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)

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.
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PMID:Identification of nucleotide binding sites in the poliovirus RNA polymerase. 775 55

Cerastocytin, a thrombin-like enzyme from the venom of the desert viper, Cerastes cerastes, has been purified to homogeneity by fast performance liquid chromatography (FPLC) on Mono-Q and Mono-S columns. It is a basic protein (isoelectric point higher than 9) made of a single polypeptide chain of 38 kDa. Its N-terminal polypeptide sequence shows strong similarities with other thrombin-like enzymes from snake venoms. Nanomolar concentrations of cerastocytin induce aggregation of blood platelets. This activity is inhibited by chlorpromazine, theophylline and mepacrine, as in the case of platelet aggregation stimulated by low doses of thrombin. Cerastocytin also possesses an amidolytic activity measured with the thrombin chromogenic substrate S-2238. The platelet aggregating activity and the amidolytic activity of cerastocytin were inhibited by PMSF, TPCK, TLCK and soybean trypsin inhibitors, suggesting that cerastocytin is a serine proteinase. On the other hand, both amidolytic activity and platelet aggregating activity of cerastocytin were unaffected by hirudin or by antithrombin III in the presence of heparin. High concentrations of cerastocytin (1-10 microM) also cleaved prothrombin and Factor X.
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PMID:Cerastocytin, a new thrombin-like platelet activator from the venom of the Tunisian viper Cerastes cerastes. 776 51

The nematophagous fungus Verticillium chlamydosporium secreted several proteases in submerged culture in which soya peptone was the sole carbon and nitrogen source. One protease, VCP1 (M(r) 33,000, pI 10.2), was purified 14-fold from culture filtrates to apparent homogeneity using preparative isoelectric focusing in free solution, and shown to rapidly hydrolyse the chymotrypsin substrate Suc-(Ala)2-Pro-Phe-pNA and elastin. VCP1 had a Km for Suc-(Ala)2-Pro-Phe-pNA of 4.3 x 10(-5) M and a kcat of 5.8 s-1. It was highly sensitive to PMSF and TPCK, but only moderately sensitive to chicken egg-white and soya bean trypsin inhibitors. VCP1 degraded a wide range of polymeric substrates, including Azocoll, hide protein, elastin, casein and albumin, and accounted for most of the non-specific protease activity detected in culture filtrates. The purified enzyme hydrolysed proteins in situ from the outer layer of the egg shell of the host nematode Meloidogyne incognita and exposed its chitin layer. VCP1 was secreted by several isolates of V. chlamydosporium and V. lecanii, pathogens of nematodes and insects respectively, but not plant-pathogenic species of Verticillium. These observations suggest that VCP1 or similar enzyme(s) may play a role in the infection of invertebrates.
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PMID:The nematophagous fungus Verticillium chlamydosporium produces a chymoelastase-like protease which hydrolyses host nematode proteins in situ. 800 May 41

In the pathogenesis of sparganosis, proteases have been considered to play important roles in tissue migration and parasite feeding. Several bands of proteolysis were observed when crude extracts of Spirometra mansoni plerocercoid (sparganum) were examined using gelatin substrate gel at neutral pH, of which two proteases of 198 and 104 kDa were purified by two chromatographic steps, and a 36 kDa protease was purified by gelatin-affinity and DEAE-anion exchange chromatography. All the purified proteases exhibited optimal activity at pH 7.5 and 0.1 M Tris-HCl. Proteolytic activities at 198 and 104 kDa were inhibited specifically by serine protease inhibitors and 4-(amidinophenyl)methanesulfonyl fluoride (APMSF, 0.5 mM) and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK, 1 mM), which strongly suggested that these two proteases were trypsin-like proteases. The activity of the 36 kDa protease was inhibited by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK, 1 mM) and chymostatin (0.1 mM), and was potentiated in 10 mM Ca2+ which showed that the protease had a chymotrypsin-like property. All the proteases were Schiff (PAS) positive. Proteases of 198 and 104 kDa degraded collagen completely within 24 h. The 36 kDa enzyme cleaved human recombinant interferon-gamma (rIFN gamma) and bovine myelin basic protein. In addition, all the purified proteins elicited strong antibody responses in the infected patients.
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PMID:Characterization of three neutral proteases of Spirometra mansoni plerocercoid. 802 61

