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)

A previously defined immunoglobulin M(kappa) monoclonal antibody reacting with a surface epitope of Mycoplasma hyorhinis is shown in this report to mediate specific, complement-dependent mycoplasmacidal activity. Immunoblot analysis of mycoplasma components and their tryptic cleavage products showed that the epitope recognized was present on a protein with an apparent molecular weight of 23,000 (p23) and on a limit tryptic fragment of this protein with an apparent molecular weight of 18,000 (p18). Both p23 and p18 are shown by Triton X-114 phase fractionation to partition efficiently into the hydrophobic detergent phase. Other antigens bearing epitopes not expressed at the cell surface were present among the numerous hydrophilic proteins found in the aqueous phase. The external orientation and membrane association of the p23 antigen were further established by demonstrating that trypsin treatment of intact mycoplasmas generated the antigenic p18 fragment, which remained tightly associated with the organism. These results localize an epitope responsible for antibody-mediated mycoplasma killing onto a specific, surface-exposed region of an integral membrane protein of this organism. Since the monoclonal antibody used in this study does not bind to the surface of all strains of M. hyorhinis, the epitope identified also defines a structural marker of antigenic surface variation within this species, a feature previously observed during serological classification of the organism. Analysis of the antigenic and structural features of the p23 surface antigen may therefore be useful in establishing mechanisms of surface antigen variation among integral membrane proteins of mycoplasmas that could dictate important antigenic characteristics recognized during chronic disease caused by these agents.
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PMID:Triton X-114 phase fractionation of an integral membrane surface protein mediating monoclonal antibody killing of Mycoplasma hyorhinis. 243 31

The compartmentation of fast-transported proteins that possess sulfated tyrosine residues--sulfoproteins--has been examined for further resolution of the possible significance of sulfated tyrosine in routing and delivery of fast-transported proteins. In vitro fast axonal transport of [35S]methionine- or 35SO4-labeled proteins was measured in dorsal root ganglion neurons for analysis of protein compartmentation en route and in synaptic regions. When membrane fractions were exposed to Na2CO3 for separation of "lumenal" and peripheral membrane proteins from integral components of the membrane, approximately 20% of the [35S]methionine incorporated into fast-transported proteins was present in a carbonate-releasable form in the axon, whereas 53% of the incorporated 35SO4 was released by carbonate. Eighty percent of the 35SO4 in this releasable fraction was acid labile, typical of sulfate ester-linked to tyrosine. Sulfoproteins were also detected in synaptosomes and were released into the extracellular medium in a calcium-dependent fashion, an observation suggesting that fast-transported sulfoproteins are secreted. Of the remaining 47% of the fast-transported 35SO4-labeled proteins resistant to carbonate treatment (the integral membrane protein fraction), nearly 60% of the 35SO4 was acid labile. Other membrane stripping agents, such as 0.1 M NaOH, 0.5 M NaCl, or mild trypsin treatment, failed to remove acid-labile 35SO4-labeled species from carbonate-treated membrane. Quantitative comparisons of several of the most abundant sulfoproteins resolved via two-dimensional gel electrophoresis confirmed that approximately 7% of each of the species remained associated with carbonate-treated membranes, presumably as integral membrane components.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Complex compartmentation of tyrosine sulfate-containing proteins undergoing fast axonal transport. 243 47

