Co-purification of Pea and Bean Leaf Soluble Auxin-binding Proteins with Ribulose-1,5-Bisphosphate Carboxylase

Abstract

Soluble auxin-binding proteins (ABPs) were purified to constant specific activity from bean and pea leaves by a procedure involving (NH4)2SO4 fractionation, anion exchange chromatography and gel filtration. Pea and bean ABPs exactly co-purify with ribulose-1,5-bisphosphate carboxylase (RuBPCase) Auxin-binding sites are associated with membranes isolated from coleoptiles and primary leaves of Zea mays (1, 2, 5-7, 13, 19, 20, 26-28, 31), epicotyls and roots of Pisum sativum (8), soybean hypocotyls (36), mung bean hypocotyls (18), and zucchini hypocotyls (16). Solubilization of such auxin-binding sites from Z. mays has been achieved (5, 7, 31). The solubilized microsomal auxin-binding protein from Z. mays has a specificity for natural and synthetic auxins that parallels the ligand specificity of the membrane-associated protein and the biological activities of the auxin analogues tested (5). On current evidence this membraneassociated auxin-binding protein may well be the (an) auxin receptor. In the absence of evidence for an auxin-induced functional change in this protein the possibility exists that this protein has other than a receptor function. Apparently soluble auxinbinding proteins have been reported (15,21,29,32) Primary bean seedling leaves were excised from seedlings 8-14 days after sowing the seed, washed with distilled H20, and sliced with scissors. All subsequent operations were carried out at 0-4 C.The sliced leaves were suspended in five volumes of an extraction medium containing 50 mm Tris (Cl-, pH 8.0), 10 mm EDTA, 0.1% (v/v) 2-mercaptoethanol, 0.5 mm PMSF, and 0.2% (v/v) ethanol, and homogenized at stop I in a Waring Blendor for I min. The homogenate was filtered through Miracloth and the filtrate centrifuged at 35,000g for 40 min. The supernatant was brought to 30% (NH4)2SO4 saturation, the resulting precipitate being collected by centrifugation and discarded. The resulting supernatant was brought to 50% (NH4)2SO4 saturation and the precipitate collected by centrifugation and dissolved in a minimum volume of buffer A containing 50 mm Tris (Cl-, pH 8.0), 10 mM EDTA, and 0.1% (v/v) 2-mercaptoethanol. This solution was applied to a column (2.5 cm2 x 70 cm) of Ultrogel AcA-34 and eluted with buffer A (Fig. la). The IAA-binding fractions were pooled and applied to a column (4.0 cm2 x 9.0 cm) of DEAE-Sephacel equilibrated with buffer A. The DEAE-Sephacel column was eluted with a linear gradient from 0 to 0.25 M (NH4)2SO4 (in buffer A) (Fig. Ib). The active fractions were pooled, concentrated by precipitation at 80%1o (NH4)2SO4 saturation, and reapplied (in buffer A) to the Ultrogel AcA-34 column and eluted in buffer A. One protein peak associated with a constant specific activity IAA-binding activity peak is observed at this stage (Fig. Ic). The purification schedule is presented in Table I. The same scheme was applied to the purification of ABP from the leaves of pea seedlings. Pea leaves were harvested 10-14 days after sowing the seed. Variants of this basic purification schedule were emp...

Demonstration of Auxin Binding to Strawberry Fruit Membranes

“…The pH optimum for the 120 K membrane fraction is 4.0 (Fig. 2), while the pH optima for vegetative tissues range from 5.0 in zucchini (12) and tobacco pith callus (32), 5.5 in com (1,23), to 8.0 in mung bean (13,33). The physiological relevance ofthis low pH optimum is unknown, but fruits in general appear to have a low pH optimum for auxin binding as evidenced by pH optima of 3.75 for cucumber fruit and 3.5 for bean and tomato fruit CM fraction (16,18).…”

A Binding Resolution

“…Since ligand binding and receptor activation are usually the rate-limiting steps in signal transduction, the number of binding sites should be small. The highly abundant protein ribulose-bisP carboxylase www.plantphysiol.org/cgi/doi/10.1104/pp.109.136606 binds indole-3-acetic acid saturably (Wardrop and Polya, 1980), but clearly there are no other data, either physiological or genetic, supporting the model that this protein is an auxin receptor.…”

Ligand Specificity of Bean Leaf Soluble Auxin-binding Protein

“…Royal Windsor) essentially as described previously (32). Because of the instability of bean ABP after chromatography on DEAESephacel this chromatographic step was replaced with a further gel filtration through a column (2.5 cm2 x 70 cm) of Ultrogel AcA-34 as described previously (32).…”

“…Because of the instability of bean ABP after chromatography on DEAESephacel this chromatographic step was replaced with a further gel filtration through a column (2.5 cm2 x 70 cm) of Ultrogel AcA-34 as described previously (32). Pea ABP was purified from about 10-day-old leaves of peas (Pisum sativum var.…”

“…Pea ABP was purified from about 10-day-old leaves of peas (Pisum sativum var. Greenfeast) as described previously (32). Materials.…”