Formation and stability of vincristine-tubulin complex in kidney cytosols. Role of GTP and GTP hydrolysis.
Vincristine-tubulin complex formed in the 100,000 g fraction of mouse kidney dissociated rapidly at 37 degrees in the absence of guanosine-5'-triphosphate (GTP). In the presence of 2 mM GTP, there was a substantial (2.8-fold) increase in complex stability; NaF (100 mM) but not beta-glycerophosphate (1 mM) also reduced the rate of dissociation. Further, complex was stabilized by other ribonucleoside-5'-triphosphates (but not their respective 5'-monophosphates), and a nonhydrolyzable analogue of GTP. Stability of the VCR-tubulin complex formed in cytosol from kidney and separated from unbound VCR and GTP by gel filtration was influenced by the concentration of GTP. These results appear not to be a consequence of denaturation of tubulin during incubation, as VCR binding activity remained constant under experimental conditions both in the presence and after the removal of GTP. Further, the rate of formation of the VCR-tubulin complex in kidney was also influenced by the concentration of GTP and was increased by the addition of NaF. In the absence of added GTP, virtually no complex was isolated. ATP, CTP, or ITP has little effect on complex formation, suggesting that the effect may be GTP specific. These data suggest that the destabilizing activity in cytosols prepared from mouse kidney, and the failure to form a stable VCR-tubulin complex in kidney, are in part the consequence of rapid hydrolysis of GTP by a pyrophosphohydrolase. Direct measurement of the hydrolysis of GTP showed that the activity in kidney (9.26 nmol/min/mg protein) was 9.3-fold greater than in tumor extracts.
Bowman
L C
LC
Division of Hematology/Oncology, St. Jude Children's Research Hospital, Memphis, TN 38101.
Houghton
J A
JA
Houghton
P J
PJ
D000818:Animals; D001665:Binding Sites; D003600:Cytosol / Q000378:metabolism*; D005260:Female; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D008808:Mice, Inbred CBA; D014404:Tubulin / Q000378:metabolism*; D014750:Vincristine / Q000378:metabolism*
D014404:Tubulin; D014750:Vincristine; D006160:Guanosine Triphosphate