scholarly journals Characterization of the mutant α-mannosidase in bovine mannosidosis

1978 ◽  
Vol 175 (3) ◽  
pp. 1013-1022 ◽  
Author(s):  
Lynda J. Burditt ◽  
Nigel C. Phillips ◽  
Donald Robinson ◽  
Bryan G. Winchester ◽  
Neil S. Van-De-Water ◽  
...  
Keyword(s):  
Specific Activity ◽  
Relative Proportion ◽  
Gel Filtration ◽  
Residual Activity ◽  
Deae Cellulose ◽  
Mutant Enzyme ◽  
Ph Optimum ◽  
Normal Tissues ◽  
Km Value ◽  
Normal Enzyme

Residual acidic α-mannosidase, varying in amount up to approx. 15% of normal values, can be measured in various organs of a calf with mannosidosis. The highest specific activity and relative proportion of residual activity were found in the liver. Chromatography on DEAE-cellulose showed that the residual activity was associated with two components, which were eluted at comparable positions with those found in normal tissues. The residual activity had a lower thermal stability and a higher Km value for a synthetic substrate than did the normal enzyme. No differences in molecular weight or electrophoretic mobility between normal acidic α-mannosidase and the residual activity were observed by gel filtration and electrophoresis on cellulose acetate respectively. The isoelectric focusing profiles for the α-mannosidase in the normal and pathological livers were very similar. It is suggested that a mutant enzyme, resulting from a mutation in a structural gene, accounts for the residual acidic α-mannosidase in mannosidosis. The mutant enzyme, which cross-reacts with antiserum raised against normal bovine acidic α-mannosidase, is present at a decreased concentration compared with the normal enzyme. There is a correlation between the concentrations of residual activity and cross-reacting material in mannosidosis. α-Mannosidase with a pH optimum of 5.75 and which is activated by Zn2+ was also detected in the liver of the calf with mannosidosis. However, it is probably not a product of the defective gene because addition of Zn2+ indicated that it was also present in normal tissues.

Purification and characterization of rat epididymal neutral β-galactosidase and its changes during in vivo development

1993 ◽  
Vol 71 (1-2) ◽  
pp. 22-26 ◽  
Author(s):  
Pratima Dutta ◽  
Gopal C. Majumder
Keyword(s):  
Concanavalin A ◽  
Sexual Maturity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Tissue Homogenate ◽  
Affinity Column ◽  
Ph Optimum

A neutral β-D-galactosidase has been partially purified from rat epididymis and characterized. The enzyme having molecular mass of approximately 50 kilodaltons has been purified 400-fold by using calcium phosphate gel adsorption, DEAE-cellulose chromatography, Sephadex G-100 gel filtration, and concanavalin A - agarose affinity chromatography. Although the neutral enzyme binds to the concanavalin A affinity column, the activity could be eluted with α-methyl mannoside only if the buffer contained salt (NaCl) at a concentration as high as 0.3 M. The enzyme was of cytosolic origin, since 90% of the total enzymic activity of the tissue homogenate was recovered in the soluble fraction of these cells. The neutral β-galactosidase was not dependent on metal ions for its activity and it had a pH optimum of 7.0. Zn2+, p-chloromercuribenzoate, Hg2+, and Pb2+ served as potent inhibitors of the enzyme. There was a marked increase (approximately fourfold) in the specific activity of the neutral β-galactosidase during sexual maturity of epididymis in vivo.Key words: neutral β-galactosidase, rat epididymal, cytosolic, developmental, sexual maturity.

scholarly journals Mannosidosis in Angus cattle. The enzymic defect

1974 ◽  
Vol 137 (2) ◽  
pp. 363-371 ◽  
Author(s):  
N. C. Phillips ◽  
D. Robinson ◽  
B. G. Winchester ◽  
R. D. Jolly
Keyword(s):  
Gel Filtration ◽  
Residual Activity ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Starch Gel Electrophoresis ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Angus Cattle ◽  
Starch Gel ◽  
Component C

Normal calf α-mannosidase activity exists in at least three forms separable by chromatography on DEAE-cellulose and by starch-gel electrophoresis. Two components, A and B, have optimum activity between pH3.75 and 4.75, but component C has an optimum of pH6.6. Components A and B are virtually absent from the tissues of a calf with mannosidosis and the residual activity is due to component C. The acidic and neutral forms of α-mannosidase differ in their molecular weights and sensitivity to EDTA, Zn2+, Co2+ and Mn2+. An acidic α-mannosidase component (pH optimum 4.0) accounts for most of the activity in normal plasma but it is absent from the plasma of a calf with mannosidosis. Although the acidic α-mannosidase component is probably related to tissue components A and B, it can be distinguished from them by ion-exchange chromatography and gel filtration. The optimum pH of the low residual activity in the plasma from a calf with mannosidosis is pH5.5–5.75. The results support the hypothesis that Angus-cattle mannosidosis is a storage disease caused by a deficiency of lysosomal acidic α-mannosidase activity.

scholarly journals Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp

1989 ◽  
Vol 262 (2) ◽  
pp. 409-416 ◽  
Author(s):  
G A Saravani ◽  
D A Cowan ◽  
R M Daniel ◽  
H W Morgan
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Serine Proteinase ◽  
Gel Permeation Chromatography ◽  
Exchange Chromatography ◽  
Acetate Buffer ◽  
Ph Optimum ◽  
Low Concentrations ◽  
Low Ionic Strength

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3′-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.

