Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Soybean nodulin-26, a homologue of bovine eye lens major intrinsic protein (MIP-26), is an integral protein of the peribacteroid membrane in symbiotic root nodules. It comprises 271 amino acids with six potential transmembrane domains and lacks an amino-terminal signal sequence. A full-length nodulin-26 cDNA and its various deletion derivatives were transcribed in vitro after linking them to bacteriophage T3 promoter. In vitro translation of these transcripts in a rabbit reticulocyte lysate, in the presence or absence of canine pancreatic microsomal membranes, suggested that nodulin-26 is cotranslationally inserted into the microsomes without a cleavable signal peptide. The first two transmembrane domains (103 amino acids) of the protein are sufficient for microsomal membrane insertion. Membrane-translocated nodulin-26 binds to Con-A and is sensitive to endoglycosidase-H treatment, suggesting that it is glycosylated. Native nodulin-26 from root nodules retains its sugar moiety as it, too, binds to Con-A. Chemical cleavage mapping at cysteine residues, a trypsin protection assay, and the Con-A binding affinity of nodulin-26 suggested that both the NH2 and COOH termini of this protein are on the cytoplasmic surface of the peribacteroid membrane, while the glycosidic residue is on the surface of the membrane facing the bacteroids. In vitro phosphorylation experiments showed that nodulin-26 is a major phosphorylated protein in the peribacteroid membrane. This phosphorylation is mediated by a Ca(2+)-dependent, calmodulin-independent protein kinase located in the peribacteriod membrane. Externally supplied acid phosphatase dephosphorylates this protein, but alkaline phosphatase does not. Based on its homology with several eukaryotic and prokaryotic channel-type membrane proteins, nodulin-26 may form a channel translocating specific molecules to the bacteroids during endosymbiosis in legume plants.
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PMID:Topology and phosphorylation of soybean nodulin-26, an intrinsic protein of the peribacteroid membrane. 162 42

The lens junction protein (MIP26), and its trypsin cleavage product (MIP21), isolated from calf fiber cells, are incorporated into liposomes and the permeability and gating of the resulting channels are studied spectrophotometrically by an osmotic swelling assay. Liposomes incorporated with either protein and loaded with Dextran T-10 swell when placed in isotonic or hypertonic KCl, sucrose or polyethyleneglycol (PEG), indicating the presence of channels permeable to molecules as large as MW 1500. In the absence of calmodulin (CaM), the permeability of either MIP26 or MIP21 channels is not altered by Ca++. On the contrary, MIP26-CaM channels reversibly close in the presence of Ca++ (10(-5)M). Preliminary experiments show channel closure with lowered pH (5.5) as well. While MIP26-CaM channels close to all the permeants tested, MIP21-CaM channels close only partially with Ca++, becoming impermeable to large probes (PEG) while remaining permeable to sucrose and KCl. This indicates that the trypsin-cleaved C-terminal arm of MIP26 is the channel gate. Evidence from spectrophotofluorometry and circular dichroism spectroscopy indicates that activated CaM changes the conformation of isolated MIP26, suggesting that channel occlusion could result from a change in protein configuration.
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PMID:Permeability and gating of lens gap junction channels incorporated into liposomes. 241 Jan 93

Lens gap junction channels are studied in a reconstituted system obtained by incorporating into liposomes, with or without calmodulin, the lens junction protein (MIP26) and its trypsin-cleaved product (MIP21) that lacks the C-terminal arm. Channel permeability is studied with an osmotic swelling assay. MIP26 and MIP21 liposomes swell in sucrose or polyethyleneglycol with or without Ca++ indicating the presence of large channels. Without Ca++, MIP26 and MIP21 liposomes swell in both permeants. With Ca++, MIP26-calmodulin liposomes do not swell in either permeant, indicating complete channel closure, while MIP21-calmodulin liposomes swell in sucrose but not in polyethyleneglycol. This suggests that the C-terminal arm participates in channel gating.
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PMID:Is the C-terminal arm of lens gap junction channel protein the channel gate? 241 98

Calf lens fiber plasma membranes, containing only the intrinsic membrane protein MP26 and its maturation product MP22 were treated with proteolytic enzymes such as trypsin, protease V8 from S. aureus or with chemical agents as CNBr in formic acid. The cleavage products, purified by electrophoresis, were analysed for their amino acid composition and N-terminal sequences. Proteolysis gave rise to peptides which were mainly shortened at the C-terminal end of the molecules. While the V8 protease produced a fragment with a similar N-terminal sequence as the maturation product MP22, trypsin yielded another cleavage product. Chemical hydrolysis yielded large fragments (11-15 kDa) with hydrophobic N-terminal sequences. Our results suggest that MP26 is characterised by an N-terminal signal sequence and possesses other hydrophobic domains which could function as untranslocated insertion sequences.
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PMID:Sequence analysis of peptide fragments from the intrinsic membrane protein of calf lens fibers MP26 and its natural maturation product MP22. 388 55

