Acid Protease Activity During Germination of Microcysts of the

Transcription

1 JOURNAL OF BACTERIOLOGY, Jan. 1976, p Copyright i 1976 American Society for Microbiology Vol. 125, No. 1 Printed in U.S.A. Acid Protease Activity During Germination of Microcysts of the Cellular Slime Mold Polysphondylium pallidum DANTON H. O'DAY Erindale College and Department of Zoology, University of Toronto, Mississauga, Ontario, Canada L5L 1C6 Received for publication 4 August 1975 Extracts of dormant microcysts of Polysphondylium pallidum demonstrate ph optima for the hydrolysis of casein at 3.5 and 6.0. During germination the intracellular ph 6.0 caseinolytic specific activity undergoes a three- to fourfold increase, whereas the ph 3.5 specific activity does not change significantly. The ph 6.0 protease is also active on azo-albumin, revealing the same developmental pattern with this substrate. Both acid protease activities are excreted during the germination process. Addition of purified nonspecific protease to cultures speeds up germination, suggesting that the excreted protease may play a role in removal of the microcyst wall. When cycloheximide is added to cultures, complete germination (emergence) is stopped whereas the ph 6.0 protease activity still accumulates to between 50 and 60% of the maximum control activity. Although this suggests that post-translational controls might mediate the accumulation of a portion of the ph 6.0 protease increase, mixing and dilution experiments with cell extracts do not reveal the differential presence of soluble activators or inhibitors of this activity at different developmental stages. The presence of tightly bound enzyme-inhibitor complexes for protease B in dormant microcysts has not been ruled out and is currently under study. Microcyst formation can be induced by subjecting the amoebae of certain species of cellular slime molds to high osmotic conditions (2, 13). The mature cyst is essentially a condensed amoebal cell surrounded by a bilayered cell wall (5). The microcyst cell wall contains cellulose, some undefined glucose polymers, lipid, and an extremely large amount of protein (14; M. Biihlmann, Thesis, University of Zurich, 1971). When dormant microcysts are placed in a sterile, non-nutrient medium, germination is rapid and synchronous (3, 11). During germination the cell wall undergoes an initial loosening followed by partial or complete dissolution which allows emergence of an individual amoebae from each microcyst (5, 11). Several hydrolytic enzymes undergo specific intracellular and extracellular changes in activity during microcyst germination, suggesting that they may function in cell wall removal (11). In this study we present the initial characterizations of acid protease activity in cell extracts of cellular slime molds with an analysis of their intracellular and extracellular patterns during germination of Polysphondylium pallidum microcysts. Experiments involving augmentation of extracellular protease support the concept that extracellular acid proteases function in cell wall removal. 8 MATERIALS AND METHODS Culture methods and samples preparation. Microcysts of Polysphondylium pallidum strain WS-320 were formed in the modified axenic culture of Sussman (12) as detailed previously (11). Cultures containing 95 to 100% microcysts after 7 days were used in these experiments. Microcysts were pelleted by centrifugation, resuspended at 107 cells/ml in 10-ml volumes of sterile 0.01 M potassium phosphate buffer, ph 6.5 (excystment medium), in 50-ml Erlenmeyer flasks, and placed on a rotary shaker (11). When cycloheximide (400 ug/ml; Sigma Chemical Co.) was used it was dissolved in the excystment medium before microcysts were added. The morphological aspects of germination were monitored by phase contrast microscopy. Samples for enzyme assays were removed at specified intervals and cells and culture fluid were frozen unseparated (total activity) or they were separated into cell and culture fluid fractions (extracellular activity) before freezing in plastic-capped test tubes using a methanol-dry ice mixture. Protease assay. Extracts for protease assay were prepared by subjecting cell and total (cell plus supernatant) samples to sonic oscillation on ice for 2 min with a Bronwill Sonifier (Bronwill Industries) operating at maximal output. The resulting crude brei was assayed for its enzyme and protein content. Supernatant samples were thawed and assayed directly. Caseinolytic activity was assayed at 37 C using 0.5 ml of 1% casein (BDH) in distilled water, in a total reaction volume of 1.0 ml. All buffers were used at a

