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 complete amino acid sequence of the coat protein of RNA bacteriophage PRR1 is presented. After thermolysin digestion, 26 peptides were isolated, covering the complete coat protein chain. Their alignment was established in part using automated Edman degradation on the intact protein, in part with overlapping peptides obtained by enzymic hydrolysis with trypsin, pepsin, subtilisin and Staphylococcus aureus protease, and by chemical cleavage with cyanogen bromide and N-bromosuccinimide. To obtain the final overlaps, a highly hydrophobic, insoluble tryptic peptide was sequenced for seven steps by the currently used manual dansyl-Edman degradation procedure, which was slightly modified for application on insoluble peptides. PRR1 coat protein contains 131 amino acids, corresponding to a molecular weight of 14534. It is highly hydrophobic, and the residues with ionizable side chains are distributed unevenly: acidic residues are absent in the middle third of the sequence, whereas a clustering of basic residues occurs between positions 44 and 62. PRR1 coat protein was compared with the coat proteins of RNA coliphages MS2 and Q beta, and the minimum mutation distance was calculated for both comparisons. It is highly probable that PRR1. Q beta and MS2 share a common ancestor. The basic region present in the three coat proteins is recognized as an essential structural feature of RNA phage coat proteins.
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PMID:The primary structure of the coat protein of the broad-host-range RNA bacteriophage PRR1. 10 28

The nucleic acid binding and unwinding properties of wild-type Escherichia coli ribosomal protein S1 have been compared to those of a mutant form and a large trypsin-resistant fragment, both reported recently [J. Mol. Biol. 127, 41-45 (1979) and J. Biol. Chem. 254, 4309-4312 (1979). The mutant (m1-S1) contains 77% and the fragment (S1-F1) 66% of the polypeptide chain length (approximately 600 amino acid residues) of protein S1. The mutant is active in protein synthesis in vitro; the fragment, although retaining one or more of the functional domains of S1, is inactive in protein synthesis. We find that m1-S1 is is almost as effective as S1 in binding to poly(rU), phage MS2 RNA and simian virus 40 (SV40) DNA, and in unfolding poly(rU) and the helical structures present in MS2 RNA and phi X174 viral DNA. S1-F1, however, binds to poly(rU) and denatured SV40 DNA, but not to MS2 RNA. It unfolds neither poly(rU), nor the residual secondary structure of MS2 RNA or phi X174 viral DNA. Thus, there appears to be a correlation between the loss in ability of S1 to unwind RNA and the loss in its ability to function in protein synthesis.
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PMID:Nucleic acid binding and unfolding properties of ribosomal protein S1 and the derivatives S1-F1 and m1-S1. 23 41

The A-protein of coliphage MS2 was purified to a state of sufficient homogeneity to study its primary structure. The NH2-terminal sequence was determined for the first 8 residues. Comparison with the reported sequence of R17 protein (Weiner, A. M., Platt, T., and Weber, K. (1972) J. Biol. Chem. 247, 3242-3251) shows a difference at position 6 where alanine in R17 is replaced by threonine in MS2. The COOH-terminal sequence was shown to be -Arg-Leu-Ser-Arg, confirming the existence of UAG as the termination codon of the maturation protein (Comtreras, R., Ysebaert, M., Min Jou, W., and Fiers, W. (19731 Nature New Biol. 241, 99-101; Vandekerckhove, J., Nolf, F., and Van Montagu, M. C. (1973) Nature New Biol. 241, 102; Remaut E., and Fiers, W. (1972) J. Mol. Biol. 71, 243-261). Peptides obtained by enzymatic hydrolysis with trypsin were fractionated by a combination of gel filtration and paper electrophoresis and chromatography. Thirty-eight peptides were analyzed for amino acid composition and sequence. They provide information for 312 of the 393 residues of the A-protein polypeptide chain.
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PMID:Sequence of the A-protein of coliphage MS2. I. Isolation of A-protein, determination of the NH2- and COOH-terminal sequences, isolation and amino acid sequence of the tryptic peptides. 91 36