The simian rotavirus SA11 genome segment 10 codes for a nonstructural glycoprotein, NS28, that has been hypothesized to be involved in budding of viral particles into the endoplasmic reticulum (ER) membrane. Previous studies had suggested that NS28 is an integral membrane protein of the ER, possibly a transmembrane protein. We have examined the topography of NS28 inserted in microsomal membranes following cell-free translation of genome segment 10 transcripts. These transcripts were obtained either by hybrid selection of mRNA synthesized by the endogenous viral RNA polymerase or by in vitro transcription of genome segment 10 cDNA using SP6 polymerase. Full-length and truncated gene 10 transcripts were translated in a cell-free system supplemented with dog pancreatic microsomes. The existence of a cytoplasmic domain of the translation product was demonstrated by protease protection experiments. An 18,000 (18K) mol wt glycosylated polypeptide was protected from digestion with proteinase K and trypsin, whereas chymotrypsin digestion yielded a 23K mol wt glycosylated polypeptide. Correlation of these biochemical data with the known sequence of NS28 suggests that a 10K mol wt hydrophilic, carboxy-terminal fragment (from amino acid number 86 to amino acid number 175) of this glycoprotein is exposed on the cytoplasmic side of the ER membrane. A model of how NS28 folds in the ER membrane is proposed.
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PMID:Topography of the simian rotavirus nonstructural glycoprotein (NS28) in the endoplasmic reticulum membrane. 283 61

The M2 protein of influenza A virus is a small integral membrane protein of 97 residues that is expressed on the surface of virus-infected cells. M2 has an unusual structure as it lacks a cleavable signal sequence yet contains an ectoplasmic amino-terminal domain of 23 residues, a 19 residue hydrophobic transmembrane spanning segment, and a cytoplasmic carboxyl-terminal domain of 55 residues. Oligonucleotide-mediated deletion mutagenesis was used to construct a series of M2 mutants lacking portions of the hydrophobic segment. Membrane integration of the M2 protein was examined by in vitro translation of synthetic mRNA transcripts prepared using bacteriophage T7 RNA polymerase. After membrane integration, M2 was resistant to alkaline extraction and was converted to an Mr approximately equal to 7,000 membrane-protected fragment after digestion with trypsin. In vitro integration of M2 requires the cotranslational presence of the signal recognition particle. Deletion of as few as two residues from the hydrophobic segment of M2 markedly decreases the efficiency of membrane integration, whereas deletion of six residues completely eliminates integration. M2 proteins containing deletions that eliminate stable membrane anchoring are apparently not recognized by signal recognition particles, as these polypeptides remain sensitive to protease digestion, indicating that in addition they do not have a functional signal sequence. These data thus indicate that the signal sequence that initiates membrane integration of M2 resides within the transmembrane spanning segment of the polypeptide.
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PMID:Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus. 283 32

The cellular distribution, membrane orientation, and biochemical properties of the two major NaOH-insoluble (integral) plasma membrane proteins of Euglena are detailed. We present evidence which suggests that these two polypeptides (Mr 68 and 39 kD) are dimer and monomer of the same protein: (a) Antibodies directed against either the 68- or the 39-kD polypeptide bind to both 68- and 39-kD bands in Western blots. (b) Trypsin digests of the 68- and 39-kD polypeptides yield similar peptide fragments. (c) The 68- and 39-kD polypeptides interconvert during successive electrophoresis runs in the presence of SDS and beta-mercaptoethanol. (d) The 39-kD band is the only major integral membrane protein evident after isoelectric focusing in acrylamide gels. The apparent shift from 68 to 39 kD in focusing gels has been duplicated in denaturing SDS gels by adding ampholyte solutions directly to the protein samples. The membrane orientation of the 39-kD protein and its 68-kD dimer has been assessed by radioiodination in situ using intact cells or purified plasma membranes. Putative monomers and dimers are labeled only when the cytoplasmic side of the membrane is exposed. These results together with trypsin digestion data suggest that the 39-kD protein and its dimer have an asymmetric membrane orientation with a substantial cytoplasmic domain but with no detectable extracellular region. Immunolabeling of sectioned cells indicates that the plasma membrane is the only cellular membrane with significant amounts of 39-kD protein. No major 68- or 39-kD polypeptide bands are evident in SDS acrylamide gels or immunoblots of electrophoresed whole flagella or preparations enriched in flagellar membrane vesicles, nor is there a detectable shift in any flagellar polypeptide in the presence of ampholyte solutions. These findings are considered with respect to the well-known internal crystalline organization of the euglenoid plasma membrane and to the potential for these proteins to serve as anchors for membrane skeletal proteins.
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PMID:Properties and topography of the major integral plasma membrane protein of a unicellular organism. 313 63

