Substances from cultured soybean cells which stimulate or inhibit acetylene reduction by free-living Rhizobium japonicum [culture conditions, submerged culture, surface, Streptomyces protease, Daucus carota, Psophocarpus tetragonolobus]

The effect of cultured soybean (Glycine max (L.) Merr. cv. Acme) cells and extracts thereof on acetylene reduction by Rhizobium japonicum 61A76 was determined under two culture conditions. In the first (submerged culture) Rhizobium growing on solid medium were submerged in an aqueous layer containing soybean cell suspension or extract; both soybean cells and crude cell extract inhibited free living rhizobial nitrogenase measured by acetylene reduction. In the second culture condition (surface culture) Rhizobium was grown on the surface of an agar medium which contained expressed soybean cell sap (7.4% v/v), which caused an increase in free living rhizobial nitrogenase activity. Larger concentrations (12.9 to 19.4% v/v) of soybean cell extract inhibited acetylene reduction also in surface culture. Inhibition in submerged culture or surface culture was found after autoclaving of cells or extract, or treating the extract with pH extremes or with Streptomyces protease. Each of these treatments destroyed stimulatory activity of cell extract in surface cultures. Fractionation of the soybean cell extract on a Biogel P-6 column showed that there are three inhibitory fractions and at least one stimulatory fraction. Using column data and the fact that stimulatory and inhibitory factors were dialysable against 0.1 M phosphate buffer, we estimated the molecular weights of the factors. The stimulatory factor has a molecular mass (M(, r)) between 6,000 and 14,000. The inhibitory factors have molecular masses less than 6,000. Crude extracts from cultured carrot (Daucus carota (L) cv. Danvers) cells gave results similar to those seen with soybean cell extract. Extracts from cultured winged bean (Psophocarpus tetragonolobus (L.) DS. cv. TPt2) cells gave inhibition but no stimulation