scholarly journals Partial purification of topoisomerase IB from serum of diabetic patients and study it's kinetic properties and molecular weight

2019 ◽  
Vol 23 (10) ◽  
pp. 46
Author(s):  
Saif M. Hasan ◽  
Firas T. Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Maximum Velocity ◽  
Partial Purification ◽  
Diabetic Patients ◽  
Kinetic Properties ◽  
Topoisomerase Ib ◽  
Electrophoresis Method

This study was done to partially purification of  topoisomerase IB from serum of diabetic patients using Gel filtration technique, by using Sephadex G 100 gel. A single peak in fraction four has been obtained, and the degree of purification (17.1) fold, enzyme yield (108.2%) and specific activity (0.189ng/mg). Kinetics studies for the partial purified enzyme were carried out which showed optimal concentration of  substrate which was (0.1ng/ml), Michael's - Menten constant (Km=0.033ng) and maximum velocity (Vmax=0.90 ng/ml), while optimum Temperature was (37C°) and optimum pH was (7.5). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method, in presence of polyacrylamide  gel and sodium dodecyl sulphate (SDS-PAGE) which showed that the approximated molecular weight was (66KD).   http://dx.doi.org/10.25130/tjps.23.2018.168 

scholarly journals Study of some hormones and Partial purification of prolidase from serum of women with polycystic ovary syndrome

2019 ◽  
Vol 24 (7) ◽  
pp. 59
Author(s):  
Reemy M. Mohamed Saleh ◽  
Firas T . Maher ◽  
Nagham Q. Kadhim
Keyword(s):  
Molecular Weight ◽  
Polycystic Ovary Syndrome ◽  
Specific Activity ◽  
Polycystic Ovary ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Ovary Syndrome ◽  
Electrophoresis Method ◽  
High Level

This study was done by partially purification of prolidase from serum of patients with polycystic ovary syndrome by Gel filtration technique, and using sephadex G100 gel as a stationary phase. The degree of purification (15.1) fold, enzyme yield (95.5%) and specific activity (0.00176 IU/I), were carried out .Kinetics studies for the partial purified enzyme technique showed optimal concentration of substrate which was 5 mmol/l Km = 0.66ng and Vmax =0.80 mM, while optimum Temperature was (35C°) and optimum pH was (8). The molecular weight of the partial purified enzyme has been determined by gel electrophoresis method , in presence of polyacrylamide gel and sodium dodecyl sulphate (SDS_PAGE) which showed that the approximates molecular weight was (54KD),we found high level of prolactin in the patient with polycystic ovary syndrome which was(24.03) when in the control was( 10.09),the value of TSH in the patient was( 17.08) which is high value and in the control was( 1.49), the value of T4 in the patient was (100.2) and in control was (118.4),the value of T3 in the patient was (0.3)and in control was (1.3).Testosterone in patient was (0.391) and in control was (0.206).    http://dx.doi.org/10.25130/tjps.24.2019.130

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.

Partial purification of a boar sperm membrane protein inducing sperm agglutinating antibody

1990 ◽  
Vol 10 (2) ◽  
pp. 131-139
Author(s):  
Oyewole Adeyemo ◽  
E. O. Okegbile ◽  
O. O. Olorunsogo
Keyword(s):  
Molecular Weight ◽  
Membrane Protein ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Boar Sperm ◽  
Sperm Membrane ◽  
Triton X

For the development of immunological contraception, attention is being concentrated on the possibility of using a sperm membrane antigen. Boar sperm membrane was extracted with triton-X 100 and fractionated by Sephadex G-150 column chromatography. The glycosylated and nonglycosylated portions of protein peaks from the gel filtration were obtained by fractionating on concanavalin A-Sepharose and eluting the bound protein with 0.3 M methyl mannoside. A glycosylated fraction was found to induce sperm agglutinating antibodies in rabbit. The partially purified protein has a molecular weight of 30 kilodaltons, as determined by sodium dodecyl polyaccyrlamide gel electrophoresis. Further work is planned on the histochemical determination of the origin of this protein and species cross-activity of the antibody.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

scholarly journals Phosphoenolpyruvate carboxylase from the crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. Purification, molecular and kinetic properties

1978 ◽  
Vol 175 (2) ◽  
pp. 391-406 ◽  
Author(s):  
R Jones ◽  
M B Wilkins ◽  
J R Coggins ◽  
C A Fewson ◽  
A D B Malcolm
Keyword(s):  
Phosphoenolpyruvate Carboxylase ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Maximum Velocity ◽  
Single Band ◽  
Kinetic Properties ◽  
Subunit Structure

Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.

scholarly journals Purification and properties of 6-phosphogluconate dehydrogenase from rabbit mammary gland

1975 ◽  
Vol 151 (2) ◽  
pp. 263-270 ◽  
Author(s):  
S A Betts ◽  
R J Mayer
Keyword(s):  
Molecular Weight ◽  
Mammary Gland ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Phosphogluconate Dehydrogenase ◽  
Purification And Properties ◽  
Competitive Inhibitors ◽  
Final Purification Step

1. 6-Phosphogluconate dehydrogenase from rabbit mammary gland was purified to homogeneity by the criterion of polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. The molecular weight of the subunit is 52 000. The enzyme was purified 150-fold with a final specific activity of 20 mumol of NADP+ reduced/min per mg of protein and overall yield of 3%. The molecular weight of the native enzyme is estimated to be 104 000 from gel-filtration studies. The final purification step was carried out by affinity chromatography with NADP+-Sepharose. 2. The Km values for 6-phosphogluconate and NADP+ are approx. 54 muM and 23 muM respectively. 3. Citrate and pyrophosphate are competitive inhibitors of the enzyme with respect to both 6-phosphogluconate and NADP+. 4. MgCl2 affects the apparent Km for NADP+ at saturating concentrations of 6-phosphogluconate.

Molekulare und kinetische Charakterisierung der Xanthin-Dehydrogenase aus dem phototrophen Bakterium Rhodopseudomonas capsulata / Molecular and Kinetic Characterization of Xanthine Dehydrogenase from the Phototrophic Bacterium Rhodopseudomonas capsulata

1981 ◽  
Vol 36 (11-12) ◽  
pp. 933-941 ◽  
Author(s):  
Werner Aretz ◽  
Herwig Kaspari ◽  
Jobst-Heinrich Klemme
Keyword(s):  
Molecular Weight ◽  
Uric Acid ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Xanthine Dehydrogenase ◽  
Rhodopseudomonas Capsulata ◽  
Kinetic Properties ◽  
Effective Electron ◽  
Enzyme Preparations ◽  
Phototrophic Bacterium

Abstract The structural and kinetic properties of xanthine dehydrogenase (EC 1.2.1.37) from the facultative phototrophic bacterium Rhodopseudomonas capsulata were studied. The enzyme was fully induced when hypoxanthine or xanthine, but less effectively when uric acid served as nitrogen source during growth. The enzyme was purified about 2300-fold from cells grown photosynthetically with hypoxanthine as N-source by using ammoniumsulfate precipitation, gel filtration, ion-exchange and affinity chromatography. The molecular weight as determined by gel filtration throug Sephacryl S-300 was 345000. Subunit analysis by sodium dodecyl sulfate gel electrophoresis suggested a composition of four identical subunits with a molecular weight of 84000. The enzyme contained 2 flavin, 2 molybdenum and 8 iron-sulfur groups per mol. The turnover number with hypoxanthine and NAD as substrates was 12000 min-1. Hypoxanthine, 1-methylhypoxanthine, 8-azahypoxanthine, xanthine, 1-methylxanthine, 2-hydroxypurine, 6,8-di-hydroxypurine, 5-azacytosine and 5-azauracil served as electron donors. The most effective electron acceptor was NAD. The kinetic constants (Km) were (in μm): 52.5 (hypoxanthine); 32.5 (xanthine) and 61.2 (NAD). Various purine compounds inhibited the enzyme competitively in respect to hypoxanthine as substrate. Although reduction of uric acid to xanthine was not detected by using purified enzyme preparations, in vitro-experiments with 14C-labelled uric acid indicated that the enzyme xanthine dehydrogenase participates in uric acid degradation in Rps. capsulata. According to their electrophoretic mobilities, the xanthine dehydrogenases isolated from hypoxanthine-and uric acid-grown cells were identical.

scholarly journals Extraction and Purification of Organophosphorus hydrolase Enzyme from Soil Microorganism Pseudomonas diminuta

2017 ◽  
Vol 2 (4) ◽  
pp. 416 ◽  
Author(s):  
Maheshwari D. T. ◽  
Thygaraj Varsha ◽  
N. S. Kumar
Keyword(s):  
Specific Activity ◽  
Organophosphorus Compounds ◽  
Sodium Dodecyl ◽  
Maximum Velocity ◽  
Maximum Activity ◽  
Soil Microorganism ◽  
Organophosphorus Hydrolase ◽  
Kinetic Properties ◽  
Extraction And Purification ◽  
Sds Page

