Arginine-Specific Cysteine Proteinase from Porphyromonas gingivalis as a Convenient Tool in Protein Chemistry

2001 ◽  
Vol 382 (9) ◽  
pp. 1399-1404 ◽  
Author(s):  
A. Banbula ◽  
P. Mak ◽  
M. Smoluch ◽  
J. Travis ◽  
J. Potempa
Keyword(s):  
Porphyromonas Gingivalis ◽  
Cysteine Proteinase ◽  
Enzymatic Digestion ◽  
Specific Protein ◽  
Protein Chemistry ◽  
Protein Cleavage ◽  
Peptide Bonds ◽  
Highly Active ◽  
High Concentrations ◽  
Triton X

Abstract RgpB, a cysteine proteinase produced by Porphyromonas gingivalis, exhibits proteolytic activity selectively directed against peptide bonds containing an arginine residue in the P1 position. Here we show that this enzyme can be used for very efficient and specific protein cleavage. RgpB is highly active even at high concentrations of denaturing agents, including urea (up to 6 M) and SDS (0.1%), both of them being commonly used for solubilization of insoluble proteins and peptides. Moreover, RgpB is able to digest polypeptide chains in buffers supplemented with 1% Triton X-100, 1% octyl or decylpyranoside, detergents employed for the enzymatic digestion of proteins transferred onto nitrocellulose membranes. These features render RgpB a suitable tool for use in protein chemistry.

scholarly journals Degradation of Host Heme Proteins by Lysine- and Arginine-Specific Cysteine Proteinases (Gingipains) ofPorphyromonas gingivalis

2001 ◽  
Vol 183 (19) ◽  
pp. 5609-5616 ◽  
Author(s):  
Aneta Sroka ◽  
Maryta Sztukowska ◽  
Jan Potempa ◽  
James Travis ◽  
Caroline Attardo Genco
Keyword(s):  
Human Serum ◽  
Porphyromonas Gingivalis ◽  
Heme Proteins ◽  
Cysteine Proteinase ◽  
Cysteine Proteinases ◽  
Normal Human ◽  
Minimal Media ◽  
High Concentrations ◽  
Extensive Degradation ◽  
Β Chain

ABSTRACT Porphyromonas gingivalis can use hemoglobin bound to haptoglobin and heme complexed to hemopexin as heme sources; however, the mechanism by which hemin is released from these proteins has not been defined. In the present study, using a variety of analytical methods, we demonstrate that lysine-specific cysteine proteinase of P. gingivalis (gingipain K, Kgp) can efficiently cleave hemoglobin, hemopexin, haptoglobin, and transferrin. Degradation of hemopexin and transferrin in human serum by Kgp was also detected; however, we did not observe extensive degradation of hemoglobin in serum by Kgp. Likewise the β-chain of haptoglobin was partially protected from degradation by Kgp in a haptoglobin-hemoglobin complex. Arginine-specific gingipains (gingipains R) were also found to degrade hemopexin and transferrin in serum; however, this was observed only at relatively high concentrations of these enzymes. Growth ofP. gingivalis strain A7436 in a minimal media with normal human serum as a source of heme correlated not only with the ability of the organism to degrade hemoglobin, haptoglobin, hemopexin, and transferrin but also with an increase in gingipain K and gingipain R activity. The ability of gingipain K to cleave hemoglobin, haptoglobin, and hemopexin may provide P. gingivalis with a useable source of heme for growth and may contribute to the proliferation of P. gingivaliswithin periodontal pockets in which erythrocytes are abundant.

scholarly journals Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line.

1985 ◽  
Vol 100 (5) ◽  
pp. 1540-1547 ◽  
Author(s):  
B Lathrop ◽  
E Olson ◽  
L Glaser
Keyword(s):  
Growth Factor ◽  
Cell Growth ◽  
Fibroblast Growth Factor ◽  
Cell Line ◽  
Calf Serum ◽  
Specific Protein ◽  
Fibroblast Growth ◽  
Mouse Muscle ◽  
Differentiated Cells ◽  
High Concentrations

