Kinetic Studies of Na+/K+-ATPase in Tissue Aerobic Thyroid Patients

Na+/K+-ATPase is a prevalent enzyme that maintains the Na+ and K+ gradients across the cell membrane by transporting three Na+ out and two K+ into the cell, the aim of this study is to provide detailed mechanistic insights, potentially with important effects on physiological regulation of active Na and K transport in tissues of Aerobic Thyroid Patient. Thyroid tissues were obtained from a 35 year old patients, the operation was carried out at the Al-Hadi Specialist Hospital in Samarra city, the sample was stored at -20ºC until used. The purification protocol included Salt Precipitation, Ion Exchange Chromatography, Gel Filtration and Electrophoresis, a spectrophotometric method was used to determine the enzyme activity. kinetic parameters was also obtained for the enzyme. Partial purification of Na+/K+-ATPase revealed two isoenzymes (I ,II). The purity of separated isoenzymes were proved by SDS-PAGE electrophoresis. The kinetic characteristics of Na+/K+-ATPase showed that optimum substrate concentration about 1.5mM, Km 1.052mM, and Vmax 6.062, optimum temperature was 37 ºC, optimum pH 7.4 and optimum time in 25 min. Na+/K+-ATPase purified from Thyroid tissue has distinct kinetic characteristic that reflects the importance of intracellular regulation of specific Na+/K+-ATPase pump which gives cells the ability to precisely coordinate to their physiological requirements .

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Identification of cytosolic protein tyrosine kinases of human prostate by renaturation after SDS/PAGE

Biochemical Journal ◽

10.1042/bj2840653 ◽

1992 ◽

Vol 284 (3) ◽

pp. 653-658 ◽

Cited By ~ 12

Author(s):

Y Durocher ◽

A Chapdelaine ◽

S Chevalier

Keyword(s):

Tyrosine Kinases ◽

Cell Function ◽

Guanidine Hydrochloride ◽

Gel Filtration ◽

Exchange Chromatography ◽

Protein Tyrosine Kinases ◽

Partial Purification ◽

Major Band ◽

Protein Tyrosine ◽

Sds Page

The identification of protein tyrosine kinases (PTKs) was successfully achieved by renaturation in gels after SDS/PAGE. To this effect, samples were mixed with a PTK substrate, namely the polydispersed co-polymer of glutamic acid and tyrosine [poly(Glu, Tyr), M(r) from 30,000 to 94,000], and were simultaneously submitted to electrophoresis. Following guanidine hydrochloride denaturation, renaturation and phosphorylation with [gamma-32P]ATP, kinase activity was detected by autoradiography. When applied to cytosol from human hyperplastic prostate, eleven protein kinases were detected, among which one major (M(r) 50,000) and two minor proteins (M(r) 40,000 and 38,000) were identified as PTKs by the presence of phosphotyrosine. Incubation of the gel in hot alkali after glutaraldehyde cross-linking almost completely eliminated the detection of non-PTK enzymes. On the other hand, in the absence of poly(Glu,Tyr), no PTK activity was detected. Partial purification of cytosolic PTKs indicates that the native M(r) of the major phosphotransferase was 44,000, as estimated by gel filtration following ammonium sulphate precipitation and anion-exchange chromatography. Upon renaturation after electrophoresis, this fraction showed only one major band active on poly(Glu,Tyr) which was associated with the polypeptide of M(r) 50,000. This enzyme was also identified following two-dimensional electrophoresis and renaturation in the presence of poly(Glu,Tyr), allowing the determination of a pI in the range 7.5-7.8. Thus PTKs can be easily renatured following electrophoresis and rapidly identified on the basis of their M(r) and pI in both crude or partially purified preparations. With the crucial role played by PTKs in the activation of cell function and carcinogenesis, this procedure could be useful in the identification of such enzymes and in distinguishing them from their substrates in gels.

