Partial purification of dog angiotensinogen.

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.

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

2019 ◽  
Vol 16 (3(Suppl.)) ◽  
pp. 0740
Author(s):  
Al Samurai Et al.
Keyword(s):  
Kinetic Characteristic ◽  
Thyroid Tissue ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Old Patients ◽  
Physiological Regulation ◽  
Sds Page ◽  
Intracellular Regulation

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 .                                                                                             

Studies on an Inhibitor of Plasminogen Activation in Human Serum

1973 ◽  
Vol 30 (02) ◽  
pp. 414-424 ◽  
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.

Plasminogen-Activator in Human Early Milk: Its Partial Purification and Characterization

1981 ◽  
Vol 45 (02) ◽  
pp. 121-126 ◽  
Author(s):  
Utako Okamoto ◽  
Noboru Horie ◽  
Yoko Nagamatsu ◽  
Jun-Ichiro Yamamoto
Keyword(s):  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Partial Purification ◽  
Order Kinetic ◽  
Diisopropyl Fluorophosphate ◽  
Step Procedure ◽  
Activity Band ◽  
C 50 ◽  
Gel Isoelectric Focusing

SummaryMilk plasminogen-activator was partially purified from human transitional milk collected at about 10 days after delivery, by a five-step procedure involving chloroform treatment, ammonium sulfate precipitation, and column chromatography on Sephadex G-150, CM Sephadex C-50 and DEAE Sephadex A-50. This gave milk-activator with a maximum purification factor of about 2,400-fold with respect to the skimmed milk. The CM Sephadex-step preparation showed, on polyacrylamide gel electrophoresis, a single plasminogen-activator activity band located between the bands of albumin and prealbumin of human serum. This preparation exhibited no kinin forming activity. The activator hydrolyzed acetyl-glycyl-L-lysine methyl ester with similar order kinetic constants to urokinase, and was inhibited strongly by diisopropyl-fluorophosphate. The molecular weight of the activator as estimated by gel filtration was approximately 86,000, the isoelectric points as estimated by gel isoelectric focusing were pH 7.2, 6.9 and 6.6, and the activator activity was not quenched by antiurokinase globulin, indicating that the milk-activator is a different entity from urokinase.

scholarly journals Reuse of Newspaper As An Adsorbent For Cu (II) Removal By Citric Acid Modification

2018 ◽  
Vol 156 ◽  
pp. 02012 ◽  
Author(s):  
Mardiah ◽  
Rif’an Fathoni ◽  
Pratiwi Pudyaningtyas ◽  
Hamdania Gamu ◽  
Rinaldy
Keyword(s):  
Citric Acid ◽  
Adsorption Process ◽  
Kinetic Studies ◽  
Order Reaction ◽  
Acid Modification ◽  
Pseudo First Order Reaction ◽  
First Order ◽  
Pseudo Second Order ◽  
Heavy Metal Adsorbent ◽  
The Impact

High Consumption of paper, bring the impact of the waste paper itself. And the utilization of the paper is limited to recycled products and crafts, whereas paper such as newspaper still contains cellulose that can be potential to be used as a heavy metal adsorbent. In this study, newspaper was dissolved in sodium bicarbonate to reduce various impurities and then was reacted with citric acid (CA). The modified adsorbent was characterized by FTIR and was tested for adsorb Cu(II) in artificial solution. After adsorption process, the solution was filtered and analysed using Atomic Absorption Spectrophotometer (AAS). The adsorption experimental data was fitted to Langmuir, Freundlich, Tempkin, and Dubinin-Radushkevich for equilibrium model and was fitted to pseudo first order reaction and pseudo second order reaction for kinetic studies. The result showed that CA-modification newspaper able to remove heavy metals Cu(II) in solution.

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.

