Purification and Properties of Phosphoglyceromutase from Sheep Muscle

1971 ◽  
Vol 49 (11) ◽  
pp. 1183-1194 ◽  
Author(s):  
Eric James ◽  
R. O. Hurst ◽  
T. G. Flynn
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Enzymic Activity ◽  
Sulfhydryl Groups ◽  
Cellulose Acetate Electrophoresis ◽  
Absolute Requirement ◽  
Protein Components ◽  
Sheep Muscle

Phosphoglyceromutase (2,3-diphospho-D-glycerate phosphotransferase, EC 2.7.5.3) has been purified from sheep muscle. The enzyme has a specific activity of 828 units/mg and is stable for several months at 0–2 °C in 0.1 μ phosphate buffer, pH 7.0. The Km for 2,3-diphosphoglycerate (DPGA) is 0.003 mM; the Km for 3-phosphoglycerate is 9.0 mM. A small amount of DPGA-phosphatase activity was associated with the enzyme.At pH 5.4 and 7.0 sheep phosphoglyceromutase was shown to be homogeneous by sedimenting as a single sharp peak in the ultracentrifuge and by the appearance of a single band on both disc gel and cellulose acetate electrophoresis. The sedimentation coefficient of the enzyme at pH 7.0 was 4.1 S, the diffusion constant 7.21 × 10−7 cm2/s, and the molecular weight calculated from both the-sedimentation and gel filtration data was of the order of 51 000.Disc gel electrophoresis of the enzyme at pH 9.3 revealed the presence of three protein components which were shown to be charge isomers.Titration of the enzyme with p-chloromercuribenzoate indicated that 4.0 sulfhydryl groups were present per mole. Reaction with 5,5′-dithio-bis-(2-nitrobenzoate) showed that one of the sulfhydryl groups may be an absolute requirement for enzymic activity.

Multiple Forms of Phosphoglyceromutase from Chicken Breast Muscle

1972 ◽  
Vol 50 (10) ◽  
pp. 1132-1142 ◽  
Author(s):  
Eric James ◽  
R. O. Hurst ◽  
T. G. Flynn
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Breast Muscle ◽  
Heat Inactivation ◽  
Chicken Breast ◽  
Multiple Forms ◽  
Active Components ◽  
Net Charges

Phosphoglyceromutase (2,3-diphospho-D-glycerate: 2-phospho-D-glycerate phosphotransferase, EC 2.7.5.3) has been purified from both frozen and fresh chicken breast muscle. During purification it was found that substrate, 3-phospho-D-glycerate stabilized the enzyme against heat inactivation to almost the same extent as did the cofactor 2,3-diphospho-D-glycerate.Phosphoglyceromutase prepared from frozen chicken breast muscle separated into three peaks of activity (I, II, and III) following chromatography on DEAE-Sephadex in 0.05 μ phosphate buffer, pH 8.0, using a 0.0–0.4 M NaCl gradient. Each peak of activity was shown by polyacrylamide disc gel electrophoresis at pH 9.3 to contain two enzymically active components (isoenzymes Ia Ib, IIa IIb, and IIIa IIIb). Isoenzymes in the same peak had the same specific activity. Phosphoglyceromutase prepared from fresh chicken breast muscle yielded only one peak of activity following chromatography on DEAE-Sephadex. This peak contained two enzymically active components corresponding to isoenzymes Ia and Ib. Additional peaks of activity were not produced when phosphoglyceromutase from fresh muscle was subjected to freezing and thawing.Isoenzyme Ia and mixtures of Ia and Ib, IIa and IIb, and IIIa and IIIb were homogeneous in the ultra-centrifuge sedimenting as single peaks. The sedimentation coefficient obtained for isoenzyme Ia and for Ia and Ib combined was 4.15 S, the diffusion constant 6.62 × 10−7 cm2/s, and the molecular weight calculated from both gel filtration and sedimentation data was of the order of 59 000. These results were confirmed by charge isomer studies which also showed that the isoenzymes of phosphoglyceromutase from frozen chicken breast muscle were proteins of the same size but different net charges.

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 Human plasma uridine diphosphate galactose-glycoprotein galactosyltransfertase. Purification, properties and kinetics of the enzyme-catalysed reaction

1977 ◽  
Vol 167 (3) ◽  
pp. 621-628 ◽  
Author(s):  
A Bella ◽  
J S Whitehead ◽  
Y S Kim
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Gel Filtration ◽  
Amino Sugar ◽  
Enzymic Activity ◽  
Transferase Activity ◽  
Uridine Diphosphate ◽  
Absolute Requirement ◽  
Km Value ◽  
Alpha Lactalbumin

The soluble galactosyltransferase of human plasma catalysed the transfer of galactose from UDP-galactose to high- and low-molecular-weight derivatives of N-acetylglucosamine, forming a beta-1-4 linkage. The enzyme was purified by using (NH4)2SO4 precipitation and affinity chromatography on an alpha-lactalbumin-Sepharose column. The galactosyltransferase was maximally bound to this column in the presence of N-acetylglucosamine, and the enzyme was eluted by omitting the amino sugar from the developing buffer. The molecular weight of the enzyme was estimated to be 85000 by gel filtration. The assay conditions for optimum enzymic activity was 30 degrees C and pH7.5. Mn2+ ion was found to be an absolute requirement for transferase activity. The Km for Mn2+ was 0.4 mM and that for the substrate, UDP-galactose, was 0.024 mM. The Km for the acceptors was 0.21 mM for alpha1-acid glycoprotein and 3.9 mM for N-acetylglucosamine. In the presence of alpha-lactalbumin, glucose became a good acceptor for the enzyme and had a Km value of 2.9 mM. Results of the kinetic study indicated that the free enzyme reacts with Mn2+ under conditions of thermodynamic equilibrium, and the other substrates are added sequentially.

