Interaction of dynamin with microtubules: its structure and GTPase activity investigated by using highly purified dynamin.
We purified a large amount of dynamin with high enzymatical activity from rat brain tissue by a new procedure. Dynamin 0.48 mg was obtained from 20 g of rat brain. The purity of dynamin was almost 98%. Dynamin plays a role of GTPase rather than ATPase. In the absence of microtubules, Michaelis constant (Km) and maximum velocity (Vmax) for dynamin GTPase were 370 microM and 0.25 min-1, respectively, and in their presence, both were significantly accelerated up to 25 microM and 5.5 min-1. On the other hand, the ATPase activity was very low in the absence of microtubules, and even in their presence, Km and Vmax for dynamin ATPase were 0.2 mM and 0.91 min-1. Despite slow GTPase turnover rate in the absence of microtubules, binding of GTP and its nonhydrolizing analogues was very fast, indicating that GTP binding step is not rate limiting. Dynamin did not cause a one-directional consistent microtubule sliding movement just like kinesin or dynein in the presence of 2 mM ATP or 2 mM GTP. We observed the molecular structure of dynamin with low-angle rotary shadowing technique and revealed that the dynamin molecule is globular in shape. Gel filtration assay revealed that these globules were the oligomers of 100-kDa dynamin polypeptide. Dynamin bound to microtubules with a 1:1 approximately 1.2 molar ratio in the absence of GTP. Quick-freeze deep-etch electron microscopy of the dynamin-microtubule complex showed that dynamin decorates the surface of microtubules helically, like a screw bolt, very orderly and tightly with 11.4 +/- 0.9 (SD)nm period. Contrary to the previous report, microtubules make bundles by the attachment of the dynamin helixes around each adjacent microtubule, and no cross-bridge formation was observed.
Maeda
K
K
Department of Anatomy and Cell Biology, School of Medicine, University of Tokyo, Japan.
Nakata
T
T
Noda
Y
Y
Sato-Yoshitake
R
R
Hirokawa
N
N
D000595:Amino Acid Sequence; D000818:Animals; D001923:Brain Chemistry; D017301:Ca(2+) Mg(2+)-ATPase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D034281:Dynamins; D005613:Freeze Etching; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011487:Protein Conformation; D051381:Rats
D006150:Guanine Nucleotides; D017301:Ca(2+) Mg(2+)-ATPase; D020558:GTP Phosphohydrolases; D034281:Dynamins