Maria Wróbel

Transamination and transsulphuration of L-cysteine in Ehrlich ascites tumor cells and mouse liver. The nonenzymatic reaction of L-cysteine with pyruvate.

1. The activity of cysteine aminotransferase (CAT), 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese is much lower in Ehrlich ascites tumor cells (EATC) than in mouse liver. 2. Contrary to mouse liver homogenate, no synthesis of sulphane sulphur-containing compounds from L-cysteine is observed in EATC homogenate. 3. 2-Methyl-thiazolidine-2,4-dicarboxylic acid (CP), 2-methyl-thiazolidine-4-carboxylic acid (CA) and thiazolidine-4-carboxylic acid (CF) can be used as sources of low molecular-weight thiol compounds both in EATC and mouse liver homogenate. 4. Pyruvate formed from phosphoenolpyruvate (PEP) in EATC homogenates reacts with L-cysteine (L-CYS) to CP.

The level of sulfane sulfur in the fungus Aspergillus nidulans wild type and mutant strains

The interdependence of the sulfane sulfur metabolism and sulfur amino acid metabolism was studied in the fungus Aspergillus nidulans wild type strain and in mutants impaired in genes encoding enzymes involved in the synthesis of cysteine (a precursor of sulfane sulfur) or in regulatory genes of the sulfur metabolite repression system. It was found that a low concentration of cellular cysteine leads to elevation of two sulfane sulfurtransferases, rhodanase and cystathionine γ-lyase, while the level of 3-mercaptopyruvate sulfurtransferase remains largely unaffected. In spite of drastic differences in the levels of biosynthetic enzymes and of sulfur amino acids due to mutations or sulfur supplementation of cultures, the level of total sulfane sulfur is fairly stable. This stability confirms the crucial role of sulfane sulfur as a fine-tuning regulator of cellular metabolism.

Effect of mercury ions on cysteine metabolism in Xenopus laevis tissues

The effect of mercury ions on the level of cysteine, glutathione, sulfane sulfur, and on the activity of rhodanese, 3-mercaptopyruvate sulfurtransferase (MPST) and γ-cystathionase in brain, heart muscle, liver, kidneys, testes and skeletal muscle of adult Xenopus laevis was investigated. Frogs of both sexes were exposed for 7 or 14 days to 1.353mgL(-1) (ppm) of mercury chloride (HgCl(2)) dissolved in water. The activity of the investigated enzymes participating in cysteine metabolism depends on cysteine in their active sites. Mercury ions can bind to -SH groups and, therefore, lower the activity of enzymes and change the level of sulfane sulfur, a product of l-cysteine desulfuration. The effect of mercury was found to depend on the time of exposure and the kind of tissue. In the liver, the main site of glutathione biosynthesis, the ratio of GSH to GSSG was essentially unchanged. The total glutathione level was decreased after 7 days of exposure to mercury, similarly as the activity of rhodanese. Sulfane sulfur levels were significantly increased after a shorter duration, while they decreased after a longer time of exposure. The kidney, brain and testes were able to enhance the level of GSH, probably thanks to high γ-glutamyltranspeptidase activity. These tissues showed an increased value of GSH/GSSG ratio during the shorter exposure to mercury. The activity of sulfurtransferases was decreased, especially after the longer exposure to mercury. In the heart and skeletal muscle, the level of GSH, sulfane sulfur, and the activity of the investigated sulfurtransferases was diminished after 14 days of exposure to Hg. It can be concluded that the main mechanism of toxic Hg activity is generation of reactive oxygen species in cells due to depleted GSH level, and a decreased sulfurtransferases activity either by blocking or oxidation of their -SH groups, what in consequence results in a diminished sulfane sulfur levels in tissues, especially the heart and testes.

Hydrogen sulfide generation from L-cysteine in the human glioblastoma-astrocytoma U-87 MG and neuroblastoma SHSY5Y cell lines

Hydrogen sulfide (H2S) is endogenously synthesized from l-cysteine in reactions catalyzed by cystathionine beta-synthase (CBS, EC 4.2.1.22) and gamma-cystathionase (CSE, EC 4.4.1.1). The role of 3-mercaptopyruvate sulfurtransferase (MPST, EC 2.8.1.2) in H2S generation is also considered; it could be important for tissues with low CTH activity, e.g. cells of the nervous system. The expression and activity of CBS, CTH, and MPST were detected in the human glioblastoma-astrocytoma (U-87 MG) and neuroblastoma (SHSY5Y) cell lines. In both cell lines, the expression and activity of MPST were the highest among the investigated enzymes, suggesting its possible role in the generation of H2S. The RP-HPLC method was used to determine the concentration of cystathionine and alpha-ketobutyrate, products of the CBS- and CTH-catalyzed reactions. The difference in cystathionine levels between cell homogenates treated with totally CTH-inhibiting concentrations of dl-propargylglycine and without the inhibitor was used to evaluate the activity of CBS. The higher expression and activity of CBS, CTH and MPST in the neuroblastoma cells were associated with more intensive generation of H2S in the presence of 2 mM cysteine. A threefold higher level of sulfane sulfur, a potential source of hydrogen sulfide, was detected in the astrocytoma cells in comparison to the neuroblastoma cells.

Is Development of High-Grade Gliomas Sulfur-Dependent?

