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)

In this study, we sequenced a new type I ribosome-inactivating protein, trichoanguina, from the seeds of Trichosanthes anguina (snake gourd). Trichoanguina is a basic glycoprotein having an apparent molecular mass of 35.0 kD and possessing strong ribosome-inactivating activity. Trichoanguina was cleaved with cyanogen bromide and partially digested with thermolysin, chymotrypsin, trypsin and Staphylococcus aureus V8 protease. The subsequent peptide fragments were separated by SDS-polyacrylamide gel electrophoresis, followed by electroblotting to polyvinylidene difluoride membranes and then sequencing. The sequencing of trichoanguina was completed, consisting of 245 amino acid residues. The sequencing of trichoanguina revealed a considerable homology to trichosanthin and alpha-trichosanthin, which are known as abortifacient, ribosome-inactivating and antihuman immunodeficiency virus proteins, with 46.7% and 55.6% amino acid identities, respectively. The sequence conserves two active sites: Glu-158 and Arg-161. Copyright 1996 S. Karger AG, Basel
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PMID:Amino Acid Sequence of Trichoanguina, a Ribosomal-Inactivating Protein from Trichosanthes anguinea Seeds. 1172 98

HIV-1(LAV-1) particles were collected by ultracentrifugation, treated with subtilisin, and then purified by Sepharose CL-4B column chromatography to remove microvesicles. The lysate of the purified human immunodeficiency virus type 1 (HIV-1) particles was subjected to two-dimensional (2D) gel electrophoresis and stained, and the stained spots were excised and digested with trypsin. The resulting peptide fragments were characterized by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Twenty-five proteins were identified as the proteins inside the virion and the acid-labile formyl group of an amino terminal proline residue of HIV-1(LAV-1) p24(gag) was determined by MALDI-TOF MS before and after weak-acid treatments (0.6 N hydrochloric acid) and confirmed by post-source decay (PSD) of the N-formylated N-terminal tryptic peptide (N-formylated Pro(1)-Arg(18)). The role of formylation has been unclear so far, but it is surmised that the acid-labile formylation of HIV-1(LAV-1) p24(gag) may play a critical role in the formation of the HIV-1 core for conferring HIV-1 infectivity.
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PMID:Acid-labile formylation of amino terminal proline of human immunodeficiency virus type 1 p24(gag) was found by proteomics using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. 1205 74

We describe a protocol for preparative-scale purification of the fusion protein of the human immunodeficiency virus type 1 (HIV-1), gp41, from cells overexpressing the viral envelope proteins and from HIV-1 isolates. In the first step, the proteins were extracted from the membrane in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. The extract was then subjected to separation by continuous elution electrophoresis using a nonionic or zwitterionic detergent in the mobile elution buffer, which results in the simultaneous exchange of SDS with that detergent. The separated proteins were obtained in an SDS-free buffer containing either Brij, 3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) or Triton X-100 and could then be subjected to subsequent purification steps like isoelectric focusing in the second dimension or immunoaffinity chromatography. The dilute protein fraction was concentrated and applied on a 10 mL immunoaffinity column packed with anti-gp41 monoclonal antibody immobilized on protein-G sepharose. The protein was eluted from the column at pH 2.7 and obtained in pure form in amounts of 30-50 micrograms that constituted a yield of 1%. The pure gp41 could not be sustained in solution in the absence of detergent and was not susceptible to proteolytic digestion by trypsin. The identification of the protein and the degree of purity was confirmed indirectly using surface enhanced laser desorption ionization-time of flight-mass spectrometry (SELDI-TOF-MS). The possible application of this approach for the isolation of integral membrane proteins with the propensity to undergo spontaneous folding and aggregation is being discussed.
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PMID:Improved separation of integral membrane proteins by continuous elution electrophoresis with simultaneous detergent exchange: application to the purification of the fusion protein of the human immunodeficiency virus type 1. 1217 85

