The isolation of wall-bound proteins regulating yield threshold tension in glycerinated hollow cylinders of cowpea hypocotyl

To isolate and purify the factor regulating the yield threshold tension (y) through acidification of the cell wall, proteins were extracted from hypocotyls of Vigna unguiculata L. Their effects on the pH-dependencies of the wall extensibility (phi) and y were examined with reconstitution experiments by incorporating them into the heat-denatured glycerinated hollow cylinders (GHCs). The wall mechanical properties of the reconstituted GHCs were determined using stress-strain experiments. Only the proteins extracted with 1 kmol m(-3) of NaCl from the wall of elongation region restored the pH-dependencies of phi and y once extinguished with heat-denaturation, but proteins extracted from the other cell constituents or from the mature region of hypocotyl affect neither properties. Fractionation of the wall-bound proteins by a hydrophobic column chromatography showed that the two different fractions affected phi or y independently. The sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed that the active fraction which restored the pH-dependency of y still consists of two proteins of 30 and 32 kDa after purification by the sequential fractionation with cation-exchange and gel filtration. These two proteins were named as 'yieldin 30' and '32'. Western blotting analysis using the rabbit-antiserum against the cucumber expansin indicated that the yieldins are independent of cucumber expansin.