Purification and molecular properties of glycogen phosphorylase b from trout white muscle

1993 ◽  
Vol 71 (5-6) ◽  
pp. 308-312 ◽  
Author(s):  
Hossein Mehrani ◽  
Kenneth B. Storey
Keyword(s):  
Molecular Weight ◽  
Oncorhynchus Mykiss ◽  
Glycogen Phosphorylase ◽  
White Muscle ◽  
Specific Activity ◽  
Fish Muscle ◽  
Maximum Activity ◽  
Substantial Contribution ◽  
Physiological Concentration ◽  
Glycogen Phosphorylase B

Glycogen phosphorylase b (EC 2.4.1.1) was isolated from white skeletal muscle of rainbow trout (Oncorhynchus mykiss) and purified 214-fold to a final specific activity of 135 U/mg protein (assayed in the direction of glycogen breakdown at 21 °C) by using glycogen – concanavalin A, DEAE-Sephadex, and 3′,5′-cAMP affinity chromatography. Purified phosphorylase b was a dimer with a native molecular weight of 193 000 and a subunit molecular weight of 87 000. Michaelis constants for glycogen, phosphate, and AMP were 128 μM, 31 mM and 142 μM, respectively, at pH 7.2; maximum activity of the enzyme was obtained at pH 7.5 and 25 °C. Glucose and ATP behaved as phosphorylase b inhibitors; glucose inhibition decreased at lower pH values. IMP did not affect the enzyme. The catalytic properties of trout phosphorylase b indicate that the enzyme would be virtually inactive at the physiological concentration of substrates and activators found in resting trout white muscle, but changes in cellular pH, ATP, Pi, and AMP levels during burst muscle work could allow phosphorylase b to augment phosphorylase a activity and make a substantial contribution to overall glycogenolysis in working trout white muscle.Key words: Oncorhynchus mykiss, regulation of glycogenolysis, fish muscle exercise, fish white muscle metabolism.

Glycogen phosphorylase in fish muscle: demonstration of three interconvertible forms

1990 ◽  
Vol 258 (2) ◽  
pp. C344-C351 ◽  
Author(s):  
H. Schmidt ◽  
G. Wegener
Keyword(s):  
Glycogen Phosphorylase ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Muscular Activity ◽  
Fish Muscle ◽  
Kinetic Properties ◽  
Phosphorylase Activity ◽  
Tissue Extracts

White skeletal muscle of crucian carp contains a single isoenzyme of glycogen phosphorylase, which was purified approximately 300-fold to a specific activity of approximately 13 mumol.min-1.mg protein-1 (assayed in the direction of glycogen breakdown at 25 degrees C). Tissue extracts of crucian muscle produced three distinct peaks of phosphorylase activity when separated on DEAE-Sephacel. Peaks 1 and 3 were identified, in terms of kinetic properties and by interconversion experiments, as phosphorylase b and a, respectively. Peak 2 was shown to be a phospho-dephospho hybrid. The three interconvertible forms of phosphorylase were purified and shown to be dimeric molecules at 20 degrees C. At 5 degrees C, a and the hybrid tended to form tetramers. The Mr of the subunit was estimated to be 96,400 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hybrid is kinetically homogeneous, and its kinetic properties are intermediate between those of b and a forms. The b, hybrid, and a forms of phosphorylase can be isolated from rapidly frozen muscle of crucian but in different proportions, depending on whether fish were anesthetized or forced to muscular activity for 20 s. Muscle of anesthetized crucian had 36, 36, and 28% of phosphorylase b, hybrid, and a forms, respectively, whereas the corresponding values for exercised fish were 12, 37, and 51%. Results suggest that three interconvertible forms of phosphorylase exist simultaneously in crucian muscle and that hybrid phosphorylase is active in contracting muscle in vivo.

Control of glycogenosis and effects of exercise on phosphorylase kinase and cAMP-dependent protein kinase in rainbow trout organs

1993 ◽  
Vol 71 (11-12) ◽  
pp. 501-506 ◽  
Author(s):  
Hussein Mehrani ◽  
Kenneth B. Storey
Keyword(s):  
Skeletal Muscle ◽  
Rainbow Trout ◽  
Protein Kinase ◽  
Oncorhynchus Mykiss ◽  
Glycogen Phosphorylase ◽  
White Muscle ◽  
Swimming Exercise ◽  
Phosphorylase Kinase ◽  
Dependent Protein Kinase ◽  
Dependent Protein

