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)

Protein insertion into mitochondrial outer membrane (OM) vesicles isolated from Neurospora crassa has recently been reported. The N. crassa OM vesicles retained the features of the intact mitochondria concerning the dependency of insertion on the receptor protein [A. Mayer et al. (1993) J. Cell Biol. 121, 1233-1243]. In this study, OM vesicles were purified from bovine adrenal cortex mitochondria, and unilamellar proteoliposomes were reconstituted from OM vesicles using heptyl beta-thioglucoside. Both OM vesicles and the reconstituted outer membrane vesicles (ROM) were able to import porin, but unable to import the precursor of adrenodoxin, which translocates across both the outer and inner membranes of intact mitochondria. Porin insertion into both OM vesicles and ROM was inhibited in the presence of purified recombinant adrenodoxin precursor and also by ATP depletion, and was dependent on the trypsin-sensitive membrane surface factor, suggesting that the purified OM vesicles as well as ROM retained the properties of the intact OM concerning porin insertion. The protein import machinery of OM seems to be functional for the outer membrane protein without the participation of the inner membrane. The successful reconstitution of the protein import activity from solubilized OM will pave the way for further biochemical characterization of the protein import machinery of OM.
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PMID:Reconstitution of import-competent outer membrane vesicles from mammalian mitochondria. 779 73

A recombinant plasmid containing ompK36, the gene coding for the Klebsiella pneumoniae outer membrane protein OmpK36, was constructed by transposon mutagenesis and subcloning. Clones were identified in a cosmid library in Escherichia coli on the basis of their reaction with antiserum against the OmpK36 protein and by the presence in gel electrophoretic analysis of a band in E. coli outer membranes migrating with a mobility corresponding to 36 kDa. The ompK36-encoded protein exhibited characteristic properties of porins, such as heat modifiability and resistance to trypsin. The sequence of the gene revealed that OmpK36 is a close relative of the enterobacterial porin family, with a high degree of homology with E. coli OmpC, PhoE, and OmpF. On the basis of the structures of OmpF and PhoE porins, determined previously by X-ray analysis, it appears likely that the three-dimensional structure of OmpK36 also contains the motif of a 16-stranded beta-barrel, with long loops on one end and short turns on the other. Like the OmpC porin from E. coli, OmpK36 contains a long insertion in loop 4. The results of a binding study of complement component C1q to OmpK36 and the analysis of the OmpK36 model suggest that C1q binding sites are covered by the lipopolysaccharide core in the native porin.
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PMID:A porin from Klebsiella pneumoniae: sequence homology, three-dimensional model, and complement binding. 786 62

Although no binding of porin-precursor to trypsin-pretreated rat liver mitochondria was detectable, its integration into these mitochondria was observed to some extent, possibly by the bypass-import system first demonstrated in fungus mitochondria. A topographical study of this bypass-import system demonstrated that import of porin occurs at contact sites between the outer and inner membranes. Furthermore, antibodies to 29 kDa outer membrane protein, a component of the import machinery in contact sites for precursors of ornithine aminotransferase and sulfite oxidase, inhibited the integration of porin, suggesting that the 29 kDa protein is involved in the imports of most mitochondrial precursor proteins.
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PMID:Integration of porin into the outer mitochondrial membrane through the common import machinery located in contact sites. 848 60

The porin-associated protein of Rhodospirillum rubrum FR1 was found to contain a peptidoglycan binding motif. A partial fragment of 179 amino acids, obtained by cleavage of PAP with trypsin, Asp-N protease, and CNBr, was sequenced. Substantial sequence homology was found of the C-terminal part (residues 126-179) of porin-associated protein with OmpA, the peptidoglycan-associated lipoprotein of several bacteria, protein F of Pseudomonas aeruginosa, and PIII of Neisseria gonorrhoeae, the latter being also a porin-associated protein. The 179 amino acid fragment comprised about 67% of the mass spectrometrically determined total mass of PAP of 27850 Da.
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PMID:A peptidoglycan binding domain in the porin-associated protein (PAP) of Rhodospirillum rubrum FR1. 867 70

The major diffusion channel in the outer membrane of Haemophilus influenzae type b (Hib) is porin (341 amino acids; Mr 37 782). The Hib porin gene was cloned and overexpressed in Bacillus subtilis. Recombinant Hib porin (Bac porin), having aggregated into inclusion bodies, was purified under denaturing conditions and subsequently refolded. To compare Bac porin that is intrinsically devoid of lipooligosaccharides versus native Hib porin, the properties of Bac porin were assessed by the following four criteria: circular dichroism spectroscopy, channel formation in planar bilayers, resistance to trypsin digestion and formation of the conformational epitope recognized by an anti-Hib porin monoclonal antibody. We conclude that in the absence of lipooligosaccharides, Bac porin was refolded into a functional form which closely resembled the structure of Hib porin.
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PMID:Purification and refolding of recombinant Haemophilus influenzae type b porin produced in Bacillus subtilis. 877 68

