Intracellular and extracellular acetylglucosaminidase activity during microcyst formation in Polysphondylium pallidum

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1973 by Academic Press, Inc. in any form resewed

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Experimental Cell Research 79 (1973) 186-l 90

INTRACELLULAR ACETYLGLUCOSAMINIDASE FORMATION

AND

EXTRACELLULAR

ACTIVITY

DURING

IN POL YSPHO ND YLIUM

MICROCYST

PALLID

UM

D. H. O’DAY Erindale College, University of Toronto, Mississauga, Ont., Canada

SUMMARY The specific activity of acetylglucosaminidase (EC 3.2.1.30) doubles during microcyst differentiation. The increase in activity is apparently due to concomitant protein synthesis since addition of cycloheximide at the time of increase stops further accumulation of the enzyme. The enzyme activity is a product of two isozymes which do not appear to change in relative activity during the differentiation process. Acetylglucosaminidase is excreted during microcyst development but the excretion is not stopped by cycloheximide. A cystless mutant of P. pallidurn, NG-6, demonstrates higher specific activities but a relatively normal intracellular and extracellular pattern of accumulation of acetylglucosaminidase. Thus a normal pattern of biochemical differentiation of this enzyme can occur in the absence of normal morphological differentiation.

Microcyst differentiation is an alternate pathway of development for the cellular slime molds [2]. Usually, slime mold cells undergo a multicellular development and construct a fruiting body (morphogenesis) containing spores and stalk cells [3]. However, under high osmotic conditions the individual amoebae differentiate as microcysts [17]. This differentiation process seemsto require continuous protein synthesis since it is reversibly stopped at all times by addition of cycloheximide [13], a preferential inhibitor of protein synthesis in slime mold cells [lo, 161.Microcysts possess a cell wall consisting of two layers of different texture [6] and containing cellulose, glycogen, lipid and protein [18]. Biochemical differentiation occurs during microcyst formation. cr-Mannosidase increasesin specific activity during this differentiation process and the increase seems to Exptl Cell Res 79 (1973)

require concurrent protein synthesis [ 131. Certain lysosomal acid hydrolases including a-mannosidase [ 131 and acid phosphatase [15] are secreted from the P. pallidurn cells during microcyst differentiation. Hydrolytic enzymes are also excreted from another slime mold Dictyostelium discoideum [I] and from the protozoan Tetrahymena pyriformis [l I] but the function of this enzyme excretion is still unknown. This paper documents the intracellular and extracellular pattern of another glycolytic enzyme, acetylglucosaminidase, during microcyst differentiation. This enzyme may function in the metabolism of the extracellular polysaccharide of the cellular slime molds [7] which contains large amounts of Nacetylglucosamine [5]. A summary of portions of this work has previously appeared in abstract form [12].

Microcysts of P. pallidurn METHOD

187

AND MATERIALS

Culture methods and microcyst formation Stock cultures of P. pallidurn strains WS-320 (wild tvDe: kindlv suoohed bv K.B. Raoer) and NG-6 (aggregateless-cystiess mutant; isolated’ and kindly donated bv D. W. Francis) were grown on E. coli B/r on 0.2 % Cerophyl agar plates. To obtain amoebae for encvstment. snores (WS-320) or amoebae (NG-6) were scraped off the stock plates, placed in a culture medium of autoclaved E. coli B/r (approx. lo9 cells/ ml) in 0.01 M potassium phosphate buffer (pH 6.5) and grown on a rotary shaker for 3 (WS-320) or 6 (NG-6) days. The cells were harvested by centrifugation for 5 min at 3 000 rpm in an International Table Centrifuge (Model HNS, Head Cat. No. 805), resuspended in fresh phosphate buffer and shaken overnight for the consumption of remaining bacteria. The amoebae were again harvested by centrifugation as described and finally resuspended to a concentration of 1.0 x 10’ cells/ml in 0.2 M sucrose in the phosphate buffer (encystment medium) to initiate microcyst differentiation [6]. Samples were removed hourly with a sterile loop and examined by phase microscopy for the presence of cysts.

