Question

(A) ASSEMBLY 45 + phalloidin C15 - phalloidin E-15 actin (uM) (B) DISASSEMBLY + phalloidin 100 - phalloidin 50 0 20 time after dilution (minutes) Figure 16-11 Effects of phalloidin on actin filaments (Problem 16-28). (A) Growth rates at the minus ends of acrosomal bundles in the presence and absence of phalloidin. (B) Disassembly of actin filaments upon dilution in the presence and absence of phalloidin.

Isolated bundles of actin filaments from the acrosomal processes of Limuluspolyphemus(horseshoe crab) sperm have readily distinguishable plus ends (tapered) and minus ends (blunt).  Assembly at the ends of such bundles was used to determine the mechanism of action of phalloidin, which has a marked effect on actin assembly. When phalloidin is mixed with actin in a molar ratio of at least 1:1, the growth rate increases at both ends, as shown for minus ends in Fig. 16-11A.  Because growth rate = kon[actin] – koff, these plots have the form y = mx + b, so that the slope of the line equals konand the y intercept = -koff.

1.By analyzing the on and off rates, decide how phalloidin increases the growth rate of actin filaments.  Explain your reasoning.

2.In Fig. 16-11B, actin filaments grown in the presence or absence of phalloidin were diluted in the absence of actin monomers and their disassembly was assayed.  Do these results confirm or contradict your conclusions form part A? Explain your answer.

3.What is the critical concentration for actin assembly at the minus end in the absence of phalloidin?  In the presence of phalloidin?

4.Propose a molecular mechanism for the effects of phalloidin on actin assembly, considering the molar ratio above and the data in Fig. 16-11B.

Answer 1

3. The critical concentration for actin assembly at the minus end in absence of phalloidin is 2 µM

and in presence is 0.5 µM, because from these points we can measure the first actin molecules per second.