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)

Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 nM [gamma-32P]ATP. Phosphorylation of a 45-kDa endogenous protein was detected within 1 min and increased linearly for approximately 20 min. Unlike what was seen with [gamma-32P]ATP, in the presence of [32P]orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45-kDa protein was reduced by 80-90% in the presence of 1 microM unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 mM PO4(3-). Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 mM KCl or 100 microM glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45-kDa protein. The phosphorylation was found to be insensitive to MgCl2 but inhibited in the presence of MnCl2 or NaF and in the absence of CaCl2. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80-90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto-protein enzymes and intracellular kinases.
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PMID:Identification of an ectokinase activity in cerebellar granule primary neuronal cultures. 796 20

Technological advances in the field of mass spectrometry (MS) are providing powerful analytical means for the investigation of proteins and peptides. In the present work we have used pneumatically assisted electrospray (ion-spray) MS for the biochemical characterization of recombinant human catechol O-methyltransferase (rhCOMT). hCOMT could be expressed in Escherichia coli in large quantities but in two forms of different size, both enzymically active. Electrospray MS analysis showed that the smaller rhCOMT protein had a molecular mass of 24352 +/- 2 Da, corresponding to the calculated value for native hCOMT (without the initiating methionine), whereas that mass of the larger protein was of 25775 +/- 4 Da. To investigate the molecular differences between the two proteins, they were digested with trypsin and the peptides produced analysed by electrospray MS. Neither protein apparently contained disulfide bridges and the observed molecular masses of the tryptic peptides corresponded to the calculated values. It was possible to determine, however, that the larger protein contained an extended C-terminus with the correct sequence GPGSEAGP plus an additional stretch, EDLR. This C-terminal extension resulted from ribosomal frameshift at the codon of the last proline (CCC, rare codon in prokaryotes). In fact, rightward frameshifting would produce a hCOMT form with an additional stretch of 11 amino acid (EDLRSHHHHHH) and the calculated molecular mass of this protein (25773.5 Da) is in good agreement with our experimental result. The differential reactivity of the cysteine residues of the correct rhCOMT enzyme, in the presence and in the absence of S-adenosyl-L-methionine (AdoMet) and MgCl2, was also studied. 5-Iodoacetamido fluorescein (5-IAF) was used as thiol-modifying reagent. Under the conditions used, 5-IAF rapidly inactivated rhCOMT but the presence of AdoMet and MgCl2 partially protected it from inactivation. The 5-IAF-labeled tryptic peptides were separated by HPLC and then submitted to electrospray MS and tandem MS. Several cysteine residues appeared to be readily available to chemical modification by 5-IAF. Incorporation of 5-IAF occurred to a larger extent into Cys32, Cys68, Cys94 and Cys172. AdoMet and MgCl2 markedly reduced the label incorporation into Cys68 and Cys94, therefore suggesting that these residues belong to a region at or near the binding site of AdoMet.
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PMID:Mass spectrometric analysis of human soluble catechol O-methyltransferase expressed in Escherichia coli. Identification of a product of ribosomal frameshifting and of reactive cysteines involved in S-adenosyl-L-methionine binding. 802 Apr 75

A 5-kDa polypeptide, pseudothionin Solanum tuberosum 1 (Pth-St1), which was active against Clavibacter michiganensis subspecies sepedonicus, a bacterial pathogen of potatoes, has been purified from the buffer-insoluble fraction of potato tubers by salt extraction and HPCL. Pth-St1 was also active against other potato pathogens tested (Pseudomonas solanacearum and Fusarium solani). The N-terminal amino acid sequence of this peptide was identical (except for a N/H substitution at position 2) to that deduced from a previously reported cDNA sequence (EMBL accession number X-13180), which had been misclassified as a Browman-Birk protease inhibitor. Pth-St1 did not inhibit either trypsin or insect alpha-amylase activities, and, in contrast with true thionins, did not affect cell-free protein synthesis or beta-glucuronidase activity. Northern-blot and tissue-print analyses showed that steady-state mRNA levels were highest in flowers (especially in petals), followed by tubers (especially in the epidermal cell layers and in leaf primordia), stems and leaves. Infection of leaves with a bacterial pathogen suspended in 10 mM MgCl2 switched off the gene, whereas mock inoculation with 10 mM MgCl2 alone induced higher mRNA levels.
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PMID:Pseudothionin-St1, a potato peptide active against potato pathogens. 803 86

