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:3.4.21.64 (proteinase K)
4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Although Neisseria meningitidis can use haemoglobin as an iron source in vitro, the mechanism of haemoglobin-iron uptake is unknown. Using a biotinylated human haemoglobin probe in a solid-phase dot-binding assay, haemoglobin-binding activity was detected in total membranes derived from meningococci grown under iron-limited but not iron-sufficient conditions. In competition binding experiments, bovine and human haemoglobin could abrogate binding. In contrast, no binding inhibition was seen with ferric nitrate, protoporphyrin IX, and iron-loaded human transferrin. The ability of both haemin and catalase, a nonhaemoglobin haem-containing compound, to inhibit binding competitively suggested that the ligand recognized by the binding protein is the haem moiety. Scatchard plot analysis revealed a heterogeneous receptor population. Limited proteolysis with proteinase K abolished binding activity, suggesting a haemoglobin-protein interaction. Detection of activity in a whole-cell binding assay demonstrated that this haemin-binding protein was surface exposed. In a limited survey of meningococcal strains, the presence of haemoglobin-binding activity in all isolates indicated that expression of this binding protein is not serogroup specific.
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PMID:Identification of an outer-membrane haemoglobin-binding protein in Neisseria meningitidis. 148 30

We recently identified a 26-kDa hemin-repressible outer membrane protein (Omp26) expressed by the periodontal pathogen Porphyromonas gingivalis. We report the localization of Omp26, which may function as a component of a hemin transport system in P. gingivalis. Under hemin-deprived conditions, P. gingivalis expressed Omp26, which was then lost from the surface after a shift back into hemin-rich conditions. Experiments with 125I labeling of surface proteins to examine the kinetics of mobilization of Omp26 determined that it was rapidly (within less than 1 min) lost from the cell surface after transfer into a hemin-excess environment. When cells grown under conditions of hemin excess were treated with the iron chelator 2,2'-bipyridyl, Omp26 was detected on the cell surface after 60 min. One- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analyses using purified anti-Omp26 monospecific polyclonal immunoglobulin G antisera established that Omp26 was heat modifiable (39 kDa unheated) and consisted of a single protein species. Immunogold labeling of negatively stained and chemically fixed thin-section specimens indicated that Omp26 was associated with the cell surface and outer leaflet of the P. gingivalis outer membrane in hemin-deprived conditions but was buried in the deeper recesses of the outer membrane in hemin-excess conditions. Analysis of subcellular fractions of P. gingivalis grown either in hemin-excess or hemin-deprived conditions detected Omp26 only in the cell envelope fraction, not in the cytoplasmic fraction or culture supernatant. Limited proteolytic digestion of hemin-deprived P. gingivalis with trypsin and proteinase K verified the surface location of Omp26 as well as its susceptibility to proteolytic digestion. Heat shock treatment of hemin-excess-grown P. gingivalis also resulted in Omp26 translocation onto the outer membrane surface even in the presence of hemin. Furthermore, hemin repletion of heat-shocked, hemin-deprived P. gingivalis did not result in Omp26 translocation off the outer membrane surface, suggesting that thermal stress inactivates this transmembrane event. This newly described outer membrane protein appears to be associated primarily with the outer membrane, in which it is exported to the outer membrane surface for hemin binding and may be imported across the outer membrane for intracellular hemin transport.
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PMID:Localization of a Porphyromonas gingivalis 26-kilodalton heat-modifiable, hemin-regulated surface protein which translocates across the outer membrane. 152 61

Transport of iron(III) hydroxamates across the inner membrane into the cytoplasm of Escherichia coli is mediated by the FhuC, FhuD and FhuB proteins and displays characteristics typical of a periplasmic-binding-protein-dependent transport mechanism. In contrast to the highly specific receptor proteins in the outer membrane, at least six different siderophores of the hydroxamate type and the antibiotic albomycin are accepted as substrates. A fhuB mutant (deficient in transport of substrates across the inner membrane) which overproduced the periplasmic FhuD 30-kDa protein, bound [55Fe] iron(III) ferrichrome. Resistance of FhuD to proteinase K in the presence of ferrichrome, aerobactin, and coprogen indicated binding of these substrates to FhuD. FhuD displays significant similarity to the periplasmic FecB, FepB, and BtuE proteins. The extremely hydrophobic FhuB 70-kDa protein is located in the cytoplasmic membrane and consists of two apparently duplicated halves. The N- and C-terminal halves [FhuB(N) and FhuB(C)] were expressed separately in fhuB mutants. Only combinations of FhuB(N) and FhuB(C) polypeptides restored sensitivity to albomycin and growth on iron hydroxamate as a sole iron source, indicating that both halves of FhuB were essential for substrate translocation and that they combined to form an active permease. In addition, a FhuB derivative with a large internal duplication of 271 amino acids was found to be transport-active, indicating that the extra portion did not disturb proper insertion of the active FhuB segments into the cytoplasmic membrane. A region of considerable similarity, present twice in FhuB, was identified near the C-terminus of 20 analyzed hydrophobic proteins of periplasmic-binding-protein-dependent systems.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Iron(III) hydroxamate transport across the cytoplasmic membrane of Escherichia coli. 183 Feb 9