Multiple molecular forms of avian aldolases. IV. Purification and properties of chicken (Gallus domestkus) brain aldolase

1970 ◽  
Vol 48 (3) ◽  
pp. 322-333 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Specific Activity ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Breast Muscle ◽  
Molecular Forms ◽  
Chicken Breast ◽  
Rabbit Brain ◽  
Muscle Enzymes ◽  
Ph Optimum ◽  
Chicken Brain

Aldolase (fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) was purified from chicken (Gallus domesticus) brain tissue. The enzyme was shown to be homogeneous according to the following criteria: purification to a constant specific activity following sequential chromatography on DEAE and Sephadex, sedimentation velocity analysis, and electrophoresis on cellulose acetate strips.Several properties of the enzyme were determined including the Stokes radius (47 Å), diffusion constant (D020 w = 4.6 × 10−7 cm2/s), sedimentation coefficient (s020 w = 8.0), and molecular weight (155 000). The enzyme has a broad pH optimum centered around 7.2. The apparent Michaelis constants for fructose 1,6-diphosphate and fructose 1-phosphate were 7 × 10−5 M and 3 × 10−2 M, respectively. The activity ratio with the above two substrates was 30.Many of the molecular properties of this enzyme are similar to those of the rabbit brain enzyme and the muscle enzymes from both chickens and rabbits. The enzymic properties of chicken brain aldolase correspond more closely to those of the rabbit brain enzyme than they do to chicken breast muscle aldolase. The amino acid composition of chicken brain aldolase was found to be quite different from chicken breast muscle aldolase with respect to certain amino acids (methionine, cysteine, tryptophan, histidine, proline, aspartate, valine, and phenylalanine).

Multiple Molecular Forms of Avian Aldolases. V. Purification and Molecular Properties of Chicken (Gallus domesticus) Liver Aldolase

1971 ◽  
Vol 49 (6) ◽  
pp. 647-657 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Sedimentation Velocity ◽  
Gallus Domesticus ◽  
Molecular Forms ◽  
Velocity Analysis ◽  
Multiple Molecular Forms ◽  
Stokes Radius

Aldolase (fructose-1,6-diphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) was purified from chicken liver. The enzyme was shown to be homogeneous according to the following criteria: purification to a constant specific activity following sequential chromatography on carboxymethyl-Sephadex and Sephadex G-200, electrophoresis on cellulose acetate strips, sedimentation velocity analysis, absence of 10 other glycolytic enzymes, and immunodiffusion in agar.The sedimentation coefficient (s°20w 8.0), Stokes radius (47 Å), diffusion constant (D°20w 4.0 × 10−7 cm2/s), and molecular weight (160 000) are similar to those of rabbit liver aldolase and the muscle and brain enzymes from both chickens and rabbits.

Multiple molecular forms of avian aldolases. I. Crystallization and physical properties of chicken (Gallus domesticus) breast muscle aldolase

1969 ◽  
Vol 47 (5) ◽  
pp. 517-526 ◽  
Author(s):  
Ronald R. Marquardt
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Diffusion Constant ◽  
Sedimentation Coefficient ◽  
Double Diffusion ◽  
Glycolytic Enzyme ◽  
Gallus Domesticus ◽  
Molecular Forms ◽  
Rabbit Muscle ◽  
Multiple Molecular Forms

Aldolase (fructose 1,6-diphosphate-D-glyceraldehyde 3-phosphate lyase, EC 4.1.2.13) was purified and crystallized from chicken (Gallus domesticus) breast muscle.The crystalline enzyme is homogeneous according to the following criteria: purification to a constant specific activity, electrophoresis on cellulose acetate strips, absence of five other glycolytic enzyme activities, and immunodiffusion in agar.The sedimentation coefficient, diffusion constant, and molecular weight of the chicken enzyme are the same as for rabbit muscle aldolase. The ultraviolet spectra of the two proteins are the same. Electrophoretic comparison between the rabbit and chicken enzymes revealed a slightly different rate of migration.Antibodies directed against the pure chicken enzyme were prepared, and the reaction with pure chicken and rabbit aldolase was followed using the precipitin and double diffusion tests. A very pronounced reaction was observed between anti-serum and the chicken enzyme; the rabbit enzyme, in contrast, did not cross-react with the anti-serum.