To analyze the mechanisms of glycogen phosphorylase control in organs of the rainbow trout Oncorhynchus mykiss, activities of glycogen phosphorylase kinase (GPK) and cAMP-dependent protein kinase (PKA), as well as levels of cAMP, were quantified. The complete cascade for activating glycogen phosphorylase was present in trout organs and all components were activated in white skeletal muscle and liver during exhaustive swimming exercise. GPK and PKA showed the highest activities in the liver, being three- and four-fold higher than corresponding activities in white muscle. Exercise stimulated a 60% increase in GPK activity in the liver and a 40% rise in white muscle. Furthermore, the amount of active PKA rose from 12 to 21% in the liver and from 32 to 57% in white muscle after exhaustive exercise and the cellular levels of cAMP increased by 50% in the liver and 70% in white muscle of exercised fish. Other organs (heart, gill, brain, kidney) showed little or no change in these parameters as a result of exhaustive exercise. GPK activity in liver, muscle, and heart extracts was strongly stimulated by in vitro incubation with the catalytic subunit of mammalian PKA, activity rising by 6- to 7-fold in white muscle extracts and 2- to 2.6-fold in liver and heart extracts. This occurred in extracts from both control and exercised fish and suggested that even in fish exercised to exhaustion, the maximal enzymatic potential for activation of glycogenolysis was not expressed. Other modes of GPK activation were not apparent, for the enzyme in crude extracts was stimulated only by incubation with cAMP and did not respond to cGMP or Ca2+ + phorbol 12-myristate 13-acetate. The data indicate that the cAMP-activated, PKA- and GPK-mediated cascade is key to the activation of glycogenolysis in both the skeletal muscle and liver during burst swimming exercise by trout.Key words: exercise, glycogen phosphorylase kinase, protein kinase A, cAMP, Oncorhynchus mykiss, control of glycogenolysis.

Pyruvate kinase from the moderate halophile, Vibrio costicola

1977 ◽  
Vol 55 (8) ◽  
pp. 825-833 ◽  
Author(s):  
Eveline de Médicis ◽  
Bertrand Rossignol
Keyword(s):  
Molecular Weight ◽  
Potassium Chloride ◽  
Pyruvate Kinase ◽  
Specific Activity ◽  
Chloride Concentration ◽  
Maximum Velocity ◽  
Maximal Activity ◽  
Maximum Activity ◽  
Moderate Halophile ◽  
Exclusion Chromatography

Pyruvate kinase (EC 2.7.1.40)from Vibrio costicola was purified to homogeneity. The tetramer had a molecular weight of 270 000 measured by exclusion chromatography on Sepharose 4B and was dissociated into apparently identical subunits with a molecular weight of 65 000 measured by SDS–polyacrylamide gel.The kinetic parameters Vm, K0.5, and n were measured at variable KCl concentrations. Both affinity and activity were inhibited when potassium chloride concentration was at 0.025 M, a fact which indicates that low potassium concentration is required for activity. Maximal activity and affinity were obtained at 0.125 M KCl. Potassium chloride appeared as an allosteric inhibitor in the range of 0.125 M to 1.025 M. Above 1.025 M KCl, both maximum velocity and affinity were decreased. NaCl had the same effect as KCl. The pH profiles of K0.5 and Vm of the phosphoenolpyruvate dependence revealed two main pK values: at 6.4 which controls affinity, and at 8.6, which controls activity.The activity of the enzyme showed a hysteretic response to salt. The specific activity measured under fixed standard conditions of temperature and salt concentrations showed a reversible variation which depended upon the preincubation conditions of temperature and salt concentration. The maximum activity tested was reached after low (0.1 M) or high (2 M) KCl preincubation at room temperature.

scholarly journals Purification and Characterization of the Glucose Oxidase from Penicillium notatum

2018 ◽  
Vol 9 (01) ◽  
Author(s):  
Baydaa A. Hassan ◽  
Mohammed A. Jebor ◽  
Zahra M. Ali
Keyword(s):  
Molecular Weight ◽  
Glucose Oxidase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Purification And Characterization ◽  
Penicillium Notatum ◽  
Oxidase Enzyme ◽  
Filtration Technique

This study aims to purification and characterization of the glucose oxidase enzyme from Penicillium notatum, the enzyme was purified by ammonium sulfate precipitation (60%), dialysis and gel filtration chromatography using sephadex G-200, A trial for the purification of glucose oxidase using gel filtration technique resulted in one type of glucose oxidase with specific activity of (62.382 U/mg) with (7.385 folds) purification. the purified glucose oxidase had a maximum activity at pH = 5.5, 45 °C, glucose oxidase was stable with pH values ranging between (5 – 6) and the enzyme was maintained the activity when it incubated into (25 -35) °C for 15 minutes, analyses of the glucose oxidase for molecular weight was carried out by PAGE and SDS-PAGE electrophoresis, which revealed 78 KDa, also molecular weight of the glucose oxidase was achieved by gel filtration technique and was found 87 KDa this means that enzyme consisting of only one subunit, the Km and Vmax value of glucoamylase (B) were (19.6 mM, 7.5 mM/min ) respectively using different concentration of glucose.

