Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Immature Xenopus laevis oocytes contain large quantities of a 7S ribonucleoprotein particle containing transcription factor IIIA (TFIIIA) and 5S RNA in a 1:1 molar ratio. We have reconstituted RNPs containing 5S RNA and either intact TFIIIA or proteolytic fragments that represent progressive C-terminal deletions of the protein. A partial trypsin digestion fragment encompassing the amino terminal seven zinc fingers of TFIIIA rebinds 5S RNA with nearly the same affinity as intact TFIIIA. We have compared the RNase protection patterns resulting from binding of intact and deleted forms of TFIIIA. RNAse protection assays using cobra venom nuclease were performed on complexes reconstituted with 5' and 3' end-labeled 5S RNA. Similar experiments with 3' end-labeled 5S RNA were performed with nuclease alpha-sarcin. With both nucleases, nucleotides in helix V of 5S RNA show more complete protection from nuclease cleavage when the RNA is bound to intact TFIIIA than when it is bound to a 20 kDa tryptic fragment of TFIIIA lacking the C-terminal portion of the protein. These results suggest that fingers 8 and 9 of TFIIIA interact with the distal portion of helix V in the 5S RNA.
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PMID:The carboxyterminal zinc fingers of TFIIIA interact with the tip of helix V of 5S RNA in the 7S ribonucleoprotein particle. 182 69

alpha-Sarcin is a single polypeptide chain protein which exhibits antitumour activity by degrading the larger ribosomal RNA of tumour cells. We describe the interaction of a alpha-sarcin with lipid model systems. The protein specifically interacts with negatively-charged phospholipid vesicles, resulting in protein-lipid complexes which can be isolated by ultracentrifugation in a sucrose gradient. alpha-Sarcin causes aggregation of such vesicles. The extent of this interaction progressively decreases when the molar ratio of phosphatidylcholine increases in acidic vesicles. The kinetics of the vesicle aggregation induced by the protein have been measured. This process is dependent on the ratio of alpha-sarcin present in the protein-lipid system. A saturation plot is observed from phospholipid vesicles-protein titrations. The saturating protein/lipid molar ratio is 1:50. The effect produced by the antitumour protein on the lipid vesicles is dependent on neither the length nor the degree of unsaturation of the phospholipid acyl chain. However, the aggregation is dependent on temperature, being many times higher above the phase transition temperature of the corresponding phospholipid than below it. The effects of pH and ionic strength have also been considered. An increase in the ionic strength does not abolish the protein-lipid interaction. The effect of pH may be related to conformational changes of the protein. Binding experiments reveal a strong interaction between alpha-sarcin and acidic vesicles, with Kd = 0.06 microM. The peptide bonds of the protein are protected against trypsin hydrolysis upon binding to acidic vesicles. The interaction of the protein with phosphatidylglycerol vesicles does not modify the phase transition temperature of the lipid, although it decreases the amplitude of the change of fluorescence anisotropy associated to the co-operative melting of 1,6-diphenyl-1,3,5-hexatriene (DPH)-labelled vesicles. The results are interpreted in terms of the existence of both electrostatic and hydrophobic components for the interaction between phospholipid vesicles and the antitumour protein.
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PMID:Study of the interaction between the antitumour protein alpha-sarcin and phospholipid vesicles. 270 1

The complete amino acid sequence of the single polypeptide chain of cytotoxin restrictocin has been determined. Its structure was established by automated Edman degradation of the intact molecule reduced and [14C]carboxymethylated and of fragments obtained by chemical cleavage of the protein with cyanogen bromide and BNPS-skatole and by enzymatic cleavage of the polypeptide chain with trypsin. The molecule consists of 149 amino acid residues with a calculated relative molecular mass of 16836. The protein presents two disulfide bridges, one between cysteine residues at positions 5 and 147 and the other one formed by cysteine residues at positions 75 and 131. The amino acid sequence of restrictocin shows a high degree of homology (86%) with that of the cytotoxin named alpha-sarcin.
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PMID:The primary structure of the cytotoxin restrictocin. 647 66

