ISSN:
0730-2312
Keywords:
calmodulin
;
dynein
;
ATPase
;
anion
;
solubilization
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
,
Medicine
Notes:
The basal ATPase activity of 30S dynein, whether obtained by extraction of ciliary axonemes with a high (0.5 M NaCl) or low (1 mM Tris-0.1 mM EDTA) ionic strength buffer is increased by NaCl, NaNO3, and Na acetate, with NaNO3 causing the largest increase. The calmodulin-activated ATPase activity of 30S dynein is also increased by addition of NaCl, NaNO3, or Na acetate, but the effects are less pronounced than on basal activity, so that the calmodulin activation ratio (CAR) decreases to 1.0 as salt concentration increases to 0.2 M. These salts also reduce the CAR of 14S dynein ATPase to 1.0 but by strongly inhibiting the calmodulin-activated ATPase activity and only slightly inhibiting the basal activity. Sodium fluoride differs both quantitatively and qualitatively from the other three salts studied. It inhibits the ATPase activity of both 14S and 30S dyneins at concentrations below 5 mM and, by a stronger inhibition of the calmodulin-activated ATPase activities, reduces the CAR to 1.0. Na acetate does not inhibit axonemal ATPase, nor does it interfere with the drop in turbidity caused by ATP and extracts very little protein from the axonemes. NaCl and, especially, NaNO3, cause a slow decrease in A350 of an axonemal suspension and an inhibition of the turbidity response to ATP. NaF, at concentrations comparable to those that inhibit the ATPase activities of the solubilized dyneins, also inhibits axonemal ATPase activity and the turbidity response. Pretreatment of demembranated axonemes with a buffer containing 0.25 M sodium acetate for 5 min followed by extraction for 5 min with a buffer containing 0.5 M NaCl and resolution of the extracted dynein on a sucrose density gradient generally yields a 30S dynein that is activated by calmodulin in a heterogeneous manner, ie, the “light” 30S dynein ATPase fractions are more activated than the “heavy” 30S dynein fractions. These results demonstrate specific anion effects on the basal and calmodulin-activated dynein ATPase activities, on the extractability of proteins from the axoneme, and on the turbidity response of demembranated axonemes to ATP. They also provide a method that frequently yields 30S dynein fractions with ATPase activities that are activated over twofold by added calmodulin.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/jcb.240250403
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