Plant clathrin-coated vesicles (CCV), suitably protected against proteolysis, were isolated from zucchini hypocotyls, and screened for the presence of adaptin-like polypeptides using monoclonal antibodies prepared against alpha, beta(beta') and gamma-adaptins of bovine brain. An immunoreactive polypeptide in plant CCV was only detected in the case of the beta(beta')-adaptin antibody. This polypeptide has a molecular mass of 108 kDa in SDS-PAGE, and gives rise to a major cleavage product of 70 kDa after proteolysis with trypsin. Gel filtration of 0.75 M MgCl2-dissociated coat proteins showed that the plant beta(beta')-type adaptin eluted with other polypeptides in a manner similar to the adaptor complexes of brain CCV. Upon subsequent hydroxyapatite chromatography the immunoreactive polypeptide eluted in fractions corresponding to Golgi (HA-I) rather than plasma membrane (HA-II) brain adaptor complexes. In addition, this polypeptide did not shift to a higher molecular mass when subjected to urea-SDS-PAGE. Confirmation of the presence of a beta-type adaptin in plants was provided by dot and Southern blotting experiments using genomic DNA from zucchini hypocotyls and a beta-adaptin cDNA clone from human fibroblasts.
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PMID:Identification of a beta-type adaptin in plant clathrin-coated vesicles. 805 48

Large percentages of Toxoplasma gondii tachyzoites could be induced to display two types of movement associated with active invasive behavior by exposing them for 1 min to 0.002% trypsin in phosphate-buffered saline (PBS). The motile activity, consisting of clockwise rotation around the posterior end (about 20 revolutions per min) and twirling-gliding over a poly-L-lysine substrate (1.2 +/- 0.2 microns/s standard deviation), was observed and recorded by video-enhanced contrast microscopy. The number of active tachyzoites reached a maximum 1 min after trypsinization; the motile response of the population lasted for about 5 min. Activation was prevented by soybean trypsin-inhibitor, and could not be induced again in previously treated specimens. Electronmicroscopy of trypsinized tachyzoites fixed in the presence of ruthenium-red revealed discrete discontinuities of the plasma membrane, which sealed within 90 min after washing with PBS. Treated tachyzoites were able to invade cultured epithelial cells with a higher relative infectivity than that of untreated parasites. Perfusion of trypsinized tachyzoites with 1 mM of either CaCl2 or MgCl2 and 1 mM ATP increased the number of activated parasites to over 60%; on the other hand, all induced motility was inhibited or blocked by agents that chelate divalent cations. The present preparation, which provided the first serial illustrations of T. gondii movements induced by a defined chemical stimulus, may offer a useful experimental model for the study of motility in this parasite.
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PMID:Divalent cation and ATP dependent motility of Toxoplasma gondii tachyzoites after mild treatment with trypsin. 808 4

A processing protease for the human immunodeficiency virus type I (HIV-I) envelope glycoprotein gp160 precursor has been purified to homogeneity from the post-nuclear membrane fraction of a human T4+ lymphocyte clone. Most of the processing activity was found to be present in the fractions of endoplasmic reticulum and Golgi apparatus of the cells. The purified enzyme has a monomeric structure with a molecular mass of 26 +/- 3 kDa, as judged by gel-permeation liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The purified enzyme converted gp160 to gp120 and gp41, showing a pH optimum of 6.5-7.0. Direct amino acid sequencing of the amino terminus of the product gp41 revealed that the cleavage site of gp160 was between Arg511 and Ala512. The enzyme activity was inhibited by trypsin-type protease inhibitors, but was not affected by CaCl2, MgCl2 or chelating agents. The properties of the purified enzyme are clearly distinct from those of processing proteases reported previously. Although the significance of the enzyme in vivo is not currently certain, judging from its cleavage specificity and subcellular localization, this endopeptidase appears to be a processing enzyme for the human immunodeficiency virus type I gp160 precursor protein in human T cells.
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PMID:Processing protease for gp160 human immunodeficiency virus type I envelope glycoprotein precursor in human T4+ lymphocytes. Purification and characterization. 809 9

The location of linker histones H1 and H5 in chicken erythrocyte chromatin was studied as a function of the fiber structure by the use of proteolytic enzymes immobilized onto Immobilon membranes. The immobilization of trypsin and chymotrypsin creates proteolytic probes, specific respectively to the terminal portions of the molecules or to the phenylalanine in the globular domain, that are incapable of penetrating into the interior of the condensed fiber. The chromatin fiber was studied in three different conformations: open zig-zag (in Tris buffer), closed zig-zag (upon addition of 10 mM-NaCl), or 30 nm fiber (upon addition of 0.35 mM-MgCl2). The results from digestion experiments performed on linker histones either in chicken erythrocyte chromatin, or free in solution or bound in mononucleosomes revealed several features relevant to linker histone location: (1) histone H5 is more protected than histone H1 in the fiber; (2) the N and C-terminal portions of histone H1 do not change their accessibility, and hence their location, upon compaction of the fiber; this behavior of H1 is in contrast to that of histone H5, whose tails become significantly internalized in the 30 nm fiber; (3) phenylalanine in the globular domain of both H1 and H5 is inaccessible (buried) both in the fiber and in the mononucleosomal particle. Sedimentation velocity measurements performed during the course of trypsin digestion demonstrate that the conformation of the fiber is highly sensitive to even a few cuts in some of the linker histone molecules; hence, the linker histones are an important factor in the organization of the fiber in all its different condensation states.
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PMID:On the location of histones H1 and H5 in the chromatin fiber. Studies with immobilized trypsin and chymotrypsin. 844 56

