Catalase is an essential enzyme in the decomposition of intracellular hydrogen peroxide (H₂O₂). In yeast Saccharomyces cerevisiae cells, two different types of catalase have been found, which were designated catalase type A and catalase type T. The two enzymes are encoded by different genes (CTA1 and CTT1 respectively), possessing independent control and being localized in different compartments: catalase T is a cytoplasm enzyme whereas catalase A is localized in peroxisomes. The molecular weights of the biologically active, homotetrameric enzymes are 170-190 kD and 225-250 kD for catalase A and T respectively.

Studies with wild type yeast Saccharomyces cerevisiae cultivated on mixture of glucose and H₂O₂ showed an enhanced level of mitochondrial cytochrome c peroxidase. These results suggested that mitochondria possess an independent mechanism for decomposition of exogenous H₂O₂. They are also site of endogenous O₂- production via autooxidation of ubisemiquinone and electron transport proteins and H₂O₂ formed by its spontaneous or enzymatic dismutation. The scavenge of these reactive oxygen species is realized by mitochondrial antioxidant system, represented by Mn containing superoxide dismutase, Cu/Zn one found in the intermembrane space of the mitochondria and cytochrom c peroxidase. This well known mechanism for detoxification was added by discovery of catalase enzyme in mitochondria of mesophyll cells of tabacco (Nicotiana sylvestris) and - in rat heart mitochondria.

These observations directed us to investigate mitochondria for the presence of catalase activity in three different strains of yeast Saccharomyces cerevisiae. Our results clearly indicated that a mitochondrial catalase enzyme is present in vivo and suggested the existence of an additional cellular compartment for localization of this protein in yeast cell.

Catalase activity and electrophoretic profile of the enzyme

Catalase enzyme has been investigated during cultivation of three different yeast strains Saccharomyces cerevisiae NBIMCC 582, NBIMCC 583 and NBIMCC 584 on YPD medium containing 2% glucose, 1% peptone, and 1% yeast extract, which is widely used in yeast studies and provides comprehensive experiences on this enzyme. Growth of the strains has been performed for 72 h and samples have been withdrawn at 6^th h, 12^th h, 48^th h, and 72^nd h. It was shown that glucose is exhausted at the 12^th hour of cultivation (Figure 1). The highest catalase activity was measured after 48 h of cultivation (Table 1). This increase in catalase activity after the consumption of glucose in the nutrient medium could be due to the availability of different amino compounds in YPD medium, which are metabolized by fungi through hydrogen producing oxidase. A correlation of the catalase activity with the accumulation of its specific substrate (H₂O₂) obviously takes place.

Crude extracts from cells of Saccharomyces cerevisiae 583 strain of different physiological state was prepared by cell wall disruption, which was carried out enzymatically with zymolyase. The cell debris was removed by centrifugation at 1000 x g for 10 min, and then cell free extracts were frozen and thawed 3 times in order to disrupt mitochondria, and were centrifuged again at 15 000 x g for 10 min.

An electrophoretic analysis was performed with the obtained extracts (Figure 2). After 6 hours growth, only the band of catalase T has been visualized. The next electrophoretic analyses of catalase have been performed at 12, 48 and 72 hours of cultivation, when glucose from the nutrient medium has been thoroughly consumed. An interesting fact is the appearance of the second band of catalase A at 12 and 48 hours of cultivation and its disappearance at 72 h. The band, present during all cultivation period is that of catalase T.

The above mentioned two catalase enzymes act in the cells of Saccharomyces cerevisiae, decomposing H₂O₂ accumulated during oxidative processes in the cytosole and correlating with the Cu/Zn superoxide dismutase activity (Table 1). There is not clear evidence about such kind of mechanism placed in mitochondria and coupling with Mn SOD.