Relationship between volume of bathing medium and ectoenzyme activity in monolayers of cultured BPAEC

1996 ◽  
Vol 271 (3) ◽  
pp. L464-L469
Author(s):  
A. Papapetropoulos ◽  
L. A. Elmore ◽  
J. D. Catravas
Keyword(s):  
Maximum Velocity ◽  
Order Reaction ◽  
First Order Reaction ◽  
Reaction Volume ◽  
Reaction Conditions ◽  
Bathing Medium ◽  
First Order ◽  
Pulmonary Capillary ◽  
Ace Activity

It is unclear whether all or a fraction of the capillary plasma volume (Vcp) serves as the reaction volume (Vr) for pulmonary capillary endothelial ectoenzymes, in vivo. Cultured endothelial cell (EC) monolayers provide a convenient model for studying EC-bound enzyme-Vr relationships. Because the Michaelis-Menten parameter [maximum velocity of enzyme reaction (Vmax) = E x kcat/Vr, where E is enzyme mass and kcat is the constant of product formation] is inversely proportional to Vr, we hypothesized that increasing the volume of medium (Vm) bathing EC monolayers would proportionally reduce the calculated Vmax (or Vmax/K(m), where K(m) is the Michaelis constant) values of an ectoenzyme reacting with a substrate only if, and as long as, Vm = Vr. To test this hypothesis, studies were performed in bovine pulmonary arterial EC grown to confluence. Activities of angiotensin-converting enzyme (ACE) and 5'-nucleotidase (NCT) were assayed in Earle's balanced salts solution utilizing [3H]benzoyl-Phe-Ala-Pro ([3H]BPAP) and 5'-[14C]AMP as substrates, respectively. Under first-order reaction conditions and at constant substrate concentrations ([BPAP] = 15 nM, [AMP] = 1 microM), Vmax/K(m) ratios of ACE and NCT declined to 20% of their original values, as Vm increased from 0.6 to 2 ml. ACE activity was also studied at constant substrate mass (BPAP = 7 pmol) under first-order reaction conditions. Again, enzyme activity (Vmax/K(m)) declined proportionally to increasing Vm. Under zero-order reaction conditions ([BPAP] = 250 microM), ACE activity (Vmax) was similarly related to Vm. Linear regression analyses revealed that ACE or NCT would recognize up to at least 3 ml Vm, a volume vastly exceeding that of Vcp in a section of the capillary bed composed of an equivalent number of ECs, thus suggesting that Vcp could serve as the reaction volume for pulmonary capillary EC ectoenzymes in vivo.

scholarly journals Sesquiterpene α-Farnesene Synthase: Partial Purification, Characterization, and Activity in Relation to Superficial Scald Development in Apples

2000 ◽  
Vol 125 (1) ◽  
pp. 111-119 ◽  
Author(s):  
H.P. Vasantha Rupasinghe ◽  
Gopinadhan Paliyath ◽  
Dennis P. Murr
Keyword(s):  
Metal Ion ◽  
Maximal Activity ◽  
Exchange Chromatography ◽  
Cytosolic Fraction ◽  
Skin Tissue ◽  
Hill Coefficient ◽  
Partial Purification ◽  
Farnesyl Pyrophosphate ◽  
Superficial Scald

To decipher the relation between α-farnesene metabolism and the development of superficial scald in apples, trans,trans-α-farnesene synthase, the enzyme that catalyzes the conversion of farnesyl pyrophosphate to α-farnesene, was partially purified from skin tissue of `Delicious' apples (Malus ×domestica Borkh.) and characterized. Total and specific activities of the enzyme were higher in the cytosolic fraction than in membrane fractions. α-Farnesene synthase was purified 70-fold from the cytosolic fraction by ion exchange chromatography and gel permeation, and the native molecular weight was estimated to be 108,000. The enzyme had optimal activity at a pH of 5.6 and absolutely required a divalent metal ion such as Mg2+ or Mn2+ for activity. It exhibited allosteric kinetics, S(0.5) for farnesyl pyrophosphate being 84±18 μmol·L-1, and a Hill coefficient (nH) of 2.9, indicating the number of subunits to be two or three. Enzyme activity was highest between 10 and 20 °C, while 50% of the maximal activity was retained at 0 °C. In vivo α-farnesene synthase activity was minimal at harvest, then increased rapidly during 16 weeks storage in air at 0 °C, and decreased during further storage. Activity of α-farnesene synthase, α-farnesene content, and conjugated triene alcohol (the putative scald-causing oxidation product of α-farnesene) content in skin tissue were not correlated to the inherent nature of scald susceptibility or resistance in 11 apple cultivars tested.

scholarly journals Mouse Uterine Alkaline Phosphatase: Improved Purification by Affinity Chromatography and Further Characterization of the Enzyme

1980 ◽  
Vol 33 (3) ◽  
pp. 279 ◽  
Author(s):  
RN Murdoch ◽  
Louise E Buxton ◽  
DJ Kay
Keyword(s):  
Alkaline Phosphatase ◽  
Affinity Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Apparent Molecular Weight ◽  
Anion Exchange Chromatography ◽  
Pregnant Mouse ◽  
Exchange Chromatography ◽  
Enzyme Preparations

An improved procedure for the purification of alkaline phosphatase from about 10 g of day 7 pregnant mouse uterine tissue is described. Following homogenization, the procedure involved solubilization and extraction with 0�8% (v/v) Triton X-lOO and 20% (v/v) n-butanol, ammonium sulfate precipitation, concanavalin A-Sepharose 4B affinity chromatography, DEAE-cellulose anion-exchange chromatography and Sephacryl S200 gel filtration. On subjecting 2162-fold purified enzyme preparations to polyacrylamide-gel electrophoresis, a single band of protein coincident with the zone of enzyme activity and having an apparent molecular weight of 205 OOO� lOOOO was identified. Affinity chromatography yielded the largest increase in purity of any step in the procedure and established the glycoprotein nature of the uterine enzyme.

scholarly journals Evidence for N-linked glycosylation in Toxoplasma gondii

1993 ◽  
Vol 291 (3) ◽  
pp. 713-721 ◽  
Author(s):  
M Odenthal-Schnittler ◽  
S Tomavo ◽  
D Becker ◽  
J F Dubremetz ◽  
R T Schwarz
Keyword(s):  
Toxoplasma Gondii ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Surface Glycoprotein ◽  
Common Precursor ◽  
The Common ◽  
Oligosaccharide Structures

In this paper we report experiments demonstrating the presence of N-linked oligosaccharide structures in Toxoplasma gondii tachyzoites, providing the first direct biochemical evidence that this sporozoan parasite is capable of synthesizing N-linked glycans. The tachyzoite surface glycoprotein gp23 was metabolically labelled with [3H]glucosamine and [3H]mannose. Gel-filtration chromatography on Bio-Gel P4 columns produced four radiolabelled N-linked glycopeptides which were sensitive to peptidase-N-glycanase F, but resistant to endoglycosidases H and F. Using chemical analysis and exoglycosidase digestions followed by Dionex-high-pH anion-exchange chromatography and size fractionation on Bio-Gel P4 we show that gp23 has N-linked glycans in the hybrid- or complex-type structure composed of N-acetylgalactosamine, N-acetylglucosamine and mannose and devoid of sialic acid and fucose residues. In addition, the sensitivity of glycopeptides from glycoprotein extracts to endoglycosidases H and F revealed the in vivo synthesis of oligomannose-type structures by T. gondii tachyzoites. We have extended these findings by demonstrating the ability of T. gondii microsomes to synthesize in vitro a glucosylated lipid-bound high-mannose structure (Glc3Man9GlcNAc2) that is assumed to be identical with the common precursor for N-glycosylation in eukaryotes.

Characterization and partial purification of a broad spectrum antibiotic AS-48 produced by Streptococcus faecalis

1986 ◽  
Vol 32 (10) ◽  
pp. 765-771 ◽  
Author(s):  
A. Gálvez ◽  
M. Maqueda ◽  
E. Valdivia ◽  
A. Quesada ◽  
E. Montoya
Keyword(s):  
Broad Spectrum ◽  
Gel Filtration ◽  
Basal Medium ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Gram Negative Bacteria ◽  
Broad Spectrum Antibiotic ◽  
Streptococcus Faecalis ◽  
Group D

Streptococcus faecalis S-48 produces a broad spectrum antibiotic, active against Gram-positive and Gram-negative bacteria. This substance is produced in solid and liquid media and also in a defined basal medium. It is sensitive to protease, pronase, or trypsin, heating at 70 °C, and alkaline pH, but resistant to treatment with lipase, lysozyme, alkaline phosphatase, DNAase, RNAase, acidic or neutral pHs, and also lower temperatures (60 °C). Several organic solvents cause precipitation, but not inactivation. This antibiotic has been partially purified by gel filtration and further ion-exchange chromatography. Its molecular weight has been estimated close to 2000. The biological activity of this antagonistic substance against the selected indicator strains, Streptococcus faecalis S-47 and Escherichia coli U-9, is bactericidal. The characterization of this substance, initially classified as a bacteriocin, indicates that it is an antibiotic of peptidic nature. The significance of antibiotic occurrence in group D of the genus Streptococcus is also discussed.

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