Porphyromonas gingivalis W50, W83, A7A1-28, and ATCC 33277 were investigated for their abilities to lyse sheep, human, and rabbit erythrocytes. All of the P. gingivalis strains studied produced an active hemolytic activity during growth, with maximum activity occurring in late-exponential-early-stationary growth phase. The enzyme was cell bound and associated with the outer membrane. Fractionation of P. gingivalis W50 localized the putative hemolysin almost exclusively in the outer membrane fraction, with significant hemolytic activity concentrated in the outer membrane vesicles. Ca2+ and Mg2+ ions significantly increased the expression of hemolytic activity. Hemolytic activity was inhibited by proteinase K, trypsin, the proteinase inhibitors Na-P-tosyl-L-lysine chloromethyl ketone and benzamidine, the metabolic inhibitor M-chlorophenyl-hydrazone, and iodoacetate. KCN and sodium azide (NaN3) only partially inhibited P. gingivalis hemolytic activity, while antiserum to whole cells of each of the P. gingivalis strains had a significant inhibitory effect on hemolytic activity. The P. gingivalis W50 hemolysin was inhibited by cysteine, dithiothreitol, and glutathione at concentrations of at least 10 mM; at low concentrations (i.e., 2 mM), dithiothreitol did not completely inhibit hemolytic activity. Heating to temperatures above 55 degrees C resulted in an almost complete inhibition of hemolytic activity. The effect of heme limitation (i.e., iron) on hemolysin production indicated that either limitation or starvation for heme resulted in significantly increased hemolysin production compared with that of P. gingivalis grown in the presence of excess heme.
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PMID:Hemolytic activity in the periodontopathogen Porphyromonas gingivalis: kinetics of enzyme release and localization. 203 55

Due to its extreme insolubility, Fe3+ is not transported as a monoatomic ion. In microbes, iron is bound to low molecular weight carriers, designated siderophores. For uptake into cells of Escherichia coli Fe3+ siderophores have to be translocated across two membranes. Transport across the outer membrane is receptor-dependent and energy-coupled; transport across the cytoplasmic membrane seems to follow a periplasmic binding protein-dependent transport mechanism. In support of this notion we demonstrate specific binding of the Fe3+ hydroxamate compounds ferrichrome, aerobactin, and coprogen, which are transported via the Fhu system, to the periplasmic FhuD protein, and no binding of the transport inactive ferrichrome A, ferric citrate, and iron sulfate. About 10(4) ferrichrome molecules were bound to the FhuD protein of cells which overproduced plasmid-encoded FhuD. Binding depended on transport across the outer membrane mediated by the FhuA receptor and the TonB protein. Binding to FhuD was supported by the exclusive resistance of FhuD to proteinase K in the presence of the transport active hydroxamates. The overproduced precursor form of the FhuD protein was not protected by the Fe3+ hydroxamates indicating a conformation different to the mature form. The FhuD protein apparently serves as a periplasmic carrier for Fe3+ hydroxamates with widely different structures.
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PMID:Iron (III) hydroxamate transport into Escherichia coli. Substrate binding to the periplasmic FhuD protein. 225 1

A simple procedure is described for the preparation of photosystem I (PSI) particles from Triton X-100-solubilized thylakoid membranes of the unicellular cyanobacterium Synechococcus 6301. The purified PSI complex contained the full complement of antenna chlorophylls, 130 +/- 5/P700, displayed the electron paramagnetic resonance signals characteristic of iron-sulfur centers X, A, and B, and had a protein/chlorophyll ratio of 2.9. Determination of the polypeptide composition, utilizing a uniformly 14C-labeled complex, showed that it contained polypeptides of 70, 18, 17.7, 16, and 10 kDa, in a molar ratio of 4.0:0.7:1.0:0.5:1.6. The relative amount of the lower molecular weight polypeptides showed progressive decrease with increase in Triton X-100 concentration and time of exposure to detergent. Consequently, it is proposed that in vivo the composition of the complex is [70 kDa]4 [18 kDa]1 [17.7 kDa]1 [16 kDa]1 [10 kDa]2. Relative to 130 mol of chlorophyll a, the PSI complex contained 16 mol of carotenoids, 13.7 +/- 1.0 g atoms of Fe, and 12.2 +/- 1.1 g atoms of labile sulfide. The properties of complexes fully depleted of the low-molecular weight polypeptides by treatment with sodium dodecyl sulfate or with proteinase K are also described.
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PMID:Characterization of a cyanobacterial photosystem I complex. 298 Dec 24