The major membrane protein of the bovine lens fiber cell is a 26-kilodalton (kD) protein (MP26), which appears to be a component of the extensive junctional specializations found in these cells. To examine the arrangement of MP26 within the junctional membranes, various proteases were incubated with fiber cell membranes that had been isolated with or without urea and/or detergents. These membranes were analyzed with electron microscopy and SDS-PAGE to determine whether the junctional specializations or the proteins were altered by proteolysis. Microscopy revealed no obvious structural changes. Electrophoresis showed that chymotrypsin, papain, and trypsin degraded MP26 to 21-22 kD species. A variety of protease treatments, including overnight digestions, failed to generate additional proteolysis. Regions on MP26 which were sensitive to these three proteases overlapped. Smaller peptides were cleaved from MP26 with V8 protease and carboxypeptidases A and B. Protein domains cleaved by these proteases also overlapped with regions sensitive to chymotrypsin, papain, and trypsin. Specific inhibition of the carboxypeptidases suggested that cleavage obtained with these preparations was not likely due to contaminating endoproteases. Since antibodies are not thought to readily penetrate the 2-3 nm extracellular gap in the fiber cell junctions, antibodies to MP26 were used to analyze the location of the protease-sensitive domains. Antisera were applied to control (26 kD) and proteolyzed (22 kD) membranes, with binding being evaluated by means of ELISA reactions on intact membranes. Antibody labeling was also done following SDS-PAGE and transfer to derivatized paper. Both assays showed a significant decrease in binding following proteolysis, with the 22 kD product showing no reaction with the anti-MP26 sera. These investigations suggest that MP26 is arranged with approximately four-fifths of the primary sequence "protected" by the lipid bilayer and the narrow extracellular gap. One-fifth of the molecule, including the C-terminus, appears to be exposed on the cytoplasmic side of the membrane.
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PMID:Arrangement of MP26 in lens junctional membranes: analysis with proteases and antibodies. 635 May 92

The major intrinsic protein from spinach chloroplast membranes, the light-harvesting chlorophyll a/b-protein complex, contains two distinct polypeptides of Mr 23,500 and 26,000 and 31% lipid by weight, comprising five diacyl lipids and seven chlorophylls, together with some carotenoids, per 26,000-Mr polypeptide. The chlorophyll a/b ratio is 1.1. Low-temperature fluorescence emission spectra of the light-harvesting complex revealed a major peak at 681 nm with a shoulder of variable intensity at 695 nm. The 695-nm emission has been correlated with a progressive aggregation of the complex into two-dimensional, semi-crystalline sheets. To determine the role of the light-harvesting complex in cation-dependent thylakoid stacking, the purified complex has been quantitatively incorporated into liposomes containing the four major chloroplast diacyl lipids using a simple freeze-thaw technique. The proteoliposomes appeared largely as unilamellar vesicles, with diameters between 0.1 and 0.8 micron. Freeze-fracture analysis showed intramembrane particles of 8-10 nm corresponding to the incorporated complex. Both monovalent and divalent cations caused an immediate aggregation of the proteoliposomes, which was reversed at low cation concentrations and was largely inhibited by prior trypsin treatment. Since lipid vesicles themselves showed none of these effects, it is concluded that surface-exposed polypeptide regions of the light-harvesting complex are directly involved in thylakoid stacking in vivo.
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PMID:The role of the light-harvesting chlorophyll a/b protein complex in chloroplast membrane stacking. Cation-induced aggregation of reconstituted proteoliposomes. 739 45

This study examines the high capacity binding of intact and carboxyl-terminal-truncated alpha A(alpha A) crystallin to two types of lens membrane preparations; membrane stripped of extrinsic protein and some lipid by extraction with urea and alkali and unextracted membrane isolated by centrifugation of total water insoluble protein on a sucrose gradient (native membrane). High capacity binding of alpha A crystallin to the urea-treated membrane was seen once the alpha A substrate concentration reached about 1 mg/ml of media. The membrane bound up to one mg of alpha A per mg of intrinsic protein (MP26) at a concentration of 5 mg alpha A/ml media, binding 5 to 10 times greater than that seen by others at saturation of the high affinity but low capacity binding sites. No apparent differences were seen between high capacity binding of carboxyl terminal-truncated alpha A (by trypsin) and intact alpha A, although each crystalline could antagonize binding of the other. However, once membrane bound, neither crystallin appeared to grossly displace the other. Using the carboxyl terminal-truncated alpha crystallin as a model substrate, native membrane was seen to have a higher capacity to bind the truncated alpha crystallin than urea-extracted membrane and binding was better correlated with the preexisting alpha A content of the native membrane than its MP26 content. An artificial native membrane was prepared by prebinding the truncated alpha A to urea-extracted membrane. This preparation bound more intact alpha A than urea-extracted membrane bearing no prebound crystallin. We conclude that lens native membrane possesses a high capacity to bind alpha crystallins and that this binding could be mediated through protein-protein interactions with alpha crystallin bound in situ to the membrane as extrinsic protein.
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PMID:High capacity binding of alpha crystallins to various bovine lens membrane preparations. 830 13

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387-392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.
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PMID:Distinct biochemical and topological properties of the 31- and 27-kilodalton plasma membrane intrinsic protein subgroups from red beet. 973 51

The combination of laser capture microdissection and mass spectrometry represents a powerful technology for studying spatially resolved proteomes. Moreover, the compositions of integral membrane proteomes have rarely been studied in a spatially resolved manner. In this study, ocular lens tissue was carefully dissected by laser capture microdissection and conditions for membrane protein enrichment, trypsin digestion, and mass spectrometry analysis were optimized. Proteomic analysis allowed the identification of 170 proteins, 136 of which were identified with more than one peptide match. Spatial differences in protein expression were observed between cortical and nuclear samples. In addition, the spatial distribution of post-translational modifications to lens membrane proteins, such as the lens major intrinsic protein AQP0, were investigated and regional differences were measured for AQP0 C-terminal phosphorylation and truncation.
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PMID:Spatial differences in an integral membrane proteome detected in laser capture microdissected samples. 1848 32