Effects of some antibiotics on glucose 6-phosphate dehydrogenase in sheep liver

In vitro effects of penicillin, sulbactam, cefazolin, and amikacine on the activity of the enzyme glucose-6-phosphate dehydrogenase in sheep liver were investigated. Glucose 6-phosphate dehydrogenase was purified from sheep liver, using a simple and rapid method. The purification consisted of two steps, preparation of homogenate and 2’, 5’-ADP Sepharose 4B affinity chromatography. As a result of the two consecutive procedures, the enzyme, having the specific activity of 11.76 EU/mg proteins, was purified with a yield of 35.72% and 1.913 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS-PAGE showed a single band for the enzyme. In addition, I50 values of the antibiotics were determined by plotting activity % vs. antibiotic concentrations. I50 values were 17.71 mM for penicillin, 27.38 mM for sulbactam, 28.88 mM for cefazolin, and 30.59 mM for amikacine.

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The Effects of Some Antibiotics on Sheep Lens Glucose 6-Phosphate Dehydrogenase in Vitro

European Journal of Ophthalmology ◽

10.1177/112067210301300207 ◽

2003 ◽

Vol 13 (2) ◽

pp. 155-161 ◽

Cited By ~ 8

Author(s):

Ş. Beydemir ◽

D.N. Kulaçoğlu ◽

M. Çiftçi ◽

Ö.I. Küfrevioğlu

Keyword(s):

Specific Activity ◽

Gentamicin Sulfate ◽

Vancomycin Hydrochloride ◽

Dehydrogenase Enzyme ◽

Postmortem Studies ◽

Cefazolin Sodium ◽

In Vitro Effects ◽

Glucose 6 Phosphate Dehydrogenase ◽

Sepharose 4B

Purpose To investigate the in vitro effects of gentamicin sulfate, vancomycin hydrochloride, sodium cefazolin and ceftriaxone on glucose 6-phosphate dehydrogenase enzyme (G6PD) purified from sheep lenses. Methods G6PD was purified from sheep lenses with a yield of 66.8% and a specific activity of 7.8 U/mg proteins, and 10,400-fold using ammonium sulfate fractionation and 2′,5′-ADP Sepharose 4B affinity gel. The enzyme activity was determined by Beutler's method. Results Gentamicin sulfate and vancomycin hydrochloride strongly inhibited the enzyme in vitro. The concentrations causing 50% inhibition (IC50) were 15.34, and 8.0 mM, respectively. Conversely, cefazolin sodium strongly activated this enzyme, and ceftriaxone caused milder activation. Conclusions If a patient with G6PD deficiency requires gentamicin sulfate or vancomycin hydrochloride, routine ophthalmic did not inhibit this enzyme. Postmortem studies are now needed to investigate the activity of G6PD and how it is affected by these antibiotics.

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Purification and characterization of β-N-acetylhexosaminidase I2 from human liver

Biochemical Journal ◽

10.1042/bj2340157 ◽

1986 ◽

Vol 234 (1) ◽

pp. 157-162 ◽

Cited By ~ 15

Author(s):

N N Dewji ◽

D R De-Keyzer ◽

J L Stirling

Keyword(s):

Gel Electrophoresis ◽

Human Liver ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Gel Filtration ◽

Deae Cellulose ◽

Alpha Subunit ◽

Gradient Gel Electrophoresis ◽

Sepharose 4B

beta-N-Acetylhexosaminidase I2 was purified from human liver by a combination of concanavalin A chromatography, DEAE-cellulose chromatography, gel filtration and affinity chromatography on 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-beta-D-glucopyranosylamine coupled to CNBr-activated Sepharose 4B. Its specific activity was 130 mumol/min per mg of protein compared with values of 150 and 320 mumol/min/mg of protein for beta-N-acetylhexosaminidases A and B purified from the same tissue. Km values for I2, A and B were 1.0 mM, 0.8 mM and 0.74 mM respectively. On gradient gel electrophoresis under non-denaturing conditions, hexosaminidase I2 behaved similarly to A and appeared to have an Mr between 100 000 and 110 000. beta-N-Acetylhexosaminidase I2 was resolved into two major polypeptides, of Mr 56 000 and 29 000, on SDS/polyacrylamide-gel electrophoresis under denaturing conditions. Immunoblotting with anti-(hexosaminidase alpha-subunit) serum confirmed that the 56 000-Mr component was the alpha-subunit and anti-(hexosaminidase B) serum reacted with the 29 000 Mr component. beta-N-Acetylhexosaminidase I2 more closely resembles form A than B, but the features of its structure that allow it to be separated from A on the basis of net charge have not yet been found.

