A proteolytic pseudomonad from skin lesions of rainbow trout (Salmo gairdnerii). II. Some properties of the proteinase

1968 ◽  
Vol 14 (8) ◽  
pp. 875-880 ◽  
Author(s):  
M. F. Li ◽  
Carol Jordan
Keyword(s):  
Rainbow Trout ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Skin Lesions ◽  
Complexing Agents ◽  
Bivalent Cations ◽  
Wide Ph Range ◽  
Ph Range ◽  
Trout Muscle ◽  
Cellulose Column

An extracellular proteinase from a pseudomonad pathogenic to rainbow trout was purified 33-fold by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G 75 gel filtration. The purified enzyme was active over a wide pH range, from pH 5.0 to 10.0. Heating at 98 °C for 1 h did not completely inactivate the enzyme. Its observed temperature optimum was 45 °C. Michaelis–Menten constants were found to be 0.625% for casein and 0.263% for rainbow trout muscle albumin. Activation energies calculated for these substrates were 8.1 kcal and 11.4 kcal per mole, respectively. The involvement of bivalent cations and free sulfhydryl groups in the enzymatic activity was demonstrated by the inhibition caused by metal-complexing agents and p-chloromercuribenzoate, respectively.

Characterization of an isozyme of S-adenosylmethionine synthetase from rat liver

1984 ◽  
Vol 62 (5) ◽  
pp. 276-279 ◽  
Author(s):  
C. H. Lin ◽  
W. Chung ◽  
K. P. Strickland ◽  
A. J. Hudson
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Aromatic Amino Acids ◽  
Adenosylmethionine Synthetase ◽  
Cellulose Column

An isozyme of S-adenosylmethionine synthetase has been purified to homogeneity by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and gel filtration on a Sephadex G-200 column. The purified enzyme is very unstable and has a molecular weight of 120 000 consisting of two identical subunits. Amino acid analysis on the purified enzyme showed glycine, glutamate, and aspartate to be the most abundant and the aromatic amino acids to be the least abundant. It possesses tripolyphosphatase activity which can be stimulated five to six times by S-adenosylmethionine (20–40 μM). The findings support the conclusion that an enzyme-bound tripolyphosphate is an obligatory intermediate in the enzymatic synthesis of S-adenosylmethionine from ATP and methionine.

A PROTEOLYTIC PSEUDOMONAD FROM SKIN LESIONS OF RAINBOW TROUT (SALMO GAIRDNERII): I. CHARACTERISTICS OF THE PATHOGENIC EFFECTS AND THE EXTRACELLULAR PROTEINASE

1967 ◽  
Vol 13 (4) ◽  
pp. 405-416 ◽  
Author(s):  
M. F. Li ◽  
Carol Flemming
Keyword(s):  
Rainbow Trout ◽  
Subcutaneous Injection ◽  
Skin Lesions ◽  
Muscular Tissue ◽  
Fluorescent Pseudomonad ◽  
Extracellular Proteinase ◽  
Bacterial Proteinase ◽  
High Degree ◽  
Trout Muscle ◽  
Live Organism

A fluorescent pseudomonad producing a powerful extracellular proteinase and closely resembling but not identical with Pseudomonas fluorescens was isolated from skin lesions of rainbow trout. Subcutaneous injection of the live organism into healthy frogs caused a condition typical of redleg disease, followed by death, and its subcutaneous injection into healthy rainbow trout caused the formation of large, necrotic, swollen areas filled with fluid. The organism was re-isolated from the deliberately infected animals and histological examinations showed a high degree of destruction of the muscular tissue in the affected areas. The pathogenic effect observed was apparently due to the extracellular bacterial proteinase. Growth of the pseudomonad was insignificant at 3 °C and 37 °C, was slight at 8 °C, and optimal between 15 and 25 °C, and the culture filtrates possessed strong proteolytic activity against casein, hemoglobin, and rainbow trout muscle albumin. The production of this proteinase was dependent on the growth of the organism and the composition of the growth medium.

