Purification and characterization of exocellular proteases produced by a clinical isolate and a laboratory strain of Pseudomonas aeruginosa

1980 ◽  
Vol 26 (1) ◽  
pp. 77-86 ◽  
Author(s):  
S. E. Jensen ◽  
L. Phillippe ◽  
J. Teng Tseng ◽  
G. W. Stemke ◽  
J. N. Campbell
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Aeruginosa ◽  
Clinical Isolate ◽  
Protease Activity ◽  
Gel Filtration ◽  
Laboratory Strain ◽  
Exchange Chromatography ◽  
The Other ◽  
Protease Production ◽  
Peptide Bonds

Exocellular protease production was examined in two separate strains of Pseudomonas aeruginosa, one a clinical isolate and the other a laboratory strain. Both strains produced two separate proteases (proteases 1 and 2) which were indistinguishable from one strain to the other. The two proteases were purified by a two-step procedure of gel filtration chromatography followed by ion-exchange chromatography. Proteases 1 and 2 were shown to be distinct serologically and unrelated by physicochemical parameters examined. Protease 1 was the major exocellular protein produced and contributed about 95% of the total protease activity of the culture. It was estimated to have a molecular weight of 34 850 and was also shown to contain 10% glucosamine by weight. Protease 2, in contrast, had an estimated molecular weight of 52750 and contained no detectable carbohydrate. Proteases 1 and 2 were both stimulated by Ca2+, and Mg2+ and inhibited by Co2+Zn2+, and 1,10-o-phenanthroline. Protease 1 was also inhibited by EDTA. In addition to protease activity, both proteases 1 and 2 demonstrated elastase activity as well as a limited collagenase activity. Specificity of the two proteases against synthetic peptides was, however, quite different. Protease 1, but not protease 2, showed a preference for peptide bonds in which the amino group was contributed by an amino acid with a hydrophobic R group.

scholarly journals The multiple forms of α-D-mannosidase in human plasma

1979 ◽  
Vol 179 (3) ◽  
pp. 583-592 ◽  
Author(s):  
S Hirani ◽  
B Winchester
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
The Other ◽  
Multiple Forms ◽  
Simple Correlation ◽  
Multiple Components

The acidic alpha-D-mannosidase in human plasma closely resembles liver acidic alpha-D-mannosidase in its affinity for concanavalin A-Sepharose, molecular weight and resolution into multiple components on DEAE-cellulose. A combination of chromatography on concanavalin A-Sepharose and gel filtration on Sephadex G-200 and Sepharose 6B suggests that four forms of intermediate alpha-D-mannosidase, which differ either in their molecular weight of affinity for concanavalin A, exist in human plasma. A practical classification and nomenclature for the multiple forms of intermediate alpha-D-mannosidase in plasma based on molecular weight and affinity for concanavalin A is proposed. Multiple forms of intermediate alpha-D-mannosidase were also observed by ion-exchange chromatography on DEAE-cellulose, but there was not a simple correlation between these forms and those obtained with the other separation procedures. The form of intermediate alpha-D-mannosidase least abundant in plasma, approx. 7% of the activity, has very similar properties to the neutral alpha-D-mannosidase in human liver. In contrast, the other three forms of intermediate alpha-D-mannosidase, which account for over 90% of the activity, do not appear to be present in liver, except perhaps in trace amounts.

scholarly journals Interaction between S-Type Pyocins and Microcin-II-Like Bacteriocins in Pseudomonas aeruginosa

2021 ◽  
Vol 83 (3) ◽  
pp. 72-80
Author(s):  
O.B. Balko ◽  
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Aeruginosa ◽  
Ion Exchange ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Activity Increase ◽  
Activity Indices