The reformation of functioning organelles at the end of mitosis presents a problem in vesicle targeting. Using extracts made from Xenopus laevis frog eggs, we have studied in vitro the vesicles that reform the nuclear envelope. In the in vitro assay, nuclear envelope growth is linear with time. Furthermore, the final surface area of the nuclear envelopes formed is directly dependent upon the amount of membrane vesicles added to the assay. Egg membrane vesicles could be fractionated into two populations, only one of which was competent for nuclear envelope assembly. We found that vesicles active in nuclear envelope assembly contained markers (BiP and alpha-glucosidase II) characteristic of the endoplasmic reticulum (ER), but that the majority of ER-derived vesicles do not contribute to nuclear envelope size. This functional distinction between nuclear vesicles and ER-derived vesicles implies that nuclear vesicles are unique and possess at least one factor required for envelope assembly that is lacking in other vesicles. Consistent with this, treatment of vesicles with trypsin destroyed their ability to form a nuclear envelope; electron microscopic studies indicate that the trypsin-sensitive proteins is required for vesicles to bind to chromatin. However, the protease-sensitive component(s) is resistant to treatments that disrupt protein-protein interactions, such as high salt, EDTA, or low ionic strength solutions. We propose that an integral membrane protein, or protein tightly associated with the membrane, is critical for nuclear vesicle targeting or function.
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PMID:A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro. 339 6

The lectin Maclura pomifera agglutinin (MPA) binds to the apical surface of pulmonary alveolar type II but not type I cells. We show that MPA binds to a single membrane glycoprotein in type II cells with a molecular mass of 230 kDa in the rabbit and 200 kDa in the rat. The glycoprotein has an abundance of terminal N-acetylgalactosamine residues. It is a hydrophilic integral membrane protein suggesting that it has an extensive extramembrane domain or is an ion channel. The glycoprotein is similar in rat and rabbit, with the exception that the rat glycoprotein is partially sialylated and is trypsin sensitive. The MPA-binding glycoprotein represents a new integral membrane marker of the apical domain of the pulmonary alveolar type II cell.
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PMID:Identification and characterization of the pulmonary alveolar type II cell Maclura pomifera agglutinin-binding membrane glycoprotein. 341 17

We determined the activity of IL 1, obtained from various human monocyte subcellular compartments, when associated with liposomes. Soluble IL 1, bound to the outer surface of lyophilized liposomes, stimulated responsive target cells. However, this activity was not preserved when soluble IL 1 was incorporated into the inner chambers of classical liposomes. In contrast, monocyte plasma membranes that exhibited IL 1 activity had the same level of activity when presented on lyophilized liposomes and when incorporated inside the classical liposomes. However, monocyte plasma membranes bound to the outer surface of the liposomes exhibited greater activity than the monocyte membrane IL 1 itself in its soluble form. This suggests that membrane IL 1 is an integral membrane protein, readily integrated into the lipid bilayers. Like soluble IL 1, the expression of IL 1 activity present in the cytosol of activated monocytes was decreased by incorporation into liposomes, but was high and active when presented on lyophilized liposomes. The best artificial cell reconstitution was obtained with lyophilized liposomes in association with monocyte cytosol and plasma membranes. When an unactivated monocyte compartment was mixed with one from an activated monocyte, the signal was equal to that of the activated cell compartment alone. The IL 1 activity of activated cell fractions associated with lyophilized liposomes was determined, and an increase of IL 1 activity for both plasma membranes and cytosol was observed, whereas a decrease of the signal was obtained for the lysosomal compartment. Endoplasmic reticulum showed no IL 1 activity, even after trypsin treatment. The highest activity after trypsin treatment was recovered in the cytosol associated with lyophilized liposomes, suggesting that molecules obtained after this treatment were able to bind tightly to the lipid bilayers.
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PMID:Liposomes expressing IL 1 biological activity. 349 85

