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 adaptation of facultative intracellular bacteria to host macrophages involves regulation of the synthesis of bacterial proteins. We analyzed the protein synthesis of Francisella tularensis LVS growing intracellularly in the macrophage-like murine cell line J774 and extracellularly in culture medium. After pulse-labeling with [35S] methionine and separation by one- and two-dimensional polyacrylamide gel electrophoresis, induction of a few proteins during intracellular growth was demonstrated. One of them, a 23-kDa protein, was prominently induced in the macrophages and also when extracellularly growing F. tularensis was exposed to hydrogen peroxide. After isolation of the 23-kDa protein from a preparative two-dimensional gel, a 22-amino-acid N-terminal peptide and two peptides obtained by trypsin digestion were sequenced. Based on the sequences, degenerate oligonucleotides were constructed for use as primers in a PCR. Hybridization of amplified DNA to XbaI-digested LVS DNA identified the gene of the 23-kDa protein in a 1.3-kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame encoding a putative protein of a calculated molecular mass of 22.2 kDa. The open reading frame was preceded by a sequence typical of ribosome-binding sites in Escherichia coli. The amplified gene was successfully expressed by the pTrc99A vector in E. coli under control of the trc promoter. The gene product showed the same mobility and immunoreactivity as the 23-kDa protein of F. tularensis. The deduced amino acid sequence showed no significant homology with protein sequences in current data banks. Thus, intracellular growth of F. tularensis in macrophages was associated with prominent upregulation of a novel 23-kDa protein.
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PMID:Identification of proteins of Francisella tularensis induced during growth in macrophages and cloning of the gene encoding a prominently induced 23-kilodalton protein. 916 49

Using the three-dimensional structures of thrombin and the leech-derived tryptase inhibitor (LDTI), which does not inhibit thrombin, we were able to construct three LDTI variants inhibiting thrombin. Trimming of the inhibitor reactive site loop to fit thrombin's narrow active site cleft resulted in inhibition constants (Ki) in the 10 nM concentration range; similar values were obtained by the addition of an acidic C-terminal peptide corresponding to hirudin's tail to LDTI. Combination of both modifications is additive, resulting in very strong inhibition of thrombin (Ki in the picomolar range). On the one hand, these results confirm the significance of the restricted active site cleft of thrombin in determining its high cleavage specificity; on the other, they demonstrate that sufficient binding energy at the fibrinogen recognition exosite can force thrombin to accept otherwise unfavorable residues in the active site cleft. The best inhibitor thus obtained is as effective as hirudin in plasma-based clotting assays.
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PMID:Structure-based design of a potent chimeric thrombin inhibitor. 924 61

The effect of lambda-toxin, a thermolysin-like metalloprotease of Clostridium perfringens, on the inactive epsilon-prototoxin produced by the same organism was examined. When the purified epsilon-prototoxin was incubated with the purified lambda-toxin at 37 C for 2 hr, the 32.5-kDa epsilon-prototoxin was processed into a 30.5-kDa polypeptide, as determined by SDS-polyacrylamide gel electrophoresis. A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD50) of the prototoxin with and without lambda-toxin treatment were 110 and 70,000 ng/kg of body weight, respectively. The lethal activity of the prototoxin activated by lambda-toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD50 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively. The epsilon-toxin gene was cloned and sequenced. Determination of the N-terminal amino acid sequence of each activated epsilon-prototoxin revealed that lambda-toxin cleaved between the 10th and 11th amino acid residues from the N-terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues. The molecular weight of each activated epsilon-prototoxin was also determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The C-terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C-terminus of the prototoxin, suggesting that removal of not only N-terminal but also C-terminal peptide is responsible for activation of the prototoxin.
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PMID:Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides. 927 98

An endogenous lipoxygenase inhibitor, purified from the cytosol of human epidermoid carcinoma A431 cells, was analyzed by N-terminal microsequencing and mass spectrometric analysis. The inhibitor was purified by SDS-PAGE, then subjected to in-gel CNBr cleavage and trypsin digestion. The N-terminal sequence data obtained from a 6-8 kDa band of in-gel CNBr cleavage and the three isolated peptides of in-gel trypsin digestion, and the C-terminal peptide sequence from matrix-assisted laser desorption ionization mass spectrometry matched the sequence of human phospholipid hydroperoxide glutathione peroxidase. The purified inhibitor exhibited peroxidase activity using phosphatidylcholine hydroperoxides as the substrate. We therefore concluded that the lipoxygenase inhibitor present in A431 cells was a phospholipid hydroperoxide glutathione peroxidase.
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PMID:Identification of a lipoxygenase inhibitor in A431 cells as a phospholipid hydroperoxide glutathione peroxidase. 953 8

Protoporphyrinogen oxidase (EC 1-3-3-4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the betaalphabeta ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the Km for protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.
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PMID:The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides. 972 41

