Abstract
Radioactive tubulin subunit incorporation into porcine and dogfish shark brain microtubules which are at steady state has been found to result primarily from a diffusional reaction, in which subunits are incorporated although there are an equal number of tubulin subunit additions to and losses from each of the two microtubule ends in a unit of time. Treadmilling is very inefficient, and the Wegner s-values are equal to 0.0005-0.001. At steady state there are approximately 5000 (pig brain) or 2500 (dogfish brain) tubulin subunits lost from the two microtubule ends/s; an equivalent number of subunit addition reactions maintains a constant microtubule mass. The rate constants for subunit loss and addition with porcine brain microtubules, determined from analysis of the steady state rate for radioactive subunit incorporation, are much larger than those measured previously, when the rate constants were determined from the disassembly rate following perturbation of the steady state by dilution (Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899). To account for this discrepancy it is suggested that at steady state the microtubule is capped by a short finite length of tubulin-GTP subunits, which undergo extremely facile association and dissociation (2500-5000 subunits/microtubule/s). This cap would be rapidly lost following dilution so that the observed rate only measures the relatively slow loss of tubulin-GDP subunits (120 subunits/microtubule/s) which had been in the interior of the microtubule; this is not equal to the rate constant for subunit loss (and addition) from the ends at steady state. Because of this, previous estimates of the Wegner s-value for treadmilling which utilized dilution for determining the steady state molecular rate constants for subunit loss are believed to be too high.
Highlights
poration may also contribute to subunit incorporation into the main body
It has been previously found that dogfish shark brain microtubular protein preparations are primarily composed of tubulin
The absorbance profiie froma fast green-stained sodium dodecylsulfate gel of the protein used inthe kinetic studies described here is shown in Fig
Summary
It has been previously found that dogfish shark brain microtubular protein preparations are primarily composed of tubulin, with relatively low amounts of a single high molecular weight protein (see gel scan 2a in Ref. 15). Analysis of the rate for the radioactive subunit incorporation requires corrections for: 1)changes in the specific activity of the E-site r3H]GTP as nonradioactive nucleotide is released as aresult of subunit dissociation from the microtubules, and 2) the finite length of the microtubules resulting in some of the microtubules becoming fully labeled during the incorporation study, so that the number of the microtubules which can contribute to net radioactivity incorporation decreases. The curve describingthe time course for radioactive subunit incorporation for the experiment shown in Fig. 2 has been corrected for changes in specific activity, and variation in the fraction of the microtubules which participate in the reaction, in Table I, columns 3 and 6, respectively. Results witphorcine brain tubulinwere analyzed (Fig6. ) and in thciasse there aresignificant positive deviations from linearity in the plot of radioactive subuniitnscorporatevdersus ( 9 & (Fig. 6B)
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