Isolation and some properties of extracellular heat-stable lipases fromPseudomonas fluorescensstrain AFT 36

1983 ◽  
Vol 50 (1) ◽  
pp. 77-89 ◽  
Author(s):  
Patrick F. Fox ◽  
Leszek Stepaniak
Keyword(s):  
Phosphate Buffer ◽  
Lipolytic Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Lipase Production ◽  
Maximum Activity ◽  
Temperature Range ◽  
Milk Serum ◽  
Heat Stable ◽  
Ph Range

SummaryAeration increased the growth and lipase production in milk byPseudomonas fluorescensstrain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented ∼ 71 % of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8·0 and 35 °C; it had aKmon tributyrin of 3·65 mM. and was inhibited by concentrations of substrate > ∼ 17 mM. The enzyme was very stable over the pH range 6–9; it was relatively heat-labile in phosphate buffer in the temperature range 60–80 °C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100–150 °C: theDvalues at 150 °C were ∼ 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the correspondingZvalues in the temperature range 100–150 °C were ∼ 40 and ∼ 42 °C and theEafor inactivation were 7·65 × 104J mol-1and 6·97 × 104J mol-1respectively.

scholarly journals Recovery of dolichyl diphosphate oligosaccharide in methanolic aqueous phase prepared from rat liver microsomal fractions

1986 ◽  
Vol 236 (3) ◽  
pp. 913-916
Author(s):  
M Sarkar ◽  
S Mookerjea
Keyword(s):  
Aqueous Phase ◽  
Rat Liver ◽  
Liver Microsomes ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Rat Liver Microsomes ◽  
Endogenous Protein ◽  
Phase Map ◽  
Chloroform Methanol ◽  
Cellulose Column

The synthesis of dolichyl diphosphate oligosaccharide was studied by incubating rat liver microsomes (microsomal fractions) with GDP-[14C]mannose, UDP-glucose, UDP-N-acetylglucosamine and [3H]dolichol phosphate. The labelled products obtained by the first step of extraction of the microsomes in methanolic aqueous phase (MAP fraction in chloroform/methanol/water; 3:2:1, by vol.) and in CMW fraction (chloroform/methanol/water; 10:10:3, by vol.) obtained by extraction of the interphase after the first step of extraction were analysed on a DEAE-cellulose column. With the progress of incubation, the radioactivity in unchanged GDP-mannose decreased, whereas the labelled dol-P-P-oligo in the MAP fraction increased about 5-6-fold. The lipid oligosaccharide in this fraction accounted for about 50-60% of the GDP-mannose used, whereas the recovery of the labelled lipid oligosaccharide in the CMW fraction was about 10%. The lipid oligosaccharide from both reactions after mild acid hydrolysis were analysed by gel filtration on Bio-Gel P-4. The oligosaccharide from the MAP fraction gave a peak of higher Mr distinctly separate from the lower-Mr peak obtained from the CMW fraction. Microsomes incubated with labelled lipid oligosaccharide from the MAP fraction showed incorporation of the label into endogenous protein.

Pectic enzymes produced by Pseudomonas fluorescens, an organism associated with “pink eye” disease of potato tubers

1972 ◽  
Vol 50 (12) ◽  
pp. 2479-2488 ◽  
Author(s):  
S. S. Hagar ◽  
G. A. McIntyre
Keyword(s):  
Pseudomonas Fluorescens ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Pectin Methylesterase ◽  
Viscosity Reduction ◽  
Cellulose Acetate Electrophoresis ◽  
Pectic Enzymes ◽  
Ph Range ◽  
Two Peaks ◽  
Layer Chromatography

