Spectrophotometric determination of aluminium and indium with 2,2',3,4-tetrahydroxy-3',5'-disulphoazobenzene.

2,2',3,4-Tetrahydroxy-3',5'-disulphoazobenzene (tetrahydroxyazon 2S) has been synthesized for the first time. This reagent has been used for the spectrophotometric determination of aluminium and indium ions. The method is very sensitive and selective for the direct determination of aluminium and indium. The optimum pH and absorbance of complexes formed of tetrahydroxyazon 2S with aluminium and indium are 5; 500 nm and 495 nm for Al and In, respectively. The system obeys Beer's law at 0.05-1.6 microg mL(-1) of aluminium and 0.06-2.1 microg mL(-1) of indium concentration. The molar absorptivity is 6.42 x 10(4)L mol(-1)cm(-1) for aluminium and 7.70 x 10(4)L mol(-1)cm(-1) for indium. The molar compositions of the complexes are 1:1 at optimum conditions. Alkaline and alkaline earth elements, halogens, thiourea, ascorbic acid, Cd(II), Pb(II), Mn(II), Zn(II), Co(II), Ni(II), Cr(III), Bi(III), La(III), Si(IV) do not interfere this method. The method can be applied to the direct spectrophotometric determination of trace amounts of aluminium in steel, alloys, waste water, river waters, spring water and ground water. The method was also successfully applied to the indium determination in artificial mixture.

Quantum chemical studies on protonation of some substituted thiazole derivatives.

The acidity constants, pK(a) values for protonation of some substituted thiazole derivatives were calculated by using AM1 and PM3 basis sets of semi-empirical methods and B3LYP/6-31G(d) basis sets of density functional theory (DFT) calculated physical and thermodynamic parameters. Correlation search among the experimental and calculated acidity constants, pK(a) values, revealed that the best correlation exist between the experimental and ab initio calculated pK(a) values with a regression of R(2)=0.98.

Comparison of NAT1, NAT2 and GSTT2-2 activities in normal and neoplastic human breast tissues.

In this study, arylamine N-acetyltransferases, NATs (E.C.2.3.1.5) and glutathione-S-transferase-T2-2, GSTT2-2 (E.C.2.5.1.18) enzyme activities in the breast tumor and surrounding tumor-free tissues of 22 female breast cancer patients with infiltrating ductal carcinoma were measured. The possible impacts of grade of malignancy, chemotherapy treatment, estrogen receptor status and menopausal status on all enzyme activities were evaluated. The results showed that, both NAT2 and GSTT2-2 display significant differences between tumor and tumor-free breast tissues, while no difference was observed in NAT1. Grade of malignancy seems to be positively associated with NAT1 and negatively associated with GSTT2-2. Though, both NAT2 and GSTT2-2 have increased mean tumor activities, the grade of malignancy, chemotherapy status, menopausal status or estrogen receptor status are not correlated statistically.

Arylamine N-acetyltransferase activities in human breast cancer tissues.

N-Acetyltransferase activities were determined in tumor (12 malignant and 6 benign) and control (non-cancerous) breast tissues from 18 female patients. The activities of matched 12 malignant tumor and control tissue cytosols showed 6 rapid, 4 intermediate and 2 slow acetylators based on p-aminobenzoic acid (NAT1) and sulfamethazine (NAT2) as substrates. Compared to the activities of slow acetylators, the rapid acetylators exhibited mean apparent Vmax values about 5- and 50-fold greater for p-aminobenzoic acid and sulfamethazine, respectively. No correlation was observed between the blood and breast tissue N-acetyltransferase (NAT1 and NAT2) activities. When the mean apparent N-acetyltransferase activities of the malignant and benign breast tumor tissues were compared, the results showed an increased activity for both p-aminobenzoic acid (PABA) and sulfamethazine (SMZ) acetylation in the malignant tissues compared to benign ones, and also control tissues showed lower activities compared to tumor tissues. Moreover, the mean NAT2 activity was about 2-fold greater in the malignant tissues when compared to NAT1 activity.

Characterization of extracellular beta-lactamases from penicillin G-resistant cells of Streptococcus thermophilus.