Secretion of Plasminogen Activators by Cultured Bovine Endothelial Cells: Partial Purification, Characterization and Evidence for Multiple Forms

1981 ◽  
Vol 45 (03) ◽  
pp. 219-224 ◽  
Author(s):  
W E Laug
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Plasminogen Activators ◽  
Maximum Activity ◽  
Partial Purification ◽  
Diisopropyl Fluorophosphate

SummaryEndothelial cells were obtained from the aortae of newborn calves and cloned. High plasminogen activator (PA) activity was detected in the supernatant medium and the cell lysates of confluent cultures. The PA activity in the growth medium increased steadily during 12 hrs of incubation, indicating active enzyme secretion by these cells. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the concentrated medium demonstrated the presence of four plasminogen activators with apparent molecular weights of 77,000 (±3000), 43,000 (±2000), 26,000 (±1500) and 14,500 (±1500) respectively. The 43,000, 26,000 and 14,500 molecular weight forms could be converted to radioactive derivates by active site labeling with 3H diisopropyl fluorophosphate (3H DFP) while the 77,000 Dalton form took up only traces of this radioactively labeled compound. The 43,000 molecular weight form was partially purified by means of salt precipitation and gel filtration. This enzyme preparation activated plasminogen by proteolytic cleavage with maximum activity at pH 7.5-8.5 and demonstrated a specific activity of 80,000 CTA (Committee on Thrombolytic Agents) units/mg protein when tested on 125I-fibrin in the presence of plasminogen. This PA was rapidly and irreversibly inhibited by diisopropyl fluorophosphate (DFP), suggesting that it was a serine protease. The partially purified enzyme was extremely labile at temperatures from 0-60° C, but could be stabilized by lowering the pH to 3 or by the addition of albumin.

Preparation and Properties of Human Prothrombin Complex

1970 ◽  
Vol 24 (03/04) ◽  
pp. 325-333 ◽  
Author(s):  
G. H Tishkoff ◽  
L. C Williams ◽  
D. M Brown
Keyword(s):  
Molecular Weight ◽  
Proteolytic Enzyme ◽  
Enzyme Inhibitor ◽  
Specific Activity ◽  
Crystalline Form ◽  
Sedimentation Equilibrium ◽  
Crystalline Complex ◽  
Interaction Product ◽  
Proteolytic Enzyme Inhibitor ◽  
Purification Scheme

SummaryAs a corollary to our previous studies with bovine prothrombin, we have initiated a study of human prothrombin complex. This product has been isolated in crystalline form as a barium glycoprotein interaction product. Product yields were reduced compared to bovine product due to the increased solubility of the barium glycoprotein interaction product. On occasion the crystalline complex exhibited good yields. The specific activity of the crystalline complex was 1851 Iowa u/mg. Further purification of human prothrombin complex was made by removal of barium and by chromatography on Sephadex G-100 gels. The final product evidenced multiple procoagulant activities (II, VII, IX and X). The monomeric molecular weight determined by sedimentation equilibrium in a solvent of 6 M guanidine-HCl and 0.5% mercaptoethanol was 70,191 ± 3,057 and was homogeneous with respect to molecular weight. This product was characterized in regard to physical constants and chemical composition. In general, the molecular properties of human prothrombin complex are very similar to the comparable bovine product. In some preparations a reversible proteolytic enzyme inhibitor (p-aminophenylarsonic acid) was employed in the ultrafiltration step of the purification scheme to inhibit protein degradation.

scholarly journals Probing the ionization state of substrate α-d-glucopyranosyl phosphate bound to glycogen phosphorylase b

1995 ◽  
Vol 308 (3) ◽  
pp. 1017-1023 ◽  
Author(s):  
I P Street ◽  
S G Withers
Keyword(s):  
Active Site ◽  
Glycogen Phosphorylase ◽  
Ph Dependence ◽  
Substrate Inhibition ◽  
19F Nmr ◽  
Ionization State ◽  
Nmr Studies ◽  
Substrate Analogues ◽  
Glycogen Phosphorylase B ◽  
Pka Value

The ionization state of the substrate alpha-D-glucopyranosyl phosphate bound at the active site of glycogen phosphorylase has been probed by a number of techniques. Values of Ki determined for a series of substrate analogue inhibitors in which the phosphate moiety bears differing charges suggest that the enzyme will bind both the monoanionic and dianionic substrates with approximately equal affinity. These results are strongly supported by 31P- and 19F-NMR studies of the bound substrate analogues alpha-D-glucopyranosyl 1-methylenephosphonate and 2-deoxy-2-fluoro-alpha-D-glucopyranosyl phosphate, which also suggest that the substrate can be bound in either ionization state. The pH-dependences of the inhibition constants K1 for these two analogues, which have substantially different phosphate pK2 values (7.3 and 5.9 respectively), are found to be essentially identical with the pH-dependence of K(m) values for the substrate, inhibition decreasing according to an apparent pKa value of 7.2. This again indicates that there is no specificity for monoanion or dianion binding and also reveals that binding is associated with the uptake of a proton. As the bound substrate is not protonated, this proton must be taken up by the proton.

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