Sample harvest Samples were removed, using sterile technique, at specific intervals and centrifuged for 5 min at 3 000 rpm in an International Table Centrifuge (model HNS, Head Cat. No. 805) to pellet the whole cells. The supernatant was decanted and the cells resuspended in 1.0 ml of distilled water. Both sunernatant and cells were stored at -20°C for assay at a later date. For the experiments with cycloheximide, the inhibitor was added in solution through a sterile Millipore filter (0.45 p) to a final cont. of 200 pg/ml in the encystment medium.

Acetylglucosaminidase

assay

Cells were thawed and disrupted by sonication for 2 min with a Bronwill sonicator. The crude cell extract was assayed. Supematants were thawed and assayed directly. The acetylglucosaminidase activity was assayed using 8 x 1O-3 M para-nitrophenyl-l\r acetyl-p-n-glucosaminide in 1.Ox 1O-2 M acetate buffer (pH 5.0). These conditions had been determined as optimal by prior experimentation. The reaction was initiated with sample and stopped after 30 min with 2 vol of 1.O M Na&O,. The released p-nitropheno1 was determined from the absorbance at 420 nm. A unit of activity was defined as pM p-nitrophenol (product) released per minute. Protein was determined using the method of Lowry et al [9] using bovine serum albumen as a standard. The spec. act. of acetylglucosaminidase was defined as units/mg of protein.

Electrophoresis Crude cell extracts in 10% sucrose were layered directly on 5 % acrylamide gels (5 cm) and electro-

2. Abscissa: hours; ordinate: intracellular acetylglucosaminidase (units/mg protein). Fig. 1. The intracellular pattern of acetylglucosaminidase spec. act. during microcyst differentiation. P. pallidurn cells were harvested from the encystment culture at hourly intervals, frozen and later assayed for enzyme activity and protein content as described in Methods and Materials. Fig. 2. Effect of cycloheximide on acetylglucosaminidase accumulation during microcyst differentiation. Cells were harvested at 2 h intervals from control cultures ( l ) or cultures treated with 200 mg/ml cycloheximide at 0, 0 h, a, 6 h, n , 9 h, frozen and later assayed for enzyme and protein content as described in Methods and Materials. Figs I,

nhoresed according to the method of Davis 141.The chamber buffer contained 0.1 M sodium phosphate buffer (pH 7.2) and the gels contained 0.05 M sodium phosphate buffer (pH 7.2). Electrophoresis was carried out at 5 mA/gel for 150 min at 3-5°C. After the run the gels were-sectioned to 2 mm slices and each slice was assayed for acetylglucosaminidase activity using 0.5 ml of 8 x lo-% M p-nitrophenyl-N-acetyl-Bacetylglucosaminide in 0.15 M acetate buffer. After 90 min the reaction was stopped with 3.0 ml of 1 M Na,CO, and the released p-nitrophenol determined at 420 nm. The activity for each slice (p-nitrophenol released/90 min) was converted to per cent of maximum to allow a comparison of the relative amounts of each isozyme at different stages of microcyst differentiation.

RESULTS Kinetics of intracellular accumulation

acetylglucosaminidase

High levels of acetylglucosaminidase are present in amoebae (0 h) before their addition Exptl Cell Res 79 (1973)

188 D. H. O’Day to the encystment medium (fig. 1). This activity decreases slightly when the cells are placed in the sucrose-buffer solution but after a few hours begins to increase to a maximal spec. act. of about 40 at 12 h of development. The activity then decreases. When cycloheximide is added to the differentiating cells at 0 h the increase in activity is not observed (fig. 2). When cycloheximide is added at later intervals of 6 and 9 h partial increases in activity occur but do not reach the maximum observed in the controls. Other work has shown that cycloheximide preferentially inhibits protein synthesis in slime mold cells [16, lo]. Mixtures of 0, 6 and 12 h cell extracts in the various possible combinations produce specific activities equal to the sum of the individual activities. Thus the increase cannot be explained in terms of the presence or absence of specific activators or inhibitors at different stages. The use of the inhibitor cycloheximide indicates that the increase in acetylglucosaminidase activity requires concomitant protein synthesis.