2 VOL. 125, 1976 final concentration of 0.05 M. The reaction was initiated by addition of 0.1 ml of enzyme extract and stopped by addition of 1.0 ml of 10% trichloroacetic acid. The resulting precipitate was removed by filtration, and the amount of released peptides and amino acids in the filtrate was determined by the Lowry method (8). For each reaction mixture a corresponding background control blank was prepared. This blank, which consisted of the reaction mixture minus enzyme extract, was incubated and stopped with the enzyme reaction mixtures. After the trichloroacetic acid was added, 0.1 ml of the corresponding enzyme extract was added and the filtrate was retrieved and treated as for the enzyme samples. This background activity was subtracted from the enzyme sample activity before specific activity was calculated. The protein content of the sample was determined using the Lowry procedure with bovine serum albumin (fraction V, Sigma Chemical Co.) as a standard. Specific enzyme activity with casein was defined as units per milligram of protein where 1 U of enzyme activity liberates 1 mg of protein equivalents per h. When azo-albumin (type I, Sigma) was used, the method of Tomarelli et al. (15) was modified so that the reaction was carried out at 37 C in a final volume of 1 ml containing 10 mg of azo-albumin per ml in 0.05 M acetate buffer at ph 5.5. The reaction was started with the addition of 0.1 ml of enzyme sample and was stopped with 1.0 ml of 10% trichloroacetic acid. After filtering, 1.5 ml of 0.5 N NaOH was added to 1.5 ml of filtrate and the absorbancy at 440 nm was read. The background control consisted of incubated reaction mixture to which enzyme sample was added after the trichloroacetic acid. The equivalents of azo-albumin hydrolyzed were determined from a standard curve of azo-albumin. Specific activity was defined as micrograms of azo-albumin hydrolyzed per milligram of protein per hour. Effect of enzymes on germination. For the analysis of the effects of specific enzymes on germination, nonspecific protease from Streptomyces griseus (type VL Sigma) and cellulase from Aspergillus niger (type I, Sigma) were used alone or together at final concentrations of 1 mg per ml each. The enzymes were dissolved in the excystment medium before microcysts were added, and the percentage of germination was determined after 3 h by phase contrast microscopy. Controls consisted of cultures which had no enzyme added or which had been autoclaved after addition of protease or cellulase to inactivate the enzymes. RESULTS Characteristics of acid protease activity. Extracts of dormant microcysts (0 h), swollen microcysts (3 h), and excysted amoebae (6 h) all showed ph optima at 3.5 and 6.0 with casein as substrate (Fig. 1). For simplicity, these enzyme activities will be designated protease A (ph 3.5 optimum) and protease B (ph 6.0 optimum). Thus protease A was routinely assayed with 0.05 M acetate buffer (ph 3.5) and protease B was assayed with 0.05 M phosphate buffer (ph 6.0). ACID PROTEASE IN POLYSPHONDYLIUM 9 With casein, protease A appeared to be the major proteolytic activity in germinating cells. Both of these activities were also observed in the extracellular medium during germination (Fig. 2). Although it is assumed that the extracellular ph FIG. 1. Effect of ph on hydrolysis of casein by extracts of germinating microcysts of P. pallidum. Cells from germinating cultures were collected at 0 h (0), 3 h (A), and 6 h (0) and assayed for their caseinolytic activity as detailed in the text. For comparative purposes the data are expressed as percentage of maximum activity units. Acetate buffer (0.05 M) was used in the range from ph 3.0 to 5.5 and phosphate buffer (0.05 M) was used from ph 6.0 to 7.5. ph FiG. 2. Effect of ph on hydrolysis of casein by the extracellular medium after germination of microcysts. After 6 h of germination, the cells were pelleted from cultures, and the caseinolytic activity of the medium was determined at different ph values as detailed in the text. For comparative purposes the data are presented as percentage of maximum activity units. The buffers used were 0.05 M acetate (ph 3.0 to 5.5) and 0.05 M phosphate (ph 6.0 to 7.5).

