Asparaginase Isolated from Developing Seeds of Pisum sativum

Asparaginase (EC 3.5.1.1) was isolated from the developing seed of Pisum sativum. The enzyme is dependent upon the presence of K+ for activity, although Na+ and Rb+ may substitute to a lesser extent. Maximum activity was obtained at K+ concentrations above 20 millimolar. Potassium ions protected the enzyme against heat denaturation. The enzyme has a molecular weight of 68,300. Asparaginase activity developed initially in the testa, with maximum activity (3.6 micromoles per hour per seed) being present 13 days after flowering. Maximum activity (1.2 micromoles per hour per seed) did not develop in the cotyledon until 21 days after flowering. Glutamine synthetase and glutamate dehydrogenase were also present in the testae and cotyledons but maximum activity developed later than that of asparaginase. Potassium-dependent asparaginase activity was also detected in the developing seeds of Viiafaba, Phaseols multiflorus, Zea mays, Hordeum vulgare, and two Lupius varieties. No stimulation of activity was detected with the enzyme isolated from Lupinus polyphylus, which has previously been shown to contain a K+-independent enzyme. Asparagine is a major nitrogen storage and transport compound in plants (1, 11, 18, 19). In Lupinus asparagine may account for 700%o of the nitrogen transported to the pod and seed in the phloem although only 7 to 10%1o of the residues of the seed protein are asparagine. When [U-'4C], amide-['5NJasparagine was supplied via the phloem to developing lupin seeds the amide nitrogen was rapidly converted to a wide variety of amino acids in the seed protein (1). It was suggested that asparagine was metabolized by the enzyme asparaginase (L-asparagine amido-hydrolase, EC 3.5.1.). Asparaginase has been purified and characterized from Lupinus polyphyllus (9); however, the enzyme was not found in the majority oflupin varieties tested nor in other legumes, e.g. Pisum, Phaseolus, or Vicia (8), a result confirmed by other workers in Pisum (2) and soya bean (23). Two other possible routes of asparagine metabolism have been considered (7); the first possibility that asparagine could substitute for glutamine in the GOGAT2 reaction has not ' This work was supported in part by a grant to L. S. from the Fundarao de Amparo a Pesquisa de Estado do Sao Paulo. 'Abbreviations: GOGAT: glutamate synthase; GS: glutamine synthetase; GDH: glutamate dehydrogenase. been confirmed with cell free extracts from a variety of sources (2, 14, 23, 28). The second route, in which asparagine is transaminated to give a-ketosuccinamic acid which subsequently breaks down to release ammonia, has been demonstrated in soya bean (24) and pea leaves (12) but we have been unable to find asparagine transamination in maturing cotyledons of P. sativum. Despite this lack of positive evidence for asparagine utilization we have confirmed a previous report (16) that isolated developing pea cotyledons grown in culture can utilize asparagine as the sole nitrogen source for extensive synthesis of storage protein (10). Studies with enzyme inhibitors suggest that asparagine is first metabolized to ammonia which is reassimilated by the GS/GOGAT pathway (15). This paper reports the results of a further search for enzymic evidence for asparagine breakdown and describes the isolation, from developing seeds of Pisum sativum and a range of other plants, of an asparaginase which is totally dependent upon the presence of K+ ions. MATERIALS AND METHODS