Binding of Enzymes to Avena Coleoptile Cell Walls

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Abstract
Aqueous suspensions of Avena coleoptile cell walls are capable of firmly binding added pectinesterase in amounts up to approximately 200 times that native to the cell walls. This is true for both native cell walls and walls in which the pectic methyl ester groups have been removed by pectinesterase. The enzyme remained bound through exhaustive water washing but is readily eluted with 0.15 N NaCl at pH 7.5. The binding is apparently an ionic one. The pectinesterase-binding capacity of cell walls is not influenced by added indoleacetic acid. The auxin is also without effect on the pectinesterase activity of tobacco pith homogenates. Large amounts of pepsin, peroxidase, [alpha]-chymotrypsin and its diisopropyl fluorophosphate inhibition product are similarly firmly bound to Avena coleoptile cell walls. As much as 0.85 mg [alpha]-chymotrypsin and 2.0 mg pepsin are bound per mg of cell walls. These proteins are elutable by the method used for pectinesterase. Neither heating of cell walls in suspension nor treatment with ethylenediaminetetraacetate changes their binding capacity for pectinesterase or [alpha]-chymotrypsin. This sequestering agent also fails to liberate the bound enzymes. Intact Avena coleoptile sections do not bind added pectinesterase. The pH of cell walls over the range of pH 3.8 to 7.5 does not influence their binding capacity for pectinesterase. The binding capacity for [alpha]-chymotrypsin is maximal at pH 6.4. Pectinesterase and a-chymotrypsin are bound to different sites in the cell wall since saturation with one does not influence the binding of the other. Saturation of cell walls with diisopropyl fluorophosphate-inhibited [alpha]-chymotrypsin decreases the binding capacity for [alpha]-chymotrypsin by 40%. The specific removal of pectic material from cell walls by highly purified polygalacturonase results in only partial reduction in the binding capacity for pectinesterase and [alpha]-chymotrypsin. Cell walls treated with pectinase, which removes polysaccharides in addition to pectic material, still retain approximately 15% of their binding capacity for these 2 enzymes. Incubation of Avena coleoptile sections at pH 4.4 where auxin is most effective and at pH 7.4 where it is but little effective showed no difference in the amount of pectinesterase accompanying the cell walls or any significant difference in the degree of esterification of the pectic material in the cell walls. No evidence has been obtained to support the view that the effect of auxin in promoting the elongation of cells is mediated through an effect on pectinesterase.