Partial Characterization of Pyruvate Decarboxylase Isolated From Germinating Pea Seeds

1980 ◽  
Vol 7 (1) ◽  
pp. 35 ◽  
Author(s):  
S Leblova ◽  
J Valik
Keyword(s):  
Sodium Phosphate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Pyruvate Decarboxylase ◽  
Temperature Stability ◽  
Hill Coefficient ◽  
Ph Optimum ◽  
The Hill

Pyruvate decarboxylase (EC 4.1.1.1), isolated from 4-day-old germinating peas, was precipitated from a sodium phosphate extract when (NH4)2SO4 was increased from 15 to 30% saturation, desalted on Sephadex G-25 or by dialysis for 24 h, and then chromatographed on a DEAE-cellulose column. This procedure increased the specific activity of the enzyme 120-fold compared with the sodium phosphate extract. The behaviour of the enzyme during gel filtration indicates that it has a high molecular weight. The pea enzyme exhibits a sigmoid dependence on the pyruvate concentration; reaction velocity is half-maximal at a substrate concentration of 1.8 mM and the Hill coefficient is 1.8. Thiamin pyrophosphate (TPP) is the coenzyme, which is relatively firmly bound to the apoenzyme, but can be removed by dialysis for 48 h. The apoenzyme is activated optimally at 2 mM TPP and inhibited by concentrations above 4 mM. The pH optimum for pea pyruvate decarboxylase is 5.8 and maximal temperature stability occurs at 48°C.

Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts

1984 ◽  
Vol 4 (6) ◽  
pp. 1003-1012
Author(s):  
R L Nelson ◽  
P E Branton
Keyword(s):  
Chicken Embryo ◽  
Protein Phosphatases ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Soluble Form ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Phosphoprotein Phosphatases ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G phosphorylated by pp60src as substrate. Crude cell extracts contained ca. 70% of the activity in the soluble form and ca. 30% associated with a crude membrane fraction. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII)--comprising ca. 20, 45, and 35%, respectively, of the total activity--were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity.

scholarly journals Purification and characterization of acid phosphatase from a germinating black gram (Vigna mungo L.) seedling

2011 ◽  
Vol 63 (3) ◽  
pp. 747-756 ◽  
Author(s):  
A.K.M. Asaduzzaman ◽  
Habibur Rahman ◽  
Tanzima Yeasmin
Keyword(s):  
Acid Phosphatase ◽  
Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Inhibitory Effect ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Black Gram ◽  
Km Value

An acid phosphatase has been isolated and purified from an extract of a germinating black gram seedling. The method was accomplished by gel filtration of a germinating black gram seedling crude extract on sephadex G-75 followed by ion exchange chromatography on DEAE cellulose. The acid phosphatase gave a single band on SDS-polyacrylamide slab gel electrophoresis. The molecular weight of the acid phosphatase determined by SDS-polyacrylamide slab gel electrophoresis was estimated to be 25 kDa. The purified enzyme showed maximum activity at pH 5 and at temperature of 55?C. Mg2+, Zn2+ and EDTA had an inhibitory effect on the activity of the acid phosphatase. Black gram seedling acid phosphatase was activated by K+, Cu2+ and Ba2+. The Km value of the enzyme was found to be 0.49 mM for pNPP as substrate.

scholarly journals Purification and characterization of cytosolic pyruvate kinase from banana fruit

2000 ◽  
Vol 352 (3) ◽  
pp. 875-882 ◽  
Author(s):  
William L. TURNER ◽  
William C. PLAXTON
Keyword(s):  
Pyruvate Kinase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Banana Fruit ◽  
Ph Optimum ◽  
Cytosolic Ph ◽  
Pep Carboxylase ◽  
Sds Page ◽  
Optimal Efficiency

Cytosolic pyruvate kinase (PKc) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7µmol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240kDa homotetramer composed of subunits of 57kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of Vmax/Km for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH6.9 and 7.5. PKc activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg2+ and K+ respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg2+ and K+ (Km values of 0.098, 0.12, 0.27 and 0.91mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PKc by L-glutamate. The allosteric features of banana PKc are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807Ő816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.

