A COMPARISON OF ALPHA-AMYLASES FROM THE LATEX OF THREE SELECTED SPECIES OF EUPHORBIA (EUPHORBIACEAE)

A technique for the partial purification of a-amylases from latex of Euphorhbia heterophylla, E. marginata, and E. tirucalli is described. The enzymes were found to be similar to other higher plant amylases using the criteria of molecular weight, pH characteristics, kinetics, number of isozymes, and blue value-reducing value patterns. Carbohydrases other than a-amylases were not detectable in latex. The amylases were employed to examine their capacity to digest latex starch grains which are common components of the laticiferous cell in this genus. Laticifer starch grains are not susceptible to in vitro amylolysis. Removal of the starch grain membrane with Triton X-100, damaging the grain, or treating the grains with a-amylases from diverse biological sources had little effect upon hydrolysis. Grains incubated with pullulanase followed by a-amylase caused a slight but significant increase in hydrolysis of raw laticifer starch grains. These studies indicate that the nonarticulated laticifer in Euphorbia is a cul de sacc for certain primary and secondary metabolic products and that the indigestible and morphologically complex starch grains in the latex have evolved to function in a secondary role within the laticifer. MANY SPECIALIZED PLANT products have been identified in the latex exudate of the nonarticulated laticifer in the genus Euiphor-biai. These products include rubber, alkanes, glycerides (Nielsen et al., 1977), C28-C30 triterpenes and their esters (Ponsinet and Ourisson, 1968), various polyfunctional polycyclic diterpenes and cryptic irritants, the fatty acid esters of which are cocarcinogenic (Hecker, 1975), and starch (Mahlberg, 1975). Although much attention has been directed toward isolation and identification of these ergastic substances, very little is known about the enzymatic or structural proteins present in latex. In a previous paper we studied the accumulation and degradation of starch in Euphorbia, and it was determined that laticifer starch grains form a metabolically inaccessible polysaccaride pool in the specialized cytoplasm of the laticifer (Biesboer and Mahlberg, 1978). We noted that a-amylases, which can effect a rapid fragmentation of the entire starch molecule by cleaving a-D-(1 -> 4) linkages, also occurred in substantial quantities in latex. The purpose of the present study is to: 1) de' Received for publication 24 March 1980; revision accepted 11 June 1980. 2 Present address: Dept. of Botany, 220 Biological Sciences Center, University of Minnesota, St. Paul, Minnesota 55108. The authors thank D. McCracken, Illinois State University, for his suggestions during the course of the experimental work. This study was supported by NSF grant DEB-78-02045. scribe a general technique for the partial purification of active enzymes from latex; 2) clarify what carbohydrases besides a-amylase (1 ,4-glucan-4-glucanohydrolase, EC 3.2.1.1.) are present in latex; 3) compare and partially characterize a-amylases from latex of several selected species of Euphorbia, and 4) determine if latex starch grains are susceptible to a-amylase digestion. MATERIALS AND METHODS-Sources of latex-Latex was taken from greenhouse or garden grown populations of Euphorbia heterophylla L., E. marginata Pursh., and E. tirucalli L., by scarification of stem or petiolar regions of the plants. Exuded latex was collected directly into graduated vials containing cold 0.1 M citrate-phosphate buffer (pH 5.9) and 0.2% calcium acetate (buffer; latex, 1:1) and maintained at 2 C. Purification procedure-The cold latexbuffer suspension was centrifuged at 30,000 x g for 1 hr or slightly longer until three distinct fractions appeared in the centrifuge tube. The hard, upper rubber layer and milky-white lower layer were carefully removed and discarded. The remaining clear latex serum fraction was retained, and cold acetone (-5 C) was added to it (2:1, v/v) to precipitate the proteins. The resulting precipitate, separated from the remaining serum by centrifugation, was air dried and subsequently dissolved in cold 0.2% calcium acetate in 0.1 M citrate-phosphate buffer