Murat Pekmez

Antioxidant activity of whey protein fractions isolated by gel exclusion chromatography and protease treatment.

Whey proteins were isolated from whey powder by a combination of gel exclusion chromatography and protease (pepsin or trypsin) treatment. Whey solution (6g/dl) was applied to Sephadex G-200 column chromatography and three fractions were obtained. Gel electrophoresis (SDS-PAGE) was used to identify the fractions; the first one contained immunoglobulins and bovine serum albumin, the second contained beta-lactoglobulin and alpha-lactalbumin whereas the third fraction contained small peptides. We have also subjected the whey filtrate to proteases (pepsin and trypsin). Treatment with proteases showed that beta-lactoglobulin can be obtained after hydrolysis of the second fraction with pepsin. When the whey filtrate was treated with pepsin and then applied to Sephadex G-200 column chromatography three fractions were obtained; the first one was bovine serum albumin, the second was beta-lactoglobulin and the third fraction contained small peptides. After trypsin treatment only two fractions were obtained; the first one was serum albumin and the second fraction was an alpha-lactalbumin rich fraction. We have determined the antioxidant activity of the fractions using an assay based on the measurement of superoxide radical scavenging activity. Our results showed that among the three fractions, the first fraction had the highest superoxide radical scavenging activity. Also, protease treatment of the second fraction resulted in an increase in the antioxidant activity.

Intracellular scavenging activity of Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid) in the fission yeast, Schizosaccharomyces pombe

The ability of Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), a water-soluble vitamin E analogue, to prevent oxidative damages is well characterized, but the mechanisms underlying it remain unclear. The protective effect of Trolox pre-treatment on H2O2-induced toxicity might be attributed to the decreased cellular permeability to H2O2 or in vitro scavenging activity of Trolox, induction of antioxidant enzymes or the direct scavenging activity of Trolox. The results obtained rule out the first and second possibilities and intracellular scavenging activity was found to be the mechanism whereby Trolox confers protection. This was confirmed by measuring protein oxidation (levels), and the observed decrease in proteasomal activity indicated that the decrease in protein carbonyls was due to Trolox scavenging activity rather than proteasome activation. In conclusion, the intracellular scavenging activity of Trolox is a key protective mechanism against H2O2. These findings obtained in Schizosaccharomyces pombe, a good model organism for eukaryotic cells, can be used as standard protocols for investigating the antioxidant activity of pure or complex potential antioxidants.

Hydrogen peroxide-induced oxidative damages in Schizosaccharomyces pombe

Oxidative stress causes damage to proteins, lipids and nucleic acids, and thereby compromises cell viability. Some of the oxidative stress markers in an eukaryotic model organism, fission yeast Schizosaccharomyces pombe, were evaluated in this study. Intracellular oxidation, protein carbonyls, lipid peroxidation and reduced glutathione (GSH) levels were investigated in H2O2-treated and non-treated control cells. It was observed that increased H2O2 concentration proportionally lowered the cell number and increased the intracellular oxidation, lipid peroxidation and protein carbonyl levels in S. pombe. A dose-dependent decrease in GSH level was also detected. The fission yeast S. pombe is best known for its contribution to understanding of eukaryotic cell cycle control. S. pombe displays a different physiology from Saccharomyces cerevisiae in several ways and is thus probably more closely related to higher eukaryotes. The purpose of this study was to provide some data about the effects of hydrogen peroxide on the proteins and lipids in the fission yeast. The data obtained here is expected to constitute a basis for the further studies on redox balance and related processes in yeast and mammalian cells.

Comparison of antioxidant capacity, protein profile and carbohydrate content of whey protein fractions.

Whey is used as an additive in food industry and a dietary supplement in nutrition. Here we report a comparative analysis of antioxidant potential of whey and its fractions. Fractions were obtained by size exclusion chromatography, before and after enzymatic digestion with pepsin or trypsin. Superoxide radical scavenging, lipid peroxidation inhibition and cupric ion reducing activities of different fractions were checked. Peptides were detected by SDS-PAGE and GC-MS was used to determine carbohydrate content of the fractions. All samples showed antioxidant activity and the second fraction of the trypsin hydrolysate showed the highest superoxide radical scavenging activity. CUPRAC value of this fraction was two-times higher than that of whey filtrate. The first fraction of the pepsin hydrolysate was the most effective inhibitor of lipid peroxidation. Each sample exhibited a different polypeptide profile. Different percentages of carbohydrates were identified in whey filtrate and in all second fractions, where galactose was the major component.

Investigation of the relationship between oxidative stress and glucose signaling in Schizosaccharomyces pombe.

