Purification and characterization of an extracellular exopolygalacturonase from Geotrichum lactis

1991 ◽  
Vol 37 (12) ◽  
pp. 974-977 ◽  
Author(s):  
C. Pardo ◽  
M. A. Lapeña ◽  
M. Gacto

Geotrichum lactis ATCC 48590 produced extracellular polygalacturonase (EC 3.2.1.67) in media containing pectate, pectin, or galacturonic acid as inducers. The synthesis of the enzyme was strongly repressed by glucose. The polygalacturonase was purified 80-fold by ammonium sulphate precipitation, Sephadex G-100 filtration, and DEAE Sephadex ion-exchange chromatography. Polyacrylamide gel electrophoresis with copolymerized substrate indicated that the isolated material was a single enzyme with polygalacturonase activity. The main product of enzyme action was galacturonic acid. The enzyme shows a molecular weight close to 53 000 by gel filtration and degrades preferentially de-esterified substrates. Km values for pectate and pectin (64% esterified) were 0.09 and 0.49 mg mL−1, respectively. The optimum pH for enzyme activity was 5.0, while the optimum temperature was 40 °C. The polygalacturonase was precipitated by concanavalin A – Sepharose, and treatment with endoglycosidase H reduced its precipitation by the lectin, suggesting that the enzyme is a glycoprotein. In addition to being found extracellularly, the polygalacturonase is also present in the periplasm of the cells. A different form of the polygalacturonase showing a lower molecular weight was located inside the cells. Key words: polygalacturonase, pectic enzymes, Geotrichum lactis.

1979 ◽  
Vol 177 (1) ◽  
pp. 9-19 ◽  
Author(s):  
Gabriel M. Umezurike

1. Filtrates from cultures of different ages of Botryodiplodia theobromae Pat. were fractionated by gel filtration, ion-exchange chromatography and polyacrylamide-gel electrophoresis. 2. Five cellulases (C1, C2, C3, C4 and C5) were found, and their molecular weights, estimated by gel filtration, were 46000–48000 (C1), 30000–35000 (C2), 15000–18000 (C3), 10000–11000 (C4) and 4800–5500 (C5). 3. Cellulase C5 was absent from old culture filtrates. 4. Cellulase C1 had little or no activity on CM-cellulose (viscometric assay), but degraded cotton flock and Whatman cellulose powder to give cellobiose only. 5. The other components (C2–C5) produced cellobiose and smaller amounts of glucose and cellotriose from cellulosic substrates and were more active in lowering the viscosity of CM-cellulose. 6. The ratio of activities assayed by viscometry and by the release of reducing sugars from CM-cellulose increased with decrease in the molecular weights of cellulases C2–C5. 7. Cellobiose inhibited the activities of the cellulases, but glucose stimulated at low concentrations although it inhibited at high concentrations. 8. A high-molecular-weight β-glucosidase (component B1, mol.wt. 350000–380000) predominated in filtrates from young cultures, but a low-molecular-weight enzyme (B4, mol.wt. 45000–47000) predominated in older filtrates. 9. Intermediate molecular species of β-glucosidase (B2, mol.wt. 170000–180000; B3, mol.wt. 83000–87000) were also found. 10. Cellulases C2–C5 acted in synergism with C1, particularly in the presence of β-glucosidase.


1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall

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.


1981 ◽  
Vol 59 (4) ◽  
pp. 256-261 ◽  
Author(s):  
J. Tremblay ◽  
G. Thibault ◽  
J. Gutkowska ◽  
R. Boucher ◽  
J. Genest

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/f0) of 1.95 was calculated from the molecular weight and Stokes radius.Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat α1-macroglobulin or human α2-macroglobulin


1994 ◽  
Vol 298 (3) ◽  
pp. 727-732 ◽  
Author(s):  
K Ford ◽  
J Waltho ◽  
D Hornby

We have previously reported the identification of a novel activity residing in the nuclear fraction of mammalian cells that selectively binds and hydrolyses deoxyribonucleoside triphosphates. Incubation of this protein with [alpha-32P]dATP leads to the appearance of a retarded band relative to free dATP when the reaction is analysed on non-denaturing polyacrylamide gels. We now show that the retarded species comprises the product of dATP hydrolysis (dADP or dAMP) bound to an as yet unidentified species. We have termed this complex the ‘product-nucleotide binding particle’ or PNBP*. Through a combination of continuous elution polyacrylamide-gel electrophoresis and gel-filtration chromatography, we demonstrate that the hydrolytic activity (dNTPase) is distinct from the radiolabelled species detected in gel-retardation experiments. T.l.c. confirms that the labelled product does not share RF values associated with a range of mono-, di- and tri-phosphate deoxyribonucleotide standards, and gel-filtration experiments suggest a molecular mass for PNBP* of between 2.5 and 3 kDa. The ability of purified PNBP* to retain its nucleotide ligand after a number of denaturing processes suggests that the ligand is covalently bound. The recovery of dNTPase activity from both gel-filtration and ion-exchange chromatography reveals that the as yet unliganded PNBP* (or a precursor form) is associated with the dNTPase enzyme as part of the active complex, prior to addition of dATP.


