Assembly of Microtubules from Tubulin Bearing the Nonhydrolyzable Guanosine Triphosphate Analogue GMPPCP [Guanylyl 5'-(![]()
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- 作者：Rick B. Dye and Robley C. Williams ; Jr.
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：November 12, 1996
- 年：1996
- 卷：35
- 期：45
- 页码：14331 - 14339
- 全文大小：443K
- 年卷期：v.35,no.45(November 12, 1996)
- ISSN：1520-4995
文摘
The growth and shortening of microtubules in guanosinetriphosphate- (GTP-) mediated dynamicinstability has previously been observed to occur at rates which areremarkably variable (Gildersleeve etal., 1992, Chrétien et al., 1995). Neighboring microtubulesobserved simultaneously can grow or shortenat different rates, and a particular microtubule can undergo changes inrate with the passage of time.This paper addresses the question whether this variability has itsorigin in processes that involve GTPhydrolysis or whether it results from variations in the structure ofmicrotubules that are independent ofGTP hydrolysis. Tubulin was prepared with the nonhydrolyzable GTPanalogue GMPPCP [guanylyl5'-(
chars/beta2.gif" BORDER=0 ALIGN="middle">,chars/gamma.gif" BORDER=0 >-methylenediphosphonate)] bound to its exchangeablenucleotide-binding site and with GTP at itsnonexchangeable site. Extensive measurements of length changeswere obtained by DIC microscopy.Microtubules formed from the GMPPCP tubulin exhibited only growth.No shortening events wereobserved. Growth occurred at highly variable rates,indistinguishable from those exhibited by GTP tubulin.Subsequent analysis of nucleotides by high-pressure liquidchromatography (HPLC) revealed that someof the GTP that was initially present at the N-site underwenthydrolysis to produce microtubule-boundguanosine diphosphate (GDP). Despite this unexpected finding, onecan conclude that variability of growthrate certainly occurs independently of dynamic instability and probablydoes not involve hydrolysis ofGTP at the E-site.
chars/beta2.gif" BORDER=0 ALIGN="middle">,chars/gamma.gif" BORDER=0 >-methylenediphosphonate)] bound to its exchangeablenucleotide-binding site and with GTP at itsnonexchangeable site. Extensive measurements of length changeswere obtained by DIC microscopy.Microtubules formed from the GMPPCP tubulin exhibited only growth.No shortening events wereobserved. Growth occurred at highly variable rates,indistinguishable from those exhibited by GTP tubulin.Subsequent analysis of nucleotides by high-pressure liquidchromatography (HPLC) revealed that someof the GTP that was initially present at the N-site underwenthydrolysis to produce microtubule-boundguanosine diphosphate (GDP). Despite this unexpected finding, onecan conclude that variability of growthrate certainly occurs independently of dynamic instability and probablydoes not involve hydrolysis ofGTP at the E-site.