<p class="p1">Synthetic organophosphorus compounds are highly toxic hence, widely used as pesticides, insecticides and chemical warfare agents. Organophosphorus hydrolase (OPH) is an organophosphotriester hydrolyzing enzyme; effectively hydrolyze a range of organophosphate esters. The objective of the present study was extraction and purification of OPH enzyme from Pseudomonas <em>diminuta </em>bacteria (soil microorganism) and to study kinetic properties of the purified enzyme. Enzyme was extracted and purified from bacteria by ammonium sulphate precipitation and ion exchange chromatography. Purity of an enzyme was determined by sodium dodecyl sulphate-polyacryamide gel electrophoresis (SDS-PAGE). Purified OPH enzyme specific activity was found to be 27.7 fold of 32.8U/mg protein, molecular weight of 72 Kda and it is a homodimer since it has shown a single band in SDS-PAGE separation. Maximum activity of the free OPH enzyme was found at Optimum pH 7.5 and temperature 35<span class="s2"><sup>o</sup></span>C with the incubation time of 10 min. Michaelis constant (K<span class="s3">m</span>) and maximum velocity (V<span class="s3">max</span>) values of free OPH enzyme for methyl parathion as substrate was found to be 286.2μM and 2.5 μM/min respectively.</p>

scholarly journals Isolation of lysyl hydroxylase, an enzyme of collagen synthesis, from chick embryos as a homogeneous protein

1980 ◽  
Vol 189 (2) ◽  
pp. 247-253 ◽  
Author(s):  
T M Turpeenniemi-Hujanen ◽  
U Puistola ◽  
K I Kivirikko
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Polymeric Form ◽  
Lysyl Hydroxylase ◽  
Enzyme Form

Two procedures are reported for the purification of lysyl hydroxylase, both procedures involving (NH4)2SO4 fractionation, affinity chromatography on concanavalin A-agarose and elution of the column with ethylene glycol. The additional steps in procedure A consist of gel filtration and chromatography on a hydroxyapatite column, and in procedure B of affinity chromatography on collagen linked to agarose and gel filtration. The best preparations obtained with either of the two procedures were pure when examined by sodium dodecyl sulphate-polyacrylamide-disc-gel or slab-gel electrophoresis, but about half of the preparations obtained by procedure A had minor contaminants. The specific activity of a typical preparation purified by procedure B was 13 4000 times that of the 15 000 g supernatant of the chick-embryo homogenate, with a recovery of about 4%. The molecular weight of the pure enzyme was bout 200 000 by gel filtration, and that of the enzyme subunit about 85 000 by sodium dodecyl sulphate/polyacrylamide-disc-gel or slab-gel electrophoresis. It is suggested that the active enzyme is a dimer consisting of only one type of monomer, and that a previously described enzyme form with an apparent molecular weight of about 550 000 is a polymeric form of this dimer. The catalytic-centre activity of the pure enzyme, as determined with a saturating concentration of a synthetic peptide substrate and under conditions specified, was about 3-4 mol/s per mol.

scholarly journals Isolation and some properties of troponin T kinase from rabbit skeletal muscle

1980 ◽  
Vol 189 (2) ◽  
pp. 219-226 ◽  
Author(s):  
N B Gusev ◽  
A B Dobrovolskii ◽  
S E Severin
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Skeletal Muscles ◽  
Specific Activity ◽  
Troponin T ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Serine Residue ◽  
Troponin Complex ◽  
Rabbit Skeletal Muscles

A method for isolation of troponin T kinase (ATP-protein phosphotransferase, EC 2.7.1.37) from rabbit skeletal muscles in proposed. The method gives a 7000-10 000-fold purification and results in an enzyme with specific activity of 400-800-nmol x min-1 x mg-1 of protein. The molecular weight of tropin T kinase as determined by gel filtration exceeds 500 000. Electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate revealed that isolated preparations of the enzyme consisted of at least three distinct proteins with apparent mol.wt. of 50 000, 46 000 and 31 000. The enzyme phosphorylates isolated troponin T at a rate which exceeds the phosphorylation rates of casein, phosvitin, histones, phosphorylase b and protamine 5-30-fold. Within the whole troponin complex, only troponin T is phosphorylated by the enzyme. The enzyme phosphorylates only the N-terminal serine residue of troponin T, i.e. the site that is normally phosphorylated in the whole troponin complex isolated from rabbit skeletal muscles.

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