Abstract
We have examined the properties of microtubules formed in the presence of GTP, 5'-guanylyl imidodiphosphate (GMPP(NH)P), and 5'-guanylyl methylenediphosphate (GMPP(CH2)P) to identify features of the assembly or disassembly reactions uniquely related to hydrolysis. The assembly of microtubules with GTP or GMPP(NH)P was similar in terms of rates and extents of assembly, the length distributions, and podophyllotoxin-induced depolymerization. The greater rapidity of GMPP(CH2)P-supported assembly, however, resulted in shorter, more numerous microtubules and the rate of podophyllotoxin-induced depolymerization was consistent with an increased number of concentration of microtubules. Experiments with GTP or analogue incorporation and release indicated that GTP-tubule turnover corresponded to a rate of about 8% of the microtubule protein taken up or released per h. With GMPP(NH)P- and GMPP(CH2)P-tubules, the rates of label uptake by unlabeled microtubules were considerably lower than observed with guanosine triphosphate. We suggest that exchange experiments can reflect contributions from head-to-tail polymerization and polymer length redistribution, but it is not as yet possible to evaluate the relative contributions of each process.
Highlights
GMPP(NH)P- and GMPP(CH2)P-tubules, the rates of that the observations of Weisenberg and Deery ( 6 ) reflect label uptake by unlabeled microtubules were consid- kinetic rather thanthermodynamic properties of microtubules erably lower than observed with guanosine triphos- prepared with GTP and GMPP(NH)P.Likewise, tubules prephate
GMPP(NH)P and GMPP(CH2)P canreplace GTP in the assemblyprocess [4,5,6,7], but nonehas presented a comparative study in terms of microtubulength measurements.The time courses of such polymerizationevents are shown in Fig.1, A, C, and E
There is generally good agreement that GTP hydrolysis attends theassembly of brain microtubules, and it is clear that nonhydrolyzable analogues can promote assembly
Summary
GMPP(NH)P- and GMPP(CH2)P-tubules, the rates of that the observations of Weisenberg and Deery ( 6 ) reflect label uptake by unlabeled microtubules were consid- kinetic rather thanthermodynamic properties of microtubules erably lower than observed with guanosine triphos- prepared with GTP and GMPP(NH)P.Likewise, tubules prephate. An important feature of microtubule behavior is the interaction of guanine nucleotides at theexchangeablenucleotide site of tubulin, and a number of studies already suggest that GTP hydrolysis at that site occurs as polymerization proceeds [1,2,3]. Nonhydrolyzable analogues such aGs MPP(NH)P’ and GMPP(CH2)Pcan support microtubule assembly [4,5,6,7], and it is relevant to inquire about the differences between microtubules assembled with GTP and the nonhydrolyzable analogues.
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