Purification and Characterization of Kallikrein from Plasma of Patients with Acute Pancreatitis

1981 ◽  
Vol 60 (2) ◽  
pp. 199-205 ◽  
Author(s):  
Naotika Toki ◽  
Hiroyuki Sumi ◽  
Sumiyoshi Takasugi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Purification And Characterization ◽  
Α2 Macroglobulin ◽  
Sepharose 4B

1. A kallikrein-like enzyme in plasma of patients with acute pancreatitis was further purified by successive hydroxyapatite/cellulose and Sepharose-4B column chromatography. 2. By these procedures 0.26 mg of purified enzyme with a specific activity of 215 S-2266 chromozyme units/mg of protein was obtained from 10 ml of original plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and had an apparent molecular weight of 31 000 as measured by gel filtration on Sephadex G-200. 4. It was confirmed immunologically that this enzyme was pancreatic kallikrein, which is distinct from plasma kallikrein, and that it could combine with α2-macroglobulin only in the presence of trypsin.

scholarly journals Purification and Characterization of a Dimethoate-Degrading Enzyme of Aspergillus niger ZHY256, Isolated from Sewage

2001 ◽  
Vol 67 (8) ◽  
pp. 3746-3749 ◽  
Author(s):  
Yu-Huan Liu ◽  
Ying-Cheng Chung ◽  
Ya Xiong
Keyword(s):  
Aspergillus Niger ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Degrading Enzyme

ABSTRACT A dimethoate-degrading enzyme from Aspergillus nigerZHY256 was purified to homogeneity with a specific activity of 227.6 U/mg of protein. The molecular mass of the purified enzyme was estimated to be 66 kDa by gel filtration and 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was found to be 5.4, and the enzyme activity was optimal at 50°C and pH 7.0. The activity was inhibited by most of the metal ions and reagents, while it was induced by Cu2+. The Michaelis constant (K m ) andV max for dimethoate were 1.25 mM and 292 μmol min−1 mg of protein−1, respectively.

Purification and Characterization of an Acetyl Xylan Esterase from Bacillus pumilus

1998 ◽  
Vol 64 (2) ◽  
pp. 789-792 ◽  
Author(s):  
Giuliano Degrassi ◽  
Benedict C. Okeke ◽  
Carlo V. Bruschi ◽  
Vittorio Venturi
Keyword(s):  
Gel Electrophoresis ◽  
Bacillus Pumilus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Acetyl Xylan Esterase ◽  
Purification And Characterization ◽  
Michaelis Constant ◽  
Naphthyl Acetate

ABSTRACT Bacillus pumilus PS213 was found to be able to release acetate from acetylated xylan. The enzyme catalyzing this reaction has been purified to homogeneity and characterized. The enzyme was secreted, and its production was induced by corncob powder and xylan. Its molecular mass, as determined by gel filtration, is 190 kDa, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single band of 40 kDa. The isoelectric point was found to be 4.8, and the enzyme activity was optimal at 55°C and pH 8.0. The activity was inhibited by most of the metal ions, while no enhancement was observed. The Michaelis constant (Km ) andV max for α-naphthyl acetate were 1.54 mM and 360 μmol min−1 mg of protein−1, respectively.

scholarly journals Purification and characterization of 3-dehydroquinase from Escherichia coli

1986 ◽  
Vol 239 (3) ◽  
pp. 699-704 ◽  
Author(s):  
S Chaudhuri ◽  
J M Lambert ◽  
L A McColl ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Catalytic Properties ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Permeation Chromatography ◽  
Purification And Characterization ◽  
Gel Permeation

A procedure has been developed for the purification of 3-dehydroquinase from Escherichia coli. Homogeneous enzyme with specific activity 163 units/mg of protein was obtained in 19% overall yield. The subunit Mr estimated from polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 29,000. The native Mr, estimated by gel permeation chromatography on Sephacryl S-200 (superfine) and on TSK G3000SW, was in the range 52,000-58,000, indicating that the enzyme is dimeric. The catalytic properties of the enzyme have been determined and shown to be very similar to those of the biosynthetic 3-dehydroquinase component of the arom multifunctional enzyme of Neurospora crassa.