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Pemurnian Parsial dan Karakterisasi Enzim Xilanase dari Bakteri Laut Bacillus safencis strain LBF P20 Asal Pulau Pari Jakarta

Jurnal Agritech ◽

10.22146/agritech.17004 ◽

2017 ◽

Vol 37 (1) ◽

pp. 31

Author(s):

Fitria Fitria ◽

Nanik Rahmani ◽

Sri Pujiyanto ◽

Budi Raharjo ◽

Yopi Yopi

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Exchange Chromatography ◽

Partial Purification ◽

Centrifugal Filter ◽

Sds Page ◽

Enzyme Specific Activity ◽

Hydrolysis Of

Enzyme xylanase (EC 3.2.1.8) is widely used in various industrial fields for the hydrolysis of xylan (hemicellulose) into xylooligosaccharide and xylose. The aims of this study were to conduct partial purification and characterization of xylanase from marine Bacillus safencis strain LBF P20 and to obtain the xylooligosaccharide types from xylan hydrolysis by this enzyme. Based on this research, the optimum time for enzyme production occurred at 96 hours with the enzyme activity of 6.275 U/mL and enzyme specific activity of 5.093 U/mg. The specific activities were obtained from precipitation by amicon® ultra-15 centrifugal filter devices, gel filtration chromatography and anion exchange chromatography that were increased by 15.07, 34.7, and 96.0 U/mg. The results showed that the highest activity at pH 7, temperature of 60 °C, and stable at 4 °C. Type of xylooligosaccharide produced by this study were xylohexoses, xylotriose, and xylobiose. SDS-PAGE analysis and zimogram showed that the molecular weight of xylanase protein were about 25 kDa. ABSTRAKEnzim xilanase (EC 3.2.1.8) digunakan dalam hidrolisis xilan (hemiselulosa) menjadi xilooligosakarida dan xilosa. Penelitian ini bertujuan untuk melakukan purifikasi parsial dan karakterisasi xilanase dari bakteri laut Bacillus safencis strain LBF P20 serta uji hidrolisis untuk mengetahui jenis xilooligosakarida yang dihasilkan oleh enzim tersebut. Berdasarkan hasil penelitian, waktu optimum untuk produksi enzim terjadi pada jam ke 96 dengan aktivitas enzim sebesar 6,275 U/mL dan aktivitas spesifik enzim sebesar 5,093 (U/mg). Aktivitas spesifik enzim hasil pemekatan dengan amicon® ultra-15 centrifugal filter devices, kromatografi filtrasi gel dan kromatografi penukar anion mengalami peningkatan berturut-turut sebesar 15,1; 34,7 dan96,0 U/mg. Hasil karakterisasi menunjukkan aktivitas tertinggi pada pH 7, suhu 60 °C dan stabil pada suhu 4 °C. Analisis SDS-PAGE dan zimogram menunjukkan berat molekul protein xilanase berkisar 25 kDa. Jenis gula reduksi yang dihasilkan yaitu xiloheksosa, xilotriosa, dan xilobiosa.

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Purification and characterization of cysteine-S-conjugate N-acetyltransferase from pig kidney

Biochemical Journal ◽

10.1042/bj3170213 ◽

1996 ◽

Vol 317 (1) ◽

pp. 213-218 ◽

Cited By ~ 13

Author(s):

Achim AIGNER ◽

Martina JÄGER ◽

Ralf PASTERNACK ◽

Peter WEBER ◽

Dirk WIENKE ◽

...

Keyword(s):

Proteinase Inhibitors ◽

Cysteine Proteinase ◽

Gel Filtration ◽

Protein Band ◽

Exchange Chromatography ◽

Polyclonal Antiserum ◽

Partial Purification ◽

Ph Optimum ◽

Sds Page

Microsomal cysteine-S-conjugate N-acetyltransferase catalyses the N-acetylation of various S-substituted cysteines in liver and kidney. We describe here the purification and more detailed characterization of this enzyme catalysing the final reaction of mercapturic acid biosynthesis, and thus playing a crucial role in the detoxicating metabolism of many xenobiotics. The solubilization of cysteine-S-conjugate N-acetyltransferase by deoxy-BIGCHAP [N,N´-bis-(3-d-gluconamidopropyl)deoxycholamide] was the prerequisite for partial purification by means of anion-exchange chromatography. The molecular mass of the enzyme was determined by gel filtration. A polyclonal antiserum was raised against the excised protein band from SDS/PAGE and purified antibodies were used for the complete purification of native cysteine-S-conjugate N-acetyltransferase by immunoaffinity chromatography. A dimeric form of the enzyme was sometimes detected on SDS/PAGE, depending on the degree of purification. For further characterization of cysteine-S-conjugate N-acetyltransferase, the stability of catalytic activity, the pH optimum and Km values were determined. The inhibitory effects of various agents were tested, revealing a substantial, yet not complete, loss of cysteine-S-conjugate N-acetyltransferase activity after treatment with cysteine proteinase inhibitors or probenecid under various conditions.

