In vitro binding of IAA to plasma membrane-rich fractions containing Mg++-activated ATPase from mung bean hypocotyls

Abstract

Cell homogenates of dark-grown mung bean hypocotyls were fractionated into six fractions (L-0, L-l to L-5) by stepwise sucrose density-gradient centrifugation. The majority (ca. 84%) of Mg⁺⁺-activated ATPase activity of the 10,000 × g pellet was localized in the L-0 (1.03 ≤ d ≤ 1.14) and L-l (1.14 ≤ d ≤ 1.16) fractions. Over 40% of the vesicular membrane in the L-0 fraction and 60% of the L-l fraction could be stained with phosphotungstic acid (PTA)-chromic acid, a selective staining for the plant plasma membrane. In vitro binding of ¹⁴C-IAA to the fraction components was the greatest in the L-l fraction among the six. The binding of ¹⁴C-IAA to the L-l fraction in vitro was markedly interfered with by the presence of a high concentration of cold IAA (2 × 10⁻⁴M). However, it was not affected by the IAA analogues IPA, IBA and IAN. This indicates that IAA highly specifically binds to the L-l fraction. In vitro specific binding of ¹⁴C-IAA to L-l and L-0 was decreased with an increasing acidity from pH 8.0 to 5.0. In vitro binding of ¹⁴C-IAA to L-l and L-5 was further enhanced when these fractions were isolated from sections pretreated with 10⁻⁵M cold IAA for 60 min