The regulation of creatine phosphokinase (CPK) expression by polypeptide growth factors has been examined in the clonal mouse muscle BC3H1 cell line. After arrest of cell growth by exposure to low concentrations of serum, BC3H1 cells accumulate high levels of muscle-specific proteins including CPK. The induction of this enzyme is reversible in the presence of high concentrations of fetal calf serum, which cause quiescent, differentiated cells to reenter the cell cycle. Under these conditions, the rate of CPK synthesis is drastically reduced. We show in the present communication that either pituitary-derived fibroblast growth factor (FGF) or brain-derived FGF are as effective as serum in repressing the synthesis of CPK when added to quiescent, differentiated cells. The decrease in the rate of synthesis of CPK occurs within 22 h after the addition of pituitary FGF to the cells. Pituitary FGF had very little effect, if any, on the rate CPK degradation. The overall rate of protein synthesis and the pattern of synthesis of the major polypeptides made by these cells was not altered by the addition of FGF. Although pituitary FGF was mitogenic for BC3H1 cells, the rate of cell growth was not absolutely correlated with the extent of repression of CPK. Brain-derived FGF fully repressed CPK induction under conditions where it showed no significant mitogenic activity. These results show that the expression of a muscle-specific protein, CPK, can be controlled by a single defined polypeptide growth factor in fully differentiated cultures, and that initiation of cell division is not required for their regulation to take place.

scholarly journals Purification and properties of l-3-glycerophosphate dehydrogenase from pig brain mitochondria

1980 ◽  
Vol 192 (1) ◽  
pp. 9-18 ◽  
Author(s):  
I R Cottingham ◽  
C I Ragan
Keyword(s):  
Isoelectric Focusing ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Brain Mitochondria ◽  
Exchange Chromatography ◽  
Glycerophosphate Dehydrogenase ◽  
Pig Brain ◽  
Purification And Properties ◽  
High Concentrations ◽  
Triton X

L-3-Glycerophosphate dehydrogenase (EC 1.1.99.5) was purified from pig brain mitochondria by extraction with deoxycholate, ion-exchange chromatography and (NH4)2SO4 fractionation in cholate, and preparative isoelectric focusing in Triton X-100. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single subunit of mol.wt. 75 000. The enzyme contains non-covalently bound FAD and low concentrations of iron and acid labile sulphide. No substrate reducible e.p.r. signals were detected. The conditions of purification, particularly the isoelectric focusing step, lead to considerable loss of FAD and possibly iron-sulphur centres. It is therefore not possible to decide with certainty whether the enzyme is a flavoprotein or a ferroflavoprotein. The enzyme catalyses the oxidation of L-3-glycerophosphate by a variety of electron acceptors, including ubiquinone analogues. A number if compounds known to inhibit ubiquinone oxidoreduction by other enzymes of the respiratory chain failed to inhibit L-3-glycerophosphate dehydrogenase, except at very high concentrations.

scholarly journals The characterization and interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous

1982 ◽  
Vol 201 (3) ◽  
pp. 515-521 ◽  
Author(s):  
P S J Cheetham ◽  
P Dunnill ◽  
M D Lilly
Keyword(s):  
Cell Membrane ◽  
Membrane Lipids ◽  
Cholesterol Oxidase ◽  
Hydrophobic Region ◽  
Hydrophobic Domain ◽  
Solubilized Enzyme ◽  
High Concentrations ◽  
Triton X ◽  
Catalytic Functions ◽  
Hydrophobic Anchor

The physical properties and the methods used for interconversion of three forms of cholesterol oxidase extracted from Nocardia rhodochrous by treatment with Triton X-100, trypsin or buffer alone provide evidence that these forms differ chiefly in the possession or absence of a hydrophobic anchor region connected by a trypsin-sensitive region. The hydrophobic domain normally integrates the enzyme into the cell membrane and confers amphipathic properties on the solubilized enzyme, causing adsorption to hydrophobic resins, aggregation when detergent is removed and formation of mixed micelles with detergent and cholesterol resulting in surface-dilution kinetic behaviour and activation by relatively high concentrations of water-miscible solvents. By contrast, only the enzymic fragment is extracted with trypsin and it behaves as a conventional soluble enzyme and does not aggregate or interact with hydrophobic resins, detergents or water-miscible solvents. As no phospholipid could be detected in the enzyme extracts, the detergent appears to act as a substitute for the cell-membrane lipids that would normally interact with the hydrophobic region. This cholesterol oxidase is an example of a prokaryotic enzyme possessing two closely associated catalytic functions, dehydrogenase and isomerase activities, and an anchoring function.

scholarly journals Identification of a novel protein (G210) specific to the Golgi apparatus.