Most of the research about viral interactions with human chromosomes was done during the sixties and early seventies and very few was performed after the human immunodeficiency virus (HIV) appearance as an epidemic in the eighties. The objective of this work was to estimate if particular chromosomal changes follow the infection of homosexual males by HIV and to determine if the lifestyle, habits, sexual practices, of our sample of male homosexuals predisposes them to chromosomal abnormalities at a higher rate than the background level of cytogenetic damage the general population has. This was a double blinded case-control study, 17 individuals positive for HIV antibodies (HIV+) detected by enzyme-linked immunosorbent assay (ELISA) and confirmed by western blot (WB) were the cases, and 17 individuals negative for HIV antibodies (HIV-) the controls. These men were a very homogeneous population in terms of age, social status, lifestyles, drug abuse, sexual practices and education. Blood was collected between September 1988 and October 1989. Fresh whole blood was cultured in duplicate for 72 hr. Cell harvest followed conventional methods. Once all cell cultures were gathered, the tubes were picked up at random and air dried chromosome preparations were trypsin-giemsa banded (GTG) after overnight incubation at 60 C degrees. The percentage of gaps and breaks these men had was not different from the reported for the general population, nor were there significant difference among both groups (O.R. = 1.8) in items of amount of chromosomal fragility. The distinction among them was at the level of the specific chromosomal sites where the gaps and breaks located, being sites at 2p21 and at 3p21 four times more frequent among HIV+. These probably represent viral modification sites on chromosomes which are known to look like non-staining gaps which are caused by the virus or viral products. This presumption is supported by an earlier report of repeated breaks at 3p21.1, in fact this was the most common lesion site in this study of chromosomal aberrations of male homosexuals and the authors even considered the probability of "a new type of chromosome marker". Furthermore, years later the CKR5 structural gene was mapped to human chromosome 3p21. This gene codes for the chemokine receptor 5 (CKR5) protein which serves as a secondary receptor on CD4+ T lymphocytes for certain strains of HIV-1. It is possible that this gene was being transcribed in HIV+ men and the consequent "staggering" of DNA contributed to the production of gaps and breaks at 3p21.
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PMID:Chromosomal defects in 34 male homosexuals, half of them with HIV antibodies. 1229 63

A new trypsin-chymotrypsin Inhibitor, with an N-terminal sequence showing some differences from the previously reported trypsin-chymotrypsin inhibitor, was isolated from the broad bean Vicia faba. The inhibitor was a peptide with a molecular mass of 13 kDa. It was adsorbed on Affi-gel blue gel and CM-Sepharose. It exerted antifungal activity toward Mycosphaerella arachidicola and Physalospora piricola. In addition, the trypsin-chymotrypsin inhibitor elicited a mitogenic response from mouse splenocytes and inhibited the activity of human immunodeficiency virus-1 reverse transcriptase.
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PMID:A new peptidic protease inhibitor from Vicia faba seeds exhibits antifungal, HIV-1 reverse transcriptase inhibiting and mitogenic activities. 1252 42

An association between human immunodeficiency virus type I (HIV-1) protease inhibitors (PIs) and galactorrhoea/hyperprolactinemia adverse effect has recently been reported in four HIV-1-infected women treated with PIs (indinavir, nelfinavir, ritonavir or saquinavir). This could be explained by a direct effect of ritonavir and saquinavir on anterior pituitary prolactin (PRL) release, and/or an indirect effect of PIs on the secretion of hypothalamic dopamine, which is the main PRL inhibitory factor. Anterior pituitaries were explanted from adult male Wistar rats, the cells were trypsin dispersed, plated into multiwell cultures and incubated for 1 h with either ritonavir or saquinavir (0.01 nM-1μM). PRL release into the incubation medium was evaluated by radioimmunoassay. Hypothalamic neuronal endings (synaptosomes) were prepared by tissue homogenization, incubated with ³H-dopamine, substituting for the endogenous dopamine pool, and perfused with ritonavir or saquinavir, both basally and during depolarization (K⁺ 15 mM)-induced dopamine release. Beta-emission from 2 min perfusate fractions, corresponding to ³H-dopamine release, was detected by liquid scintillation scanning. We found that both ritonavir and saquinavir are able to significantly stimulate PRL secretion, with saquinavir slightly more effective than ritonavir. On the contrary, both protease inhibitors do not modify either basal or depolarization-induced dopamine release. We can speculate that HIV PIs despite a high affinity for the catalytic site of HIV protease, could also bind to and inhibit homologous mammalian proteins in the anterior pituitary that are involved in PRL secretion.
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PMID:Ritonavir and Saquinavir directly stimulate anterior pituitary prolactin secretion, in vitro. 1259 90

Antibody (Ab) nucleophilic reactivity was studied using hapten and polypeptide antigens containing biotinylated phosphonate diester groups (covalently reactive antigen analogs, CRAs). Polyclonal IgG from healthy donors formed covalent adducts with a positively charged hapten CRA at levels superior to trypsin. Each of the 16 single chain Fv clones studied expressed a similar reactivity, indicating the V domain location of the nucleophiles and their broad distribution in diverse Abs. The formation of hapten CRA-Fv adducts was correlated with Fv proteolytic activity determined by cleavage of a model peptide substrate. Despite excellent nucleophilicity, proteolysis by IgG proceeded at lower rates than trypsin, suggesting that events occurring after nucleophilic attack on the substrate limit the rate of Ab proteolysis. The extracellular domain of the epidermal growth factor receptor with phosphonate diester groups at Lys side chains and a synthetic peptide corresponding to residues 421- 431 of human immunodeficiency virus glycoprotein (gp) 120 with the phosphonate diester at the C terminus formed covalent adducts with specific polyclonal and monoclonal Abs raised by immunization with epidermal growth factor receptor and synthetic gp120-(421- 436) devoid of phosphonate diester groups, respectively. Adduct formation was inhibited by extracellular domain of the epidermal growth factor receptor (exEGFB) and synthetic gp120-(421- 436) devoid of phosphonate groups, suggesting that the nucleophiles are located within the antigen binding sites. These results suggest the innate character of the Ab nucleophilic reactivity, its functional coordination with non-covalent adaptive binding interactions developing over the course of B cell maturation, and novel routes toward permanent inhibition of Abs.
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PMID:Broadly distributed chemical reactivity of natural antibodies expressed in coordination with specific antigen binding activity. 1266 70