The insect-selective neurotoxin (BmK IT) of scorpion Buthus martensi Karsch was first reduced and S-alkylated, and then digested by TPCK-trypsin and Staphylococcus aureus V-8 Protease. The enzymatic peptides were purified on TLC-plastic sheet and submitted to determine their amino acid compositions and sequences. The sequence of the 70 amino acid residues of BmK IT was established with reference to the primary structure of AaH IT, another excitatory insect-selective toxin from the venom of North African scorpion Androctonus australis Hector. About 75% of the homologous sequence was found in the molecules of BmK IT and AaH IT. It is obvious that the results contribute toward better understanding of the molecular structure characteristics, structure/activity relationship of scorpion insect-selective toxins, and they can serve as the molecular basis for utilizing the toxins as a tool to clarify molecular mechanism involved in channel gating, and to infer the possibility of developing them as new selective bioinsecticides.
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PMID:Amino acid sequence of an excitatory insect-selective toxin (BmK IT) from venom of the scorpion Buthus martensi Karsch. 806 86

Acetylcholinesterase from human caudate nucleus and partial thalamus was purified by using Con A-Sepharose, short-arm and long-arm ligand Sepharose affinity chromatographies. SDS-PAGE of the purified AChE under the reduced condition showed one main band, corresponding to a molecular weight of 66 kD. The purified AChE with a specific activity of 3384 U/mg protein represented 20% activity of the homogenate supernatant. Analysis of purified AChE by gradient slab PAGE and DISC-PAGE with activity staining revealed the existence of monomer, dimer, tetramer, hexamer and octomer of the enzyme. The isoelectric point of AChE ranged between pH 5.6 and 6.0. Con A-Sepharose affinity chromatography retained most of the applied AChE activity implying that the enzyme is a kind of glycoprotein. The isolated human brain AChE had no cross-immunoreactivity with 3F3 and weak cross-immunoreactivity with 2G8 and 1H11 anti-Torpedo AChE antibodies. Balb/c mice were immunized with human cerebellum AChE purified with Con A and short-arm ligand affinity chromatographies. The antiserum produced showed strong cross-immunoreactivity with Torpedo AChE but weak cross-immunoreactivity with human RBC membrane AChE. The purified human brain striatum AChE was reduced and alkylated, and then hydrolyzed by immobilized TPCK-treated trypsin. Trypsin peptides in the hydrolysate was separated by RP-HPLC. Several large peptide peaks and numbers of small peaks were observed. The large peaks showed obvious immunoreactivity with the mouse anti human cerebellum AChE antiserum.
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PMID:Purification and properties of acetylcholinesterase from human brain. 813 33

A reverse-phase HPLC method for the analysis of tryptic digests of recombinant porcine growth hormone (pGH) has been developed and validated. Digestion was performed at 4 degrees C for a 20-hr period with TPCK-treated trypsin at a 1:20 (w/w) trypsin:pGH ratio. Gradient elution HPLC, using an Aquapore RP300 C8 column, was incorporated for separation of the digestion products and peak identification was carried out by mass spectrometry (MS). The digestion procedure and subsequent chromatography were linear in the initial concentration range of 4.55-45.46 microM (100 to 1000 micrograms/mL) pGH. The variability in the fragment retention times was low and the normalized peak area variability was less than 5% for all but three of the fragments. The utility of the trypsin digestion and chromatography procedures has been demonstrated by assessing chemical changes in pGH induced by incubation at elevated pH. Upon incubation of pGH in 0.2 M Tris buffer at pH 9 (ionic strength adjusted to 0.5 with NaCl) and 37 degrees C over a period of 400 hr, significant degradation in the regions corresponding to the digestion fragments T23-T25 (residues 181-182 linked by a disulfide bond to residues 184-191), T9 (residues 96-108), and T5-T18 (residues 43-64 linked by a disulfide bond to residues 158-166) was observed. The disappearance of the peaks corresponding to fragments T23-T25 and T9 both displayed apparent first-order degradation kinetics over the time period investigated with half-lives of 131 and 154 hr, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Validation of a peptide map for recombinant porcine growth hormone and application to stability assessment. 827 10