A genomic library of Mycoplasma hyopneumoniae was constructed by cloning random DNA fragments approximately 300 base pairs long in a fusion expression plasmid, pEx29, containing the N terminus of the phage MS2 polymerase under the control of the PL promoter of phage lambda. Clones that produced fusion proteins carrying surface-specific antigenic determinants were identified by using antiserum raised in a pig by intranasal inoculation of viable mycoplasmas. Rabbit antisera produced against gel-purified fusion proteins synthesized in Escherichia coli were analyzed by Western blotting to identify antigenically related mycoplasma components. Distinct mycoplasma proteins termed P90, P68, P50, P30, and P26 were identified. Evidence for the surface location of P90, P68, and P50 was provided by their sensitivity to trypsin and their comigration with lactoperoxidase-catalyzed iodinated proteins of intact mycoplasmas. Immune electron microscopy, performed with antiserum against the hybrid MS2-mycoplasma protein produced in E. coli and corresponding to P90, also showed that its antigenic determinant is associated with the mycoplasma surface.
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PMID:Surface proteins of Mycoplasma hyopneumoniae identified from an Escherichia coli expression plasmid library. 241 Mar 63

About 45% of the rotavirus SA11 VP3 gene was inserted into a thermoinducible expression plasmid under the control of phage lambda PL promoter. The primary translation product predicted on the basis of the plasmid construction was a hybrid protein in which the 98 amino-terminal amino acids of phage MS2 polymerase were followed by amino acids 42 to 387 of the VP3 protein, which included the region containing the cleavage sites associated with trypsin enhancement of infectivity. On induction, a polypeptide that had the expected mol. wt. and contained VP3-related amino acid sequences as judged by immunological criteria, was synthesized to a level representing about 15% of the total bacterial protein. When a bacterial lysate enriched for the fusion polypeptide was injected into mice, it induced antibodies which inhibited haemagglutination and neutralized SA11 rotavirus infectivity.
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PMID:Synthesis in Escherichia coli and immunological characterization of a polypeptide containing the cleavage sites associated with trypsin enhancement of rotavirus SA11 infectivity. 302 94

Ribosomes from gram-positive Micrococcus luteus contain an acidic protein (ML-S1). ML-S1 has been purified by chromatography of ribosomes on a poly(U)-Sepharose column and the purified protein has a mobility in sodium dodecyl sulphate/polyacrylamide gels similar to that of ribosomal protein S1 of Escherichia coli (apparent Mr 72,000). Protein ML-S1 reacted with E. coli anti-S1 serum with an immunological partial-identity reaction. ML-S1 also reacted with antibodies raised against two structural domains of E. coli S1 (the N-terminal ribosome-binding domain and central and C-terminal nucleic-acid-binding domain). Weak reaction with antiserum to the nucleic-acid-binding domain of E. coli S1 was observed. ML-S1 was digested with trypsin under mild and exhaustive conditions. Mild digestion resulted in the production of a trypsin-resistant core (ML-S1F1) like E. coli S1. The fragment pattern obtained after exhaustive digestion differed appreciably from that obtained with E. coli S1. ML-S1 bound to poly(U) as strongly as E. coli S1 and also showed appreciable binding to denatured DNA. Addition of ML-S1 to S1-depleted ribosomes from E. coli and M. luteus markedly stimulated the poly(U)-directed polyphenylalanine synthesis. Phage MS2-RNA-dependent translation was also found to be stimulated by ML-S1 although to a much lesser extent than the stimulation by E. coli S1. At a molar excess of ML-S1 to ribosomes the protein showed a similar inhibitory effect to E. coli S1 on polypeptide synthesis. Our data indicate that ML-S1 retained the structural domains important for its function despite certain structural differences from E. coli S1.
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PMID:Structural and immunochemical characterization of a ribosomal protein from gram-positive Micrococcus luteus which is functionally homologous to Escherichia coli ribosomal protein S1. 311 53

