Etsuko Masuyama

Application of stains-all staining to the analysis of axonemal tubulins: identification of β-tubulin and β-isotubulins

The cationic dye, Stains-all, is known to stain brain beta-tubulin blue and alpha-tubulin red (Serrano, L. et al. (1986) J. Biochem. Biophys. Methods 12, 281-287; Serrano, L. et al. (1989) Biochem. Int., 19, 235-246). The present experiments show that this stain can also be applied to detect beta-tubulin in axonemal tubulins from various sources such as cilia of protozoa, sperm flagella of echinoderm, and sperm flagella of mollusc. Furthermore, these experiments showed that it selectively stains isoforms of axonemal beta-tubulin blue following isoelectric focusing, whereas those of alpha-tubulin are stained red. These results indicate that Stains-all staining is a useful tool for electrophoretic analysis of axonemal tubulins.

Flagellar adenosine triphosphatases from annelid spermatozoa: Electrophoretic identification of dyneins

Abstract Axonemes of sperm flagella were prepared from the annelid, Tylorrhynchus heterochaetus . Dialysis of the axonemes against 1 m m Tris-HCl buffer (pH 8.3)-0.1 m m EDTA-0.1 m m dithiothreitol (Tris-EDTA solution) caused disintegration of typical 9 + 2 microtubules into each doublet, resulting in extraction of one-third of the protein and almost all ATPase activity. Agarose polyacrylamide gel electrophoresis of the extract showed the presence of three kinds of dyneins actively stained for ATPase (designated as bands I, II, and III) and two non-ATPase proteins (bands IV, V). The polypeptide components of each dynein molecule and intact axoneme were analyzed by subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis to obtain the following results: (1) In the highmolecular-weight region, the intact axonemes yield two major polypeptides with molecular weights of 365,000 and 345,000 (designated as bands A and B, respectively) and three minor polypeptides, 310,000, 290,00, and 270,00 (C1, C2, C3). (2) All three dyneins contain A-band polypeptide as a common polypeptide component. In addition, band I dynein and band II dynein also contain B and C1 polypeptides, and C3 polypeptide, respectively, as high-molecular-weight components. (3) Band III dynein also contains four polypeptides in the lower molecular-weight region, which migrate similarly with those of 21 S dynein from sea urchin sperm flagella or 18 S dynein from Chlamydomonas .

Isolation and properties of the axopodial cytoskeleton of a heliozoan, Echinosphaerium akamae

The axopodia of a large heliozoan, Echinosphaerium akamae, were efficiently liberated from the cell body by treatment with 65% D(2)O solution containing 5 mM MgCl(2),2.1 mM EGTA, 1 mM KCl and 5 mM HEPES (pH 6.9). After D20 treatment, the cell bodies were removed by centrifugation at a low speed for 30 sec and the resulting supernatant was recentrifuged at 10,000 × g for 10 min. The axopodia were obtained as the pellet fraction without any contamination from the cell body. The isolated axopodia maintained their regular arrangement of cytoskeletal microtubules and were shortened by treatment with Ca(2+). When the isolated axopodia was subjected to SDS-polyacrylamide gel electrophoresis, two major protein bands were detected. The molecular weights of the proteins, tentatively identified as the heliozoan tubulins, were estimated to be about 46 and 50 kD, and an antibody against rat brain tubulin reacted with only the 46 kD protein species.

Application of a new slot comb to electrophoretic analysis

In the present experiment, a new slot comb was designed in order to form a wide and sloped sample well on the stacking gel of electrophoresis. Using this slot comb, a gradient of the reagent layer of interest can be easily formed transversely on the gel that is perpendicular to the direction of electrophoresis. Thus, the protein sample overlaid on the agent migrates across the gradient layer during electrophoresis to produce a continuous electrophoretic band reflecting the interaction between the protein and reagent. This new slot comb (tentatively called slope comb) was applied to the following two experiments. In the first experiment, in combination with this comb and a reducing agent, 2-mercaptoethanol, the reducing steps of cross-linked axonemal proteins with o-iodosobenzoic acid (OIBA) were analyzed electrophoretically, enabling visualization of the reducing pattern of each axonemal protein in a single experiment. The results obtained indicate that alpha and beta tubulins are cross-linked differently by OIBA. In the second experiment, the formation of the Ca2+ gradient layer using this slope comb could electrophoretically differentiate the Ca2+ sensitivity of three Ca(2+)-binding proteins.