2 t

i

4, 5. Abscissa: hours; ordinate: extracellular acetylglucosaminidase (units/ml). Fig. 4. Kinetics of the extracellular accumulation of acetylglucosaminidase activity during microcyst formation. Samples were harvested at 2 h intervals from encystment cultures. The whole cells were precipitated by centrifugation and the supernatant was assayed for enzyme activity as described in Methods and Materials.

Figs

Isozymes of acetylglucosaminidase Two forms of acetylglucosaminidase are separable by electrophoresis in acrylamide gels under the conditions described. It is evident that no new isozyme forms appear during differentiation and it appears that the relative amounts of each isozyme form remain constant (fig. 3) during the differentiation process. Kinetics of extracellular acetylglucosaminidase accumulation

\ OO-5

5

gel slice number; ordinate: acetylglucosaminidase activity. Isozymes of acetylglucosaminidase from two different developmental stages. Cells were harvested at l , 0 and 0, 12 h from the encystment culture, frozen and later disrupted by sonication. The crude cell extracts were layered on 5 % acrylamide gels and electrophoresis was carried out for 150 min. The gels were then sliced and the gel sections assayed for acetylglucosaminidase activity. The details of the method are described in Methods and Materials.

Fig. 3. Abscissa:

Exptl Cell Res 79 (1973)

Weiner & Ashworth [19] have reported that acetylglucosaminidase is a lysosomal enzyme and it is excreted during growth of D. discoideum [l]. For this reason the extracellular encystment medium was assayed for acetylglucosaminidase activity. No enzyme activity is evident in the first few hours but after 4 h acetylglucosaminidase activity appears in the medium and increases markedly as the process continues (fig. 4) to reach about 5.4

Microcysts of P. pallidurn

189

Acetylglucosaminidase activity is also excreted from the mutant and the pattern of extracellular accumulation is similar to the normal pattern although greater amounts of activity are releasedfrom the mutant strain. DISCUSSION

Fig. 5. Effect of cycloheximide on the extracellular

accumulation of acetylglucosaminidase activity during microcyst formation. Samples from control cultures (0) and cultures treated with 200 mg/ml cycloheximide at 0 h ( 0) and 9 h ( n ) were harvested at 2 h intervals. The whole cells were pelleted by centrifugation and the enzyme activity of the medium was assayed as described in Methods and Materials.

Acetylglucosaminidase is an enzyme which doubles in spec. act. during microcyst differentiation in P. pallidum. As shown by cycloheximide inhibition studies, this enzyme requires concomitant protein synthesis for its accumulation. The increase does not seem to be explainable by the presence or absence of specific activators or inhibitors at different times. The same situation seemsto exist for the enzyme during morphogenesis of D. discoideum and Loomis [7] has shown that

units/ml at 28 h. This final value corresponds to about 63 units/mg of protein (spec. act.). The addition of cycloheximide to the differentiating cells at any time does not stop the release of the enzyme from the cells (fig. 5). Although lower levels of activity appear in the medium this is probably due to the decreased synthesis of the enzyme in the treated cells. Intracellular and extracellular acetylglucosaminidase in a cystless mutant (NG-6)

The mutant strain of P. pallidurn, NG-6, does not undergo evident morphological changes when placed in the encystment medium and thus represents a true cystless mutant [ 131.When cells of NG-6 are harvested from the encystment medium at intervals and assayed for acetylglucosaminidase activity a pattern of increase in enzyme activity like that of normal microcyst differentiation is apparent (fig. 6). The intracellular level of acetylglucosaminidase at all times is approximately double the normal level of activity.

