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.

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).

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.

Purification and Properties of Thymidylate Synthetase from Pig Thymus

1972 ◽  
Vol 50 (4) ◽  
pp. 352-362 ◽  
Author(s):  
V. S. Gupta ◽  
J. B. Meldrum
Keyword(s):  
Catalytic Activity ◽  
Sulfonic Acid ◽  
Specific Activity ◽  
Gel Filtration ◽  
Thymidylate Synthetase ◽  
Heat Inactivation ◽  
Synthetase Activity ◽  
Purification And Properties ◽  
Michaelis Constants ◽  
Sulfhydryl Compounds

Thymidylate synthetase of pig thymus has been separated into two principal forms (designated I and II, based on their order of elution) by chromatography on CM-Sephadex. By the use of (NH4)2SO4 the synthetase activity was separated into two fractions, and these were further purified by gel filtration using Sephadex G-100 and chromatography on CM-Sephadex. The highest specific activity obtained for I and II was 10.4 and 16.3 μmol of thymidine-5′-phosphate per hour per milligram of protein at 25° and pH 7.3 which represents a purification of 1680- and 2630-fold, respectively. Electrophoretically, I and II appear to be 70–80% pure. The Michaelis constants of 7.4 × 10−6 M, 1.7 × 10−5 M, and 1.8 × 10−4 M for II with respect to deoxyuridine-5′-phosphate, 5,10-methlenetetrahydrofolate, and uridine-5′-phosphate, respectively, have been determined. A double pH optima in the range of 6.6–6.8 and 7.2–7.4 in 2-N-morpholinoethane sulfonic acid buffer was exhibited by both forms. Forms I and II showed maximal catalytic activity only in the presence of sulfhydryl compounds (60 mM) and also had the ability to methylate uridine-5′-phosphate, although at a slower rate (ca. 28% and 13%, respectively) compared with the rate of methylation of deoxyuridine-5′-phosphate. Both deoxyuridine-5′-phosphate and tetrahydrofolate (to a lesser extent) afforded protection to II against heat inactivation.

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.

scholarly journals Resolution of regulated secretion into sequential MgATP-dependent and calcium-dependent stages mediated by distinct cytosolic proteins.

1992 ◽  
Vol 119 (1) ◽  
pp. 139-151 ◽  
Author(s):  
J C Hay ◽  
T F Martin
Keyword(s):  
Secretory Pathway ◽  
Kinase Inhibitors ◽  
Specific Activity ◽  
Gel Filtration ◽  
Protein Kinase Inhibitors ◽  
Active Components ◽  
Cytosolic Proteins ◽  
Regulated Secretion ◽  
Calcium Dependent ◽  
Regulated Secretory Pathway

The biochemical events and components responsible for ATP-dependent Ca(2+)-activated secretion remain to be identified. To simplify the molecular dissection of regulated secretion, we have resolved norepinephrine (NE) secretion from semi-intact PC12 cells into two kinetically distinct stages, each of which was studied separately to discern its molecular requirements. The first stage consisted of MgATP-dependent priming of the secretory apparatus in the absence of Ca2+. MgATP-dependent priming was readily reversible and inhibited by a broad range of protein kinase inhibitors. The second stage consisted of Ca(2+)-triggered exocytosis which, in contrast to priming, occurred in the absence of MgATP. Both priming and triggering were found to be dependent upon or stimulated by cytosolic proteins. The priming and triggering activities of cytosol were functionally distinct as indicated by differing thermolability. Furthermore, active components in cytosol resolved by gel filtration were found to support either priming or triggering, but not both. For both priming and triggering reactions, several peaks of activity were detected; one of each type of factor was partially purified from rat brain cytosol, and found to be enriched for stage-specific activity. Two partially purified factors exhibiting stage-specific activity, a approximately 20-kD priming factor and approximately 300-kD triggering factor, were able to support regulated secretion as effectively as crude cytosol when used sequentially in the partial reactions. Further characterization of stage-specific cytosolic factors should clarify the nature of MgATP- and Ca(2+)-dependent events in the regulated secretory pathway.

scholarly journals Evaluation of Inactivation Kinetics and Thermodynamic Properties of Peroxidase from Cucumis sativus for Biotechnological and Industrial Applications

2021 ◽  
pp. 46-62
Author(s):  
Iweha Christian Chukwunyere ◽  
Ugwoke Faith Ifeanyi ◽  
Ogara Lydia Amaechi ◽  
Nweze Justus Amuche ◽  
Ogbonna Kenneth Ejike ◽  
...  
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Velocity ◽  
Industrial Applications ◽  
Heat Inactivation ◽  
Inactivation Kinetics ◽  
Order Kinetic ◽  
Two Phases ◽  
Ph Range ◽  
Kinetics Of

The unpurified enzyme gave specific activity of 51.36 μ/mg and thereafter it was subjected to two phases of purification process of salt precipitation and gel filtration. The precipitating agent was ammonium sulphate while Sephadex-G100 served as purification matrix. The purification fold achieved after precipitation and filtration was 3.3 and with corresponding specific activities of 34.22 μ/mg and 116.31 μ/mg. The substrate used for the assay was o-dianisidine. Within 40-80°C of temperature, the kinetics of the peroxidase inactivation was evaluated. The results from assays showed that cucumber peroxidase conformed to the hypothesis of  Michealis-Menten Theory. From the Lineweaver-Burk plot, Michaelis Constant (Km) and maximum velocity (Vmax) were evaluated and  obtained 5.02mg/ml and 11.57μmol/min respectively. The heat induced inactivation gave biphasic curves, where initial rise in temperature was rapidly succeeded with much slower decrease. A first-order kinetic behaviour was observed for cucumber peroxidase heat inactivation. The k values of between 3.49×10-2 to 8.38×10-2 min-1 was obtained while the Z value was found to be 22.3°C. Decrease in k values with rise in temperature suggests that cucumber peroxidase was rapidly inactivated at elevated temperature. The slope of Arrhenius plot gave the activation energy of 127.99KJMol-1K-1. Also evaluated were thermodynamic constants (ΔH, G Δ, ΔS) for inactivation of peroxidase at variable temperatures. Cucumber peroxidase activity was observed to be pH sensitive and stable within pH range of 5.6-8. Further decrease or increase from this range resulted to decrease in peroxidase stability.

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.

scholarly journals Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

2019 ◽  
Vol 81 (1) ◽  
Author(s):  
Rahma R. Z. Mahdy ◽  
Shaimaa A. Mo’men ◽  
Marah M. Abd El-Bar ◽  
Emad M. S. Barakat
Keyword(s):  
Metal Ions ◽  
Kinetic Parameters ◽  
Biochemical Characterization ◽  
Galleria Mellonella ◽  
Fat Body ◽  
Specific Activity ◽  
Larval Instar ◽  
Gel Filtration ◽  
Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

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