Ribonucleotide diphosphate reductase from human metastatic melanoma.
Cell free extracts from metastases of human melanoma contain a highly active ribonucleoside diphosphate reductase (RR) which uses guanosine diphosphate (GDP) as substrate and deoxythymidine triphosphate (dTTP) as effector. No activity could be detected in these extracts when cytidine diphosphate (CDP) was used as the substrate with adenosine triphosphate (ATP) as effector. The activity of this RR required the presence of either magnesium or calcium: there was a time lag before cell extracts from melanotic melanoma metastases showed full activities, but extracts from amelanotic tumors showed normal kinetics in the presence of these divalent cations. By contrast to other RRs, the activities in cell-free extracts could not be inhibited by hydroxyurea (10(-2) M). Even though an activity related free radical could be detected by electron paramagnetic resonance spectroscopy at 77 degrees K, the signal could not be quenched by 10(-2) M of this free radical trap. However, after ammonium sulphate fractionation, enzyme activity from melanotic melanoma was inhibited by 66% in 1 h. In the presence of substrates, effector and cofactors, the radical signal at g = 2.009 was also quenched by 60%; in the absence of substrate, effectors and cofactors, this signal was unaffected. These results indicate that two different free radicals must be present on melanoma RR. One is present in the resting enzyme, and the other is used during catalytic activity. The thiolate-active site of RR from melanoma was inhibited by the new nitrosourea anti-tumour drug fotemustine (IC50 = 10(-4) M as determined from a dose-response study).(ABSTRACT TRUNCATED AT 250 WORDS)
Schallreuter
K U
KU
Department of Dermatology, University of Hamburg, Germany.
Elgren
T E
TE
Nelson
L S
LS
MacFarlan
S
S
Yan-Sze
I
I
Hogenkamp
H P
HP
D000970:Antineoplastic Agents / Q000494:pharmacology; D002474:Cell-Free System; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008545:Melanoma / Q000201:enzymology*; D008961:Models, Structural; D009362:Neoplasm Metastasis; D009607:Nitrosourea Compounds / Q000494:pharmacology; D009943:Organophosphorus Compounds / Q000494:pharmacology; D012262:Ribonucleoside Diphosphate Reductase / Q000378:metabolism*
D000970:Antineoplastic Agents; D046911:Macromolecular Substances; D009607:Nitrosourea Compounds; D009943:Organophosphorus Compounds; D012262:Ribonucleoside Diphosphate Reductase; C054368:fotemustine; D006918:Hydroxyurea