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Radioiodinated Human Fibrinogen. In Vitro Effects of Increasing Iodination

10.1055/s-0039-1689552 ◽

1975 ◽

Author(s):

B. E. Ly ◽

P. Kierulf

Keyword(s):

Amino Acid ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Clotting Time ◽

Terminal Amino Acid ◽

Human Fibrinogen ◽

Terminal Amino ◽

In Vitro Effects ◽

Polypeptide Chains

Fibrinogen preparations with increasing contents of iodine, ranging from 0.2 to 20 atoms of iodine per molecule fibrinogen, were obtained with the ICl method. Aggregation and shortening of the thrombin clotting time occurred when the content of iodine exceeded 3 atoms per molecule.Upon the action of thrombin, the increase in N-terminal glycine, reflecting fibrin formation, was almost identical in native and iodinated fibrinogen. At visible gelation, however, decreased amounts of N-terminal glycine were found in heavily iodinated fibrinogen, thus indicating enhanced fibrin polymerization. N-terminal analysis of heavily iodinated fibrinogen demonstrated a deficiency in N-terminal tyrosine concomitantly with the apparance of a new N-terminal amino acid, identified as mono-iodo-tyrosine.Polyacrylamide gel electrophoresis at pH 8.9 revealed an increase in mobility following extensive iodination, but no shift in the isoelectric point was observed upon isofocusing.Neither clottability nor the behaviour of fibrinogen and its subunit polypeptide chains on SDS-gel electrophoresis was affected by iodination.

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KONSENTRASI PROTEIN DAN PENENTUAN BERAT MOLEKUL EKSKRETORI/SEKRETORI L3 Ascaridia galli

Jurnal Kedokteran Hewan - Indonesian Journal of Veterinary Sciences ◽

10.21157/j.ked.hewan.v3i1.3074 ◽

2016 ◽

Vol 3 (1) ◽

Author(s):

Darmawi D ◽

Ummu Balqis ◽

Risa Tiuria ◽

Retno D Soejoedono ◽

Fachriyan H Pasaribu

Keyword(s):

Sodium Dodecyl Sulphate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Ascaridia Galli ◽

Sds Page

Penelitian ini bertujuan menentukan konsentrasi dan berat molekul protein ekskretori/sekretori larva (L3) Ascaridia galli (A. galli). Larva L3 diperoleh dari usus halus 100 ayam tujuh hari setelah pemberian dosis 6000 L2 melalui esofagus ayam. Sebanyak 5–10 L3 dikultur secara in vitro dalam setiap ml medium Rosswell Park Memorial Institute (RPMI 1640), pH 6,8, tanpa merah fenol dalam inkubator pada temperatur 37 0C dan 5% CO2 selama 3 hari. Ke dalam medium ditambahkan 100 unit ml-1 penisilin G, 100 µg ml-1 streptomisin, 5 µg ml-1 gentamisin dan 0,25 µg ml-1 kanamisin. Ekskretori/sekretori dipreparasi dari produk metabolisme L3 yang dilepaskan ke dalam medium kultur. Untuk mendapatkan protein ekskretori/sekretori, medium kultur dipekatkan dengan vivaspin 30.000 MWCO, dan kuantitas protein dihitung dengan metode Bradford. Berat molekul protein ekskretori/sekretori divisualisasikan dengan sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Hasil penelitian menunjukkan bahwa konsentrasi protein ekskretori/sekretori adalah 0,595 mg/ml dengan berat molekul 28 kDa.

