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)

Papaya glutamine cyclotransferase (PQC), present in the laticiferous cells of the tropical species Carica papaya, was purified near to homogeneity. Starting from the soluble fraction of the collected plant latex, a combination of ion-exchange chromatography on SP-Sepharose Fast Flow, hydrophobic interaction chromatography on Fractogel TSK Butyl-650 and affinity chromatography on immobilized trypsin provided a purification factor of 279 with an overall yield of 80%. In the course of the purification procedure, the two solvent accessible thiol functions located on the hydrophobic surface of the enzyme were converted into their S-methylthioderivatives. Papaya QC, a glycoprotein with a molecular mass of 33000 Da, contains a unique and highly basic polypeptide chain devoid of disulfide bridges as well as of covalently attached phosphate groups. Its absorption spectrum is dominated by the chromophores tyrosine which, nonetheless, do not contribute to the fluorescence emission of the plant enzyme. With a lambdamax of emission at 338 nm and a moderate susceptibility to be quenched by acrylamide, most of the tryptophyl residues of papaya QC appear to be sterically shielded by surrounding protein atoms. Fluorescence can thus be used to monitor unfolding of this enzyme. Preliminary experiments show that papaya QC is exceptionally resistant to chemical (guanidinium hydrochloride), acid and thermal denaturation. At first sight also, this enzyme exhibits high resistance to proteolysis by the papaya cysteine proteinases, yet present in great excess (around 100 mol of proteinases per mol of PQC) in the plant latex. Altogether, these results awaken much curiosity and interest to further investigate how the structure of this plant enzyme is specified.
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PMID:Purification and characterization of papaya glutamine cyclotransferase, a plant enzyme highly resistant to chemical, acid and thermal denaturation. 974 28

Two low-molecular cytolytic toxins (RmI and RmII) and four trypsin inhibitors were isolated from the aqueous extract of sea anemone Radianthus macrodactylus. The method of isolation involved precipitation with acetone, gel filtration on acrylex P-4, ion-exchange chromatography on CM-32 cellulose, affinity chromatography on trypsin-binding sepharose 4B, ion exchange chromatography on an Ultrapore TSK CM-3SW column, and reversed phase HPLC on a Silasorb C18 column. RmI, RmII, and JnI inhibitor displayed molecular masses 5100, 6100, and 7100 Da, respectively, when subjected to SDS-PAGE. The isoelectric points were 9.2 and 9.3 for RmI and RmII, respectively. The amino acid composition and N-terminal amino acid residue (glycine) were determined for RmI, RmII, and JnI. Both proteins were nontoxic to mice and crabs. Hemolytic activity was determined to be 25 and 20 HU/mg for RmI and RmII, respectively, and their action on erythrocyte membrane was not inhibited by exogenous sphingomyelin. RmI and RmII exhibited antihistamine activity.
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PMID:[Low-molecular cytolysins and trypsin inhibitors from sea anemone Radianthus macrodactylus. Isolation and partial characterization]. 974 12

The prenyltransferase undecaprenyl pyrophosphate synthetase (di-trans,poly-cis-decaprenylcistransferase; EC 2.5.1.31) was purified from the soluble fraction of Escherichia coli by TSK-DEAE, ceramic hydroxyapatite, TSK-ether, Superdex 200, and heparin-Actigel chromatography. The protein was labeled with the photolabile analogue of the farnesyl pyrophosphate analogue (E, E)-[1-3H]-(2-diazo-3-trifluoropropionyloxy)geranyl diphosphate and was detected on a sodium dodecyl sulfate-polyacrylamide gel as a protein with an apparent molecular mass of 29 kDa. This protein band was cut out from the gel, trypsin digested, and subjected to matrix-assisted laser desorption ionization mass spectrometric analysis. Comparison of the experimental data with computer-simulated trypsin digest data for all E. coli proteins yielded a single match with a protein of unassigned function (SWISS-PROT Q47675; YAES_ECOLI). Sequences with strong similarity indicative of homology to this protein were identified in 25 bacterial species, in Saccharomyces cerevisiae, and in Caenorhabditis elegans. The homologous genes (uppS) were cloned from E. coli, Haemophilus influenzae, and Streptococcus pneumoniae, expressed in E. coli as amino-terminal His-tagged fusion proteins, and purified over a Ni2+ affinity column. An untagged version of the E. coli uppS gene was also cloned and expressed, and the protein purified in two chromatographic steps. We were able to detect Upp synthetase activity for all purified enzymes. Further, biochemical characterization revealed no differences between the recombinant untagged E. coli Upp synthetase and the three His-tagged fusion proteins. All enzymes were absolutely Triton X-100 and MgCl2 dependent. With the use of a regulatable gene disruption system, we demonstrated that uppS is essential for growth in S. pneumoniae R6.
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PMID:Use of genomics to identify bacterial undecaprenyl pyrophosphate synthetase: cloning, expression, and characterization of the essential uppS gene. 988 62

The survival of cerebrocortical neurons from 6 to 8-day-old chick embryos was investigated in a serum-free hormone-supplemented medium. The addition of cerebral extract promoted the survival of cortical neurons in a dose-dependent manner, but induced almost no neurite outgrowth. The trophic activity was higher in the adult cerebrum than in the embryonic cerebrum. The tropic factor was partially purified from adult chicken cerebrum, and the molecular weight of the factor was estimated to be about 60 kDa. The activity for survival factor was fairly resistant to heat or trypsin treatment. When the partially purified sample was, however, treated with trypsin (1 mg/ml) for 20 h and applied to a TSK G2000 SW gel filtration column, the activity moved from 60 to 70 kDa untreated active fractions to fractions with about 10 kDa. These physicochemical properties of the survival factor suggest a new class of macromolecular trophic factors in the brain.
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PMID:Characterization and partial purification of a neurotrophic factor for cerebrocortical neurons from chicken cerebral extract. 2050 Dec 84


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