Escherichia coli hemolysin is secreted as a water-soluble polypeptide of Mr 107,000. After binding to target erythrocytes, the membrane-bound toxin resembled an integral membrane protein in that it was refractory towards extraction with salt solutions of low ionic strength. Toxin-induced hemolysis could be totally inhibited by addition of 30 mM dextran 4 (mean Mr, 4,000; molecular diameter approximately 3 nm) to the extracellular medium. Uncharged molecules of smaller size (e.g., sucrose, with a molecular diameter of 0.9 nm, or raffinose, with a molecular diameter of 1.2 to 1.3 nm) did not afford such protection. Treatment of erythrocytes suspended in dextran-containing buffer with the toxin induced rapid efflux of cellular K+ and influx of 45Ca2+, as well as influx of [14C]mannitol and [3H]sucrose. [3H]inulin only slowly permeated into toxin-treated cells, and [3H]dextran uptake was virtually nil. Membranes lysed with high doses of E. coli hemolysin exhibited no recognizable ultrastructural lesions when examined by negative-staining electron microscopy. Sucrose density gradient centrifugation of deoxycholate-solubilized target membranes led to recovery of the toxin exclusively in monomer form. Incubation of toxin-treated cells with trypsin caused limited proteolysis with the generation of membrane-bound, toxin-derived polypeptides of Mr approximately 80,000 without destroying the functional pore. We suggest that E. coli hemolysin may damage cell membranes by partial insertion into the lipid bilayer and generation of a discrete, hydrophilic transmembrane pore with an effective diameter of approximately 3 nm. In contrast to the structured pores generated by cytolysins of gram-positive bacteria such as staphylococcal alpha-toxin and streptolysin O, pore formation by E. coli hemolysin may be caused by the insertion of toxin monomers into the target lipid bilayers.
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PMID:Escherichia coli hemolysin may damage target cell membranes by generating transmembrane pores. 351 65

The recently described adherens junction-specific 135-kD protein (Volk, T., and B. Geiger, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:2249-2260) was localized along cardiac muscle intercalated discs by immunogold labeling of ultrathin frozen sections. Analysis of this labeling indicated that the 135-kD protein, adherens junction-specific cell adhesion molecule (A-CAM), is tightly associated with the plasma membrane unlike vinculin labeling, which was present along the membrane-bound plaques of the fascia adherens. In cultured chick lens cells, A-CAM was associated with Ca2+-dependent junctions that were cleaved upon a decrease of extracellular Ca2+ concentrations to less than or equal to 0.5 mM. In the chelator-separated junction, A-CAM became exposed to exogenously added antibodies or to proteolytic enzymes. Upon addition of trypsin to EGTA-treated cells, A-CAM was cleaved into three major cell-bound antigenic peptides with apparent molecular masses of 78, 60, and 46 kD, suggesting that the extracellular domain of A-CAM has a size greater than or equal to kD. Incubation of electrophoretic gels with 125I-concanavalin A (Con A) indicated that one of the major Con A-binding proteins in chicken lens membranes is a integral of 135-kD glycoprotein that was partially purified on Con A-Sepharose column and identified as A-CAM by immunoblotting. Detergent partitioning assay using Triton X-114 biphasic system was carried out to determine whether A-CAM displays properties of an integral membrane protein. This assay indicated that the intact A-CAM molecule was recovered in the buffer phase but its cell-associated tryptic peptides, which presumably lost a great part of the A-CAM extracellular extension, readily partitioned into the detergent phase. The results obtained in this and in the following paper (Volk, T., and B. Geiger, 1986, J. Cell Biol., 103:1451-1464) strongly suggest that A-CAM is a Ca2+-dependent adherens junction-specific membrane glycoprotein that is involved in intercellular adhesion in these sites.
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PMID:A-CAM: a 135-kD receptor of intercellular adherens junctions. I. Immunoelectron microscopic localization and biochemical studies. 353 54


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