Gill cilia and sperm flagella from the lamellibranch mollusc Aequipecten irradians were isolated by several methods and chemically fractionated by low-ionic-strength dialysis. These organelles differ in their forms of dynein and in the stabilities of their homologous microtubules and subsidiary structures (i.e. nexin fibres and spoke material). In flagella more than 80% of the axoneme ATPase is solubilized, appearing as a 14S peak in the ultracentrifuge, and the whole axoneme is broken down to unlinked, doublet outer fibres. In cilia only half of the ATPase is solubilized from axonemes as a 14S component. The ciliary B-tubules and one member of the central pair also dissolve, leaving the A-tubules and the other central tubule held together by nexin fibres and matrix material as a 9 + 1 singlet axoneme, to which is bound the remaining half of the ATPase. This tightly bound form of the ciliary ATPase can be removed in an enzymically active form by brief trypsin treatment which causes the breakdown of the singlet axoneme. The trypsin-solubilized ATPase behaves like native 14S dynein in the ultracentrifuge but breaks down to polypeptides when electrophoresed in the presence of sodium dodecyl sulphate (SDS).
The native 14S material containing the dynein was partially purified by column chromatography and was shown on SDS-polyacrylamide gels to be composed of 2 components. The molecular weights of these as determined by 3% acrylamide SDS-gels are 450000 and 500000, and they are not reduced by prior treatment with 8 M urea. Parallel SDS-gels loaded with identical amounts of ciliary and flagellar axonemes show very nearly identical components; the ratio of the amounts of the 500000 and 450000 mol. wt. components is about 2:1 respectively. The fractionation of ciliary and flagellar axonemes was analysed quantitatively on SDS-gels. Essentially all of the 450000 and 500000 mol. wt. components are found in the soluble fraction from dialysed flagella. The soluble fraction from cilia contains the 2 components in a 1:1 ratio, along with half the axoneme ATPase, while the singlet axoneme fraction contains the remainder of the 500000 material and the remaining half of the ATPase. On this basis it is believed that the 500000 component is the dynein ATPase, while the 450000 material represents a non-ATPase protein.
The chemical and structural differences between cilia and flagella may offer an explanation for their different modes of beat.