3 10 O'DAY activities are derived by excretion of the intracellular protease A and B activities, experiments are being carried out to verify the relatedness of the intracellular and extracellular enzyme activities. It is evident that the ph curves for activity for the ph 3.5 and 6.0 enzymes do not overlap significantly and thus determinations at one ph will probably not be affected by residual activity of the other enzyme. Initial studies on ph optima utilizing acetate, McIlvains, and phosphate buffer involved overlapping ph values to study the effects of the buffers on enzyme activity. It was found that all of these buffers yielded almost identical protease activity at the same ph values. When azo-albumin was used as substrate a ph optimum was observed at ph 5.5 (data not shown). The azo-albumin precipitated below ph 4.0, making it impossible to determine if protease A was active on this substrate. Analysis of other parameters revealed that protease A activity on casein was linear with time for over 3 h. This enzyme reaction was also linear within the range of protein concentrations utilized in these experiments (0.05 to 0.5 mg/ml). Protease B was also linear with protein concentration and with time for over 4 h. In fact, calculations done with enzyme reactions run for 4 and 24 h yielded extremely close specific activity values for protease B, suggesting that the reaction may be linear with time for at least 24 h. Samples kept at - 20 C for 10 days yielded almost identical results to samples stored only 1 or 3 days, indicating that this enzyme is stable under our conditions of storage. Developmental patterns. Protease A activity underwent little intracellular change during germination, whereas the enzyme activity accumulated extracellularly throughout germination (Fig. 3). This increase was due to an actual increase in units of enzyme activity since the protein content of cells did not change significantly during microcyst germination. The rate of accumulation of the extracellular enzyme appeared to be linear with the maximal extracellular activity (units) being equivalent to one-quarter of the original enzyme activity (units) present in the dormant microcysts. The intracellular level of protease B specific activity underwent a three- to fourfold increase during germination, reaching a specific activity of 0.53 by 6 h (Fig. 4). This enzyme activity also appeared to accumulate in the extracellular medium in a linear fashion during germination (Fig. 4). The cells excrete an amount of protease B activity (units) equivalent to half the original 4-2 >- '0,.. U 0~ hou rs FIG. 3. Intracellular and extracellular patterns of protease A during germination. Total samples were removed from germinating cultures and separated into cells and supernatant by centrifugation. The activity of cell extracts (0) and supernatant (0) on casein at ph 3.5 was determined and the specific activity was calculated as detailed in the text , 0 0~~~~~ 0 0 o o hours J. BACTERIOL. FIG. 4. Intracellular and extracellular patterns of protease B during germination. The activity of cell extracts (0) and supernatants (0) on casein at ph 6.0 was determined and the specific activity was calculated as detailed in the text.

4 VOL. 125, 1976 activity of dormant microcysts. Total activity (cells plus culture medium) patterns for protease B revealed a similar pattern to the intracellular developmental change when casein was used as substrate. The total activity of protease B underwent a partial but significant increase in specific activity in cultures that had been treated with 400 Ag of cycloheximide per ml (data not shown). This increase was due to an actual doubling in enzyme activity since the protein content of control and cycloheximidetreated cultures remained the same during germination. When azo-albumin was used as a substrate for total enzyme activity at ph 5.5, the developmental pattern was almost identical to that for the ph 6.0 caseinolytic activity, suggesting that this pattern is also due to protease B activity (Fig. 5). Of interest, again, is the increase in ph 5.5 albuminase (protease B) activity in cultures that were treated with cycloheximide. Mixing experiments. Mixing of cell extracts from 0, 3, and 6 h of germination in all possible combinations followed by enzyme assays of the mixtures yielded activity values that were extremely close to the expected totals for the mixture (Table 1). These data argue against the differential presence of soluble activators or inhibitors of protease A or B at different developmental stages. '4- u 0~ 2 4 hou rs FIG. 5. Effects of cycloheximide on total protease B albuminase activity during germination. Total samples of control (0) and cycloheximide-treated (0) cultures were removed during germination and their activity on azo-albumin at ph 5.5 was determined. The details of the method of assay and calculation of specific activity are contained in the text. 6 ACID PROTEASE IN POLYSPHONDYLIUM 11 Addition of purified protease and cellulase. When 1 mg of nonspecific protease per ml was added to cultures of germinating microcysts, the rate of emergence was greatly enhanced (Table 2). Cellulase also enhanced germination, whereas protease plus cellulase increased the rate of germination to a rate approximately equal to the sums of the increases caused by each of these enzymes separately. This enhancement was apparently due to the enzyme activity alone since heat-inactivated protease and cellulase did not increase the rate of germination. In the presence of cycloheximide the enzymes were ineffective at enhancing germination. DISCUSSION Weiner and Ashworth (16) have previously indicated the presence of lysosomal acid protease activity in growing myxamoebae of the TABLE 1. Activity of acid protease in mixed cell extractsa Protease A Protease B Sample (ph 3.5) act (ph 6.0) act (h) Observed Expected Observed Expected a Cell extracts from different stages of germination (0, 3, 6 h) were mixed in small plastic test tubes, left on ice for 30 min, then assayed for caseinolytic activity at ph 3.5 and 6.0 as described in the text. TABLE 2. Effect ofprotease and cellulase on the rate of germination of P. pallidum microcystsa Expt situation Amoebae (%) Expt 1 Expt 2 Expt 3 Control Protease Inactivated proteaseb 23 Cellulase Inactivated cellulaseb 17 Protease + cellulase Cycloheximide 5 3 Cycloheximide + enzymesc 4 4 a Cultures were set up as described in the text. The percentage of amoebae was determined after 3 h of germination. "Enzymes were inactivated by autoclaving. c Includes both cellulase and protease.

