Evidence for Lysosomal Enzymes in Acanthamoeba and Their Activity Changes During Encystment

Assays on cell-free homogenates of Acanthamoeba castellanii reveal that the three hydrolases, acid phosphatase (APase), acid deoxyribonuclease, and acid ribonuclease (RNase), possess pH optima of 5.0, 4.8, and 5.2, respectively. These enzymes exhibit an enhanced sedimentation at 20,000 x g when sucrose is in the homogenizing buffer. Treatment of homogenates with Triton X-100 increases total enzyme activity. These results suggest that the enzymes are particle-bound in lysosomes. During encystment there is a differential decrease in the activity per cell of all three enzymes, with RNase decreasing most rapidly and APase least rapidly. The specific activity of APase increases during encystment even though its activity per cell gradually decreases. OHIO J. SCI. 77(1) 28, 1977 Acanthamoeba castellanii is a free-living soil and freshwater amoeba that can be cultured axenically. Its life cycle consists of a vegetatively multiplying trophozoite which can, under unfavorable environmental conditions, differentiate into a cyst form. During encystment there is a prominent decrease in the cellular contents of DNA, RNA, protein, lipid, and glycogen; these endogenous degradations are thought to supply both energy and precursors for the synthesis of the cyst wall (Neff and Neff/l969; Griffiths, 1970). Most likely lysosomal enzymes are involved in this degradation since such enzymes act as catabolic hydrolases in a wide variety of eukaryotic differentiative processes (Dingle and Fell, 1969). This viewpoint is strengthened by the electron micrographs of Bowers and Korn (1969) which reveal the appearance of autophagic vacuoles during encystment in A. castellanii. These autophagic vacuoles stain positively for the lysosomal enzyme, acid phosphatase (APase), and contain mitochondria, various cytoplasmic debris, and glycogen particles. In order to learn more about the role that Manuscript received February 6, 1976, and in revised form July 29, 1976 (#76-13). Present address: Department of Biology, University of Missouri at Kansas City, 5100 Rockhill Road, Kansas City, Missouri 64110. lysosomal enzymes play during differentiation in A. castellanii, we have assayed the activities of three lysosomal markers, APase, acid deoxyribonuclease (DNase), and acid ribonuclease (RNase), during starvation-induced encystment. MATERIALS AND METHODS The amoeba used in this study was obtained from Robert J. Neff (designated as Acanthamoeba sp., strain 1-12) who isolated it from soil at Pacific Grove, California (Neff, 1957). Page (1967) concluded that the sarcodinid should correctly be called Acanthamoeba castellanii (Douglas, 1930). The amoeba was cultured axenically at 30°C in a growth medium devised by Neff et al (1964) that was prepared according to Byers et al (1969). Detailed procedures for counting the cells electronically and for normalizing the growth data are discussed by Byers et al (1969) and Martin (1973). The staining solution of Mattar and Byers (1971) was used for differentially counting encysting cells in a hemacytometer. Trophozoites were harvested for encystment experiments after they had grown for 72 hrs and had reached a concentration of 1.5 to 2.5 x 10 cells/ml, or after 168 hrs of growth, when they had attained a density of 4.5 to 6.0 x 10 amoebae/ml (fig. 1). They were collected aseptically by centrifugation at 1000 x g, washed in 0.15 M NaCl, and inoculated into a nutrientfree salt solution to induce a synchronous encystment. The encystment medium of Neff et al (1964) was used, but its preparation was modified (Martin, 1973) to eliminate the necessity of bubbling autoclaved encystment medium with CO2 to dissolve the precipitate of carbonates which forms during sterilization. The