Recombinant unmethylated heterogeneous nuclear ribonucleoprotein particle (hnRNP) protein A1 was enzymatically methylated by nuclear protein/histone protein methylase I [Rajpurohit, Lee, Park, Paik and Kim (1994) J. Biol. Chem. 269, 1057-1082] and the effect of methylation on several physiocochemical properties was studied. The relative binding-affinity of methylated and unmethylated protein A1 to nucleic acid was quite different. This was observed by the elution behaviour of the protein A1 on a single-stranded DNA/cellulose column; the concentration of NaCl required to release the bound protein A1 was 0.59 M for the methylated and 0.63 M for the unmethylated, respectively. Employing isoelectrofocusing, pI values of the methylated and unmethylated proteins were found to be 9.41 and 9.48, respectively. Maximum fluorescence quenching of protein A1 in the presence of coliphage MS2-RNA was found to be 40% with methylated and 45% with unmethylated. When both species of protein A1 were subjected to controlled trypsin digestion, t1/2 of the methylated protein was 1.31 min and the unmethylated, 1.63 min. The difference in their t1/2 values was much greater in the presence of MS2-RNA; 2.4 min for the former and 4.3 min for the latter, indicating that the methylated species was less stabilized by the RNA than the unmethylated. All of the above results consistently suggested that the binding-property of hnRNP protein A1 to single-stranded nucleic acid was significantly reduced subsequent to its arginine-methylation. The biological significance of this observation is discussed.
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PMID:Effect of enzymic methylation of heterogeneous ribonucleoprotein particle A1 on its nucleic-acid binding and controlled proteolysis. 781 96

We show that a simple cell-free translation system from Escherichia coli, programmed with phage MS2 RNA, is able to infect F+ E. coli cells. The plaques appearing on the E. coli host strain are morphologically indistinguishable from those derived from normal phage MS2 infection. This effect is strictly translation-dependent, since an incomplete translation system or the system inhibited by antibiotics leads to no infection. The cell-free based infection is maximal under conditions favouring the highest synthesis of maturation protein (one of the four phage-encoded proteins). The infection is abolished when RNase A or trypsin treatment is included before addition of cells. Similarly, due to RNA and maturation protein degradation, the continued incubation of the translation mixture under protein synthesis conditions significantly decreases infectivity. These findings suggest the formation of 'minimal infectious units', simple complexes of MS2 RNA and maturation protein. Here we describe the first example of bacteriophage infectious unit formation directly performed in a cell-free translation system. A possible application of this phenomenon might be the construction of newly designed RNA vector delivery systems and, moreover, could be an approach for molecular evolution studies.
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PMID:Formation of bacteriophage MS2 infectious units in a cell-free translation system. 895 35

Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.
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PMID:Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry. 980 26

Heterogeneous nuclear RNP protein A1, one of the major proteins in hnRNP particle (precursor for mRNA), is known to be posttranslationally arginine-methylated in vivo on residues 193, 205, 217 and 224 within the RGG box, the motif postulated to be an RNA binding domain. Possible effect of NG-arginine methyl-modification in the interaction of protein A1 to nucleic acid was investigated. The recombinant hnRNP protein A1 was in vitro methylated by the purified nuclear protein/histone-specific protein methylase I (S-adenosylmethionine:protein-arginine N-methyltransferase) stoichiometrically and the relative binding affinity of the methylated and the unmethylated protein A1 to nucleic acid was compared: Differences in their binding properties to ssDNA-cellulose, pI values and trypsin sensitivities in the presence and absence of MS2-RNA all indicate that the binding property of hnRNP protein A1 to single-stranded nucleic acid has been significantly reduced subsequent to the methylation. These results suggest that posttranslational methyl group insertion to the arginine residue reduces protein-RNA interaction, perhaps due to interference of H-bonding between guanidino nitrogen arginine and phosphate RNA.
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PMID:Recent advances in protein methylation: enzymatic methylation of nucleic acid binding proteins. 989 55


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