Table 2.

Effect of BFA on GTP[γS] binding to ARF1 and Golgi in the presence of cytohesin-1 or ARF-GEP

Additions	[35S]GTP[γS] bound, pmol
	No BFA	2 μg BFA
None	1.1	1.3
Cytohesin-1	4.3	4.2
ARF-GEP	2.1	1.6

ARF1 (1 μg, 50 pmol) was incubated with Golgi membrane (12.5 μg of protein) in Tris buffer (pH 8.0) containing 1 mM ATP, 5 mM creatine phosphate, 1 unit of creatine phosphokinase, 3.3 mM MgCl₂, and 64 mM NaCl without or with cytohesin-1 (1 μg) or partially purified BFA-sensitve ARF-GEP (20 μg) from Sepharose CL-6B chromatography (11), and 2 μg of BFA in 0.2% EtOH or vehicle alone in a volume of 90 μl at 21°C for 10 min. After addition of 4 μM [³⁵S]GTP[γS], samples were incubated for 40 min at 36.5°C before assay of bound [³⁵S]GTP[γS]. Control values (1.0–1.1 pmol bound in the absence of ARF with or without cytohesin-1, ARF-GEP, and/or BFA) have been subtracted.