1989 ◽  
Vol 37 (8) ◽  
pp. 1177-1182 ◽  
Author(s):  
R Hogue-Angeletti ◽  
R Y Xu ◽  
J O Gonatas ◽  
A Stieber ◽  
N K Gonatas
Keyword(s):  
Golgi Apparatus ◽  
Specific Protein ◽  
Light Microscopic Level ◽  
Gut Epithelium ◽  
Carbonate Solutions ◽  
Microsomal Membranes ◽  
Relative Molecular Weight ◽  
Immunohistochemical Studies ◽  
Triton X ◽  
Novel Protein

A monoclonal antibody, 3C9, has enabled the detection of a novel Golgi-specific protein in bovine tissues. Immunohistochemical studies at the light microscopic level have detected the 3C9 antigen only in certain cells: exocrine pancreas, gut epithelium, and thymus epithelium. Examination of gut and pancreas by immunoelectron microscopy showed a localization exclusive to the Golgi apparatus. The relative molecular weight of the antigen detected by immunoblotting is 210,000 daltons. The antigen is not extracted from microsomal membranes of bovine gut epithelium by sodium carbonate solutions. Furthermore, the 3C9 antigen enters into the detergent phase when Triton X-114 partitioning methods are used. These data strongly suggest that this novel antigen is an intrinsic membrane protein, resident in the Golgi apparatus of certain cells. Moreover, they enhance the hypothesis that the distribution of enzymes and polypeptides in the Golgi apparatus is cell specific.

Production and consumption of nitric oxide by denitrifyingFlexibacter canadensis

1995 ◽  
Vol 41 (7) ◽  
pp. 585-591 ◽  
Author(s):  
Qitu Wu ◽  
Roger Knowles ◽  
Yiu-Kwok Chan
Keyword(s):  
Nitric Oxide ◽  
Nitrite Reductase ◽  
Chelating Agent ◽  
No Production ◽  
Production And Consumption ◽  
Carbonyl Cyanide ◽  
No Consumption ◽  
High Concentrations ◽  
Ph Range ◽  
Triton X

Production and consumption of nitric oxide (NO) by Flexibacter canadensis cells under anaerobic conditions was investigated using a chemiluminescence NO analyzer. Net NO production from nitrite in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP) was pH dependent, increased in the pH range from 4.5 to 6.5, and sharply decreased at pH >6.5. CCCP inhibited NO consumption but only at pH values ≤6.5. This can explain why CCCP stimulation of NO production depends on the pH. Denitrification of nitrite at high concentrations (≥5 mM) also resulted in net NO accumulation. Diethyldithiocarbamate, a copper chelating agent, prevented not only net production of NO during the reduction of nitrite in the presence of CCCP, but also production of nitrous oxide (N2O) from nitrite in the presence of C2H2. This suggests that F. canadensis may possess a copper-type nitrite reductase. However, cytochrome cd1- and copper-containing nitrite reductase DNA probes from Pseudomonas species did not hybridize with the total DNA of F. canadensis, indicating that the nitrite reductase of F. canadensis may possess unique properties. In addition to diethyldithiocarbamate, sulfide, carbon monoxide, azide, cyanide, hydroxylamine and Triton X-100 prevented net NO production from nitrite in the presence of CCCP, and also inhibited NO consumption. C2H2, an inhibitor of N2O reductase, did not affect NO production or consumption.Key words: nitrite reductase, nitric oxide (NO), carbonyl cyanide m-chlorophenylhydrazone (CCCP), Flexibacter canadensis.

scholarly journals Kinetics and specificity of homogeneous protein disulphide-isomerase in protein disulphide isomerization and in thiol-protein-disulphide oxidoreduction

1983 ◽  
Vol 213 (1) ◽  
pp. 235-243 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Reduced Glutathione ◽  
Physiological Role ◽  
Homologous Proteins ◽  
Ph Optimum ◽  
Oxidoreductase Activity ◽  
Protein Disulphide Isomerase ◽  
Highly Active ◽  
Reducing Conditions ◽  
Order Of Magnitude ◽  
High Concentrations