Catalase in mitochondria

Mitochondrial fractions from Saccharomyces cerevisiae strains were isolated after osmotic shock of obtained with zymolyase spheroplasts. The mitochondrial fraction was isolated from the cell free extract by centrifugation at 3500 x g for 20 min. Then the crude mitochondrial pellet was carefully washed three times with buffer. Collected mitochondria were resuspended in deionized water and were lysed by freezing and thawing. They were tested for purity using measurement of the activities of several enzymes, typically located in different cellular compartments: hexokinase and glucose-6-phosphate dehydrogenase - in cytosole, D-amino oxidase and isocitric lyase - in peroxisomes and succinate dehydrogenase and Mn superoxide dismutase - in mitochondria (Table 2). Using these marker enzymes the purity of mitochondrial fraction was displayed, as the enzyme activities characteristic for the cytosole and the peroxisomes were not detected in it.

Further the mitochondrial fractions and crude extracts obtained from strains 582, 583 and 584 were subjected to acrylamide gel electrophoresis (Figure 3 a, b, c). All mitochondrial fractions contained a single band specifically stained for catalase with Rm value of 0.239, different from those measured for catalase A (Rm = 0.218) and catalase T (Rm = 0.257). At the same time the visualization of the peroxidase activity was not successful, probably due to very low activity of the protein (Figure 4). The low peroxidase activity, measured spectrophotometrically, could be due to some kind of peroxidase activity of the catalase, as it has been shown for yeast Candida boidinii.

The results obtained for the mitochondrial catalase in the three strains clearly indicated that a catalase enzyme located in the mitochondria of Saccharomyces cerevisiae exists.

Correlation between activities of mitochondrial catalase and Mn superoxide dismutase

It is well known that catalase and Cu/Zn superoxide dismutase activities correlate during cultivation of many yeasts on different substrates and conditions. It is worthwhile to study the activity of mitochondrial catalase, Mn and intercristate Cu/Zn superoxide dismutase enzymes in correlation with growth. These investigations were performed with the three Saccharomyces cerevisiae strains 582, 583 and 584 using the mitochondrial fraction obtained from different hours of cultivation. The dynamics of above mentioned enzyme activities are presented in Figure 5 a, b, c. These data clearly showed that the specific activities of catalase and Mn superoxide dismutase enzymes correlated with the growth and their maximum has been measured at 48 h of cultivation. The activity of Cu/Zn mitochondrial superoxide dismutase appears to be a constant one (3.5 + 0.02 ÷ 5.0 + 0.01 U/mg) during the whole period of cultivation.

These results suggested that the role of mitochondrial catalase is coupled mainly with the function of manganese superoxide dismutase for detoxification of mitochondria from reactive oxygen species, generated during respiratory processes within these organelles.

Determination of charge and molecular weight of Saccharomyces cerevisiae mitochondrial catalase

As it has been mentioned above, the molecular weight of catalase T is between 225 - 250 kD, and for catalase A - considerably lower 170 - 190 kD. The electrophoretic pattern shows abnormal mobility of both cytosolic catalase enzymes. Applying the method of Hedrick and Smith, 1968, investigation of their behavior in native polyacrilamide gel electrophoresis with different concentrations of acrylamide was performed (Figure 6). The results obtained (Figure 6 and Figure 7) suggest that both enzymes are charge isomers which explains why catalase A, although with lower molecular weight possess lower electrophoretic mobility value than catalase T. Evaluating the plot of relative mobility, the mitochondrial enzyme could be also considered as charge isomer. For estimation the molecular weight of the mitochondrial enzyme, comparison of its relative mobility with the ones of known proteins was done (Figure 8 and Figure 9). On this basis we could calculate the molecular weight of the mitochondrial enzyme, which is approximately 240 000 Da.

These data open the question about the origin of this new catalase protein, located in the mitochondria and its relationship to catalases A and T.

HEDRICK, J.L. and SMITH, A.J. Size and charge separation and estimation of molecular weights of proteins by disc gel electophoresis. Archives in Biochemistry and Biophysics, July 1968, vol. 126, no. 1, p. 155-164.