Envelopes of large-cell variant Coxiella burnetii, the agent of Q fever, were the starting material for purification of an outer membrane protein (OMP) oligomer with aggregate molecular mass of approximately 2 x 10(4) kDa. The oligomer was resistant to trypsin and dissociation by SDS at 100 degrees C. Reducing agents dissociated the oligomer into monomers of 29.5 and 31 kDa, which migrated as a doublet during SDS-polyacrylamide gel electrophoresis. Both monomers were reactive in an immunoblot assay with monoclonal antibodies (mAbs) 4E8 and 4D6, which were previously selected for their reactivity with purified and SDS-denatured 29.5 kDa protein. Proteoliposomes were functional in an equilibrium assay at pH 7 and a swelling assay at pH 7 and 4.5. The pores in proteoliposomes allowed the passage of arabinose, glucose, and sucrose, but restricted stachyose. Polyclonal antibodies to C. burnetii cells and the mAbs were able to bind C. burnetii at pH 7 and 4.5. The uptake of 14C-glucose at pH 4.5 was inhibited by polyclonal antibodies and mAbs after binding to cells at pH 7. The mAbs did not inhibit 14C-glucose uptake at pH 4.5 after binding to cells at pH 4.5. Although the mAbs bind C. burnetii porin epitopes before and after acid activation, the mAbs bound under acidic conditions were unable to inhibit porin function. The inhibition of porin channel function by mAbs confirms the role of porin as a permeability barrier for the subsequent active transport of glucose by C. burnetii. In another study, we showed that the 29.5 kDa OMP antigen induced active immunity against virulent challenge. This information, combined with the recent confirmation that porins are important antigens in the induction of specific protective immune responses against infection by gram-negative bacteria, suggests that humoral immunity directed against C. burnetii porins might play an important role in immunity against Q fever (human infection) and coxiellosis (animal infection), global enzootic diseases.
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PMID:Pore-forming activity of Coxiella burnetii outer membrane protein oligomer comprised of 29.5- and 31-kDa polypeptides. Inhibition of porin activity by monoclonal antibodies 4E8 and 4D6. 878 19

The major outer membrane protein, FomA, of the Gram-negative human oral pathogen Fusobacterium nucleatum functions as a porin and is assumed to act as a receptor protein in coaggregation with other oral pathogenic bacteria such as Streptococcus sanguis and Porphyromonas gingivalis. We describe here the cloning of fomA from F. nucleatum in E. coli. Using pGEM3Zf(+), three recombinant plasmids were carrying parts of the fomA gene, but none of these contained regions upstream of the coding sequence. From these plasmids a clone was constructed which contained the whole fomA gene. The ATCC 10953 fomA gene was cloned under the phosphate limitation-inducible phoE promoter, using a vector derived from pACYC184. The protein was found to be incorporated into the outer membrane of the host in an apparently normal manner, as judged by heat-modifiability, trypsin-accessibility, and accessibility to antibodies to the protein in a whole cell enzyme-linked immunosorbent assay. The cloned FomA was found to exhibit pore-forming activity.
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PMID:Cloning of the fomA gene, encoding the major outer membrane porin of Fusobacterium nucleatum ATCC10953. 893 41

The primary and atomic structures of the porin protein from Rhodobacter (Rb.) capsulatus strain 37b4 were determined several years ago by peptide sequencing and X-ray crystallography. In this work the gene encoding this porin (named porCa) was cloned and sequenced. The porin open reading frame encodes 320 amino acids-a mature protein of 300 residues (molecular mass 31 552 kDa) and a presequence of 20 amino acids. Our deduced amino-acid sequence was directly confirmed by purifying the porin protein from the same bacterial strain and sequencing the amino terminus as well as several peptides derived from trypsin digestion. However, comparison of this deduced amino-acid sequence with the published primary structure of this porin, nominally from the same strain (but cultivated for ca. 30 years in a different laboratory) reveals seven differences in the amino-acid sequence at the following positions in the mature protein (published/present): 59 (Gly/Ala), 123 (Tyr/Asn), 135 Ser/Thr), 189 (Ile/Val), 196 (Asn/His), 231 (Ala/Thr) and 238 (Ser/deleted). Surprisingly, analysis of the positioning of these mutations revealed that they are located exclusively on transmembrane strands, with two of them deeply buried within the structure. These mutations may in fact have only marginal influence on porin structure and function. Northern blot analysis revealed that porCa encodes an RNA transcript of 1070 nucleotides. No differential response in the abundance or size of this mRNA was seen upon growth under phototrophic/anaerobic vs. chemotrophic/aerobic conditions, under high or low osmotic pressure. Primer extension experiments revealed a transcription start site 73 bases upstream from the ATG translation start, juxtaposed to the identified putative promoter region. Fusion of lacZ with this putative promoter region (using a 288-bp upstream region) revealed similar promoter activity in beta-galactosidase assays under both physiological conditions tested, again suggesting that this gene is constitutively expressed. The molecular genetic characterization described in this work opens the way for structure-function studies by site-directed mutagenesis.
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PMID:Molecular characterization and organization of porin from Rhodobacter capsulatus strain 37B4. 899 88

