By John N. Abelson, Melvin I. Simon, W. E. Balch, Channing J. Der, Alan Hall
Normal Description of the Volume:Small GTPases play a key function in lots of elements of latest mobilephone biology: regulate of telephone progress and differentiation; rules of phone adhesion and mobilephone flow; the association of the actin cytoskeleton; and the legislation of intracellular vesicular delivery. This quantity plus its significant other Volumes 255 and 257 hide all biochemical and organic assays at the moment in use for reading the function of small GTPases in those facets of telephone biology on the molecular point. it's the first compendium of functional options for operating with small GTPases of the Rho group.General Description of the Series:The severely acclaimed laboratory usual for greater than 40 years, tools in Enzymology is likely one of the so much hugely revered guides within the box of biochemistry. in view that 1955, every one quantity has been eagerly awaited, usually consulted, and praised through researchers and reviewers alike. Now with greater than three hundred volumes (all of them nonetheless in print), the sequence comprises a lot fabric nonetheless appropriate today--truly a necessary book for researchers in all fields of existence sciences.
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Extra info for Small GTPases and Their Regulators, Part B: Rho Family
Segal, Biochem. J. 298, 585 (1994). 26T. Sasaki, M. Kato, and Y. Takai, J. Biol. Chem. 268, 23959 (1993).  P u r i f i c a t i o n of Rac2 from Human Neutrophils By ULLA G. KNAUS and GARY M. BOKOCH The Rac2 protein belongs to the Rho family of GTP-binding proteins and is closely related to the Racl protein (92% identity). Whereas Racl is ubiquitously expressed, Rac2 is only found in cells of myeloid origin. Studies with a human leukemia cell line (HL-60) showed a seven- to ninefold increase of Rac2 expression on differentiation to a neutrophiMike type, whereas R a c l levels rose only slightly.
After completion of the gradient, the column is further eluted with 150 ml of 1 M NaC1 in TEDMPM buffer. 5 ml are collected and assayed for GTPySbinding activity. The flow through and two overlapping peaks in the salt gradient (100-170 mM NaC1) show GTPyS binding (Fig. 1). When assayed for N A D P H oxidase activity in a cell-free system, as mentioned earlier, the first part of the GTPyS-binding peak (100-130 mM NaC1) correlates with oxidase stimulatory activity. The second part of the biphasic GTPySbinding peak represents the Rac-related proteins Rho (detected by ADPribosylation with botulinum toxin C3 ADP-ribosyltransferase) and Cdc42Hs (detected by Western blotting).
Kotani, K. Hirata, M. Katayama, and Y. Takai, J. Biol. Chem. 267, 14611 (1992). 15S. Kuroda, A. Kikuchi, K. Hirata, T. Masuda, K. Kishi, T. Sasaki, and Y. Takai, Biochem. Biophys. Res. Commun. 185, 473 (1992). 16H. Yaku, T. Sasaki, and Y. Takai, Biochem. Biophys. Res. Commun. 198, 811 (1994). 17M. Isomura, A. Kikuchi, N. Ohga, and Y. Takai, Oncogene 6, 119 (1991). 18T. Sasaki, M. Kato, and Y. Takai, Z Biol. Chem. 268, 23959 (1993). a9y. Miura, A. Kikuchi, T. Musha, S. Kuroda, H. Yaku, T. Sasaki, and Y.