No pectin methylesterase (PME) activity was observed in crude or dialyzed extracts from macerated potato tuber tissue inoculated with Pseudomonas fluorescens; however, pectic lyase (syn. polygalacturonic acid transeliminase, PATE) activity was observed. Two PATE enzymes (peaks 1 and 2) were eluted from a pH 9.4 DEAE-cellulose column using a gradient of pH 7.6 Tris-HCl buffer (0.01–0.1 M). Enzyme in peak 1 was about 6 times more active than enzyme in peak 2 based on reducing group assays, and 10 times more active in viscosity reduction of 1% Na-polypectate (NaPP) at pH 8.5. No increase in absorbancy was observed at 515 nm of clarified reaction mixtures, indicating that saturated oligouronides did not accumulate. Other properties of the two peaks: optimum pH range was 8.5–9.5, substrate preference was NaPP vs. pectin, addition of Ca2+ (0.001 M) enhanced activity while EDTA (0.001 M) decreased activity to [Formula: see text], cellulose acetate electrophoresis revealed one band of protein per peak, and heat of inactivation was 51–60C. Thin-layer chromatography of hydrolytic products from NaPP revealed unsaturated uronides and pectic fragments after 2 h hydrolysis; after 96 h hydrolysis only unsaturated uronides were observed. Molecular weight estimations by Sephadex G-200 gel filtration were about 18 000 (peak 1) and 22 500 (peak 2). Enzyme in either peak macerated 400-μ sections of potato tissue.

Effect of Streptococcal Extracellular Nuclease on the Carrier Activity of RNA for Streptolysin S

1983 ◽  
Vol 38 (1-2) ◽  
pp. 107-111 ◽  
Author(s):  
Akira Taketo ◽  
Yoriko Taketo
Keyword(s):  
Hemolytic Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Nucleotide Composition ◽  
Active Fraction ◽  
Streptolysin S ◽  
The Core ◽  
Extracellular Nuclease ◽  
Effective Component ◽  
Cellulose Column

Upon digestion with a streptococcal extracellular nuclease, yeast RNA yielded acid-insoluble core having increased carrier activity for streptolysin S. The carrier activity was found in minor fractions of the core which were eluted from a DEAE-cellulose column at higher salt concentrations. Upon gel filtration through a Sephadex G-75 column, the effective component (Fr. I) was eluted earlier than bulk oligonucleotides (Fr. II). Nucleotide composition (in mol %) of Fr. I was AMP: 21.8; GMP: 55.1; CMP: 8.2; UMP: 14.9, whereas that of Fr. II was AMP: 38.0; GMP: 33.1; CMP: 8.0; UMP: 20.9. Chromatographic patterns of SLS complex induced by Fr. I were similar to those of the toxin formed in the presence of active fraction prepared from RNase I core. Hemolytic activity of the latter complex was, like the former, unaffected by streptococcal nuclease treatment. The carrier activity of DNA digested with the nuclease was also investigated.

scholarly journals Screening and Purification of a Chymotrypsin Inhibitor from Entrolobium Saman Seeds

2010 ◽  
Vol 15 ◽  
pp. 19
Author(s):  
Maher Ali. Maqtari ◽  
A.B. Mohamed. Saad
Keyword(s):  
Molecular Weight ◽  
Enzyme Inhibitor ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Molar Ratio ◽  
Apparent Dissociation Constant ◽  
Chymotrypsin Inhibitor ◽  
Ammonium Sulphate Precipitation ◽  
Wide Ph Range ◽  
Ph Range