scholarly journals Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues

1978 ◽  
Vol 175 (3) ◽  
pp. 1051-1067 ◽  
Author(s):  
K K Mäkinen ◽  
P L Mäkinen
Keyword(s):  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Ph Optimum ◽  
Preparative Scale ◽  
Molecular Weights ◽  
Gel Permeation ◽  
Lysine Residues ◽  
Enzyme I

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

scholarly journals Purification, characterization and inhibition of human skin collagenase

1978 ◽  
Vol 169 (2) ◽  
pp. 265-276 ◽  
Author(s):  
David E. Woolley ◽  
Robert W. Glanville ◽  
Dennis R. Roberts ◽  
John M. Evanson
Keyword(s):  
Human Skin ◽  
Culture Medium ◽  
Serum Proteins ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polyacrylamide Gel ◽  
Collagen Fibrils ◽  
Ph Optimum

1. The neutral collagenase released into the culture medium by explants of human skin tissue was purified by ultrafiltration and column chromatography. The final enzyme preparation had a specific activity against thermally reconstituted collagen fibrils of 32μg of collagen degraded/min per mg of enzyme protein, representing a 266-fold increase over that of the culture medium. Electrophoresis in polyacrylamide disc gels showed it to migrate as a single protein band from which enzyme activity could be eluted. Chromatographic and polyacrylamide-gel-elution experiments provided no evidence for the existence of more than one active collagenase. 2. The molecular weight of the enzyme estimated from gel filtration and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was approx. 60000. The purified collagenase, having a pH optimum of 7.5–8.5, did not hydrolyse the synthetic collagen peptide 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg-OH and had no non-specific proteinase activity when examined against non-collagenous proteins. 3. It attacked undenatured collagen in solution at 25°C, producing the two characteristic products TCA(¾) and TCB(¼). Collagen types I, II and III were all cleaved in a similar manner by the enzyme at 25°C, but under similar conditions basement-membrane collagen appeared not to be susceptible to collagenase attack. At 37°C the enzyme attacked gelatin, producing initially three-quarter and one-quarter fragments of the α-chains, which were degraded further at a lower rate. As judged by the release of soluble hydroxyproline peptides and electron microscopy, the purified enzyme degraded insoluble collagen derived from human skin at 37°C, but at a rate much lower than that for reconstituted collagen fibrils. 4. Inhibition of the skin collagenase was obtained with EDTA, 1,10-phenanthroline, cysteine, dithiothreitol and sodium aurothiomaleate. Cartilage proteoglycans did not inhibit the enzyme. The serum proteins α2-macroglobulin and β1-anti-collagenase both inhibited the enzyme, but α1-anti-trypsin did not. 5. The physicochemical and enzymic properties of the skin enzyme are discussed in relation to those of other human collagenases.

scholarly journals A New Procedure for Purification of Crotalase From Crotalus Adamanteus Venom

1977 ◽  
Author(s):  
F.S. Markland ◽  
J. Chou ◽  
Y. Shih ◽  
H. Pirkle
Keyword(s):  
Sodium Chloride ◽  
Large Scale ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Tris Buffer ◽  
High Yield ◽  
Crude Venom ◽  
Diamondback Rattlesnake

A new procedure has been developed for large scale, rapid purification of crotalase, the thrombin-1ike enzyme from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). The three step procedure involves: (1) molecular sieve chromatography on Sephadex G-100 in 0.04 M Tris buffer containing 0.10 M sodium chloride, pH 7.1; (2) gradient elution from DEAE-cellulose with sodium acetate buffer, pH 7.0; and (3) affinity chromatography on p-aminobenzamidine Sepharose using a spacer of 6-aminohexanoic acid. Crotalase was eluted from the affinity resin by 0.05 M Tris buffer containing 0.10 M sodium chloride and 0.15 M benzamidine-hydrochloride, pH 9.0, after first washing with the Tris buffer containing 0.40 M sodium chloride. From the crude venom, pure enzyme was obtained with an overall recovery of 40-60% of clotting activity and a 90-100 fold increase in specific activity. Crotalase was shown to be pure by Polyacrylamide disk gel electrophoresis which gave one band. The molecular weight was estimated to be approximately 31,000 by gel filtration on a calibrated Sephadex G-100 column. Amino acid analysis was performed and the composition was shown to be very similar to that reported earlier (F.S. Markland and P.S. Damus, J. Biol. Chem. 246: 6460, 1971). Clotting activity of the enzyme was not inhibited by heparin, either with or without plasma, whereas, thrombin was rapidly inactivated by heparin in the presence of plasma. In conclusion, we have developed a rapid and reproducible procedure for isolation in high yield of large quantities of the thrombin-like enzyme from the venom of the eastern diamondback rattlesnake. Studies are continuing on the primary structure and possible clinical applications of this enzyme.

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