In this study, biochemical properties of two extracellular beta-lactamases produced by penicillin-resistant Streptococcus thermophilus cells were investigated. Both beta-lactamases showed specificity for penicillins but not for cephaloridins. The beta-lactamases exhibited different affinities for penicillin G. The one with the higher molecular weight (FI) had a Km value of 3.44 microM and a Vmax value of 8.33 mumol/min/mg of protein, whereas the beta-lactamase with the lower molecular weight (FII) had a Km value of 4.76 microM and a Vmax value of 3.13 mumol/min/mg of protein. Both beta-lactamases were inhibited by iodine, copper sulfate, and iron sulfate but not by EDTA. The optimal pH ranged between 6 and 7, and the optimal temperatures were between 40 and 45 degrees C for both enzymes.

Sheep tissue acetyl coenzyme A-dependent arylamine N-acetyltransferases.

Acetyl coenzyme A-dependent arylamine N-acetyltransferases (NATs), EC 2.3.1.5. were measured in sheep tissues (N = 14) using p-aminobenzoic acid (PABA) as a substrate. Specific activities of liver, kidney, and lung NATs were 5.3 x 10(-3) +/- 1.4 (mean +/- SE) nmoles.min-1.mg protein-1, 3.4 x 10(-3) +/- 1.1 nmoles.min-1.mg protein-1, 2.5 x 10(-3) +/- 1.2 nmoles.min-1.mg protein-1, respectively. Km values were 53 +/- 3 microM for liver, 34 +/- 2 microM for kidney, and 28 +/- 2 microM for lung tissue. Optimum pH for the acetylation reaction was found as 7.5. The enzyme activity was stable for at least 6 months in all tissues, when stored at -70 degrees C. The enzyme from sheep tissues were quite heat-stable. Inhibition studies showed that N-ethylmaleimide was a potent inhibitor of sheep tissue NAT enzymes.

Production of superoxide radicals by sheep liver nitrofurantoin reductase: In vitro effects of nitrofurantoin metabolites on DNA.

The reduction of nitrofurantoin by purified liver nitrofurantoin reductase was followed by the production of superoxide radicals, which were detected by the reduction of epinephrine. The conditions for the formation of superoxide radicals were optimized. The maximum superoxide radical formation occurred at approximately 3.5 mug purified reductase, with optimum pH of 7.8 and at 0.05 mm nitrofuran- toin concentration. The K(m) and V(max) values were calculated as 1.95 x 10(-2) mm and 4.81 nmol Superoxide formed per minute. An obliteration of the banding pattern was observed on the agarose gel electrophoresis of sheep liver DNA, which was incubated in the nitrofurantoin-nitrofurantoin reductase system, as a possible consequence of the generation of superoxide radicals during the reduction of nitrofurantoin by nitrofurantoin reductase.

Interindividual variability of coumarin 7-hydroxylation in a Turkish population.

One hundred healthy Turkish volunteers (70 male, 30 female) aged from 19 to 56 years were given 5 mg coumarin p.o. after an overnight fast. Urine samples were collected before and 2, 4 and 8 h after drug administration. The extent and rate of formation of 7-OH-coumarin (7OHC) was determined by the urinary excretion of the metabolite as measured with the fluorometric method. On average, 80% of 7OHC formed was excreted in 2 h. The total amount of 7OHC formed was 59.8% (21.5%) (mean and SD, n = 100, range 17-100%) of the given dose. The percentage of 7OHC excreted during the first 2 h compared with the 7OHC excretion at 8 h was a constant and stable individual characteristic for the rate of the formation of 7OHC ('2 h coumarin test'). Although four individuals had relatively slow coumarin test values (34-40%), no clear-cut polymorphism in the rate of 7OHC formation was found. However, 7OHC formation was lower in males and in cigarette smokers.

Purification and characterization of two forms of soluble NADH cytochrome b5 reductases from human erythrocytes.

1. Two forms of soluble NADH cytochrome b5 reductase were purified from human erythrocytes. Two distinct fractions both having the NADH cytochrome b5 reductase activity eluted from the second DEAE-cellulose column were further purified by ultrafiltration and 5'-ADP-agarose affinity chromatography. 2. The final preparations were purified 9070- and 4808-fold, respectively, over hemolysate. Both reductases exhibited identical electrophoretic patterns when subjected to SDS-PAGE and apparent monomer Mr of each reductase was determined to be 32,000 +/- 1300. 3. Vmax values of reductase II for the various electron acceptors, namely, 2,6-dichlorophenolindophenol, ferricyanide and cytochrome c through cytochrome b5 were found to be 1.9, 1.8 and 2 times higher than those of reductase I. 4. Some differences were noted for reductase I and reductase II fractions. Their elution profiles from a second DEAE-cellulose column were quite different and that suggested that reductase II is more acidic than reductase I. Reductase II was found to be more sensitive to heat treatment than reductase I.

