Comparisons of soybean urease isolated from seed and tissue culture.

Abstract

(Received for publication, May 10, 1978, and in revised form, September 15, 1978) Joseph C. PolaccoS and Evelyn A. Havir From the Departments of Genetics and Biochemistry, The Connecticut Agricultural Experiment Station, New Haven, Connecticut 06504 Urease was purified 500-fold to electrophoretic ho- mogeneity from ground, dry soybeans. Sodium dodecyl sulfate-acrylamide gel electrophoresis indicates a sub- unit size of 93,500 daltons which is identical with that of jack bean urease. In solutions of high ionic strength, there exists a single urease species (species 1) with a size of about 480,000 daltons based on agarose column chromatography and migration in acrylamide gels. In solutions of lower ionic strength (e.g. 3 nm phosphate), a new lighter species (species 2) predominates which has a size of about 280,000 daltons. Soybean and jack bean urease are serologically related but also contain unique antigenic determinants. The amino acid com- position profiles of soybean and jack bean urease show only small differences in the amounts of four amino acids. The urease activity purified from soybean cell suspension cultures was electrophoretically identical with the seed enzyme (both species 1 and species 2 being present in buffers of intermediate ionic strength, viz. 10 mM phosphate). Antibodies to soybean seed urease were purified by affinity chromatography. They were then separated from cross-reacting antibodies to jack bean urease by chromatography over Sepharose 4B containing covalently linked jack bean urease. The effluent antibodies no longer precipitated nor inhibited jack bean urease but inhibited ureases from soybean seed and soybean tissue culture to the same extent. In spite of this immunological evidence for a detailed sim- ilarity between soybean seed urease and urease par- tially purified from suspension culture, urease activity in crude extracts of suspension culture is heteroge- neous both with respect to size and affinity for mono- specific antiseed urease antibodies. Urease is a seed protein common to most of the Legumi- nosae. Jack bean urease, the first enzyme to be isolated to crystalline purity (l), has a high methionine content (2, 3). Generally in legume seed proteins, this amino acid is present in limiting amounts for human and monogastric animal nutri- tion (4). If the methionine content of soybean urease is similar to that of jack bean, breeding for high seed urease levels may lead to increases in seed methionine levels. In this report, we examine the similarity between soybean and jack bean urease and between the ureases of soybean seed and soybean tissue culture. Soybean seed urease was purified approximately 500-fold to electrophoretic homogeneity. Soy- * This work was supported in part by Grant PCM 78-18501 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 USC. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. bean and jack bean urease are serologically related and share a common subunit size as determined by SDS’-polyacryl- amide electrophoresis. Moreover, the amino acid profiles, including methionine content, are very similar. Urease from each source can exist in different interconvertible polymeric forms. By the criteria of electrophoretic mobility, gel filtration patterns, and inactivation by antibodies to seed urease, the purified urease of soybean seed and partially purified urease from suspension culture were identical. However, urease ac- tivity in crude extracts of suspension culture showed apparent size and immunological heterogeneity.