alpha-Sarcin is a ribosome-inactivating protein that translocates across lipid bilayers, these two abilities explaining its cytotoxic character. This protein is composed of a single polypeptide chain with two disulfide bridges. Reduction and carboxyamidomethylation of alpha-sarcin results in protein unfolding, based on the results of the spectroscopic characterization of the chemically modified protein. The absorption and fluorescence emission bands of the tryptophan residues of the modified protein appear blue- and red-shifted, respectively. Far-UV circular dichroism analysis reveals the presence of residual secondary structure (beta-strands and turns) in the alkylated protein. This retains its ability to interact with lipid bilayers. It promotes vesicle aggregation, lipid-mixing between bilayers and leakage of the intravesicular aqueous contents. The modified protein tends to abolish the phase transition of acid phospholipids as detected by differential scanning calorimetry and depolarization measurements of fluorescence-labelled vesicles. The protein gain access to vesicle-entrapped trypsin. The fluorescence emission of the tryptophan residues is blue-shifted upon interaction of the protein with the bilayers, and anthracene incorporated into the hydrophobic core of the membranes quenches the tryptophan fluorescence emission of the protein. The secondary structure of the alkylated protein interacting with lipid vesicles has been studied by infrared spectroscopy. An increase in the alpha-helix and turn contents and a concomitant decrease in the beta-structure content are observed upon interaction with the bilayers. The results obtained are discussed in terms of the structural requirements for the interaction of alpha-sarcin with lipid membranes.
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PMID:Spectroscopic characterization of the alkylated alpha-sarcin cytotoxin: analysis of the structural requirements for the protein-lipid bilayer hydrophobic interaction. 754 65

alpha-Sarcin is a cytotoxic protein produced by the mould Aspergillus giganteus. Insertion of alpha-sarcin into asolectin membranes has been demonstrated by protein labelling with photoreactive phospholipids. alpha-Sarcin added externally to tRNA-containing asolectin liposomes degrades the entrapped tRNA. Trypsin-containing asolectin liposomes were also prepared. Encapsulated trypsin degrades alpha-sarcin, even in the presence of a large excess of external hen egg-white trypsin inhibitor to prevent any alpha-sarin degradation outside the vesicles. These processes occur only with acidic phospholipids and were not observed when phosphatidylcholine vesicles were used. These results indicate that alpha-sarcin penetrates the lipid bilayer and becomes exposed to the lumen of negatively charged liposomes.
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PMID:Translocation of alpha-sarcin across the lipid bilayer of asolectin vesicles. 821 20

Internalization of rotavirus in MA104 cells was found to induce coentry of alpha-sarcin, a toxin that inhibits translation in cell-free systems and to which cells are normally impermeable. Entry of the toxin, measured by inhibition of protein synthesis at early times after infection, correlated with virus penetration leading to expression of infectivity, since toxin entry (1) was induced only by trypsin-treated triple-layered virions, to a degree dependent on the toxin and the virus concentration; (2) correlated with the degree of permissivity of different cell lines to rotavirus infection; (3) was inhibited to a similar extent as infectivity by treatment of cells with neuraminidase; and (4) was inhibited by pre- or postadsorption incubation of the virus with neutralizing monoclonal antibodies to VP7 and VP4 (VP8*). Neither the virus infectivity nor the toxin coentry was significantly affected by treatment of cells with bafilomycin A1, an inhibitor of the vacuolar proton ATPase, indicating that both events are independent of the endosomal acid pH. Virus-like particles (VLP), composed of rotavirus proteins 2/6/7/4, but not 2/6/7 or 2/6, were able to induce toxin entry as efficiently as virions. Use of genetically modified VLP in combination with the toxin coentry assay, which measures entry through a productive pathway, should allow identification of the regions of the outer capsid proteins essential for rotavirus penetration.
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PMID:Productive penetration of rotavirus in cultured cells induces coentry of the translation inhibitor alpha-sarcin. 935 54