Yersinia enterocolitica 1165 (0:8) expressed several iron-regulated proteins with molecular masses of 240, 194, 80, 79, 70, and 67 kDa. These proteins were not detected in cells grown in iron-rich conditions. Cell surface iodination indicated that the 240- and 190-kDa proteins (HMWPs) were not surface exposed, whereas the 67- and 70-kDa proteins appeared to be exposed to the cell surface. Incubation with iron protected the 67- and 70-kDa proteins from proteinase K treatment, suggesting that they may be involved in iron acquisition. Monoclonal antibodies (MAbs) were produced against the HMWPs and the 67-kDa iron-regulated protein. MAbs to the HMWPs not only recognized the 240- and 194-kDa proteins but also reacted with the 67- and 70-kDa iron-regulated proteins. Similarly, MAbs to the 67-kDa protein reacted with the 67- and 70-kDa proteins and the HMWPs, suggesting that these iron-regulated proteins are related immunologically. In addition, the MAbs recognized the 67- and 70-kDa proteins and HMWPs from other Y. enterocolitica serotypes, suggesting that the antigenic sites recognized on these iron-regulated proteins are conserved. The MAbs examined did not inhibit iron binding or iron uptake and did not provide protection against a Y. enterocolitica 1165 (0:8) infection in a systemic mouse infection model. Although these MAbs were not protective in this model, these iron-regulated proteins may play a role in iron acquisition and virulence, but the MAbs examined are probably not directed against epitopes involved in iron acquisition or virulence.
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PMID:Characterization of monoclonal antibodies to Yersinia enterocolitica iron-regulated proteins. 764 Nov 39

Several clinical isolates of Vibrio parahaemolyticus were examined for their ability to utilize either hemin or hemoglobin as a sole source of iron. Both compounds appeared to be equally good iron sources. Maximum growth was obtained at 5 microM hemin or 1.25 microM hemoglobin under the conditions tested. Using a hemin-agarose batch affinity method, the hemin-binding protein was isolated from crude total membranes of a hemin-utilizing strain, WP1, grown under iron-deficient but not under iron-sufficient conditions. This protein was identical to the 83 kDa outer membrane protein which was expressed in response to iron limitation. The protein was susceptible to proteinase K cleavage in whole cells, indicating its exposure at the cell surface. Hemin and hemoglobin, but not protoporphyrin IX, inhibited binding of the protein to hemin-agarose.
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PMID:Utilization of hemin and hemoglobin as iron sources by Vibrio parahaemolyticus and identification of an iron-repressible hemin-binding protein. 775 Jul 38

The effect of the iron-chelating compounds EDDA and BPD on polypeptide regulation in the putative oral pathogen Treponema denticola was studied. SDS-PAGE analysis of the T. denticola strains grown in the presence of EDDA or BPD, i.e. iron-limiting environmental conditions, revealed the expression of 44 and 43 kDa polypeptides in the outer sheath, a 73 kDa polypeptide in the cell membrane, and a 16 kDa polypeptide in the soluble cell fraction. The hemin-binding activity of purified outer sheaths from T. denticola TD-4 grown in the presence of 6.4 mM EDDA was significantly greater than that observed in control (absence of EDDA) outer sheaths. Both activities were inhibited by proteinase K. SDS-PAGE, LDS-PAGE and TMBZ staining revealed the 44 and 43 kDa outer-sheath polypeptides to be expressed by T. denticola strains GM-1. MS-25, ATCC 33520 and ATCC 33404 (TD-4), strains which possessed strong hemin-binding activity. The 44 kDa hemin-binding polypeptide was purified by 1% CHAPS solubilization, HPLC, and SDS-preparative electrophoresis. N'-terminal sequence analysis indicated the purified 44 kDa polypeptide to belong to a new, undescribed group of polypeptides possessing hemin-binding activity.
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PMID:Effect of iron regulation on expression and hemin-binding function of outer-sheath proteins from Treponema denticola. 781 16

Congo red was bound from solution by strains of Porphyromonas gingivalis including W50, HG189, HG184, NCTC 11834, Bg 381, WPH35, the slower brown pigmenting colonial variant W50/BR1, and the avirulent mutant W50/BE1, and by Porphyromonas endodontalis HG370 and Porphyromonas asaccharolytica B537. SDS-PAGE of whole cells of all species examined displayed a 66 kDa Congo-red-binding component which was also detected in the outer membranes of P. gingivalis W50 grown in the chemostat under both haemin limitation and haemin excess, and which corresponded to a Coomassie-blue-stained band of the same mobility. Pretreatment of haemin-excess batch-grown cells of P. gingivalis W50 with polymyxin B, which binds to lipid A, did not inhibit binding, whilst binding was enhanced in the presence of 2 M ammonium sulphate, suggesting the involvement of non-specific hydrophobic interactions. Binding was also reduced by pretreatment with trypsin and papain, and by 8-anilino-1-naphthalenesulphonic acid, which binds to hydrophobic amino acids. The 66 kDa binding component was sensitive to proteinase K digestion, and loss of Congo red staining of this band correlated with the quantitative reduction in Congo red binding by whole cells. These data, and our previous work, show that Congo red and iron protoporphyrin IX (haemin) are bound to different outer-membrane components, and that Congo red binding may be of little value as a marker to detect virulent strains of P. gingivalis or those expressing haemin-binding proteins.
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PMID:Congo red binding by Porphyromonas gingivalis is mediated by a 66 kDa outer-membrane protein. 789 13


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