Table 1.

Nucleotides enhance binding of rSsa2p with BHst 5.

Nucleotide conditions	Kd (μM)
No nucleotide	5.4 ± 0.3
ATP (1 mM)	0.4 ± 0.1
ADP (0.9 mM) + ATP (0.1 mM) and Pi (1 mM)	0.6 ± 0.2
ATPγS (1 mM)	1.3 ± 0.4

rSsa2p (5 μM) was mixed with excess BHst 5 (100 μM) without or with 1 mM ATP-Mg²⁺, 0.9 mM ADP-Mg²⁺, 0.1 mM ATP-Mg²⁺ and 1 mM P_i, or 1 mM ATPγS-Mg²⁺ in total volume of 250 μl in Buffer A to form complex. The mixture was loaded onto a FPLC Superose 12 10/300 column to separate rSsa2p–BHst 5 complexes from free BHst 5. The concentration of BHst 5 and rSsa2p was measured by densitometric scanning against quantification standards. Dissociation constants (K_d) of rSsa2p–BHst 5 complexes under each nucleotide condition were calculated using the equation K_d = [BHst 5_f][rSsa2p_f]/[rSsa2p–BHst 5].