Isolation and partial purification of mitochondrial and cytosolic rhodanese from liver of normal and p-dimethylaminoazobenzene treated mice.
Rhodanese (thiosulfate:cyanide sulfurtransferase, E.C. 2.8.1.1), an enzyme involved in heme regulation, showed distinctive mitochondrial and cytoplasmic activities in several models of tumorigenesis. To investigate the basis for these differences, the enzyme was partly purified and characterized from the mitochondrial and cytosolic liver fraction of mice treated with the carcinogen p-dimethyl-aminoazobenzene (DAB). A linear relationship between incubation time and specific activity was observed up to about 30 min for cytosolic enzyme and 15 min for mitochondrial enzyme irrespective of whether or not the enzyme was derived from treated or untreated animals. The optimum incubation temperature was 3 degrees C for the enzyme of both fractions in control animals and 30 degrees C for treated animals in both cases. In control and DAB treated animals the cytoplasmic rhodanese exhibited a maximum at a lower pH than for the mitochondrial enzyme. The enzyme showed typical Michaelis-Menten behavior with cyanide inhibition at concentrations higher than 25 mM for controls and 10 mM for treated animals for both fractions and thiosulfate inhibition at concentrations higher than 100 mM in all cases studied. Km values of 190 and 65.66 mM were obtained for thiosulfate and 6.37 and 9.79 mM for cyanide for both mitochondrial and cytosolic fractions of control animals; while Km values of 31.75 and 4.58 mM were obtained for thiosulfate and 0.61 and 1.11 mM for cyanide in both fractions of treated animals. We demonstrated differences in the kinetics for rhodanese derived from mitochondrial and cytoplasmic fractions of livers taken from tumor bearing mice. These differences might provide an explanation for the abnormalities of heme synthesis previously reported during hepatocarcinogenesis.
Vazquez
E
E
Department of Biochemistry, University of Buenos Aires, Ciudad Universitaria, Argentina.
Polo
C
C
Stedile
G
G
Schebor
C
C
Karahanian
E
E
Batlle
A
A
D000818:Animals; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D051379:Mice; D008930:Mitochondria, Liver / Q000187:drug effects / Q000201:enzymology*; D013696:Temperature; D013884:Thiosulfate Sulfurtransferase / Q000302:isolation & purification / Q000378:metabolism*; D004124:p-Dimethylaminoazobenzene / Q000494:pharmacology*
D004124:p-Dimethylaminoazobenzene; D013884:Thiosulfate Sulfurtransferase