ISSN:
0886-1544
Keywords:
cilia
;
flagella
;
ATPases
;
dynein
;
Chlamydomonas
;
Life and Medical Sciences
;
Cell & Developmental Biology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Medicine
Notes:
Extraction of isolated, demembranated flagellar axonemes of Chlamydomonas reinhardii with 0.6 M KCl solubilized 77-92% of the total axonemal Mg++ or Ca++-ATPase activity, which sedimented as 18S and 12S peaks in sucrose density gradients. The ATPases of these two peaks were further purified by hydroxyapatite (HAP) column chromatography. The ATPase activity of the 18S peak eluted from the HAP column as a single peak coinciding with the protein peak. The HAP purified 18S ATPase had a specific activity of ∼2.0 ± 0.5 μmoles Pi hydrolyzed min/mg and was associated with four high molecular weight (HMW) polypeptides of ∼ 310,000-340,000 daltons, two intermediate molecular weight (IMW) polypeptides of 78,000 and 69,000 daltons, and eight low molecular weight (LMW) polypeptides of 7,800-19,600 daltons. When the 12S sucrose gradient peak together with a trailing shoulder were chromatographed on HAP, the ATPase activity was eluted in two peaks designated 12S and 10.5S on the basis of the sedimentation properties of their associated polypeptides. The 12S peak contained a single dynein ATPase having a specific activity of ∼ 0.6 ± 0.3 μmoles Pi hydrolyzed min/mg and associated with ∼ 330,000-, 21,700-, and 18, 100-dalton polypeptides. The 10.5S peak contained several high, intermediate, and low molecular weight polypeptides; of these, on HMW polypeptide and one 28,700-dalton polypeptide correlated well with the ATPase activity. The purified ATPases had no polypeptides in common; each therefore represents a discrete dynein. Based on protein recovered in the purified fractions, 18S dynein represents ∼ 9.2% of the total axonemal protein; 12S dynein represents ∼ 4.7% of the axonemal protein.
Additional Material:
11 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/cm.970020604
