The effects of some antibiotics on sheep lens glucose 6-phosphate dehydrogenase in vitro.

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.

In vitro antioxidant properties of dantrolene sodium.

Dantrolene sodium is a skeletal muscle relaxant, which inhibits intracellular Ca2+ release from the sarcoplasmic reticulum. The aim of this study is to examine possible in vitro antioxidant effects of dantrolene sodium. For this reason, the in vitro antioxidant effects of dantrolene sodium were studied using thiocyanate methods. Additionally, the reducing power and free radical scavenging activity were determined. Dantrolene sodium showed strong antioxidant activity in the linoleic acid emulsion system. The antioxidant activity increased with an increasing amount of dantrolene sodium (50, 100, 250 microg). The 50, 100 and 250 microg samples of dantrolene sodium showed 55%, 70% and 82% inhibition on peroxidation of linoleic acid, respectively. On the other hand, the 250 microg sample of alpha-tocopherol showed 62% inhibition of peroxidation of linoleic acid. Like antioxidant activity, the reducing power of dantrolene sodium increased in a dose-dependent manner. The reducing power of dantrolene was statistically significant vs control, but lower than butylated hydroxytoluene (BHT) and quercetin. Although dantrolene sodium had free radical scavenging activity this was not statistically significant. In contrast to dantrolene sodium, quercetin and butylated hydroxyanisole (BHA) had highly potent free radical scavenging activities and those were statistically significant. According to the these results, it may be said that antioxidant effect of dantrolene sodium is more related to its antioxidant activity in linoleic acid emulsion and reducing power, than to its free radical scavenging activity. These properties may be major reasons for the inhibition of lipid peroxidation.

Effects of melatonin on enzyme activities of glucose-6-phosphate dehydrogenase from human erythrocytes in vitro and from rat erythrocytes in vivo.

The in vivo and in vitro effects of melatonin on glucose-6-phosphate dehydrogenase (G6PD) from human erythrocytes have been investigated. For this purpose, human erythrocyte glucose-6-phosphate dehydrogenase was purified, at the beginning, 13.654 times in a yield of 28% by using ammonium sulphate precipitation and 2',5'-ADP Sepharose 4B affinity gel. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was determined by the Beutler method using a spectrophotometer at 340 nm. This method was utilized for all kinetic studies. For in vitro experiments, the enzyme activity increased below 0.08 mM melatonin concentration and reached a plateau above 0.1 mM. Ten mg kg(-1)melatonin was administered intraperitonally and indicated the stimulatory effect on the enzyme. Time-dependent in vivo studies were executed for melatonin in Sprague-Dawley type rats. It was found that G6PD activity in the erythrocytes was increased by the melatonin in 1.5 and 3.5 h. These results show that both in vitro(below 0.08 mM) and in vivo pharmacological levels of melatonin increased enzyme activity in erythrocytes. The findings also indicate that melatonin may be pharmacologically useful in patients where a deficiency of the enzyme in red blood cells (RBC) causes haemolytic anaemia.

Purification of lactoperoxidase from bovine milk and investigation of the kinetic properties.

Lactoperoxidase (LPO) was purified from bovine milk using Amberlite CG 50 H+ resin, CM Sephadex C-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography. During the purification steps, the activity of enzyme was measured using 2,2'-azino-bis (3-ethylbenzthiazoline-6 sulfonic acid) diamonium salt (ABTS) as a chromogenic substrate at pH 6. Optimum pH and optimum temperature values for LPO were determined for ABTS, p-phenylendiamine, catechol, epinephrine, and pyrogallol as substrates, and then Km and Vmax values for the same substrate were obtained by means of Lineweaver-Burk graphics. The purification degree of the enzyme was controlled by SDS-PAGE and Rz (A412/A280) values. Km values, at optimum pH and 20 degrees C, were 0.197 mM, 0.063 mM, 0.64 mM, 25.2 mM, and 63.95 mM for p-phenylendiamine, ABTS, epinephrine, pyrogallol, and catechol, respectively. Vmax values, at optimum pH and 20 degrees C, were 3.5x10(-5) EU/mL, 4.0x10(-5) EU/mL, 5.8x10(-4) EU/mL, 8.4x10(-4) EU/mL, and 1.01x10(-3) EU/mL for the same substrates, respectively. p-Phenylendiamine was first found as a new substrate for LPO.

Effect of cefaperazone/sulbactam and ampicillin/sulbactam on the in vitro activity of human erythrocyte glucose-6-phosphate dehydrogenase.

We investigated the effects of the antibiotic drugs cefaperazone/sulbactam and ampicillin/sulbactam on the in vitro activity of the enzyme glucose-6-phosphate dehydrogenase (G6PD). The enzyme was purified from human erythocytes using 2',5' ADP-Sepharose 4B affinity gel. The enzymatic activity was measured spectrophotometrically at 340 nm, according to the method of Beutler. The I50 values were determined from Activity % - [Drug] graphs, and the Ki constants and inhibition types for each drug were determined using Lineweaver-Burk graphs. The I50 value was 13.5 mg/ml for cefaperazone/sulbactam and 36 mg/ml for ampicillin/sulbactam. The Ki constants were 10.16 for ampicillin/sulbactam and 38.22 for cefaperazone/sulbactam. Cefaperazone/sulbactam competitively inhibited G6PD activity, whereas ampicillin/sulbactam non-competitively inhibited the activity of the enzyme.