According to our previous results, S-type bacteriocins of Pseudomonas aeruginosa are characterized by high activity against phytopathogenic Pseudomonas syringae strains. In addition to these pyocins producing strains are able to synthesize microcin-II-like bacteriocins. Presence of interaction between these two killer factors can determine methods of their use and activity increase of bacteriocins with antiphytopathogenic properties. The aim of the work was to test possibility of interaction between S-type pyocins and microcin-II-like bacteriocins of P. aeruginosa. Methods. The objects of the study were pyocins produced by 6 P. aeruginosa strains. Killer factors in composition of induced lysates were concentrated by 70% ammonium sulphate precipitation, dialyzed through dialysis membrane with molecular weight cut-off (MWCO) 3.5 kDa. Then ion-exchange chromatography with DEAE-cellulose, gel filtration with Sephadex G-75 and ultracentrifugation at 215.000 g for 1 and 4 hours were used for their separation. Protein concentration and antimicrobial activity were determined in obtained fractions. Visualization of proteins in active fraction composition was conducted by electrophoresis according to the Laemmli method. Results. Under ion-exchange chromatography with DEAE-cellulose application elution of bacteriocins available in lysate composition occurs simultaneously. The highest indices of activity and protein concentration were in the 4th fraction, containing two protein bands with molecular weight near 58 and 9 kDa, which are typical for S5 pyocin and microcin-II-like bacteriocins of P. aeruginosa. Further gel filtration of sampled fractions through Sephadex G-75 allowed to separate noted killer factors and obtaine purified fraction containing microcin-II-like pyocins only. Application of ultracentrifugation during 1 hour didn’t precipitate studied bacteriocins, whereas during 4 hours – lead to their separation. At the same time a twofold increase of activity indices for S-type pyocins in precipitates and for microcin-IIlike killer factors – in supernatants were observed. However achieved concentration was characterized by short-term effect, since in 14 days activity of supernatants decreased by 4–16 times, and for precipitates – by 80–640 times. Then revealed tendency for activity decrease continued. Conclusions. S-type pyocins and microcin-II-like bacteriocins of P. aeruginosa interact with each other, that ensures their stabilization and protects again destruction. Application of methods that cause separation of these killer factors is inexpedient, since it results into considerable decrease of bacteriocin activity indices.

scholarly journals Low-Molecular-Mass Human Salivary Mucin, MG2: Structure and Binding of Pseudomonas aeruginosa

1993 ◽  
Vol 4 (3) ◽  
pp. 315-323 ◽  
Author(s):  
Molakala S. Reddy ◽  
Michael J. Levine ◽  
William Paranchych
Keyword(s):  
Molecular Weight ◽  
Pseudomonas Aeruginosa ◽  
Ion Exchange ◽  
Molecular Mass ◽  
Protein Interaction ◽  
Binding Assay ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Salivary Mucin

Low-molcular-mass human salivary mucin, MG2, was isolated from human submandibular-sublingual saliva (HSMSL) employing citraconylation, gel filtration, and ion-exchange chromatography. Following proteolysis with trypsin, two glycopeptides were purified. The higher molecular weight glycopeptide was highly glycosylated with O-linked units. The lower molecular weight glycopeptide was less glycosylated and contained most of the N-linked units. Interaction between components of HSMSL and pili of Pseudomonas aeruginosa was examined by an overlay binding assay. Pili were found to bind to MG2. Preliminary studies indicated that the binding may involve a protein to protein interaction.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

Effect of extractant and molecular size on the optical and chemical properties of soil humic acids

Soil Research ◽  
1969 ◽  
Vol 7 (3) ◽  
pp. 229 ◽  
Author(s):  
JHA Butler ◽  
JN Ladd
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Humic Acids ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Molecular Size ◽  
Carboxyl Content ◽  
Peptide Bonds ◽  
Molecular Weights ◽  
Amino Acid Contents

Humic acids extracted from soil with sodium pyrophosphate have greater proportions of lower molecular weight material, less acid-hydrolysable amino acid nitrogen contents, but greater carboxyl contents and extinction values (260 and 450 nm) than humic acids extracted subsequently from the same sample with alkali. Humic acids extracted with alkali from fresh soil samples have intermediate values. Extinction values at 260 nm are directly correlated with carboxyl contents for a given soil. Different crop histories have no significant effect on the measured properties of the extracted humic acids. An alkali-extracted humic acid has been fractionated by gel filtration into seven fractions of different nominal molecular weight ranges. As the molecular weights of the fractions increase, both aliphatic C-H (based on infrared absorption at 2900 cm-1) and acid-hydrolysable amino acid contents increase, whereas extinction values at 260 nm and carboxyl contents decrease. The infrared spectra of the high molecular weight fractions have peaks at 1650 and 1510 cm-1 which correlate with acid-hydrolysable amino acid contents and which correspond to amide I and II bands of peptide bonds. Alkaline hydrolysis to split peptide bonds eliminates both these peaks. The spectra also have peaks at 1720 and 1210 cm-1 which correlate with the carboxyl content.

