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)

The major outer membrane protein of Haemophilus influenzae type b (Hib) is porin (Mr 38,000, 341 amino acids). To identify antigenic determinants on Hib porin that might be exposed at the bacterial cell surface, seven mouse monoclonal anti-Hib porin antibodies were generated. The monoclonal antibodies were tested for their binding to intact cells by flow cytometry; all but one bound to the cell surface. Digestions of Hib porin with cyanogen bromide, hydroxylamine or trypsin generated fragments, the identities of which were confirmed by microsequencing of the amino termini. Following electrophoresis and immunoblotting of the fragments, the specificities of the monoclonal antibodies for their cognate sequences were determined. The porin gene ompP2 was expressed in the baculovirus expression vector system; the recombinant porin was recognized by all of the monoclonal antibodies. Deletions were created by omega mutagenesis of ompP2, generating proteins truncated after amino acids 139, 174, 182, and 264. These deletion proteins were tested for reactivities with the monoclonal antibodies, thereby establishing the boundaries of three antigenic determinants that were recognized by the monoclonals: domain (i), amino acids 104-139; domain (ii) amino acids 162-174; and domain (iii), amino acids 267-341. The biological activities of monoclonal antibodies that were representative of these three classes were tested for their bactericidal activity in complement-mediated lysis of whole cells. The monoclonal antibodies were also tested for their immunoprotective properties in the infant rat model of bacteraemia. Although the monoclonal antibodies were surface-binding, they were neither bactericidal nor protective.
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PMID:Monoclonal antibodies specific to porin of Haemophilus influenzae type b: localization of their cognate epitopes and tests of their biological activities. 137 79

The primary structure of the integral membrane protein porin from the purple bacterium Rhodobacter capsulatus was determined. The protein was cleaved with trypsin, CNBr and Asp-N protease. The peptides were isolated, sequenced and aligned to a total length of 301 residues with an Mr of 31,536. The low isoelectric point of 3.9 is confirmed by the high excess of 34 Asp and 17 Glu (16.9%) over 10 Lys, 7 Arg and 2 His (6.3%). Overall sequence similarity to other porins is not evident when using sequence alignment programs. However, a partial relationship to Neisseria porins seems to exist. The established sequence has been used as the basis for a three-dimensional structure determination by X-ray diffraction at 0.18-nm resolution. The arrangement of the sequence in the 16-stranded beta-barrel of porin is given. Some sequence-structure correlations are discussed.
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PMID:Primary structure of porin from Rhodobacter capsulatus. 165 Dec 39

We have investigated the transmembrane topology of the bovine heart mitochondrial porin by means of proteases and antibodies raised against the amino-terminal region of the protein. The antisera against the human N-terminus reacted with porin in Western blots of NaDodSO4-solubilized bovine heart mitochondria and with the membrane-bound porin in enzyme-linked immunosorbent assay (ELISA). The immunoreaction with mitochondria coated on microtiter wells showed that the amino-terminal region of the protein is not embedded in the lipid bilayer but is exposed to the cytosol. Back-titration of unreacted anti-N-terminal antibodies after their incubation with intact mitochondria demonstrated that the porin N-terminus is also exposed in "noncoated" mitochondria. No difference in antisera reactivity was observed between intact and broken mitochondria. Intact and broken mitochondria were subjected to proteolysis by specific proteases. The membrane-bound bovine heart porin was strongly resistant to proteolysis, but a few specific cleavage sites were observed. Staphylococcus aureus V8 protease gave a large 24K N-terminal peptide, trypsin produced a 12K N-terminal and an 18K C-terminal peptide, and chymotrypsin gave two peptides of Mr 19.5K and 12.5K, which were both recognized by the antiserum against the human N-terminus. Carboxypeptidase A was ineffective in cleaving the membrane-bound porin in both intact and broken mitochondria. Thus, the carboxy-terminal part of the protein is probably not exposed to the water phase. The cleavage patterns of membrane-bound porin, obtained with S. aureus V8 protease, trypsin, and chymotrypsin, showed no difference between intact and broken mitochondria, thus indicating that all porin molecules have the same orientation in the membrane. The computer analysis of the sequence of human B-lymphocyte porin suggested that 16 beta-strands can span the phospholipid bilayer. This result, together with the overall information presented, allowed us to draw a possible scheme of the transmembrane arrangement of mammalian mitochondrial porin.
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PMID:Peptide-specific antibodies and proteases as probes of the transmembrane topology of the bovine heart mitochondrial porin. 171 14