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Partial purification of dog angiotensinogen.

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1979.236.6.e655 ◽

1979 ◽

Vol 236 (6) ◽

pp. E655

Author(s):

B J Morris ◽

B Moffat ◽

I A Reid

Keyword(s):

Gel Filtration ◽

Kinetic Studies ◽

Apparent Molecular Weight ◽

Exchange Chromatography ◽

Order Reaction ◽

Partial Purification ◽

First Order ◽

Step Procedure ◽

Dog Plasma

Dog angiotensinogen was purified 450-fold from the plasma of nephrectomized dogs by a simple four-step procedure involving precipitation between 1.5 and 2.3 M ammonium sulfate, gel filtration on Sephadex G-150, ion-exchange chromatography on DE-52 cellulose, and affinity chromatography on Concanavalin A-Sepharose. The purity of the final preparation was over 50%. The preparation of dog angiotensinogen had an apparent molecular weight of 80,000 determined by gel filtration on Sephadex G-100. Kinetic studies indicated that the Km of the reaction of dog renin with partially purified dog angiotensinogen (1,840 pmol/ml) was similar to that for the reaction with angiotensinogen in diluted dog plasma (1,820 pmol/ml). Thus the purification procedures employed did not alter the affinity of dog renin for the Leu10-Leu11 bond of dog angiotensinogen. Because the concentration of angiotensinogen in dog plasma is about 700 pmol/ml, a first order reaction with respect to substrate is indicated in vivo.

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Studies on an Inhibitor of Plasminogen Activation in Human Serum

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649120 ◽

1973 ◽

Vol 30 (02) ◽

pp. 414-424 ◽

Cited By ~ 20

Author(s):

Ulla Hedner

Keyword(s):

Ion Exchange ◽

Human Serum ◽

Antithrombin Iii ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Specific Adsorption ◽

Partial Purification ◽

Plasminogen Activation ◽

Preparative Electrophoresis

SummaryA procedure is described for partial purification of an inhibitor of the activation of plasminogen by urokinase and streptokinase. The method involves specific adsorption of contammants, ion-exchange chromatography on DEAE-Sephadex, gel filtration on Sephadex G-200 and preparative electrophoresis. The inhibitor fraction contained no antiplasmin, no plasminogen, no α1-antitrypsin, no antithrombin-III and was shown not to be α2 M or inter-α-inhibitor. It contained traces of prothrombin and cerulo-plasmin. An antiserum against the inhibitor fraction capable of neutralising the inhibitor in serum was raised in rabbits.

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

Bayero Journal of Pure and Applied Sciences ◽

10.4314/bajopas.v13i2.14 ◽

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.

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Optimization of Metal Ions in Sugarcane Bagasse Fermenting Medium for the Production of Streptokinase by Streptococcus equisimilis

Science Letters ◽

10.47262/sl/9.2.132021003 ◽

2021 ◽

Vol 9 (2) ◽

pp. 24-30

Keyword(s):

Metal Ions ◽

Sugarcane Bagasse ◽

Specific Activity ◽

Gel Filtration ◽

Value Added ◽

Cost Effective ◽

Fermentation Medium ◽

Exchange Chromatography ◽

Sds Page ◽

The Cost

Streptokinase is a fibrinolytic enzyme and a product of β-hemolytic Streptococci strains. This enzyme is used as a medication to break down clots in some cases of heart disease. Streptococcus equisimilis, a species of group C Streptococci, is widely used for the production of streptokinase by fermentation technology. In this study, the sugarcane bagasse fermentation medium was optimized for metal ions (KH2PO4, MgSO4.7H2O, CaCO3 and NaHCO3) at various levels to attain the maximal production of streptokinase. Sugarcane bagasse was used due to its profuse availability and as an ideal substrate for microbial processes for the manufacturing of value-added products. The results showed that maximal streptokinase production was found at 0.04% KH2PO4, 0.04% MgSO4.7H2O, 0.15% NaHCO3 and 0.04% CaCO3. Finally, the optimized medium resulted in 84.75 U/mg specific activity and 74.5% recovery. The purification process was carried out simultaneously using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. Finally, a purified sample of streptokinase was run on SDS-PAGE and resolute 47 kDa molecular weight. The use of β-hemolytic Streptococci to obtain streptokinase is not free from health risks and is related to anaphylaxis. This study provides a way forward for the cost-effective ways to obtain streptokinase for the treatment of thrombosis.

