Vol. 29, No. 1 Printed in U.S.A.

APPLIED MICROBIOLOGY, Jan. 1975, p. 115-117 Copyright 0 1975 American Society for Microbiology

Decryptification of Acid Phosphatase in Arthrospores of Geotrichum Species Treated with Dimethyl Sulfoxide and Acetone DAVID A. COTTER,* ANITA J. MARTEL, AND PAUL MACDONALD Department of Biology, Southeastern Massachusetts University, North Dartmouth, Massachusetts 02747 Received for publication 21 October 1974

Decryptification of acid phosphatase in Geotrichum sp. arthrospores was accomplished using acetone or dimethyl sulfoxide treatment. Both dimethyl sulfoxide and acetone irreversibly destroyed the integrity of the spore membranes without solubilizing acid phosphatase.

Changes in the specific activity of acid phosphatase have been shown to occur during the life cycle of some fungi (9, 11). We wish to determine if acid phosphatase activity is correlated with arabitol and mannitol formation in the life cycle of Geotrichum sp. The present experiments were performed to develop a technique for acid phosphatase assay during differentiation of Geotrichum sp. Our initial attempts to isolate the known acid phosphatase activity from lysosome-like particles (10) in the arthrospores were unsatisfactory. Mechanical grinding followed by sonification with glass beads (1 ,um in diameter) did not produce greater than 30% disruption of arthrospores. Dimethyl sulfoxide Me2SO has been used to alter the permeability of lysosomal membranes (7, 8) and to decryptify a-glucosidase of Saccharomyces cerevisiae (1). Treatment of 48-hold washed arthrospores with this solvent did result in decryptification of acid phosphatase. The procedure consisted of growing Geotrichum sp. in E-1 medium (glucose salts) for 48 h at 23.5 C (2). The resulting arthrospores were washed twice in distilled water by centrifugation and then placed in various concentrations of Me2SO (Matheson, Coleman and Bell, Norwood, Ohio, reagent grade) in distilled water or buffer solutions for 30 min at 30 C. The total volume was 5 ml, with arthrospore concentrations ranging between 1.5 to 3.0 mg/ml. The treated spores were then washed twice with distilled water and suspended in 0.15 M sodium acetate buffer at pH 4.7. Portions of washed spores were also filtered onto Whatman glass fiber filters (GF/A, 4.25 cm) which were dried at 80 C for 24 h; such disks were used for dry weight determinations. Acid phosphatase activity was measured using a modification of Wilson's method (11). Each sample of arthrospores was prewarmed at

30 C in 0.15 M sodium acetate buffer (pH 4.7) before being added to a prewarmed solution of p-nitrophenyl phosphate in acetate buffer. The final reaction mixture contained approximately 1 mg of arthrospores and 2 mg of p-nitrophenyl phosphate per ml in 2.5 ml of sodium acetate buffer (pH 4.7). This substrate concentration is 10 times the Km value (5.6 x 10' M) for acid phosphatase of Geotrichum sp. (unpublished data). Reaction mixtures were incubated at 30 C for 30 min; 0.5-ml portions were taken at 0 time and 30 min and mixed with 5 ml of 10% Na2CO3 to stop the reaction. Arthrospores were pelleted by centrifugation, and the optical density of the Na2CO3 solutions was measured at 400 nm in a Bausch and Lomb Spectronic 20 spectrophotometer using the 0-time Na2CO.3 solutions as reference. Enzyme activity was optimal after treatment with 40 to 45% Me2SO (Fig. 1). It should be noted that the supernatant was devoid of acid phosphatase activity; the enzyme(s) was apparently still bound to spores which were now fully permeable to neutral red. Untreated viable spores contained neutral red only in discrete vesicles presumed to be lysosomes (5, 6, 10). Germination studies demonstrated that the spores which stained uniformly with neutral red at a concentration of 1:10,000 had lost their viability and were not capable of producing germ tubes; a total of 400 spores was counted in each viability determination. This result is in contrast to studies showing that animal cells remain viable after Me2SO treatment (4). Treatment of spores with 50% Me2SO quantitatively permeabilized the spore population (Table 1) but yielded less activity than treatment with 45% Me2SO (Fig. 1). The data may indicate that a fraction of the enzyme was denatured with 50% Me2SO treatment. At-

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APPL. MICROBIOL.

NOTES

tempts to increase enzyme activity using modified Me2SO treatments were successful (Table 2); however, Me2SO concentrations greater than 45% again resulted in reduced enzyme activity (data not shown). It appeared that Me2SO treatments could be used to decryptify acid phosphatase in Geotrichum sp. provided the concentrations were kept at 45%. Whereas the Me2SO treatment appeared adequate for

100r

A

TABLE 2. Effect of water and buffers on decryptification of acid phosphatase with 45% Me2SO Treatment

Control (no Me2SO) 45% Me2SO in deionized water Me2SO 45% Me2SO in 0.15 M sodium acetate buffer (pH 4.7) 45% Me2SO in 10 mM phosphate buffer (pH 6.5)

% of spores permeable to neutral red

Sp act of acid phosphatasea

0.5 94.8

0.01 1.70

97.2

1.50

91.3

1.10

a Specific activity of acid phosphatase was defined as optical density at 400 nm/mg (dry weight) of spores per 30 min.