The yeast Peptide Sensitive Channel (PSC), a cationic channel of the mitochondrial outer membrane closes with slow kinetics at potentials of either polarity. The properties of this inactivation closely resemble those of the Voltage-Dependent Anion Channel (VDAC) slow kinetics closures. Addition of trypsin to one compartment suppresses the inactivation observed when this compartment is made positive, but does not affect the inactivation observed at potentials of reverse polarity. Both sides of the channel are sensitive. The reduced form of the Mast Cell Degranulating peptide (rMCD) increases the rate of inactivation, but only when the polarity of the compartment to which it is added is positive. The effect is not reversed by washing the peptide out, but is suppressed by trypsin. The peptide can bind to both sides of the membrane. The effect of rMCD on PSC closely resembles that of the "modulator" on VDAC. The similarities between PSC and VDAC suggest that the former might be a cationic porin of the mitochondrial outer membrane possessing a structure closely related to that of VDAC.
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PMID:Inactivation of the peptide-sensitive channel from the yeast mitochondrial outer membrane: properties, sensitivity to trypsin and modulation by a basic peptide. 907 Apr 62

The purpose of this work was to investigate the mechanism of regulation of mitochondrial respiration in vivo in different muscles of normal rat and mice, and in transgenic mice deficient in desmin. Skinned fiber technique was used to study the mitochondrial respiration in the cells in vivo in the heart, soleus and white gastrocnemius skeletal muscles of these animals. Also, cardiomyocytes were isolated from the normal rat heart, permeabilized by saponin and the "ghost" (phantom) cardiomyocytes were produced by extraction of myosin with 800 mM KCl. Use of confocal immunofluorescent microscopy and anti-desmin antibodies showed good preservation of mitochondria and cytoskeletal system in these phantom cells. Kinetics of respiration regulation by ADP was also studied in these cells in detail before and after binding of anti-desmine antibodies with intermediate filaments. In skinned cardiac or soleus skeletal muscle fibers but not in fibers from fast twitch skeletal muscle the kinetics of mitochondrial respiration regulation by ADP was characterized by very high apparent Km (low affinity) equal to 300-400 microM, exceeding that for isolated mitochondria by factor of 25. In skinned fibers from m. soleus, partial inhibition of respiration by NaN3 did not decrease the apparent Km for ADP significantly, this excluding the possible explanation of low apparent affinity of mitochondria to ADP in these cells by its rapid consumption due to high oxidative activity and by intracellular diffusion problems. However, short treatment of fibers with trypsin decreased this constant value to 40-70 microM, confirming the earlier proposition that mitochondrial sensitivity to ADP in vivo is controlled by some cytoplasmic protein. Phantom cardiomyocytes which contain mostly mitochondria and cytoskeleton and retain the normal shape, showed also high apparent Km values for ADP. Therefore, they are probably the most suitable system for studies of cellular factors which control mitochondrial function in the cells in vivo. In these phantom cells anti-desmin antibodies did not change the kinetics of respiration regulation by ADP. However, in skinned fibers from the heart and m. soleus of transgenic desmin-deficient mice some changes in kinetics of respiration regulation by ADP were observed: in these fibers two populations of mitochondria were observed, one with usually high apparent Km for ADP and the second one with very low apparent Km for ADP. Morphological observations by electron microscopy confirmed the existence of two distinct cellular populations in the muscle cells of desmin-deficient mice. The results conform to the conclusion that the reason for observed high apparent Km for ADP in regulation of oxidative phosphorylation in heart and slow twitch skeletal muscle cells in vivo is low permeability of mitochondrial outer membrane porins but not diffusion problems of ADP into and inside the cells. Most probably, in these cells there is a protein associated with cytoskeleton, which controls the permeability of the outer mitochondrial porin pores (VDAC) for ADP. Desmin itself does not display this type of control of mitochondrial porin pores, but its absence results in appearance of cells with disorganised structure and of altered mitochondrial population probably lacking this unknown VDAC controlling protein. Thus, there may be functional connection between mitochondria, cellular structural organisation and cytoskeleton in the cells in vivo due to the existence of still unidentified protein factor(s).
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PMID:Study of regulation of mitochondrial respiration in vivo. An analysis of influence of ADP diffusion and possible role of cytoskeleton. 939 78


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