A chymotrypsin inhibitor was isolated and purified from the seeds of Enterolobium saman  (Leguminaceae family) by extraction with 100 mM phosphate buffer, heat treatment, ammonium sulphate precipitation, ion-exchange chromatography on DEAEcellulose and filtration through Sephadex G-75. The final preparation appeared to be homogeneous by both chromatographic and electrophoretic analyses. ESCI had a molecular weight of about 17,890 and an isoelectric point of 5.8. ESCI inhibited bovine chymotrypsin at an inhibitor-enzyme molar ratio of 1:2. The inhibition mode of chymotrypsin inhibitor was competitive on bovine chymotrypsin. Investigation has been carried out on the complex formed between chymotrypsin and chymotrypsin inhibitor by physico-chemical methods. An apparent dissociation constant (Ki) of 9.05 X 10-8 M has been calculated for the complex. This enzyme-  inhibitor complex was isolated by gel filtration on Sephadex G-75 and a molecular weight of 43.000 was estimated for the complex. The inhibitor did not have any effect on other proteinases, such as papain, bromelin, elastase, α -amylase, trypsin and pepsin. The chemical modification of lysine residues indicated that –NH2  groups are not essential for the activity of ESCI toward chymotrypsin. The inhibitor was an acidic protein and was stable over a wide pH range of 2-12 and temperature range of 10o C-97o C. 

scholarly journals Biochemical properties and positional specificity of Lipase from hepatopancreas of Tra catfish (Pangasius)

2013 ◽  
Vol 16 (4) ◽  
pp. 85-91
Author(s):  
Thy Bao Vuong ◽  
Lam Bich Tran ◽  
Duan Luu
Keyword(s):  
Ion Exchange ◽  
Lipase Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Biochemical Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Positional Specificity ◽  
Tra Catfish ◽  
Ph Range

Lipase from the hepatopancreas of Tra catfish (Pangasius) was precipitated by ammonium sulfate fractionation, purified by ion-exchange chromatography on DEAE cellulose and gel filtration on Sephadex G- 75. On substrate triolein, purified lipase has Km= 1.381 mM and Vmax= 0.063 mM/min. The lipase was stable at a pH range of 7.0- 9.0 and in temperatures of 35-50°C. At 500C the enzyme loosed 44,7% activity after 120 min. The enzyme was specific for the α- positions (1, 3) of triglyceride. In bile salt solution of 0.015M NaTC, lipase activity of the enzyme increased in 3.08 folds in comparison of sample without NaTC.

Aldehyde Dehydrogenases in Rat Liver

1972 ◽  
Vol 50 (7) ◽  
pp. 741-748 ◽  
Author(s):  
G. T. Shum ◽  
A. H. Blair
Keyword(s):  
Molecular Weight ◽  
Rat Liver ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Supernatant Fraction ◽  
Aldehyde Dehydrogenases ◽  
Low Concentrations ◽  
Enzyme I ◽  
Cellulose Column ◽  
Aldehyde Dehydrogenase Activity

Two enzymes (I and II) with NAD+-dependent aldehyde dehydrogenase activity have been separated and partially purified from the supernatant fraction of rat liver. Resolution was effected by DEAE-cellulose column chromatography. In addition to the differences in charge properties, these two proteins differ in substrate specificity, that of enzyme II being comparatively restricted. Enzyme I has a relatively sharp optimum in activity at pH 8 whereas enzyme II exhibits an optimal range between pH 8 and 9.5. Both enzymes are strongly inhibited by low concentrations of p-chloromercuribenzene sulfonic acid and this inhibition can be reversed by dithiothreitol. Both enzymes are also inhibited by arsenite; inhibition of enzyme I is enhanced by mercaptoethanol but inhibition of enzyme II is not so affected. Molecular weight estimation by gel filtration indicates each protein has a molecular weight of approximately 180 000.

scholarly journals Purification and Characterization of Carboxymethyl Cellulase from Bacillus sp. Isolated from a Paddy Field

2012 ◽  
Vol 61 (1) ◽  
pp. 51-55 ◽  
Author(s):  
PONNUSWAMY VIJAYARAGHAVAN ◽  
S.G. PRAKASH VINCENT
Keyword(s):  
Paddy Field ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Relative Molecular Mass ◽  
Bacillus Sp ◽  
Ammonium Sulphate Precipitation ◽  
Sds Page ◽  
Ph Range

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.

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