Effect of spin-labeled maleimide on 14S and 30S dyneins in solution and on demembranated ciliary axonemes

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Abstract
The effects of N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (SLM) on the pellet height response and ATPase activity of glycerinated Triton X-100 extracted cilia of Tetrahymena pyriformis were studied. Preincubation of cilia with SLM caused complete inhibition of the pellet height response and an initial increase in ATPase activity followed upon longer exposure to SLM by inhibition of ATPase. The effect of SLM on extracted 30S dynein was the reverse of that for whole cilia: ATPase activity was increased when 30S dynein was added to a mixture of ATP and SLM and inhibited when the 30S dynein was preincubated with SLM. The activity of 14S dynein was only inhibited by SLM. ESR spectra of ciliary axonemes that had reacted with SLM for various times showed that much of the covalently bound SLM was strongly immobilized even after 1 min of reaction, when ATPase activity increased 2-fold. The proportion of strongly immobilized label increased with longer times of reaction. Addition of ATP to SLM-labeled axonemes caused a small decrease in the height of the spectral peak corresponding to strongly immobilized label as compared with that of weakly immobilized label, indicating an increase in rotational freedom of some covalently bound label. ATP probably causes a conformation change affecting a sulfhydryl groups(s) involved in the mechanochemical system. .beta.-.gamma.-Methylene ATP (AMP-PCP) inhibits dynein ATPase. This analogue of ATP is no hydrolyzed by whole cilia or by the extracted dyneins and does not cause a pellet height response. With Mg2+ as divalent cation, AMP-PCP inhibits 30S dynein more than it inhibits 14S dynein; with Ca2+ the 30S dynein inhibition is reduced, and there is no inhibition of 14S dynein. Under conditions where AMP-PCP inhibited 30S dynein ATPase it was much less effective than ATP in protecting against the loss of ATPase activity by SLM. Although SLM inhibited Mg2+-activated 14S and 30S dyneins in solution, it did not inhibit ciliary ATPase activity. At least 2 SH groups are probably involved in ciliary motility and their reactivity to SH reagents depends on whether the dyneins are in situ or extracted.