We characterized γ-cystathionase, rhodanese and 3-mercaptopyruvate sulfurtransferase activities in various regions of human brain (the cortex, thalamus, hypothalamus, hippocampus, cerebellum and subcortical nuclei) and human gliomas with II to IV grade of malignancy (according to the WHO classification). The human brain regions, as compared to human liver, showed low γ-cystathionase activity. The activity of rhodanese was also much lower and it did not vary significantly between the investigated brain regions. The activity of 3-mercaptopyruvate sulfurtransferase was the highest in the thalamus, hypothalamus and subcortical nuclei and essentially the same level of sulfane sulfur was found in all the investigated brain regions. The investigations demonstrated that the level of sulfane sulfur in gliomas with the highest grades was high in comparison to various human brain regions, and was correlated with a decreased activity of γ-cystathionase, 3-mercaptopyruvate sulfurtransferase and rhodanese. This can suggest sulfane sulfur accumulation and points to its importance for malignant cell proliferation and tumor growth. In gliomas with the highest grades of malignancy, despite decreased levels of total free cysteine and total free glutathione, a high ratio of GSH/GSSG was maintained, which is important for the process of malignant cells proliferation. A high level of sulfane sulfur and high GSH/GSSG ratio could result in the elevated hydrogen sulfide levels. Because of the disappearance of γ-cystathionase activity in high-grade gliomas, it seems to be possible that 3-mercaptopyruvate sulfurtransferase could participate in hydrogen sulfide production. The results confirm sulfur dependence of malignant brain tumors.

Atomic Sulfur: An Element for Adaptation to an Oxidative Environment

During the period of rising oxygen concentration in the Earths atmosphere (Figure 1), sulfur atoms were incorporated into proteins as redox-active cysteine residues [1] and antioxidant molecules such as thioredoxin, glutathione, and glutaredoxin appeared [...].

An application of RP-HPLC for determination of the activity of cystathionine β-synthase and γ-cystathionase in tissue homogenates

The RP-HPLC-based method of determination of the activity of cystathionine β-synthase and γ-cystathionase was undertaken in mouse liver, kidney and brain. Products of the reactions, such as cystathionine, α-ketobutyrate, cysteine and glutathione, were measured using the RP-HPLC method. A difference in the cystathionine level between homogenates with totally CTH-inhibiting concentrations of DL-propargylglycine and without the inhibitor was employed to evaluate the activity of cystathionine β-synthase. Gamma-cystathionase activity was measured using DL-homoserine as a substrate and a sensitive HPLC-based assay to measure α-ketobutyrate. The results confirmed high cystathionine β-synthase activity and no γ-cystathionase activity in brain, and high γ-cystathionase activity in mouse liver. The method presented here allows for evaluating the relative contribution of CBS and CTH to generation of H2S in tissues. Additionally, it provides results, which reflect the redox status (GSH/GSSG) of a tissue.

Remembering Professor Toshihiko Ubuka (1934–2008)

Professor Toshihiko Ubuka was a distinguished Japanese biochemist, known domestically and internationally. In his scientific life, he researched various fields of sulfur biochemistry. Toshihiko Ubuka, Professor Emeritus from Okayama University Medical School, died of colon cancer on 4 April 2008, at the age of 74. Until he died, he worked in the Department of Clinical Nutrition in Kawasaki University of Medical Welfare: he was Vice-president of the University and Dean of Faculty of Health Science and Technology. He was born on 31 January 1934, in a farmer’s family in Okayama prefecture. The grandparents did the farming, but his father worked as a postal clerk and his mother was a high-school teacher. He suffered from diphtheria when he was 12 and was unable to attend school for a year because his lower limbs were paralyzed. He wanted to become a lawyer. However, in the last year of high school, when his father passed away because of stomach cancer, he decided to go to medical school. He studied medicine in Okayama University Medical School and graduated in 1959. During his studies he had also started to learn kendo, which is a traditional Japanese martial art. Until the end of his life he regularly practiced kendo. After graduation, he made a decision to be a biochemist and started studying biochemistry in Okayama University. He commenced his career as a researcher in Okayama Medical School in 1964, after obtaining his Ph.D. in biochemistry. He performed his scientific research work in the Department of Biochemistry in the Medical School of Okayama University under the supervision of Professor Shunji Mizuhara. Professor Ubuka said at every opportunity how deeply grateful he was to Dr. Shunji Mizuhara, who stimulated his interest in sulfur biochemistry. The first papers co-authored by Professor Ubuka and published in 1962–1964 focused on isovalthine (2-amino-5-carboxy6-methyl-4-thiaheptanoic acid), a branched-chain sulfur amino acid that was found in the urine of humans suffering from hypercholesterolemia by Professor Mizuhara. Ubuka (1962) described a method involving high-voltage electrophoresis combined with paper chromatography for the identification of isovalthine and its related compounds. Later, ion exchange amino acid analysis was used allowing for quantification of urinary isovalthine concentrations. In the years 1967–1968, in parallel with the similar discovery of J.C. Crawhall, Ubuka found out that normal human urine contained small quantities of mercaptolactatecysteine disulphide [S-(2-hydroxy-2-carboxyethylthio)cysteine; HCETC], and mercaptoacetate-cysteine disulphide [S-(carboxymethylthio)cysteine; CMTC]. Later on, in the 1970s, he synthesized S-(2-oxo-2 carboxyethylthio)cysteine (OCETC) and reduced it with lactate dehydrogenase to HCETC. In 1960, he married Satoko, who was his college classmate, and later they had their first child, Takayoshi. Several years later, in 1967, he went to New York with his M. Wróbel (&) Collegium Medicum, Jagiellonian University, Cracow, Poland e-mail: mbwrobel@cyf-kr.edu.pl

Rhodanese (thiosulfate:cyanide sulfurtransferase) from frog Rana temporaria.

The molecular mass of rhodanese from the mitochondrial fraction of frog Rana temporaria liver, equaling 8.7 kDa, was determined by high-performance size exclusion chromatography (HP-SEC). The considerable difference in molecular weight and the lack of common antigenic determinants between frog liver rhodanese and bovine rhodanese suggest the occurrence of different forms of this sulfurtransferase in the liver of these animals.