Purification and preliminary characterization of 2-monoacylglycerol acyltransferase from rat intestinal villus cells

1985 ◽  
Vol 63 (5) ◽  
pp. 341-347 ◽  
Author(s):  
F. Manganaro ◽  
A. Kuksis
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Cell Protein ◽  
Racemic Mixture ◽  
Small Intestinal Mucosa ◽  
Small Intestinal ◽  
Monoacylglycerol Acyltransferase

We have purified the monoacylglycerol acyltransferase from rat small intestinal mucosa to homogeneity by a combination of hydrophobic absorption, guanidine dissociation, and gel filtration. The purified enzyme gives a single band of 37 000 daltons on sodium dodecyl sulphate – polyacrylamide gel electrophoresis. The enzyme has a specific activity of about 5900 nmol/mg per hour and represents 0.12% of total cell protein, corresponding to about a 600-fold purification. The enzyme does not acylate diacylglycerols to triacylglycerols, which is consistent with the separate physical existence of the mono- and di-acylglycerol acyltransferases. The enzyme acylates the 2-monoacylglycerols to yield an essentially racemic mixture of diacylglycerols. It does not acylate glycerol 3-phosphate.

scholarly journals Purification and characterization of β-N-acetylhexosaminidase I2 from human liver

1986 ◽  
Vol 234 (1) ◽  
pp. 157-162 ◽  
Author(s):  
N N Dewji ◽  
D R De-Keyzer ◽  
J L Stirling
Keyword(s):  
Gel Electrophoresis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Alpha Subunit ◽  
Gradient Gel Electrophoresis ◽  
Sepharose 4B

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.

scholarly journals The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus

1986 ◽  
Vol 237 (2) ◽  
pp. 415-420 ◽  
Author(s):  
C R Goward ◽  
R Hartwell ◽  
T Atkinson ◽  
M D Scawen
Keyword(s):  
Large Scale ◽  
Bacillus Stearothermophilus ◽  
Specific Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Km Value ◽  
Extraneous Material ◽  
Sepharose 4B

Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34. The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000. Kinetic parameters for the enzyme were determined with a variety of substrates. The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine. The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose. The glucokinase was maximally active at pH 9.0.

scholarly journals Purification and Characterization of the Folate Catabolic Enzyme p-Aminobenzoyl-Glutamate Hydrolase from Escherichia coli

2010 ◽  
Vol 192 (9) ◽  
pp. 2407-2413 ◽  
Author(s):  
Jacalyn M. Green ◽  
Ryan Hollandsworth ◽  
Lenore Pitstick ◽  
Eric L. Carter
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Size Exclusion ◽  
Breakdown Product ◽  
Purification And Characterization ◽  
Sds Page

ABSTRACT The abg locus of the Escherichia coli chromosome includes three genes encoding proteins (AbgA, AbgB, and AbgT) that enable uptake and utilization of the folate breakdown product, p-aminobenzoyl-glutamate (PABA-GLU). We report on the purification and characterization of the p-aminobenzoyl-glutamate hydrolase (PGH) holoenzyme encoded by abgA and abgB. One-step purification was accomplished using a plasmid carrying abgAB with a hexahistidine tag on the carboxyl terminus of AbgB and subsequent metal affinity chromatography (MAC). Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed two subunits (∼53-kDa and ∼47-kDa proteins) of the expected masses of AbgB and AbgA; N-terminal sequencing confirmed the subunit identification, and amino acid analysis yielded a 1:1 ratio of the subunits. Size exclusion chromatography coupled with light-scattering analysis of purified PGH revealed a predominant molecular mass of 206 kDa and a minor component of 400 to 500 kDa. Both peaks contained PGH activity, and SDS-PAGE revealed that fractions containing activity were composed of both AbgA and AbgB. MAC-purified PGH was highly stimulated by manganese chloride. Kinetic analysis of MAC-purified PGH revealed a Km value for PABA-GLU of 60 ± 0.08 μM and a specific activity of 63,300 ± 600 nmol min−1 mg−1. Folic acid and a variety of dipeptides served as poor substrates of PGH. This locus of the E. coli chromosome may encode a portion of a folate catabolism pathway.

