I.Hamdi Ögüs

Inhibition characteristics of hypericin on rat small intestine glutathione-S-transferases

Glutathione-S-transferases constitute a family of enzymes involving in the detoxification of xenobiotics, signalling cascades and serving as ligandins or/and catalyzing the conjugation of various chemicals and drugs. The widely expressed cytosolic GST-pi is a marker protein in various cancers and its increased concentration is linked to drug resistance. GST-pi is autoregulated by S-glutathionylation and it catalyzes the S-glutathionylation of other proteins in response to oxidative or nitrosative stress. S-glutathionylation of GST-pi results in multimer formation and the breakage of ligand binding interactions with c-Jun NH(2)-terminal kinase (JNK). Another widely expressed GST enzyme, GST-alpha is assumed as a marker in hepatocellular damage, is implicated in cancer, asthma, cardiovascular disease and response to chemotherapy. Although, it was shown that hypericin binds and inhibits GST-alpha and GST-pi, the inhibition characteristics have not been investigated in detail. The aim of this study was to investigate the effects of hypericin on major GSTs; GST-alpha and GST-pi purified from rat small intestine. When GSH used as varied substrate the inhibition pattern with hypericin was uncompetitive for GST-alpha (K(i)=0.16 + or - 0.02 microM) and noncompetitive for GST-pi (K(i) = 2.46 + or - 0.43 microM). While using CDNB (1-chloro-2,4-dinitrobenzene) as the varied substrate, the inhibition patterns were noncompetitive for GST-alpha and competitive for GST-pi; K(i) values for GST-alpha and GST-pi were 1.91 + or - 0.21 and 0.55 + or - 0.07 microM, respectively. Since hypericin accumulated in cancer cells and important in photodynamic therapy (PDT), inhibition of GST-alpha and GST-pi by hypericin might increase the effectivity of the treatment. Considering that GST-pi is responsible for the drug resistance its inhibition might increase the benefit obtained from chemotherapy.

Simple, high-yield purification of xanthine oxidase from bovine milk.

Xanthine oxidase, a commercially important enzyme with a wide area of application, was extracted from fresh milk, without added preservatives, using toluene and heat. The short purification procedure, with high yield, consisted of extraction, ammonium sulfate fractionation, and DEAE-Sepharose (fast flow) column chromatography. Xanthine oxidase was eluted as a single activity peak from the column using a buffer gradient. The purification fold, specific activity and yield for the purified xanthine oxidase were 328, 10.161 U/mg and 69%, respectively. The enzyme was concentrated by ultrafiltration, although 31% of the activity was lost during concentration, no change in specific activity was observed. Activity and protein gave coincident staining bands on native polyacrylamide gels. The intensity and the number of bands were dependent on the oxidative state(s) of the enzyme; reduction by 2-mercaptoethanol decreased the intensity of the slow-moving bands and increased the intensity of the fastest-moving band. Following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two major bands (molecular masses of 152 and 131 kDa) were observed, accounting for > or = 95% of xanthine oxidase. Native- and SDS-PAGE showed that the purified xanthine oxidase becomes a heterodimer due to endogenous proteases.

A SIMPLE AND SENSITIVE METHOD FOR THE ACTIVITY STAINING OF XANTHINE OXIDASE

Abstract Xanthine oxidase is a commercially-important enzyme. Several biochemical compounds have been quantitated by xanthine oxidase. Xanthine oxidase has been used as an auxiliary enzyme in the staining of several enzymes or tissues, however, there is no direct staining method available for it, on polyacrylamide gels. Partially-purified xanthine oxidase from cow milk was used as the enzyme source for the development of an activity-staining method on polyacrylamide gels. Staining was very sensitive. Detection of 0.02 μU of the enzyme on polyacrylamide gels was possible. Staining of 0.05 μU takes about 1 min whereas staining of 0.5 μU will take less than 5 s. Addition of TEMED is not essential for activity staining but it did increase both the rate and the intensity of the staining. The stained gels must be washed with distilled water, extensively, in order to remove excess unoxidized nitroblue tetrazolium, and must be protected from light, for a clear background and sharp activity-band staining. This method might be useful for quality control of xanthine oxidase obtained from different sources.

New sample preparation method for the capillary electrophoretic determination of adenylate energy charge in human erythrocytes

The first step in the separation of adenine nucleotides from different types of tissues or cells is deproteinization. Several sample preparation methods successfully used for a number of tissues or cells failed to work with erythrocytes. Use of strong acids or bases for deproteinization resulted in a low yield due to the hydrolysis of adenine nucleotides. Moreover, the neutralization of these acids or bases increased the ionic strength, resulting in broad and overlapping peaks. In neutral salt precipitation methods, saturated salts caused clogging of the capillaries. A new deproteinization procedure method was developed. The samples were deproteinized by heating of erythrocytes in boiling distilled water at 95 degrees C for 5 min. The denatured proteins were removed by centrifugation and membrane filtration. The adenine nucleotides were then separated using a polyacrylamide coated capillary. Depending on the type, diameter, length of the capillary and the voltage applied, an average of 16.50 min was sufficient for the separation of adenine nucleotides. All adenine nucleotides were clearly resolved and gave very sharp peaks. The amount of each adenine nucleotide was calculated from the areas under the peaks and AEC values were calculated using the integrator software. The AEC value of 0.91+/-0.04 (n=10) obtained for healthy persons was in good agreement with the literature value of 0.85-0.95. These reported method for sample preparation and capillary electrophoresis is simple, fast and inexpensive compared to the previously reported sample preparation, HPLC and enzymatic methods for the determination of AEC.

Amitriptyline may have a supportive role in cancer treatment by inhibiting glutathione S-transferase pi (GST-π) and alpha (GST-α)

A tricyclic anti-depressant, amitriptyline, is a highly prescribed drug for cancer patients for mood elevation but there are limited studies about the interaction of amitriptyline with glutathione S-transferases pi (GST-π) and glutathione S-transferases alpha (GST-α). GST isozymes have been implicated in chemotherapeutic drug resistance. We demonstrated that the concentration dependent inhibition of GST-π and GST-α by amitriptyline followed inverse hyperbolic inhibition curves with IC50 values of 5.54 and 8.32 mM, respectively. When the varied substrate was GSH, amitriptyline inhibited both isozymes competitively and similar Ki values were found for GST-π (Ki = 1.61 ± 0.17 mM) and GST-α (Ki = 1.45 ± 0.20 mM). On the other hand, when the varied substrate was CDNB, the inhibition types were non-competitive for GST-π (Ki = 1.98 ± 0.31 mM) and competitive for GST-α (Ki = 1.57 ± 0.16 mM). Amitriptyline, in addition to its antidepressant effect, might also have a minor supportive role on the effectiveness of the anticancer drugs by decreasing their elimination through inhibiting GST-π and GST-α.

THE EFFECTS OF OXIDATIVE STRESS ON THE REDOX SYSTEM OF THE HUMAN ERYTHROCYTE

Erythrocytes are a group of cells which do not contain a nucleus, mitochondria and other cytoplasmic organelles. Due to the lack of a nucleus and other organelles they can not synthesize proteins and their energy metabolism depends solely on anaerobic glycolysis. The function of these highly differentiated cells is to exchange respiratory gasses between the lung and the tissues. The gas transporting protein, hemoglobin, constitutes 95% of the erythrocyte proteins. Hemoglobin consists of a nonprotein heme group and the protein, globulin. Heme contains iron. For a functional protein this iron must be in the ferrous (Fe2+) state (Mathews and van Holde, 1996; Stryer, 1988).