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PURIFICATION AND PARTIAL CHARACTERIZATION OF L-ASPARAGINASE ENZYME PRODUCED BY NEWLY ISOLATE BACILLUS SP.

IIUM Engineering Journal ◽

10.31436/iiumej.v18i2.654 ◽

2017 ◽

Vol 18 (2) ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Dzun Noraini Jimat ◽

Intan Baizura Firda Mohamed ◽

Azlin Suhaida Azmi ◽

Parveen Jamal

Keyword(s):

Specific Activity ◽

Hot Spring ◽

Gel Filtration ◽

Maximum Activity ◽

Exchange Chromatography ◽

Bacillus Sp ◽

Sds Page ◽

Fast Flow ◽

Microbial Strain

A newly bacterial producing L-asparaginase was successful isolated from Sungai Klah Hot Spring, Perak, Malaysia and identified as Bacillus sp. It was the best L-asparaginase producer as compared to other isolates. Production of L-asparaginase from the microbial strain was carried out under liquid fermentation. The crude enzyme was then centrifuged and precipitated with ammonium sulfate before further purified with chromatographic method. The ion exchange chromatography HiTrap DEAE-Sepharose Fast Flow column followed by separation on Superose 12 gel filtration were used to obtain pure enzyme. The purified enzyme showed 10.11 U/mg of specific activity, 50.07% yield with 2.21 fold purification. The purified enzyme was found to be dimer in form, with a molecular weight of 65 kDa as estimated by SDS-PAGE. The maximum activity of the purified L-asparaginase was observed at pH 9 and temperature of 60Â°C.

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Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

Avicenna Journal of Medical Biotechnology ◽

10.18502/ajmb.v13i4.7204 ◽

2021 ◽

Author(s):

Nguyen Thi My Trinh ◽

Tran Linh Thuoc ◽

Dang Thi Phuong Thao

Keyword(s):

Escherichia Coli ◽

Inclusion Bodies ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Insoluble Fraction ◽

Exchange Chromatography ◽

Native Page ◽

E Coli ◽

Sds Page ◽

Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

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Purification and Partial Characterization of β-Glucosidase from Fresh Leaves of Tea Plants (Camellia sinensis (L.) O. Kuntze)

Acta Biochimica et Biophysica Sinica ◽

10.1111/j.1745-7270.2005.00053.x ◽

2005 ◽

Vol 37 (6) ◽

pp. 363-370 ◽

Cited By ~ 16

Author(s):

Ye-Yun Li ◽

Chang-Jun Jiang ◽

Xiao-Chun Wan ◽

Zheng-Zhu Zhang ◽

Da-Xiang Li

Keyword(s):

Specific Activity ◽

Gel Filtration ◽

Maximum Activity ◽

Exchange Chromatography ◽

Optimum Activity ◽

Development Of Resistance ◽

Sds Page ◽

C 50 ◽

Tea Plants

Abstractβ-Glucosidases are important in the formation of floral tea aroma and the development of resistance to pathogens and herbivores in tea plants. A novel β-glucosidase was purified 117-fold to homogeneity, with a yield of 1.26%, from tea leaves by chilled acetone and ammonium sulfate precipitation, ion exchange chromatography (CM-Sephadex C-50) and fast protein liquid chromatography (FPLC; Superdex 75, Resource S). The enzyme was a monomeric protein with specific activity of 2.57 U/mg. The molecular mass of the enzyme was estimated to be about 41 kDa and 34 kDa by SDS-PAGE and FPLC gel filtration on Superdex 200, respectively. The enzyme showed optimum activity at 50 °C and was stable at temperatures lower than 40 °C. It was active between pH 4.0 and pH 7.0, with an optimum activity at pH 5.5, and was fairly stable from pH 4.5 to pH 8.0. The enzyme showed maximum activity towards pNPG, low activity towards pNP-Galacto, and no activity towards pNP-Xylo.

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