scholarly journals The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

1972 ◽  
Vol 130 (1) ◽  
pp. 211-219 ◽  
Author(s):  
Colin H. Self ◽  
P. David J. Weitzman
Keyword(s):  
Molecular Weight ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Molecular Size ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Low Concentrations ◽  
Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

Evaluation of dialysis treatment in uremic patients by gel filtration of serum

1990 ◽  
Vol 36 (11) ◽  
pp. 1906-1910 ◽  
Author(s):  
J Osada ◽  
T Gea ◽  
C Sanz ◽  
I Millan ◽  
J Botella
Keyword(s):  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
The Other ◽  
Control Group ◽  
Dialysis Treatment ◽  
Additional Information ◽  
Molecular Masses ◽  
Two Peaks

Abstract A group of substances of molecular masses between 300 and 1500 Da have been found to be toxic metabolites in patients with uremia. We determined the concentration in serum of these molecules in the following groups of patients: two hemodialyzed groups (one with cuprophane and the other with polyacrylonitrile dialyzers), one group treated with continuous ambulatory peritoneal dialysis, one group of nondialyzed azotemic patients, and one control group of healthy persons. Ultrafiltrates of the subjects' sera were fractionated on Sephadex G-15 followed by ion-exchange chromatography. Eluates were monitored by absorbance at 254 and 206 nm. Partially characterized peaks P1 and P2, obtained by gel filtration, correlated with the concentration of creatinine in serum; their concentrations were significantly (P less than 0.01) larger in hemodialyzed groups than in peritoneal dialyzed or in nondialyzed azotemic patients. After ion-exchange chromatography, two peaks (P'5 and P'6) correlated with serum creatinine and also were larger in hemodialyzed patients than in the other groups. Apparently, adequate discrimination is obtained by gel-filtration analysis and further analysis by ion-exchange chromatography does not provide additional information in most of the affected patients.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

Fractionation of the Color of Pure Maple and Other Sirups by Gel Filtration

1965 ◽  
Vol 48 (3) ◽  
pp. 493-497
Author(s):  
E E Stinson ◽  
C O Willits
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Cane Sugar ◽  
The Other ◽  
Low Molecular Weight ◽  
Brown Sugar ◽  
Maple Sugar ◽  
Molecular Weight Fractions ◽  
Color Fraction

Abstract The colorants of pure maple, cane and maple, refined cane sugar, and light brown sugar sirups were separated into two fractions, one of high- and the other of lowmolecular weights, by means of gel filtration. The ratio of the amounts of high- to the low-molecular weight fractions of pure maple was the lowest of the four sirups and serves as a means of differentiation from these sirups. The color fraction ratio was highest for blended cane-maple sugar sirup. Many maple sirups are also distinguished by a pink band formed on the gel filtration column.

Partial Primary Structure Of Human α2-Antiplasmin

1981 ◽  
Author(s):  
H R Lijnen ◽  
B Wiman ◽  
B Van Hoef ◽  
D Collen
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
High Performance Liquid Chromatography ◽  
High Performance ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Edman Degradation ◽  
Single Chain ◽  
Tryptic Digest

α2-Antiplasmin (α2AP), the main physiological inhibitor of plasmin in human plasma, is a single–chain glycoprotein with a molecular weight of 67,000 consisting of about 510 amino acids and containing 13 percent carbohydrate.A tryptic digest on 400 mg of reduced, carboxymethylated and citraconylated purified α2AP was performed. Peptides were separated by combinations of ion exchange chromatography, gel filtration and high performance liquid chromatography, and sequenced using the manual Edman degradation. Some peptides were further digested in order to establish overlaps. At the time of submission of this abstract we have sequenced 7 out of the approximately 21 arginyl peptides completely (each between 3 and 21 residues) and are working on the others. At present we have about 200 residues of sequence. Here we only report the stretches of 10 amino acids or more, which may be useful to compare the structure of α2AP with that of other serine protease inhibitors.

Sign in / Sign up

Export Citation Format

Share Document