Z

4

O *

5050

E

*

N

w

0 -

0

30

0

50

40 Yo Me;2SO

FIG. 1. Effects of various coracentrations of Me2SO in 10 mM phosphate buffer (piH6.5) on decryptification of acid phosphatase. The peak enzyme activity in each experiment was designaited the 100% 100o ativit actity level to compensate for differen ces in spore concentrations in the four experiments. Enzyme activities at other Me2SO concentrations u centages of the peak enzyme activity. Symbols: o, experiment 1; 0, experiment 2; AA, experiment 3; and *, experiment 4.

cedsthe

TABLE 1. Effect of increasinig concentrations of Me2SO in 10 mM phosphate Ibuffer (pH 6.5) on the Giontrichum SD. gt,& ,,& Op. viability of arthrospores a,/Ff Lumul -

Treatment

Control (no Me2SO) 30% Me2SO 35% Me2SO 40% Me2SO 45% Me2SO 50% Me2SO

%uniformly of spores Germina- Nonviable by tion (%)a spores stained (%)b neutral red

99.3 79.5 76.1 33.6 5.5 2.6

0.7 20.5 23.9 66.4 94.5 97.4

0.5 18.2 26.5 67.0 93.6 100.0

a Arthrospores were incubated on E-1 (0.4% glucose) medium at 23.5 C for 7 h. ° Calculated from percent germination data.

our purposes, we sought a more reliable technique. A modified acetone treatment (3) was found to be effective in decryptifying dormant arthrospores. Spores were suspended in 100% acetone at a concentration of 1 mg of spores/10 ml of solvent and stirred at -5 C for 5 min; the spores were then filtered onto glass fiber disks and washed with an additional 10 ml of acetone. The disks were allowed to dry at room temperature in a vacuum dessicator. The resulting acetone powders could be conveniently handled, since exact quantities of dry arthrospores could be resuspended in acetate buffer. Such suspensions possessed an acid phosphatase specific activity of 1.7, a result which compared well with the best MeSO decryptification. All enzyme activity remained associated with the arthrospores even though they were 100% uniformly stained with neutral red. The acid phosphatase activity was not affected by storing the acetone powders at - 20 C for at least 6 months. We conclude that the acetone procedure is superior to the Me2SO procedure because of better reproducibility and ease of handling. This investigation was supported by the Southeastern Massachusetts University Research Foundation.

LITERATURE CITED 1. Adams, B. G. 1972. Method for decryptification of a-glucosidase in yeast with dimethyl sulfoxide. Anal. Biochem. 45:173-146. 2. Cotter, D. A., and D. J. Niederpruem. 1971. Nutritional and temporal control of arabitol and mannitol accumulation in Geotrichum. Arch. Mikrobiol. 76:65-73. 3. Gunsalus, I. C. 1955. Extraction of enzymes from microorganisms (bacteria and yeast), p. 51-62. In Sidney P. Colowick and Nathan 0. Kaplan (ed.), Methods in enzymology, vol. 1. Academic Press Inc., New York. 4. Jacob, S. W., and D. C. Wood. 1971. Dimethyl sulfoxide (DMSO)-a status report. Clin. Med. 78:21-31.

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NOTES

5. Koenig, H. 1963. Intravital staining of lysosomes by basic dyes and metallic ions. J. Histochem. Cytochem. 11:120-121. 6. Koenig, H. 1965. The staining of lysosomes by basic dyes. J. Histochem. Cytochem. 13:20. 7. Lee, D. 1971. The effect of dimethylsulfoxide on the permeability of the lysosomal membrane. Biochim. Biophys. Acta 233:619-123. 8. Lee, D. 1972. The effect of glycerol, ethanol and dimethyl-sulfoxide on rat liver lysosomes. Biochim. Biophys.

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Acta 266:50-55. 9. Montes, L. F., and W. H. Wilborn. 1970. Acid phosphatase activity during phases of growth in Candida albicans. Int. J. Dermatol. 9:220-225. 10. Reiss, J. 1971. Lysosome-like particles in Geotrichum candidum: a cytochemical study. Z. Allg. Mikrobiol. 11:319-323. 11. Wilson, R. W. 1972. Acid and alkaline phosphatases in Schizophyllum commune. Can. J. Microbiol. 18:694-695.

Decryptification of acid phosphatase in arthrospores of Geotrichum species treated with dimethyl sulfoxide and acetone.

Decryptification of acid phosphatase in Geotrichum sp. arthrospores was accomplished using acetone or dimethyl sulfoxide treatment. Both dimethyl sulf...
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