The assembly of outer membrane proteins of Escherichia coli was examined using the OmpF porin as a model. Since this protein is made as a precursor, which is processed to a protein of Mr 37,000 before being assembled into trimers in the outer membrane, we synthesized a modified OmpF, which lacked 16 out of 22 amino acid residues from its signal sequence, in a coupled transcription-translation system. This modified protein resembled the unfolded, monomeric OmpF in its electrophoretic behavior, but much of the protein apparently existed in a more tightly folded conformation as it was recognized by a monoclonal antibody specific to a surface epitope of the native, trimeric OmpF porin. At least some conformers of this protein could be further incorporated into outer membrane or lipopolysaccharide bilayers, and assembled into trimers. The trimers formed were trypsin-resistant and heat-stable in sodium dodecyl sulfate up to 70 degrees C, thus showing the characteristics of the native trimeric protein. These results extend our earlier observation that OmpF monomer secreted by spheroplasts of E. coli can be trimerized in vitro (Sen, K., and Nikaido, H (1990) Proc. Natl. Acad. Sci. U.S. A 87, 743-747) and show that the trimerization can occur, albeit at a low efficiency, with porin monomers synthesized in vitro, presumably not contaminated by membrane fragments or other components of the cell envelope. However, comparison of trimerization efficiency of the nascent in vitro product with that of the same product already exposed to aqueous medium, as well as with that of the spheroplast-secreted product, leads us to the working hypothesis that the trimerization process in intact cells is accelerated either by accessory components or by the conformational changes accompanying the secretion through the cytoplasmic membrane and that the reactions observed in this study represent only part of the physiological process.
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PMID:Trimerization of an in vitro synthesized OmpF porin of Escherichia coli outer membrane. 204 Jun 34

The yeast mitochondrial outer membrane contains a major 70 kd protein with an amino-terminal hydrophobic membrane anchor and a hydrophilic 60 kd domain exposed to the cytosol. We now show that this protein (which we term MAS70) accelerates the mitochondrial import of many (but not all) precursor proteins. Anti-MAS70 IgGs or removal of MAS70 from the mitochondria by either mild trypsin treatment or by disrupting the nuclear MAS70 gene inhibits import of the F1-ATPase beta-subunit, the ADP/ATP translocator, and of several other precursors into isolated mitochondria by up to 75%, but has little effect on the import of porin. Intact cells of a mas70 null mutant import the F1-ATPase alpha-subunit and beta-subunits, cytochrome c1 and other precursors at least several fold more slowly than wild-type cells. Removal of MAS70 from wild-type mitochondria inhibits binding of the ADP/ATP translocator to the mitochondrial surface, indicating that MAS70 mediates one of the earliest import steps. Several precursors are thus imported by a pathway in which MAS70 functions as a receptor-like component. MAS70 is not essential for import of these precursors, but only accelerates this process.
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PMID:Protein import into yeast mitochondria is accelerated by the outer membrane protein MAS70. 217 Jan 6

A rapid colony immunoblot screening procedure was used to demonstrate the surface localization of porin protein F on bacterial colonies of Pseudomonas aeruginosa. By this method, we demonstrated that protein F was accessible to four different specific monoclonal antibodies in a wide variety of both mucoid and nonmucoid P. aeruginosa strains. Controls were performed to demonstrate that, using this procedure, only surface-exposed epitopes bound monoclonal antibodies and that nonspecific binding of monoclonal antibodies either to cells lacking protein F or to mucoid exopolysaccharide did not occur. Monoclonal antibodies MA4-4, MA2-10, and MA4-10, specific for protein F, also interacted with colonies of Pseudomonas putida and Pseudomonas syringae, whereas the protein F specific monoclonal antibody MA5-8 interacted only with P. aeruginosa strains. Using the above-named monoclonal antibodies, we investigated the antigenic structure of protein F. Monoclonal antibodies MA4-4, MA2-10, and MA4-10 bound to 29-31 kilodalton proteolytic fragments produced after papain or trypsin digestion of purified protein F or of protein F in outer membranes or intact cells. Antibody MA5-8 did not interact with proteolytically digested protein F but did interact with two of the six fragments produced after partial cyanogen bromide cleavage of protein F. Antibodies MA4-4, MA2-10, and MA4-10 did not interact with protein F after reduction of its internal disulphide bonds with 2-mercaptoethanol; in contrast, the reactivity of MA5-8 was unaffected. This data suggests that there are at least two distinct highly conserved surface epitopes on porin protein F.
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PMID:Characterization of two surface-localized antigenic sites on porin protein F of Pseudomonas aeruginosa. 240 19