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THE IN VITRO INTERCONVERSION BY MERCAPTANS OF TYPE I GLUCOSE 6-PHOSPHATE DEHYDROGENASE ISOZYMES OF RAT

Journal of Histochemistry & Cytochemistry ◽

10.1177/20.10.804 ◽

1972 ◽

Vol 20 (10) ◽

pp. 804-810 ◽

Cited By ~ 6

Author(s):

SAMUEL H. HORI ◽

SATOSHI YONEZAWA

Keyword(s):

Molecular Weight ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Intermediate Form ◽

Type I ◽

Mitochondrial Enzyme ◽

Weight Estimation ◽

Glucose 6 Phosphate Dehydrogenase

The rat glucose 6-phosphate dehydrogenase isozymes of cell sap origin designated D, F1, F2 and F3 were found to be interconvertible with each other in the presence or absence of mercaptans. The D and F3 enzymes were the oxidized, the F1 was the reduced and the F2 was the intermediate form. Molecular weight estimation by the polyacrylamide gel electrophoresis and Sephadex G-200 methods demonstrated that the D enzyme had twice the molecular weight of the F enzymes. The mitochondrial enzyme which moved as fast as the F3 enzyme of cell sap origin was not modified with mercaptans. Together with previous data, the present results caution in the interpretation of zymograms.

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purification and characterization of glucose 6-phosphate dehydrogenase from rainbow trout (oncorhynchus mykiss) erythrocytes

Veterinární Medicína ◽

10.17221/5712-vetmed ◽

2012 ◽

Vol 49 (No. 9) ◽

pp. 327-333 ◽

Cited By ~ 8

Author(s):

M. Ciftci ◽

A. Ciltas ◽

O. Erdogan

Keyword(s):

Rainbow Trout ◽

Oncorhynchus Mykiss ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Gel Chromatography ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Ammonium Sulphate Precipitation ◽

Glucose 6 Phosphate Dehydrogenase

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) from rainbow trout (Oncorhynchus mykiss) erythrocytes was purified, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure consisted of three steps: haemolysate preparation, ammonium sulphate precipitation, and 2’,5’-ADP Sepharose 4B affinity gel chromatography, which took one working day. Thanks to the three consecutive procedures, the enzyme, having the specific activity of 14.51 EU/mg proteins, was purified with a yield of 70.40% and 1 271.19-fold. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis was carried out. SDS polyacrylamide gel electrophoresis showed a single band for the enzyme. Optimal pH, stable pH, optimal temperature, molecular weight, and KM and Vmax values for NADP+ and glucose 6- phosphate (G6-P) were also determined for the enzyme. In addition, the effect of NADPH on the enzyme was investigated and Ki value and the type of inhibition were determined by means of Lineweaver-Burk graph obtained for NADPH.

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α-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro

Biochemical Journal ◽

10.1042/bj1751069 ◽

1978 ◽

Vol 175 (3) ◽

pp. 1069-1077 ◽

Cited By ~ 25

Author(s):

J Williams ◽

H Villarroya ◽

F Petek

Keyword(s):

Trifolium Repens ◽

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Deae Cellulose ◽

Disc Electrophoresis ◽

Polyacrylamide Gel ◽

Ph Optimum

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).

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A rapid purification of bovine testicular hyaluronidase by chromatography on dermatan sulphate-substituted 1,6-diaminohexane–sepharose 4B

Biochemical Journal ◽

10.1042/bj1990419 ◽

1981 ◽

Vol 199 (2) ◽

pp. 419-426 ◽

Cited By ~ 9

Author(s):

M Lyon ◽

C F Phelps

Keyword(s):

Gel Electrophoresis ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

High Yield ◽

Dermatan Sulphate ◽

Bovine Testicular Hyaluronidase ◽

Sepharose 4B ◽

Testicular Hyaluronidase

The binding of bovine testicular hyaluronidase to AH-Sepharose (1,6-diaminohexane--Sepharose) gels substituted with (1) dermatan sulphate, (2) desulphated dermatan sulphate, (3) heparin and (4) de-N/O-sulphated, re-N-acetylated heparin was investigated. Hyaluronidase was found to bind to (1) and (3), but not (2) and (4). On the basis of these observations a preparative scheme for the purification of testicular hyaluronidase was developed. This consisted of two steps: (i) chromatography on dermatan sulphate-substituted AH-Sepharose 4B; (ii) chromatography on acetylated AH-Sepharose 4B. This procedure gave hyaluronidase with a specific activity of 19.1 units (mumol/min)/mg in high yield. Polyacrylamide-gel electrophoresis at pH 4.3 revealed two components, both possessing hyaluronidase activity. Sodium dodecyl sulphate polyacrylamide-gel electrophoresis likewise revealed two close bands with approximate molecular weights of 61000 and 67200.