The protein disulphide-bond isomerization activity of highly active homogeneous protein disulphide-isomerase (measured by re-activation of ‘scrambled’ ribonuclease) is enhanced by EDTA and by phosphate buffers. As shown for previous less-active preparations, the enzyme has a narrow pH optimum around pH 7.8 and requires the presence of either a dithiol or a thiol. The dithiol dithiothreitol is effective at concentrations 100-fold lower than the monothiols reduced glutathione and cysteamine. The enzyme follows Michaelis-Menten kinetics with respect to these substrates; Km values are 4,620 and 380 microM respectively. The enzyme shows apparent inhibition by high concentrations of thiol or dithiol compounds (greater than 10 X Km), but the effect is mainly on the extent of reaction, not the initial rate. This is interpreted as indicating the formation of significant amounts of reduced ribonuclease in these more reducing conditions. The purified enzyme will also catalyse net reduction of insulin disulphide bonds by reduced glutathione (i.e. it has thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase activity), but this requires considerably higher concentrations of enzyme and reduced glutathione than does the disulphide-isomerization activity. The Km for reduced glutathione in this reaction is an order of magnitude greater than that for the disulphide-isomerization activity, and the turnover number is considerably lower than that of other enzymes that can catalyse thiol-disulphide oxidoreduction. Conventional two-substrate steady-state analysis of the thiol:protein-disulphide oxidoreductase activity indicates that it follows a ternary-complex mechanism. The protein disulphide-isomerase and thiol:protein-disulphide oxidoreductase activities co-purify quantitatively through the final stages of purification, implying that a single protein species is responsible for both activities. It is concluded that previous preparations, from various sources, that have been referred to as protein disulphide-isomerase, disulphide-interchange enzyme, thiol:protein-disulphide oxidoreductase or glutathione:insulin transhydrogenase are identical or homologous proteins. The assay, nomenclature and physiological role of this enzyme are discussed.

scholarly journals Purification and properties of an esterase from organophosphate-resistant strain of the mosquito Culex quinquefasciatus

1990 ◽  
Vol 266 (1) ◽  
pp. 83-90 ◽  
Author(s):  
A T Merryweather ◽  
J M Crampton ◽  
H Townson
Keyword(s):  
Culex Quinquefasciatus ◽  
Resistant Strain ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Starch Gel Electrophoresis ◽  
Post Translational Modification ◽  
Highly Active ◽  
High Concentrations ◽  
Starch Gels

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.

scholarly journals Discrete Proteolysis of Focal Contact and Adherens Junction Components in Porphyromonas gingivalis-Infected Oral Keratinocytes: a Strategy for Cell Adhesion and Migration Disabling

2002 ◽  
Vol 70 (10) ◽  
pp. 5846-5856 ◽  
Author(s):  
Edith Hintermann ◽  
Susan Kinder Haake ◽  
Urs Christen ◽  
Andrew Sharabi ◽  
Vito Quaranta
Keyword(s):  
Cell Adhesion ◽  
Proteolytic Activity ◽  
Porphyromonas Gingivalis ◽  
Cysteine Proteinase ◽  
Adherens Junction ◽  
Signaling Molecules ◽  
Cysteine Proteinase Inhibitor ◽  
Focal Contact ◽  
Oral Keratinocytes ◽  
Adhesive Interactions

ABSTRACT Adhesive interactions of cells are critical to tissue integrity. We show that infection with Porphyromonas gingivalis, a major pathogen in the periodontal disease periodontitis, interferes with both cell-matrix and cell-cell adhesion in the oral keratinocyte cell line HOK-16. Thus, infected cells showed reduced adhesion to extracellular matrix, changes in morphology from spread to rounded, and impaired motility on purified matrices in Transwell migration assays and scratch assays. Western blot analysis of P. gingivalis-challenged HOK-16 cells revealed proteolysis of focal contact components (e.g., focal adhesion kinase), adherens junction proteins (e.g., catenins), and adhesion signaling molecules (e.g., the tyrosine kinase SRC). Proteolysis was selective, since important components of adherens junctions (E-cadherin) or signaling molecules (extracellular signal-regulated kinases ERK1/2) were not degraded. The virulence factors gingipains, cysteine proteinases expressed by P. gingivalis, are likely responsible for this proteolytic attack, since they directly digested specific proteins in pull-down experiments, and their proteolytic activity was blocked by the cysteine proteinase inhibitor N-α-p-tosyl-l-lysine chloromethyl ketone and also by a caspase inhibitor. Proteolysis was strain dependent, such that ATCC 33277 and 381 had high proteolytic potential, whereas W50 showed almost no proteolytic activity. These findings may help explain the formation of gingival pockets between cementum and periodontal epithelium, a hallmark of periodontitis. Furthermore, they illustrate a new pathogenetic paradigm of infection whereby bacteria may disrupt the integrity of epithelia.

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