Milk forms a rich source of biologically interesting components. In particular, its protein fraction is known to encompass many kinds of biological functions. In this review we focus on antibacterial and antiviral properties of milk proteins and milk protein derivatives. The latter include chemically modified proteins and enzymatically induced peptides. If such peptides are released by enzymes present within the digestive tract (e.g. trypsin or pepsin), it is likely that they play a role in the health defense system. This is especially the case when the active fragments can survive the intestinal conditions long enough to arrive at the right place to exert their beneficial function. In the first part of this paper attention is paid to the antibacterial proteins lactoferrin, lactoperoxidase, and lysozyme. Furthermore, antibacterial peptides originating from caseins and whey proteins are described. The second part reports on studies of antiviral effects of milk proteins and derivatives thereof. Special focus is directed to the antiviral action towards the human immunodeficiency virus (HIV) and the human cytomegalovirus (HCMV). Unmodified milk proteins are generally not active against these viruses. An exception is lactoferrin, which shows significant antiviral activity against both HIV and HCMV. Several other milk proteins tested showed strong antiviral effects only after chemical modification, i.e. by making them polyanionic (for anti-HIV activity) or polycationic (for anti-HCMV activity). In a number of cases, conclusions are drawn concerning possible relationships between antibacterial/antiviral activity and molecular structure of the components described.
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PMID:Antibacterial and antiviral effects of milk proteins and derivatives thereof. 1276 35

The HB autoantigen, a 10-kDa DNA-binding protein recognized by autoantibodies only when bound to DNA, was identified by two-dimensional electrophoresis. Silver-stained protein spots corresponding to the antigen were excised from two-dimensional electrophoresis gels, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization-reflectron time of flight and nano-electrospray ionization-ion trap/mass spectrometry. Data base search identified the HB antigen as the barrier-to-autointegration factor, a cellular protein implicated in the cellular cycle that blocks autointegration and promotes intermolecular integration of retrovirus such as the Moloney murine leukemia and the human immunodeficiency type 1 virus. The physicochemical characteristics described for these proteins, their ability to bind double-stranded DNA but not single-stranded DNA, and their nuclear localization confirm that HB and barrier-to-autointegration factor are the same protein.
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PMID:Identification of the autoantigen HB as the barrier-to-autointegration factor. 1452 12

An inhibitor of the metallo-ectoenzyme, pyroglutamyl aminopeptidase II (PPII), a thyrotropin releasing hormone-specific peptidase, was identified by screening extracts from marine species of the Cuban coast-line belonging to the phylla Chordata, Echinodermata, Annelida, Mollusca, Cnidaria, Porifera, Chlorophyta and Magnoliophyta. Isolation of the inhibitor (HcPI), from the marine annelide Hermodice carunculata, was achieved by trichloroacetic acid treatment of the aqueous extract, followed by ion-exchange chromatography on DEAE Sephacel, gel filtration on Sephadex G-25 and reverse phase-HPLC. HcPI had a small apparent molecular weight (below 1000 Da) and was not a peptide. It inhibited rat PPII (a membrane preparation with 8.5mg protein/ml) with an apparent K(i) of 51 nM. HcPI did not inhibit serine (trypsin, chymotrypsin, elastase and dipeptidyl aminopeptidase IV), cysteine (papain, bromelain and pyroglutamyl aminopeptidase I), aspartic (pepsin and recombinant human immunodeficiency virus 1 protease (HIV1-PR)) nor other metallo proteinases (collagenase, gelatinase, angiotensin converting enzyme, aminopeptidase N and carboxypeptidase A). HcPI was non-toxic and active in vivo. Intraperitoneal injection of HcPI reduced mouse pituitary and brain PPII activity. Potency of the effect was higher in hypophysis and hypothalamus than in other brain regions. Intrathecal administration to male rats reduced PPII activity in the spinal cord. In conclusion we have identified a specific inhibitor of PPII that is the first M1 family zinc metallo-peptidase inhibitor isolated from marine invertebrates. It may be useful for elucidating the in vivo role of PPII in the pituitary and central nervous system.
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PMID:Purification of a specific inhibitor of pyroglutamyl aminopeptidase II from the marine annelide Hermodice carunculata. in vivo effects in rodent brain. 1459 39


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