Notizen: Physical Parameters and Possible Regulation of ζ-Aminolevulinic Acid Synthetase

1978 ◽  
Vol 33 (9-10) ◽  
pp. 796-798 ◽  
Author(s):  
Dhirendra L. Nandi
Keyword(s):  
Aminolevulinic Acid ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Axial Ratio ◽  
Enzymic Activity ◽  
Partial Specific Volume ◽  
Physical Parameters ◽  
Sucrose Density Gradient Centrifugation ◽  
Physical Measurements

Abstract Physical measurements were made on the ζ-aminolevulinic acid synthetase from Rhodopseudomonassph eroides. These include a Stokes radius of 3.8 nm, determined by gel filtration, and sedimentation coefficient of 5.46 S by sucrose density gradient centrifugation. From these measurements and the value of partial specific volume of 0.732 ml/g deter­mined from the amino acid composition, the following physical constants were calculated: molecular weight, 88000; diffusion coefficient, 5.65 × 10-7 cm2 s-1 ; frictional ratio, 1.30; axial ratio, 5.0. The enzyme is inhibited by more than 50% by glyceraldehyde 3-phosphate (1 mᴍ) , pyruvate (0 .02 ᴍ) , α-ketoglutarate (0.02 ᴍ) , urea (0 .8 ᴍ) , CoCl2 (0.2 mᴍ) and hemin (5 µᴍ). The effect of these inhibitors on the possible regulation of this enzymic activity is discussed.

scholarly journals Structure of the lysosomal neuraminidase–β-galactosidase–carboxypeptidase multienzymic complex

1990 ◽  
Vol 267 (1) ◽  
pp. 197-202 ◽  
Author(s):  
M Potier ◽  
L Michaud ◽  
J Tranchemontagne ◽  
L Thauvette
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Molecular Forms ◽  
Individual Species ◽  
Affinity Column ◽  
Large Species ◽  
Equilibrium System ◽  
Beta Galactosidase

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called ‘protective protein’, were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed

Characterization of protein-tyrosine kinase activity in the canine prostate

1991 ◽  
Vol 69 (2-3) ◽  
pp. 146-153 ◽  
Author(s):  
Claude Bourassa ◽  
Linh T. Nguyen ◽  
Kenneth D. Roberts ◽  
Simone Chevalier
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Specific Activity ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Tyrosine Kinase Activity ◽  
Protein Tyrosine ◽  
Endogenous Proteins ◽  
Protein Tyrosine Kinase Activity

Following the measurement of the phosphorylation of the substrate poly(Glu80Na,Tyr20) and the analysis of the alkali-resistant phosphorylation of endogenous proteins, the protein-tyrosine kinase of the canine prostate was partially characterized with regard to its subcellular localization, as well as certain kinetic and molecular properties. This kinase was mainly found in the cytosolic fraction (75%); however, its specific activity was similar to that of the residual enzyme present in the particulate fraction. Conditions for optimal activity of both fractions were determined. Under these conditions, several endogenous phosphoproteins (44–63 kilodaltons upon electrophoresis) were alkali resistant and phosphotyrosine was present in all of the major ones (pp63, pp57, pp52, and pp44). The particulate protein-tyrosine kinase activity was partially solubilized (58%) with 0.5% Triton X-100; this percentage was increased to 85% in the presence of 0.25 M KCl. Upon gel filtration, both cytosolic and particulate kinases showed an apparent molecular mass of 44 kilodaltons; these enzymes also phosphorylated similar major alkali-resistant phosphoproteins. The soluble protein-tyrosine kinase, with a sedimentation coefficient of 4.0S and an isoelectric point of 5.5, could be separated from arginine esterase and prostatic acid phosphatase.Key words: protein-tyrosine kinase, phosphotyrosine, prostate, arginine esterase, acid phosphatase.

Purification of Glycerol Kinase by "Dye-Ligand" Chromatography and Hydrophobic Interaction Chromatography on Bead-Cellulose Derivatives

1993 ◽  
Vol 58 (2) ◽  
pp. 445-451 ◽  
Author(s):  
Vladimír Žúbor ◽  
Albert Breier ◽  
Marta Horváthová ◽  
Dagmar Hagarová ◽  
Peter Gemeiner ◽  
...  
Keyword(s):  
Hydrophobic Interaction ◽  
Specific Activity ◽  
Hydrophobic Interaction Chromatography ◽  
Glycerol Kinase ◽  
Enzymic Activity ◽  
Cellulose Derivatives ◽  
Long Term Storage ◽  
Bead Cellulose ◽  
Term Storage

The crude extract of cytosole enzymes was obtained from homogenized cells of Saccharomyces cerevisiae by partition. The enzyme was then isolated from the lower aqueous phase displaying higher glycerol kinase activity by dye-ligand chromatography on Cibacron Blue (CB) or Remazol Brilliant Blue R (RB)-derivatized bead-cellulose, ATP being the eluent. The specific activity of glycerol kinase rised more than 10 and 7-times after affinity dye-ligand chromatography and hydrophobic interaction chromatography, respectively. Glycerol kinase obtained by the latter method was purified by CB-bead cellulose. The final preparation maintained its enzymic activity without noticeable losses during a long-term storage at 4 °C in dark.

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