The invertase mutant defective in the glucose signaling pathway of Schizosaccharomyces pombe (ird11) is resistant to glucose repression. This mutant is able to consume sucrose alongside glucose and grows in glucose-containing media with a generation time close to that of the wild type. Intracellular oxidation, protein carbonyl, and reduced glutathione levels and catalase, superoxide dismutase, and glutathione peroxidase activity were investigated in ird11, to determine the relationship between oxidative stress response and glucose signaling. The expression profiles of some genes involved in regulation of glucose repression (fbp1, fructose-1,6-bis-phosphatase; hxk2, hexokinase) and stress response (atf1 and pap1 transcription factors; ctt1, catalase; sod1, Cu,Zn superoxide dismutase) were analyzed using the quantitative real-time PCR technique. Oxidative stress response in ird11 seems to be affected by glucose signaling in a manner different from that caused by glucose deprivation.

Expression of Human A4V Mutant Cu,Zn Superoxide Dismutase in Schizosaccharomyces pombe: Investigations of its Toxic Properties

Cu,Zn superoxide dismutase (SOD1) is an antioxidant enzyme that catalyzes the removal of superoxide radicals generated in various biological oxidations. Amyotrophic lateral sclerosis (ALS) is one of the most common neurodegenerative disorders, occurring in families (FALS) and sporadically (SALS). FALS and SALS are distinguishable genetically but not clinically. More than 100 point mutations in the human SOD 1 gene have been identified that cause FALS. In order to determine the effects of mutant SOD protein, we first cloned wild-type and A4V mutant human SOD1 into Schizosaccharomyces pombe. This study shows viabilities and some antioxidant properties including SOD, catalase, proteasomal activity, and protein carbonyl levels of transformants in SOD1 deleted strain (MN415); and its parental strain (JY741) at different stress conditions. There was no more oxidative damage in the human mutant SOD carrying the transformant strain compared with other strains. These results may help to explain whether ALS progresses as a consequence of cellular oxidative damage.

Effect of α-tocopheryl succinate on the molecular damage induced by indomethacin in C6 glioma cells

Indomethacin is a member of the non-steroidal anti-inflammatory drug (NSAID) class, which has great potential for use in the treatment of glioma. However, it induces the generation of reactive oxygen species (ROS) and causes molecular damage while inducing its effects. Vitamin E is widely used in the complementary therapy of cancers. The main goal of the present study was to investigate the effects of α-tocopheryl succinate (α-TOS) against the oxidative damage induced by indomethacin in C6 glioma cells. Cells were treated with 10 μM α-TOS alone or in combination with 200 μM indomethacin for two days. The intracellular ROS level, molecular damage as revealed by lipid peroxidation and protein carbonyl formation, and the COX activity in C6 glioma cells were measured. Treatment of the cells with α-TOS and indomethacin, alone or in combination, caused the levels of ROS generation and protein damage to increase, but protected against lipid peroxidation and reduced COX activity.

Suppression of HSP70 Expression by Quercetin and Its Therapeutic Potential Against Cancer

Heat shock response is one of several survival pathways that protects cells against harsh conditions. This response mechanism, which is evolutionarily protected in all organisms, enhances the expression of heat shock proteins (HSP) that show protective properties for cells under stress conditions. High expression of many HSP is observed in cancer, and their functions aides the advancement of disease. It is known that overexpression of HSP70, a member of HSP family, in cancerous cells has been closely associated with tumor cell proliferation, apoptosis inhibition, enhanced migration and metastasis and drug resistance promotion. Therefore, targeting HSP70 in cancer treatment is very important. One of the best-studied inhibitors known for HSP70 is quercetin that is widely distributed flavonoid in the plant kingdom. Several in vivo and in vitro studies have reported the efficacy of quercetin in reducing elevated HSP70 levels in cancer therapy. It has become a focal point as an anticancer agent because of the induction of apoptosis in many different cancer cells. In this chapter, we reviewed the role of HSP70 in different cancer types and the suppressive effect of quercetin on expression of HSP70 family members. Moreover, we emphasized molecular mechanisms targeted by quercetin in cancer and its relationship to Hsp70.

Targeting of Heat Shock Proteins by Natural Products in Cancer

Initially discovered as a group of proteins showing significantly higher expression in response to heat stress, Heat shock proteins (HSPs) have gained considerable appreciation. Overwhelmingly increasing scientific evidence has highlighted the role of these proteins as molecular chaperones which trigger protein holding and folding thus facilitating freshly synthesized protein/s to achieve mature and biologically active conformation. It is becoming progressively more understandable that HSPs are involved in post-translational modification of proteins of signaling cascades, modulation of apoptosis related proteins, assembly and disassembly of transcriptional machinery. Recently emerging functional and structural data has provided new insights related to biochemical regulation of HSPs and the structural dynamics used by these proteins to act on a diverse client repertoire.