0 Fig. 6. Abscissa: hours; ordinate:

(left) intracellular acetylglucosaminidase (units/mg protein); (right) extracellular acetylglucosaminidase (units/ml). The intracellular ( l ) and extracellular ( n ) pattern of acetylglucosaminidase activity in a cystless mutant (NG-6) of P. pallidurn. Mutant cells were placed in the encystment medium and samples harvested at 2 h intervals. The whole cells were pelleted by centrifugation and the enzyme and protein content of both the cells and medium was determined as described in Methods and Materials. Exptl Cell Res 79 (1973)

190 D. H. O’Day both stalk acd spore cells accumulate identical levels of the enzyme. Thus acetylglucosaminidase is an enzyme characteristic of all cell types of cellular slime molds studied to date. Since the physiological role of the enzyme remains unknown, the relevance of the increase in acetylglucosaminidase during differentiating of stalk cells, spores and microcysts remains questionable. However, the presence of N-acetylglucosamine as a major extracellular polysaccharide component of slime mold cells [5] indicates an important role for this enzyme in polysaccharide metabolism [7]. Another glycolytic enzyme, cc-mannosidase is synthesized during microcyst differentiation [13] and mannose, a product of a-mannosidase hydrolysis, is also a component of the extracellular polysaccharide of slime mold cells [5]. Alkaline phosphatase, another enzyme which increases in activity during morphogenesis, decreases in activity during microcyst differentiation [14]. Acetylglucosaminidase is a lysosomal enzyme in D. discoideum [19] and is excreted during growth of this organism [I]. The enzyme is also excreted during growth and starvation in Tetrahymena [ 1I]. In P. pallidurn the extracellular acetylglucosaminidase activity increases markedly during microcyst differentiation and the release of the enzyme does not seem to require protein synthesis since it is not stopped by cycloheximide. Other lysosomal acid hydrolases are similarly excreted during this process including a-mannosidase [13] and acid phosphatase [15]. Again the function of these extracellular enzymes is open to conjecture. Mutant cells (NG-6) of P. pallidurn which cannot differentiate into microcysts demonstrate a higher but relatively normal pattern of accumulation of both intracellular and

Exptl Cell Res 79 (1973)

extracellular acetylglucosaminidase. Thus biochemical differentiation of this enzyme can occur in the absence of morphological differentiation. This same situation exists for certain enzymes in multicellular development as well [8]. cc-Mannosidase does not show its normal intracellular pattern of accumulation during microcyst formation in the mutant NG-6 but instead decreases in activity after several hours [ 131. It is evident then that different control mechanisms must regulate the activities of acetylglucosaminidase and E-mannosidase in microcyst differentiation. I would like to thank Linda Riley for her fine technical assistance. This research was supported by a grant from the National Research Council of Canada.

REFERENCES 1. Ashworth, J M, Symp sot exptl biol 25 (1971) 2. %skovics, J C & Raper, K B, Biol bull 113 (1957) 58. 3. Bonner. J T. The cellular slime molds. Princeton Univ Press, Princeton, N.J. (1969). 4. Davis. B J. Ann NY acad sci 121 (1964) 404. 5. Gerisdh, G: Malchow, D, Wilhelms: H &‘Liideritz, 0, Eur j biochem 9 (1969) 229. 6. Hohl, H R, Miura-Santo, L-Y & Cotter, D A, J cell sci 7 (1970) 285. 7. Loomis, W F Jr, J bact 97 (1969) 1149. 8. - Ibid 103 (1970) 375. 9. Lowry, 0 H, Rosebrough, N J, Fart-, A L, & Randall, R J, J biol them 193 (1951) 265. 10. Mizukami, Y & Iwabuchi, M, Exptl cell res 63 (1970) 317. II. Miiller, M. J cell biol 52 (1972) 478. 12. O’Day, D’H, J protozool; suppl. 19 (1972) 24. 13. - J bact 113 (1973). In press. 14. O’Day, D H‘ & ‘Francis, D W, Can j zoo1 51 (1973). In press. 15. O’Day, D H & Riley, L J. Manuscript submitted. 16. Sussman, M, Biochem biophys res comm 18 (1965) 763. 17. Toama, M A & Raper, K B, J bact 94 (1967) 1143. 18. - Ibid 94 (1967) 1150. 19. Weiner, E & Ashworth, J M, Biochem j 118 (1970) 512. Received September 15, 1972