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

Saccharomyces cerevisiae bearing a lipase cDNA from Fusarium heterosporum produced two lipases, A and B. Lipase B was significantly more stable to temperature than lipase A, but their optimum temperatures were similar. Lipase B was composed of one polypeptide (301 amino acids), and lipase A was composed of two polypeptides (275 and 26 amino acids) generated by the cleavage between Arg275 and Asp276 with a trypsin-like protease. It was suggested that the C-terminal peptide (26 amino acids) tightened the lipase structure when bound to the catalytic domain (275 amino acids) through a peptide bond. The tight structure was loosened by cleavage of the C-terminal peptide, even though the peptide interacted noncovalently with the catalytic domain, possibly through charged amino acids, in which it is rich. Deletion of the C-terminal peptide greatly decreased the lipase production by the recombinant S. cerevisiae, although its transcriptional level was the same as that of cells carrying the wild-type gene. These facts suggested that the C-terminal peptide affected the lipase production in the post-transcriptional step.
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PMID:C-terminal peptide of fusarium heterosporum lipase is necessary for its increasing thermostability. 983 17

A fucose sulfate glycoconjugate (FSG), a natural acrosome reaction-inducer, was purified from the egg jelly of the sea urchin Hemicentrotus pulcherrimus. The FSG is composed primarily of four constituents: a 120 kDa protein, a 237 kDa protein, a 258 kDa protein, and a polysaccharide-containing protein. Among them, the 120 kDa protein was thought to play a critical role in the association of other FSG constituent proteins, and therefore was characterized from a structural point of view. The protein was isolated from the carboxymethylated FSG by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, and then digested with trypsin to obtain information regarding the primary structure. Based on the partial amino acid sequences of three internal peptides (FSG120KA: LHNNEYGYGDTAAGEPELAQEEID, FSG 120KG: AIDIPAETGHYGR, and FSG120KC: RPTFDLADAVDT) and the N-terminal peptide (LHNNEYGYGDTAAGEPELAQQEID) of the 120 kDa protein obtained from intact FSG, degenerate oligonucleotide primers were synthesized and used to amplify a 297 bp cDNA fragment. This fragment enabled us to obtain the full-length cDNA (3176 bp) by 5'- and 3'-rapid amplification of cDNA ends. The deduced amino acid sequence revealed that the 120 kDa protein is composed of 663 amino acid residues including 72 cysteine residues, and hence, about 40% is presumed to be carbohydrate by weight. The 120 kDa protein plays an important role in the association of FSG constituent proteins (258 and 237 kDa) through disulfide bonds.
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PMID:Primary structure of a 120 kDa protein associated with the fucose sulfate glycoconjugate constituting the acrosome reaction-inducing substance of the sea urchin, Hemicentrotus pulcherrimus. 986 74

Voltage-dependent and calcium-sensitive K+ (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular Ca2+. In most tissues, their stimulation results in a noninactivating hyperpolarizing K+ current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane beta subunit (beta2) that yields inactivating MaxiK currents on coexpression with the pore-forming alpha subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the beta2 subunit abolished inactivation of the alpha subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle beta1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the beta2 subunit causes inactivation of the MaxiK channel alpha subunit by occluding its K+-conducting pore resembling the inactivation caused by the "ball" peptide in voltage-dependent K+ channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different beta subunits.
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PMID:Molecular basis of fast inactivation in voltage and Ca2+-activated K+ channels: a transmembrane beta-subunit homolog. 1009 76

We have previously shown that protein kinase C-beta (PKC-beta) is required for activation of tyrosinase (Park, H. Y., Russakovsky, V., Ohno, S., and Gilchrest, B. A. (1993) J. Biol. Chem. 268, 11742-11749), the rate-limiting enzyme in melanogenesis. We now examine its mechanism of activation in human melanocytes. In vivo phosphorylation experiments revealed that tyrosinase is phosphorylated through the PKC-dependent pathway and that introduction of PKC-beta into nonpigmented human melanoma cells lacking PKC-beta lead to the phosphorylation and activation of tyrosinase. Preincubation of intact melanosomes with purified active PKC-beta in vitro increased tyrosinase activity 3-fold. By immunoelectron microscopy, PKC-beta but not PKC-alpha was closely associated with tyrosinase on the outer surface of melanosomes. Western blot analysis confirmed the association of PKC-beta with melanosomes. Only the cytoplasmic (extra-melanosomal) domain of tyrosinase, which contains two serines but no threonines, was phosphorylated by the serine/threonine kinase PKC-beta. These two serines at positions 505 and 509 both are present in the C-terminal peptide generated by trypsin digestion of tyrosinase. Co-migration experiments comparing synthetic peptide standards of all three possible phosphorylated tryptic peptides, a diphosphopeptide and two monophosphopeptides, to tyrosinase-phosphorylated in intact melanocytes by PKC-beta and then subjected to trypsin digestion revealed that both serine residues are phosphorylated by PKC-beta. We conclude that PKC-beta activates tyrosinase directly by phosphorylating serine residues at positions 505 and 509 in the cytoplasmic domain of this melanosome-associated protein.
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PMID:Protein kinase C-beta activates tyrosinase by phosphorylating serine residues in its cytoplasmic domain. 1034 9


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