Effects of some medical drugs on enzyme activities of carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo.

In this study, firstly, the effects of sodium ampicillin, magnesium sulfate, and sodium dipyrone on human carbonic anhydrase (HCA) (EC 4.2.1.1.) isozymes have been investigated in vitro. Human erythrocyte CA-I and CA-II isozymes were separately purified by affinity chromatography. Inhibition or activation effects of three different medical drugs on HCA isozymes were determined using the CO(2)-Hydratase method by plotting activity %vs [medical drug]. I(50)values of the drugs exhibiting inhibition effects were found by means of these graphs. It was observed on HCA-I hydratase activity that sodium ampicillin and sodium dipyrone showed inhibition and activation effects, respectively. However, magnesium sulfate showed no effect. It was observed on HCA-II hydratase activity that sodium ampicillin and magnesium sulfate showed an inhibition effect, and sodium dipyrone showed an activation effect. In addition, in vivo studies were performed for these medical drugs in Sprague-Dawley rats. It was demonstrated that CA in erythrocytes was significantly inhibited by these drugs in 3 h.

Effects of some antibiotics on enzyme activity of glucose-6-phosphate dehydrogenase from human erythrocytes.

Inhibitory effects of some antibiotics on glucose-6-phosphate dehydrogenase from the erythrocytes of human have been investigated. For this purpose, at the beginning, erythrocyte glucose-6-phosphate dehydrogenase was purified 13.654 times in a yield of 28% by using ammonium sulphate precipitation and 2',5'-ADP Sepharose 4B affinity gel. Temperature of +4 degrees C was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. This method was utilized for all kinetic studies. Sodium ceftizoxime, sodium ampicillin, sodium cefuroxime, sodium cefazolin, sodium cefoperazone, streptomycin sulphate, gentamicin sulphate, and netilmicin sulphate were used as antibiotics. All the antibiotics indicated the inhibitory effects on the enzyme. K(i) constants for glucose-6-phosphate dehydrogenase were found by means of Lineweaver-Burk graphs. While sodium cefoperazone, gentamicin sulphate, and netilmicin sulphate showed competitive inhibition, the others displayed non-competitive inhibition. In addition, I(50) values of the antibiotics were determined by plotting activity percent vs [I]. In addition, in vivo studies were done for sodium sefuroxime in Sprague-Dawley type rats. It was found that G6PD in erythrocyte was more inhibited by the drug in 2.5 h.

Carbonic anhydrase from Vicia canencens leaves.

Carbonic anhydrases (CA: Carbonate hydrolase; E, C,4.2.1.1) from leaves Vicia canencens were purified and were characterized. The purification level of enzyme was 76-fold. The optimum temperature of this enzyme was 70 degrees C. pH optimum was 9.2. Each of the enzyme molecules was a decamer having MW of 262,000 and the subunit MW was 26,400.

A different structural feature for carbonic anhydrases in human erythrocytes.

This study presents a different structural feature for carbonic anhydrase in human erythrocytes. Carbonic anhydrase isozymes (CA-I and CA-II) were purified from an erythrocyte pool of 20 healthy subjects. For purification, Sepharose-4B-L-tyrosine-sulfanilamide affinity column was used. Resnets from 3-10% discontinuous SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band for CA-I and two distinct bands for CA-II. The molecular weights of the two bands were similar. One peak for CA-I and two peaks for CA-II were obtained in gel filtration. The enzymatic activities of the bands in question were also of different value. Native electrophoresis showed two bands for CA-I, and it showed three bands for CA-II. It can be concluded that CA-I is a polymer composed of a single promoter and CA-II has three different polymers composed of two distinct promoters, suggesting a new structural feature of human erythrocyte carbonic anhydrase isozymes.

Synthesis and investigation of inhibition effects of new carbonic anhydrase inhibitors.

Three new derivatives of 2-substituted 1,3,4-thiadiazole-5-sulfonamide have been synthesized. These compounds are 2-(3-chloropropionylamino)-1,3,4-thiadiazole-5-sulfonamide (1); 2-(2,2-dichloroacetylamino)-1,3,4-thiadiazole-5-sulfonamide (2); and 2-(3-phenylpropionylamino)-1,3,4-thiadiazole-5-sulfonamide (3). Inhibition effects of these compounds on carbonic anhydrase I and II have been investigated. By comparing I50 and Ki values of the compounds, it has been found that compound 1 is a more potent inhibitor than acetazolamide (b) on carbonic anhydrase II.

An enzymatic method for zinc determination in serum.

In this study, serum Zn(2+) content was determined by a new enzymatic method. The method depends on the reactivation of apocarbonic anhydrase proportional to the Zn(2+) content of the sample. Carbonic anhydrase was purified from bovine erythrocytes by affinity chromatography. The Zn(2+) in its structure was removed by dialysis against pyridine 2,6-dicarboxylic acid, resulting in a pure apoenzyme with a yield of 100%. The activity of the enzyme was determined by its esterase effect on 4-nitrophenyl acetate. Zn(2+) levels were determined in the serum samples obtained from 100 healthy subjects, 10 patients with cirrhosis, 12 diabetic patients and 15 patients with chronic renal failure by this enzymatic method and by atomic absorption for comparison. There was a good correlation between the two methods in all patients and controls and intraassay CV% was 2.4 and 4.2 for enzymatic and atomic absorption methods, respectively and interassay CV% as 3.9 and 6.1 respectively.