A fraction of band 3 protein, the major transmembrane protein of erythrocyte membranes, is held to the cytoskeletal protein spectrin via noncovalent interactions with the protein ankyrin (band 2.1). In this study, trypsin was used under defined conditions to selectively proteolyze ankyrin and thereby destroy the band 3-ankyrin linkage on the cytoplasmic side of erythrocyte ghost membranes. Electron paramagnetic resonance (EPR) spectroscopy, in conjunction with selective spin labeling methods, was used to monitor conformational changes occurring in cytoskeletal proteins or cell-surface carbohydrates as a result of this treatment. Treatment of RBC ghosts with TPCK-trypsin for 5 s at 0 degrees C caused an approx. 56% increase in the relevant EPR parameter of a maleimide spin label bound to spectrin (P < 0.004), indicative of increased segmental motion of the spin label and decreased protein-protein interactions. Analysis of the apparent rotational correlation time parameter tau of a spin label covalently and selectively bound to terminal sialic acid residues of glycophorin showed no significant effect from trypsin treatment. However, tau of spin label covalently and specifically bound to terminal galactose residues of cell-surface glycoconjugates of band 3 and other transmembrane glycoproteins significantly decreased with tryptic uncoupling of the ankyrin linkage (P < 0.005). These results suggest a marked conformational alteration in both cytoskeletal and transmembrane proteins as a result of uncoupling from ankyrin. Spermine (N,N'-bis(3-aminopropyl)tetramethylenediamine), a naturally occurring polyamine known to strengthen cytoskeletal protein-protein interactions (Wyse and Butterfield (1988) Biochim. Biophys. Acta 941, 141-149), was used to partially reverse the trypsin-induced cytoskeletal alterations. Addition of 2 mM spermine to ghosts previously treated with trypsin increased cytoskeletal protein-protein interactions as indicated by EPR (P < 0.002). SDS-PAGE was used to confirm the integrity of spectrin, band 3, and band 4.1 in all experiments. The results are discussed with reference to transmembrane signaling mechanisms and membrane-associated pathologies.
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PMID:Alteration of the erythrocyte membrane via enzymatic degradation of ankyrin (band 2.1): subcellular surgery characterized by EPR spectroscopy. 838 64

The identification of peptide bonds vulnerable to tissue peptidases is a valuable approach to design peptide agonists which exhibit a longer duration of action than the native molecules. Therefore, the kinetic of disappearance of rat growth hormone-releasing factor (rGRF(1-29)NH2) and the identification of its metabolites were studied in rat pituitary and hypothalamus. Synthetic rGRF(1-29)NH2 (10 microM) was incubated (0-120 min, 37 degrees C) in the presence of a pituitary (237 +/- 51 micrograms protein/ml) or hypothalamus homogenate (576 +/- 27 micrograms protein/ml). Using analytical high pressure liquid chromatography (HPLC), apparent half-lives of 22 +/- 3 min and 25 +/- 4 min were found in pituitary and hypothalamus, respectively. In both tissues, three degradation products, all less hydrophobic than the native peptide, were detected and isolated by preparative HPLC. The identification of the purified metabolites was ascertained by amino acid analysis, sequencing and chromatography with synthetic homologs. These results indicate that the main sites of cleavage in the pituitary and hypothalamus are Lys21-Leu22 (trypsin-like cleavage site), Leu14-Gly15 and Tyr10-Arg11 (chymotrypsin-like cleavage sites). TLCK and leupeptin did not affect the formation of fragment (1-21)OH while TPCK blocked the cleavage of Leu14-Gly15. The low affinity of fragment (1-21)NH2 for pituitary GRF binding sites suggests that hydrolysis of the Lys21-Leu22 bond inactivates rGRF(1-29)NH2 in this target tissue.
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PMID:Proteolytic degradation of rat growth hormone-releasing factor(1-29) amide in rat pituitary and hypothalamus. 839 7

Methyltrienolone, a synthetic steroid, was used as a photoaffinity ligand for steroid-binding proteins. The enzymatic activity of bovine adrenocortical cytochrome P-450(11) beta was inhibited by methyltrienolone in a competitive manner without exposure to light and cytochrome P-450(11) beta was photolabeled with methyltrienolone after irradiation with UV light. The addition of 11-deoxycorticosterone during photolabeling protected cytochrome P-450(11) beta from photolabeling. Photolabeled cytochrome P-450(11) beta was digested with TPCK-treated trypsin and the peptide fragments were separated with a reverse-phase HPLC system. The labeled peptide was analyzed and its amino acid sequence was determined to be Trp428-Leu429-Asp430-Arg431. Alignment of the primary structure of cytochrome P-450(11) beta with that of cytochrome P-450cam revealed that the identified sequence corresponds to the region between the beta 3-sheet and L-helix of cytochrome P-450cam. This region of mammalian cytochromes P-450 shows poor homology with that of cytochrome P-450cam, but is well-conserved, especially at Trp-428 and preceding amino acids, as the aromatic region. The present results demonstrate that the labeled sequence contributes in part to the formation of the substrate binding pocket of cytochrome P-450(11) beta which was not expected from the results of the primary sequence alignment with cytochrome P-450cam.
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PMID:Photoaffinity labeling of cytochrome P-45011 beta with methyltrienolone as a probe for the substrate binding region. 843 74


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