533 interproton distance restraints derived from NOESY spectra, combined with 33 dihedral restraints derived from 3JNH-H alpha coupling constants and 16 hydrogen bonds. The structures of the remaining inhibitors (T2-T4) were deduced to be almost identical to T1, on the basis of their similar chemical shifts and 2D spectra. The current study demonstrates that the structures of the trypsin inhibitors (T1-T4) are similar to that previously found for the chymotrypsin inhibitor, C1. Despite differences in sequence, there is conservation in backbone geometry between the reactive site loops of the two classes of inhibitors. From this, it is clear that the nature of the side chain on the primary binding residue, rather than the backbone fold, is the main determinant of the enzyme specificities of these proteinase inhibitors.
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PMID:Structures of a series of 6-kDa trypsin inhibitors isolated from the stigma of Nicotiana alata. 757 34


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