1989 ◽  
Vol 44 (1-2) ◽  
pp. 71-76 ◽  
Author(s):  
Ulrich Fischer

Abstract Chlorobium phaeobacteroides contains two soluble basic c-type cytochromes, a flavocytochrome c-552 and a small cytochrome c-555. Both electron transfer proteins were highly purified by ion exchange chromatography and gel filtration. The flavocytochrome c-552 exhibits maxima at 552 nm, 523 nm and 416 nm in the reduced state and at 409.5 nm with two shoulders at 440 nm and 480 nm in the oxidized form. The best purity index (A280/A416)obtained was 0.65. The molecular properties of this flavocytochrome are as follows: isoelectric point, pH 9.5 - 10; redox potential, +63 mV; molecular weight, 56,000. Cytochrome c-555 is a small basic hemoprotein with an isoelectric point of pH 9.5 - 10, a molecular weight of 9,500 and a midpoint redox potential of +105 mV. The best purity index {A280/A418) obtained was 0.176. The oxidized form of this cytochrome has a maximum at 411.5 nm, while the reduced state shows three maxima (α-band at 554.5 nm; β-band at 523 nm, and γ-band at 418 nm). The a-band is asymmetrical with a typical shoulder at 551 nm.


1984 ◽  
Vol 62 (2) ◽  
pp. 385-391 ◽  
Author(s):  
Sheikh Mehboob Basha

An acid phosphatase (EC 3.1.3.2) from peanut (Arachis hypogaea L.) seed has been purified 433-fold by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. The purified preparation was found to be homogeneous by electrophoresis and gel filtration. The molecular weight of the enzyme was estimated to be approximately 240 000 and it was found to be composed of six identical subunits, each with an apparent molecular weight of 42 500. Following isoelectric focusing, the isolectric point (pI) of the enzyme was found to be around pH 5.6. The apparent Km value with p-nitrophenyl phosphate as substrate was 2 ? 10−1 μM. The enzyme was inhibited by Hg2+, Fe2+, Cu2+, Zn2+, Pb2+, and F−. Higher concentrations (2–50 μM) and long incubation periods (60–90 min) with Ca2+ and Mg2+ ions were shown to activate the enzyme. This enzyme showed no effect toward phosphorylated sugars but appear to hydrolyze ATP, ADP, AMP, and β-glycerophosphate.


Author(s):  
Maher Ali. Maqtari ◽  
A.B. Mohamed. Saad

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. 


1978 ◽  
Vol 175 (1) ◽  
pp. 35-46 ◽  
Author(s):  
A J MacGillivray ◽  
C Johnston ◽  
R MacFarlane ◽  
D Rickwood

After labelling of mouse liver nuclei with [gamma-32P]ATP in vitro, 10-20% of the radioactivity incorporated into the saline-soluble nuclear and HAP2 chromatin fractions was located in a low-molecular-weight component (component 10) with pI near 4.5 in urea. By using combinations of ion-exchange chromatography, preparative thin-layer isoelectric focusing and gel filtration, this component was isolated from both nuclear fractions. Recovery from the saline-soluble fraction was poor under conditions that allow endogenous phosphatases to be active. Component 10 was shown to be a phosphoprotein on the basis of enzyme-digestion experiments and the detection of phosphoserine and phosphothreonine. The 32P radioactivity did not appear to be associated with phosphorylated basic amino acids. Its molecular weight was determined by gel chromatography and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels as approx. 10000, and tryptic digestion of the reduced carboxymethylated protein in urea yielded two 32P-labelled peptides. It has not been possible as yet to assign a function to component 10, though its similarity to other low-molecular-weight acidic proteins is discussed.


1971 ◽  
Vol 25 (03) ◽  
pp. 580-589 ◽  
Author(s):  
M Uszynski ◽  
U Abildgaard

SummaryProcedures for the separation of two inhibitors of the activation of plasminogen to plasmin by urokinase are described. Tissue thromboplastin was removed by adsorption to Al(0H)3 gel followed by ultracentrifugation. Plasminogen, plasminogen activator, a coagulation inhibitor and hemoglobin were removed by ion exchange chromatography (CM- or DEAE-Sephadex with NaCl gradients). The minor UK inhibitor is a relative basic protein with a pI of about 5.8. The major inhibitor was purified further by isoelectric focusing, preparative electrophoresis in polyacrylamide gel, and gel filtration. This inhibitor has α1-motility, the pI is about 5.2, and the molecular weight about 100,000. It inactivates urokinase progressively, but does not inhibit streptokinase, plasmin or thrombin.


1985 ◽  
Vol 50 (5) ◽  
pp. 1249-1257 ◽  
Author(s):  
Ján Blahovec ◽  
Michal Bartík ◽  
Evžen Kasafírek

Aminopeptidase B, specifically hydrolyzing the L-lysine and L-arginine derivatives of p-nitroaniline and β-naphthylamine, was isolated from bovine liver. A multistep purification procedure involving fractionation with ammonium sulfate, gel filtration on Sephadex, ion exchange chromatography on Ecteola-cellulose, and adsorption chromatography on hydroxylapatite, afforded an enzyme whose activity was approximately 240 times higher than the activity of the original material. The molecular weight of the enzyme determined by gel filtration on Sephadex G-200 was approximately 55 000. The Michaelis constant with respect to L-lysyl-p-nitroanilide was 1.2 . 10-3 mol/l.


Sign in / Sign up

Export Citation Format

Share Document