5 12 O'DAY cellular slime mold Dictyostelium discoideum. This protease apparently had a ph optimum of 2.0 or less and did not change in activity during fruiting body formation. Crude cell extracts of dormant and germinating microcysts of P. pallidum reveal two acid protease activities with ph optima of 3.5 (protease A) and 6.0 (protease B). At present the subcellular location of these enzymes has not been determined and it is not known whether these activities are due to single or multiple enzyme species. Protease A is the major protease activity but its activity in crude cell extracts does not change significantly during germination. Protease B, as assessed by activity on both casein and azoalbumin, undergoes a steady developmental increase during germination to reach a peak activity at 6.0 h which is three to four times greater than the specific activity of extracts of dormant microcysts. Although mixing experiments with cell extracts from different developmental stages and dilution studies argue against the increase in protease B as being due to the differential presence of diffusible activators or inhibitors during germination, the partial but significant increase in activity of this enzyme in the presence of cycloheximide suggests that de novo synthesis is not responsible for the total developmental increase. O'Day, Gwynne, and Blakey (Exp. Cell Res., in press) have shown that cycloheximide completely inhibits the rapid and dramatic increase in protein synthesis that occurs when microcyst germination is initiated. Several other enzymes undergo significant intracellular and extracellular changes during germination in P. pallidum (11). Of these, the developmental dase are completely prevented when cycloheximide is added at the beginning of germination (11; O'Day et al., in press). It may be possible that a tightly bound inhibitor is associated with the protease B activity present in the dormant microcysts and is removed during germination. The inhibitors of acid protease in yeast apparently bind tightly but can be removed by specific digestion techniques causing an enhancement in protease activity in cell extracts (7). We are presently investigating the presence of a tightly bound enzyme-inhibitor complex for the acid proteases in P. pallidum. It is possible to suggest a myriad of developmental roles for the intracellular acid protease activities, including (i) removal of protein inhibitors, (ii) post-translational modification of structural or enzymatic proteins, (iii) protein turnover, and (iv) source of extracellular proteases, but at present these potential functions in this system are purely speculative. J. BACTERIOL. Selective enzyme excretion is common in cellular slime molds. Extracellular enzyme activities have been detected during growth (1), starvation (10), fruiting body formation (6), microcyst formation (9), and microcyst germination (11). During microcyst germination both protease A and B activities are secreted throughout the germination sequence. It is reasonable to suggest a role for the extracellular acid protease activities in cell wall removal during germination. Chemical analysis of the microcyst wall has revealed it to be made up of lipid, cellulose, some undefined glucose polymers, and large amounts of protein (14). In fact, the protein component makes up approximately 30% of the microcyst wall structure and appears to be restricted to the inner layer of the bi-layered cell wall (Biihlmann, Thesis, University of Zurich, 1971). As reported here, addition of nonspecific proteases alone or in conjunction with cellulase significantly enhances germination. Although more protease A is excreted during germination, the ph profiles for the acid protease activities indicate that only protease B would be active extracellularly since the medium is buffered to ph 6.5. We intend to purify both acid protease species and add these to ungerminated microcysts to test their specificity in enhancing germination. It has been previously suggested that protease may be a critical enzyme for germination of spores of Dictyostelium discoideum since the spore wall contains protein, and Pronase used in sequence with cellulase will release amoebae from cycloheximide-treated