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Purification of Human Factor

10.1055/s-0039-1680659 ◽

1977 ◽

Author(s):

V.P.A. Bolhuis ◽

T.B.M. Hakvoort ◽

K. Breederveld ◽

I.A. Mochtar ◽

J.W. ten Cate

Keyword(s):

Polyethylene Glycol ◽

Gel Electrophoresis ◽

Human Factor ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Polyacrylamide Gel ◽

Factor V ◽

Native Protein ◽

Sepharose 4B ◽

Polyethylene Glycol 6000

Human factor V has been purified from cryo-supernatant by fractionated precipitation with polyethylene glycol (6000, 16-24% w/v, yield 65%), followed by gelfiltration on AcA 44 in Michaelis buffer with 50mM Ca++ (elution in Vo, yield 90%), adsorption of contaminating haptoglobinpolymers on hemoglobin bound to Sepharose-4B (yield 95%) and adsorption of the glycoproteins (mainly factor V) onto concanavaline A bound to Sepharose-4B, elution with α-methylpyranoside (4M) and gelfiltration of the factor V eluted. The purified factor V hasa specific activity of about 28 u/mg and thetotal yield is 16%. The native protein and the subunitsobtained by reduction in the presence of dodecyl sulphate and urea have been characterized by Polyacrylamide gel electrophoresis.

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Studies of Heparin Affinity to Antithrombin III and Other Proteins in Vitro and in Vivo

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651884 ◽

1977 ◽

Vol 38 (03) ◽

pp. 0685-0695 ◽

Cited By ~ 8

Author(s):

Y Takeda ◽

N Kobayashi

Keyword(s):

Gel Electrophoresis ◽

Antithrombin Iii ◽

Specific Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Heparin Binding ◽

Life Values ◽

Canine Plasma ◽

Heparin Affinity

SummaryProperties of human, canine, and porcine heparin and S-35-heparin were first studied. Their electrophoretic mobility through 10 g % polyacrylamide gels, specific activity and their filtration patterns through Sephadex G-200 columns were closely similar. Then, S-35-heparin (0.5 mg) and cold heparin (27 mg) were simultaneously injected intravenously into 5 dogs and their plasma behavior was compared. The plasma half-lives of S-35-heparin averaged 1.18 ± 0. 13 (SD) hr and was identical with that of cold heparin, but the half-lives were much shorter and averaged 0.46 ± 0.08 (SD) hr in 5 dogs when S-35-heparin alone was injected. Studies were next made of heparin affinity to proteins including canine antithrombin III (AT) by the use of Sephadex G-200 chromatography, polyacrylamide gel electrophoresis, and S-35-heparin as a tracer. It was found that S-35-heparin-binding to alpha1, beta, and gammaglobulins and fibrinogen was readily separable upon addition of 5 mg cold heparin, but that the binding to AT was inseparable by addition of cold heparin or by electrophoresis. However, in canine plasma, both S-35-heparin and cold heparin were mostly bound to proteins other than AT, suggesting that this might be the case in vivo. To further substantiate this, studies were made of the comparative behavior of I-125-labeled AT (I-125-AT) and S-35-heparin in dogs with the idea that the plasma half-lives of both should be equal if they were irreversibly bound to each other. The plasma half-lives of I-125-AT averaged 2.10 ± 0.05 (SD) days in 5 male dogs and 1.99 ± 0.04 (SD) days in 5 female dogs, and were much different from the half-life values of S-35-heparin as given above. These results indicate that heparin in vitro is more tightly bound to AT than to other proteins, that heparin in vivo is not irreversibly bound to AT and suggest that it is mostly bound to proteins other than AT in vivo.

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