The Ca(2+)-binding and structural properties of calmodulin (CaM) from the yeast Saccharomyces cerevisiae (yCaM) were analyzed by flow dialysis and NMR spectroscopy. Full-length yCaM and two truncated versions of yCaM were expressed in Escherichia coli and purified. yTR1 (residues 1-76) and yTR2 (residues 75-147) are similar to the vertebrate CaM fragments TR1 and TR2, which are generated by limited proteolysis with trypsin. As was found for the fragments of vertebrate CaM, the yCaM fragments retain native conformation and are useful for examining structure and metal-binding properties by NMR. Evidence for a short beta-sheet in each domain, as well as characteristic NOEs to aromatic residues, suggests that yCaM folds similarly to vertebrate CaM. Furthermore, although the previously considered "invariant" glycine at position 6 is replaced by a histidine in site II of yCaM, the far downfield chemical shift of His-61's amide proton suggests that this site adopts a conformation similar to that found in other EF-hand sites. Macroscopic Ca(2+)-binding constants were determined for yCaM by flow dialysis, revealing three high-affinity sites (dissociation constants were 5.2, 3.3, and 2.3 microM in the presence of 1 mM MgCl2 and 100 mM KCl). Positive cooperativity was observed among all sites. Ca2+ binding was also monitored indirectly by one-dimensional NMR. Titrations of the fragment molecules reveal that two binding sites reside in the N-terminal domain (sites I and II) and one in the C-terminal domain (site III). All three sites exhibit slow-exchange behavior in the intact protein, but site III exhibits fast-exchange behavior in the isolated C-terminal domain fragment (yTR2). Thus, an interaction between the two domains of intact yCaM affects the behavior of site III. These results with yCaM differ from those of vertebrate CaM in terms of Ca(2+)-binding stoichiometry, affinity of sites I and II, relative affinity of sites in the N- and C-terminal domains, and the exchange behaviors observed.
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PMID:Similarities and differences between yeast and vertebrate calmodulin: an examination of the calcium-binding and structural properties of calmodulin from the yeast Saccharomyces cerevisiae. 846 Dec 93

Primase from Escherichia coli is a single-stranded DNA-dependent RNA polymerase. As such, it requires magnesium to carry out catalysis. Limited tryptic digestion was used to probe the conformations of primase as a function of magnesium acetate concentration. In the absence of magnesium, trypsin cleaved primase at three sites. Magnesium acetate induced a conformational change such that one of these sites became inaccessible to trypsin digestion and a new site became trypsin accessible. The conformational change was only induced by Mg(OAc)2 and not MnCl2, CaCl2, NaOAc or LiCl, indicating a clear magnesium acetate-dependent conformational change. The effect was slightly induced by MgSO4 and MgCl2. An allosteric binding model indicates that primase binds at least two magnesiums in a cooperative manner. The data were best fit to a two-state model in which one conformation had a high affinity for magnesium, KR = 83.4 M-1, and the other state had virtually no affinity.
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PMID:Magnesium acetate induces a conformational change in Escherichia coli primase. 852 45

Actin purified from the yeast (Saccharomyces cerevisae) was polymerized faster than rabbit skeletal alpha-actin by MgCl2. The two actins polymerized at similar rates in the presence of CaCl2. Yeast actin, up to 25 microM, was not polymerized by KCl (100-300 mM); the monovalent salt also inhibited the MgCl2-induced polymerization of actin. The local structure of the subdomain-2 region in yeast actin filaments was probed by subtilisin and trypsin digestions. Loop 38-52 appeared more flexible and accessible to subtilisin in yeast than in rabbit actin. In contrast, tryptic digestions at Lys-61 and -68 occurred at the same rate for yeast and alpha-actin filaments. Modification of yeast actin by a sulfhydryl reagent CPM [7-(diethylamino)-3-(4'-maleimidophenyl)-4-methylcoumain] was specific to the Cys-374 residue; no labeling of a yeast actin mutant containing an alanine substitution for cysteine 374 was observed. The rates of Cys-374 labeling by CPM were similar for yeast and muscle actin, suggesting a similar environment for the C terminus in both polymers. In the in vitro motility assays, yeast actin required higher concentrations of heavy meromyosin (HMM) for its sliding than did the rabbit actin. At saturating concentrations of HMM, the sliding velocities of both actins were the same (3.0 microns/s). Relative forces generated by HMM with yeast and muscle actin were assessed by monitoring their in vitro motility in the presence of NEM-HMM load. The sliding of yeast actin was stopped at a level of external load (molar ratio NEM-HMM/HMM = 0.25) lower than that of muscle actin (NEM-HMM/HMM = 0.43), suggesting lower force production with yeast actin. These results are discussed in terms of the myosin cross-bridge cycle and actomyosin interactions.
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PMID:Polymerization and in vitro motility properties of yeast actin: a comparison with rabbit skeletal alpha-actin. 898 91


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