Kinetic properties of purified sheep lung microsomal NADH-cytochrome b5 reductase.

1. Lung NADH-cytochrome b5 reductase was saturated with its artificial substrate, potassium ferricyanide at approximately 0.1 mM ferricyanide concentration, and the activity of the lung enzyme was inhibited by the higher concentrations of potassium ferricyanide. Ferricyanide at 0.5 and 1.0 mM inhibited the activity of the enzyme by about 20 and 61% respectively. The apparent Km value was calculated as 13.7 microM potassium ferricyanide and 4.3 microM NADH. 2. The Michaelis constants for cytochrome b5 and NADH were determined to be 1.67 and 7.7 microM from the Lineweaver-Burk plots. These results demonstrate that affinity of the lung reductase for its natural substrate is almost 10 times higher than that for potassium ferricyanide. 3. Addition of non-ionic detergent stimulated the rate of reductase-catalyzed reduction of lung cytochrome b5 up to 8.2-fold. 4. Kinetic studies performed with lung reductase by varying NADH and cytochrome b5 concentrations at different fixed concentrations at cytochrome b5 or NADH showed a series of parallel lines indicating a "ping-pong" type of kinetic mechanism for interaction of NADH and cytochrome b5 with lung cytochrome b5 reductase.

Stimulatory effects of benzene on rabbit liver and kidney microsomal cytochrome P-450 dependent drug metabolizing enzymes.

Treatment of rabbits with benzene (880 mg/kg/day), s.c. for 3 consecutive days, caused 3.8- and 5.7-fold increases in aniline 4-hydroxylation rates of liver and kidney microsomes, respectively. Benzene treatment markedly enhanced hydroxylation rats of p-nitrophenol by liver and kidney by 7.2- and 4.2-fold, respectively. Both of these enzymes are associated with cytochrome P-450 LM3a. In contrast, the activity of benzphetamine N-demethylase, associated with P-450 LM2, was not altered significantly in either liver or kidney microsomes. Although the total cytochrome P-450 contents of liver and kidney microsomes were not altered significantly by the benzene treatment, in the case of liver microsomes, formation of a new cytochrome P-450 with an apparent Mr of 51,400 was observed on SDS-PAGE. On the other hand, in the kidney microsomes, the intensity of the bands corresponding to approximate Mr of 50,000 and 51,400 was markedly increased. The results of the present work, in combination with those of the previous work (Arinç et al. 1988), indicate the existence of tissue specificity in the induction of rabbit P-450 isozyme by benzene.

Purification of NADH-cytochrome b5 reductase from sheep lung and its electrophoretic, spectral and some other properties.

1. NADH-cytochrome b5 reductase was purified from sheep lung microsomes in the presence of non-ionic and ionic detergents, Emulgen 913 and cholate, respectively. 2. The purification procedure involved the ion-exchange chromatography of the detergent solubilized microsomes on DEAE-cellulose. 3. Further purification and concentration of lung reductase was carried out with a second DEAE-cellulose column followed by the affinity column chromatography of partially purified reductase on 5'-ADP-agarose column. 4. The specific activity of sheep lung reductase was 638 mumol ferricyanide reduced/min/mg protein and the yield was 6% of the initial activity in microsomes. 5. The SDS-polyacrylamide gel electrophoresis of the purified lung reductase showed one protein band having the monomer mol. wt of 34,500 +/- 1500. In the presence of 0.4% deoxycholate, it existed as an active dimer having a mol. wt of 68,500. 6. Trypsin treated lung reductase showed two extra protein bands of mol. wts of 28,000 and 25,000 on 10% SDS-polyacrylamide gels. 7. The purified enzyme was found to contain FAD as prosthetic group and the absorption spectrum of lung reductase showed two peaks at 390 and 461 nm which were typical for flavoproteins and a shoulder at 490 nm. 8. The maximal activity of lung reductase was observed between pH 6.5-8.0 and at pH 6.8, when ferricyanide and partially purified sheep lung cytochrome b5 was used as electron acceptors, respectively.