TY - JOUR
T1 - Formation and stability of vincristine-tubulin complex in kidney cytosols. Role of GTP and GTP hydrolysis
AU - Bowman, Laura C.
AU - Houghton, Janet A.
AU - Houghton, Peter J.
N1 - Funding Information:
Studies using human rhabdomyosarcomas growing as xenografts have indicated that the selective action of vincristine (VCR) is due to specific retention of unchanged drug in tumor tissue, with rapid elimination from most non-neoplastic tissues \[1\].N oble et al. \[2\]h ave also shown selective retention of VLB in a sensitive rat tumor in vioo. Further, persistence of Vinca alkaloid in cells exposed in vitro for short periods of time correlates well with cytotoxicity \[3, 4\]. However, the mechanism(s) that determines such differential retention between neoplastic and non-neoplastic tissues, and therefore therapeutic selectivity, has received less attention. The rapid elimination of Vinca alkaloids from many non-neoplastic tissues (of the mouse) may be a consequence of membrane or target phenomena. The former seems unlikely as initial levels of Vinca alkaloid which are cell-associated are often 5-to 15-fold higher than in tumor tissue \[1\].T he latter would imply differences between target molecules (tubulin) * Supported by awards CA 38933, CA 23099, and CA 23944 from the National Cancer Institute and by the American Lebanese Syrian Associated Charities. L. C. B. is the recipient of an ASCO Young Investigator Award. § Address correspondence to: Dr. Peter J. Houghton, Laboratories for Developmental Therapeutics, Division of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, 332 North Lauderdale, P.O. Box 318, Memphis, TN 38101.
PY - 1988/4/1
Y1 - 1988/4/1
N2 - Vincristine-tubulin complex formed in the 100,000 g fraction of mouse kidney dissociated rapidly at 37° 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 β-glycerophosphate (1 mM) also reduced the rate of dissociation. Further, complex was stabilized by other ribonucleuside-5′-triphosphates (but not their respective 5′-monophosphates), and a nonhydrolyzable 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 the 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.
AB - Vincristine-tubulin complex formed in the 100,000 g fraction of mouse kidney dissociated rapidly at 37° 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 β-glycerophosphate (1 mM) also reduced the rate of dissociation. Further, complex was stabilized by other ribonucleuside-5′-triphosphates (but not their respective 5′-monophosphates), and a nonhydrolyzable 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 the 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.
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U2 - 10.1016/0006-2952(88)90778-2
DO - 10.1016/0006-2952(88)90778-2
M3 - Article
C2 - 3355599
AN - SCOPUS:0023873966
SN - 0006-2952
VL - 37
SP - 1251
EP - 1257
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 7
ER -