Diminished permeation of beta-lactam antibiotics in a mutant (PCC23) of Pseudomonas aeruginosa, PAO503, was investigated. Resistance to beta-lactam antibiotics could not be correlated to a change in the beta-lactamase or target proteins in strain PCC23 but was correlated with decreased permeability. In liposome swelling assays, the permeability defect was associated with strain PCC23 porin. Amino acid analysis did not show significant difference of the porin of the mutant (PCC23) from that of the parent (PAO503). Changes in the behavior of isolated porin from PCC23 in migration in sodium dodecyl sulfate-polyacrylamide gels and in response to trypsin digestion as well as preferential labeling of PCC23 by a monoclonal antibody with a preference for the modified form of porin F (F) indicate that a structural alteration had occurred in this strain and correlated with the change in permeability.
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PMID:Penetration of beta-lactams through Pseudomonas aeruginosa porin channels. 244 74

Yeast porin, the major outer mitochondrial membrane protein, is synthesized without a cleavable extension peptide and post-translationally inserted into the membrane. When inserted into the membrane, it acquires resistance to externally added trypsin. To locate the sequences responsible for membrane insertion and topogenesis in the primary structure of yeast porin, we constructed several deletion and chimeric mutants of the porin cDNA. These cDNAs were expressed in vitro and the products were assayed for capacity to be correctly inserted into isolated mitochondria. It was thus found that deletion of the segment spanning residues 37-98 did not appreciably impair the insertion competence and the inserted protein became resistant to trypsin. On the other hand, the porin mutant lacking the segment consisting of residues 17-98 did not acquire the trypsin resistance, though it could bind to mitochondria specifically. Deletion of the carboxy-terminal 62 amino acid residues also abolished the capacity to be correctly inserted into mitochondria. We conclude that information required for membrane insertion and intramembranous topogenesis of the porin molecule is stored not only in the amino-terminal region but also in the carboxy-terminal portion.
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PMID:Both amino- and carboxy-terminal portions are required for insertion of yeast porin into the outer mitochondrial membrane. 246 4

The addition of maltose to the growth media of Actinobacillus pleuropneumonia (serotype 1) resulted in the induction of an outer membrane protein (OMP) with a molecular mass of 42 kDa. This protein had porin-like properties in that it was peptidoglycan-associated and was resistant to proteolysis by trypsin. A pleuropneumoniae expressing the 42 kDa OMP were unable to bind lambda phage. Similar proteins were also induced in A. pleuropneumoniae isolates representing serotypes 2 to 7 with the exception of serotype 4; however, not all isolates of any given serotype expressed a maltose-inducible OMP. Western immunoblotting using convalescent antiserae against the serotype 1 A. pleuropneumoniae indicated that the 42 kDa OMP was expressed in vivo and was cross-reactive with the maltose-inducible OMPs from other serotypes.
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PMID:Identification of a maltose-inducible major outer membrane protein in Actinobacillus (Haemophilus) pleuropneumoniae. 247 40

Co-translational insertion of liver microsomal cytochrome P-450 into the endoplasmic reticulum membrane is mediated by the signal recognition particle (SRP) and the presence in the cytochrome molecule of a signal sequence that can be recognized by SRP has been postulated. To locate this signal sequence, six hybrid cDNAs were constructed in which various segments of a cDNA for a rabbit liver cytochrome P-450 are fused with a cDNA or its fragment encoding yeast porin (an outer mitochondrial membrane protein) or with a cDNA for pre-interleukin 2 (a secretory protein) from which the 5'-terminal portion encoding most of its signal sequence had been removed. These hybrid cDNAs were inserted into an SP-6 transcription vector and transcribed in vitro. The mRNAs thus synthesized were translated in a cell-free system in the presence of rough microsomes. It was thus found that only those chimeric proteins containing (at their amino-terminal end) the amino-terminal cytochrome P-450 segments consisting of greater than or equal to 29 amino acid residues were co-translationally inserted into the membrane in an SRP-dependent fashion. These proteins were, however, neither processed nor translocated across the membrane. These findings, coupled with the observation that the major portion of these proteins, when inserted into the membrane, was degraded by trypsin, led to the conclusion that a short amino-terminal segment (less than 29 residues) of the cytochrome P-450 functions not only as an insertion signal but also as a stop-transfer sequence. This segment is, therefore, similar to the internal signal of type II plasma membrane proteins, but differs from the latter in the topogenic function.
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PMID:A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop-transfer sequence. 282 91


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