spores (4). However, the characteristics of this protease and its existence in germinating spores have not been determined. As revealed in the present study, and in work by Cotter (personal communication), protease in conjunction with cellulase will not liberate amoebae from cycloheximidetreated microcysts. This suggests that other critical enzymes, in addition to protease and cellulase, are also involved in the germination of microcysts. In a recent paper a model for microcyst germination was presented which invoked the sequential appearance of two enzyme systems (O'Day et al., in press). The appearance of system I is regulated by post-translational controls and causes the initial loosening of the microcyst wall which allows swelling. It appears that protease B may be a component of this system since its activity increases in the absence of protein synthesis and the enzyme appears extracellularly during swelling. Another candidate is cellulase. The later accumulation of system II enzymes, which effects complete

6 VOL. 125, 1976 cell wall removal, requires protein synthesis but not ribonucleic acid synthesis. In addition to other enzymes, the cycloheximide-sensitive protease B component may be one enzyme activity of this system. Future experiments will determine if the cycloheximide-insensitive and -sensitive protease B components are due to different proteolytic activities. ACKNOWLEDGMENTS I would like to thank Gale MacNab for her fine technical assistance and Mary Smith for performing some of the preliminary experiments with purified enzymes. This research was supported by a grant from the National Research Council of Canada. LITERATURE CITED 1. Ashworth, J. M., and J. Quance Enzyme synthesis in myxamoebae of the cellular slime mould Dictyostelium discoideum during growth in axenic culture. Biochem. J. 126: Blaskovics, J. C., and K. B. Raper Encystment stages of Dictyostelium. Biol. Bull. 113: Cotter, D. A., and K. B. Raper Spore germination in stains of Dictyostelium discoideum and other members of the Dictyosteliaceae. J. Bacteriol. 96: Hemmes, D. E., E. S. Kojima-Buddenhagen, and H. R. Hohl Structural and enzymatic analysis of the spore wall layers in Dictyostelium discoideum. J. Ultrastruct. Res. 41: Hohl, H. R., L. Y. Miura-Santo, and D. A. Cotter Ultrastructural changes during formation and germination of microcysts in Polysphondylium pallidum, a cellular slime mold. J. Cell Sci. 7: Killick, K. A., and B. E. Wright Trehalose synthesis during differentiation in Dictyostelium discoideum. ACID PROTEASE IN POLYSPHONDYLIUM 13 IV. Secretion of trehalase and the in vitro expression of trehalose-6-synthetase activity. Biochem. Biophys. Res. Commun. 46: Lenney, J. F., Ph. Matille, A. Wiemken, M. Schellenberg, and J. Meyer Activities and cellular localization of yeast proteases and their inhibitors. Biochem. Biophys. Res. Commun. 60: Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall Protein estimation with the Folin phenol reagent. J. Biol. Chem. 193: O'Day, D. H a-mannosidase and microcyst differentiation in the cellular slime mold Polysphondylium pallidum. J. Bacteriol. 113: O'Day, D. H The intracellular localization and extracellular release of certain lysosomal enzyme activities from amoebae of the cellular slime mold Polysphondylium pallidum. Cytobios 7: O'Day, D. H Intracellular and extracellular enzyme patterns during microcyst germination in the cellular slime mold Polysphondylium pallidum. Develop. Biol. 36: Sussman, M Growth of the cellular slime mold Polysphondylium pallidum in a simple nutrient medium. Science 139: Toama, M. A., and K. B. Raper Microcysts of the cellular slime mold Polysphondylium pallidum. I. Factors influencing microcyst formation. J. Bacteriol. 94: Toama, M. A., and K. B. Raper Microcysts of the cellular slime mold Polysphondylium pallidum. II. Chemistry of the microcyst walls. J. Bacteriol. 94: Tomarelli, R. M., J. Charney, and M. L. Harding Use of azo-albumin as substrate in the colorimetric determination of peptic and tryptic activity. J. Lab. Clin. Med. 34: Weiner, E., and J. M. Ashworth The isolation and characterization of lysosomal particles from myxamoebae of the cellular slime mold Dictyostelium discoideum. Biochem. J. 118: