Summtinary. A crude proteolytic enzyme preparation which hydrolyzes casein was tsolated from the cotyledons of squash seedlings. The presence of ethylene diamine tetraacetate or cysteine did not appreciably affect the activity of the preparation. During the coturse of germination, the level of proteiolytic activity increased in the cotyledion,s of intact embjryos through the third day and then decreased. The presence of the embryonic axis during the first 32 hours of germination was a prerequisite for the development of maximum proteolytic activity.The presence of a cytokin,in, suich as benzyladenine, kinetill, BTP [6-(benzylamino)-9-(2-tetrahydropyranyl)-9H-puirine], or phenyladenine, in the culture solution could reproduce the effect of the embryonic axis. Other growth regtulators did not pro(luce this stimul,ation. High ooncentrations (1 mM) of all growth reglulators examined were inhibitory. Various combiniations of growth regulators failed to produce any synergistic effects. 6-Methylpuirine inhibited the development of proteinase activity, and this inhibition was only partially restored by benzyladenine.A proteinase was partially purified from the cotyledons of 2-day-old squash seedlings and its synthesis w,as shown to be uinder the control of the embryonic axis.The concentration of storage protein in squash seedls, CucuCrbita naxima, from which the axes had been excised has been shown to remain unchanged during germination (7). The cotyledons of intact seeds, however, undergo a progressive decrease in the content of protein dturing germination. When excised embryonic axes were incubhated with the excised cotyledons, the pattern of protein degradation was similar to thiat observel in the intact seeds. Some suibstance(s) supplied by the embryonic axis is reqtuired for normal growth and maximum proteolytic activity or hydrolysis of storage protein in squash cotyledons (5, 7). Benzyladenine replaced the axis in promoting development of maximuim proteolytic activity, whereas gibberellin ha(l nlO effect. Puromycin and actinomycin D blocked the (levelopment of the proteolytic activity coIntrolled by the axis, indicative of (le novo synthesis of the proteinase (5).The objectives of this investigation were to (letermine A) whether growth regulators, alone or i,n combination, influence the proteolytic activity of squash cotyledons, B) the period of influence of the axis and benzyladenine on proteolytic activity development, and C) the distribultion of proteolytic ' activity between the axis and cotyledons. An intergral part of these objectives involved purification of the proteinase whose development is controlled by the axis.
Materials and MethodsPreparation and Assay of ai Cruide Proteolytic Enzyme. Seeds of squash (Cucurbita mtaxima Duichesne 'Chicago WVarted Hubbard') thiat had been selected for uniformity were soaked in distililed water for 4 houtrs at room temperature. Tlhe seed's were then placed in moi-st vermicuilite in a 300 incuibator, in the (lark. After 3 days the cotyledons were removed, andl a crude...