Isolation and Characterization of a Pancreatic Elastase from Plasma of Patients with Acute Pancreatitis

1982 ◽  
Vol 62 (3) ◽  
pp. 321-328 ◽  
Author(s):  
Naotika Toki ◽  
Sumiyoshi Takasugi ◽  
Hiroyuki Sumi
Keyword(s):  
Acute Pancreatitis ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Isolation And Characterization ◽  
Bean Trypsin Inhibitor

1. An elastase-like enzyme in plasma of patients with acute pancreatitis was purified by DEAE-cellulose column chromatography and polyacrylamide-gel disc electrophoresis. 2. In this way 0.24 mg of purified enzyme with a specific activity of 3.94 succinyl-l-alanyl-l-alanyl-l-alanyl-p-nitroanilide units/mg of protein was obtained from 10 ml of plasma. 3. The purified material was homogeneous as ascertained by sodium dodecyl sulphate/polyacrylamide-gel disc electrophoresis and had an apparent molecular weight of 24 000 as measured by gel filtration on Sephadex G-100. 4. This enzyme hydrolysed denatured casein and Congo Red—elastin as well as succinyl-l-alanyl-l-alanyl-l-alanyl-p-nitroanilide. Its amidolytic activity was inhibited by soya bean trypsin inhibitor, but not by aprotinin. 6. We propose that an elastase-like enzyme, probably different from elastase 1 or elastase 2, is liberated from the pancreas into blood during acute pancreatitis and becomes combined with α2-macroglobulin.

Rat intestinal angiotensin-converting enzyme: purification, properties, expression, and function

1992 ◽  
Vol 263 (4) ◽  
pp. G466-G473 ◽  
Author(s):  
R. H. Erickson ◽  
Y. Suzuki ◽  
A. Sedlmayer ◽  
I. S. Song ◽  
Y. S. Kim
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Maximal Velocity ◽  
Converting Enzyme ◽  
And Function

Angiotensin-converting enzyme [ACE (peptidyl-dipeptidase A, EC 3.4.15.1)] was purified from a total cell membrane fraction of rat intestinal mucosa. A 4,500-fold purification was achieved after affinity chromatography with lisinopril-Sepharose and gel filtration. The final preparation was judged to be homogenous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 160,000. The purified protein is a glycoenzyme containing 12% N-linked carbohydrate. Purified ACE had a specific activity of 65 U/mg protein with benzoyl-Gly-His-Leu as substrate. A kinetic analysis showed that the enzyme had the maximal velocity with substrates containing proline at the COOH-terminal end. Inhibitor studies indicated that the enzyme is a metalloprotein. Along the proximal-distal axis of the small intestine, ACE activity is most predominant in the proximal to middle portions, decreasing toward the distal end. This pattern was also observed for ACE mRNA and protein, suggesting that ACE expression is controlled at the level of mRNA. Perfusion of benzoyl-Gly-His-Leu in vivo through a segment of intestinal jejunum demonstrated that ACE is an important intestinal dipeptidyl carboxypeptidase, participating in the digestion and assimilation of dietary peptides.

Purification and characterization of an endo-β-1,3-glucanase from Trichoderma harzianum

1996 ◽  
Vol 42 (10) ◽  
pp. 1039-1044 ◽  
Author(s):  
Eliane Ferreira Noronha ◽  
Cirano José Ulhoa
Keyword(s):  
Trichoderma Harzianum ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Hydrophobic Interaction Chromatography ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Ph Optimum ◽  
Isolated Cell

β-1,3-Glucanases are produced by Trichoderma harzianum when it is grown in the presence of chitin or isolated cell wall from fungi. An endo-β-1,3-glucanase from the culture filtrate of T. harzianum was purified by gel filtration on Sephacryl S-200, followed by hydrophobic interaction chromatography on phenyl-Sepharose. A typical procedure provided 134-fold purification with a 3.6% yield. The molecular mass of the purified endo-β-1,3-glucanase was found to be approximately 36 kDa, as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis on a 10% w/v slab gel. The enzyme was active toward glucans containing β-1,3-linkages and hydrolysed laminarin to form oligosaccharides. The Km and Vmax values for β-1,3-ghicanases, using laminarin as substrate, was 1.18 mg∙mL−1 and 1.26 U∙mL−1, respectively. The pH optimum for the enzyme was pH 4.4 and maximum activity was obtained at 45–50 °C. Enzyme activity was strongly inhibited in the presence of HgCl2 and stimulated by cations such as Zn2+ and Ca2+.Key words: endo-β-1,3-glucanase, Trichoderma harzianum, purification, characterization.

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