_.V-—__v—_4Av—__~. .' -_"———-—'-—~—w w‘w‘y - A CREMECAL AND CYTGCHEMKAL ENVESYEGATmH 0F ACE?) PHOSPHATAEES 6F ?E”§’RAHYMENA PYREFORMES— W Thesis {1): the 599234!) 3% S’h 9. MECHEQ‘EAR STAW UREVERSE'PY Eemgrfi flame-r This is to certify that the thesis entitled A chemical and cytochemical investigation of acid phosphatases of Tetrahymena pyriformis W. presented by Bernard Klamer has been accepted towards fulfillment of the requirements for Ph.D. degree in 200105! I 7 r'] .- I. I,/ I ) t ,y” K “k “4-6 h’r\£’ (- Major professor Date August 16, 1960 0-169 L I B l Michig Univ A CHEMICAL AND CYTOCHEMICAL INVESTIGATION OF ACID PHOSPHATASES OF TETRAHYMENA PYRIFORMIS W by Bernard Klamer AN ABSTRACT Submitted to the School for Advanced Graduate Studies of Michigan State University of AgricultUrecand.Appliedtsctenaes. in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Department of Zoology . 1960 Approved fici’l/ 4 M Bernard Klamer ABSTRACT This investigation was undertaken for the purposes of evaluating the cytochemical methods for localization of acid phosphatase activity and to ascertain the activity of this enzyme quantitatively during periods of cellular proliferation, glucose uptake, and aging in cultures of ggtggf hymena pyriformis W. The localization patterns obtained by the Gomori procedure demon- strated that acid phosphatase activity, in cells taken during the early culture period, was confined to spherical cytoplasmic entities situated primarily in a perinuclear region. The typical localization pattern observed in cells from later in the culture period, i.e., 15 to 30 days after inoculation, was more diffuse and extended throughout most of the cytoplasm. These patterns were inhibited by treatment of cells with 0.1 M sodium fluoride and by immersion in water at 80°C for 10 minutes. Small areas with positive reactions were observed in the posterior regions of cells which were immersed in buffered control solutions containing lead nitrate. Patterns of enzyme activity obtained by use of the azo dye proce- dure were generally not as definite as those observed with the Gomori method. Numerous diazotized couplers were employed in this procedure in attempts to produce more stable dye patterns resulting from enzymatic activity. The most discrete localization patterns were observed when diazotized 3,3'-dichloro-4,4'-diamino-bipheny1 was used as a coupler and alpha-naphthyl phosphate was employed as a substrate. Patterns obtained with this procedure compared favorably with those obtained with the Gomori procedure and possessed the advantage that control cells did not exhibit positive reactions. Bernard Klamer Abstract, page 2 Quantitative determinations indicated that acid phosphatase acti- vity was at a low level during periods of rapid cellular proliferation and greatest uptake of glucose. Growth curves for each series of cul- tures were obtained by making direct cell counts prior to determination of enzyme activity. Comparison of acid phosphatase activity with the growth curves suggested that increased phosphatase activity was coin- cident with the phase of decline in which cellular autolysis occurred. The results of altering the culture media by adding various con- centrations of malonic acid, sodium malonate, and urethan supported previous observations that acid phosphatase activity was appreciably influenced by culture conditions, i.e., good growth was associated with elevated levels of phosphatase activity. Increased acid phosphatase activity also appeared to be correlated with an increase in pH of the cellular environment. The substance responsible for acid phosphatase activity in homo- genates of Tetrahymena was resolved into at least 5 components by zone electrophoresis. No differences could be discerned between the electro- phoretic pattern of enzymes from cells taken during early and late culture periods. Attempts to demonstrate beta-glucuronidase activity in Tetrahymena were unsuccessful. A CHEMICAL AND CYTOCHEMICAL INVESTIGATION OF ACID PHOSPHATASES OF TETRAHYMENA PYRIFOEMIS W by Bernard Klamer A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Sciences in Partial Fulfillment of the Requirements for the Degree of DOCTOR“OFJPHILOSOPHY Department of Zoology 1960 — ”Dr‘\ 1’ "‘l (l, (L -_,,-’ ,‘,.- L9 JAM/52 ACKNOWLEDGEMENTS The author wishes to express his sincere thanks to Dr. R. A. Fennell for his interest, direction and encouragement during the course of this investigation. Grateful thanks are also due to Drs. J. R. Shaver, A. S. Fox, and P. O. Fromm for their able assistance as committee members and tOOMrs. Bernadette Henderson, Secretary, Department of Zoology, for her many kindnesses. Special recognition is made to my wife, Lorraine, whose encour- agement and sacrifices have made this study possible. Acknowledgement is also made to the Cancer Division of the National Institutes of Health for the predoctoral fellowship, #CF-9362, during the course of this study. ‘\—— iii CURRICULUM VITAE Bernard Klamer candidate for the degree of Doctor of Philosophy Final examination: August 16, 1960 Dissertation: A Chemical and Cytochemical Investigation of Acid Phosphatases of Tetrahymena pyriformis W. Outline of Studies: Major subject: Zoology Minor subject: Physiology Biographical Items: Born, January 29, 1929, Hudsonville, Michigan Undergraduate Studies, Calvin College, 1946-50 Graduate Studies, Michigan State University, 1955-60 Experience, Member United States Army, 1951-52 Professional Membership: Society of Sigma Xi iv TABLE 9}: CONTENTS LIST OF FIGURES O O O O O O C O O O O 0 O O O O O O O O O O C 0 LIST OF TABLES o O 0 0 O O O O O I O O O C O O O 0 O O O O O 0 LIST OF PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . INTRODUCTION I O O O C O C O O O O O O I O O O O O O O O O O O O 0 MATERIALS AND METHODS. . . . . . . . . . . . . . . . . . . . RESULTS. Localization experiments. . . . . . . . . . . . . . . . . Gomori method Azo dye method Calibration curves. . . . . . . . . . . . . . . . . . . Glucose Inorganic phosphate Protein Phenolphthalein Synchronous division. . . . . . . . . . . . . . . . . . . Acid phosphatase activity of cells from various types of culture media . ... . . . . . . . . . . . . . . . . . . Series A Series B Series C Series D Series E Electrophoresis studies . . . . . . . . . . . . . . . . 16 16 20 25 25 55 TABLE 2: CONTENTS (Cont.) Beta-glucuronidase assay. . . . . . . . . . . . . . . . . . . 55 DISCUSSION 0 I O O O O O O O O O O C O O O O O O O O C O O O O O O O O 61 sum-ARY. O O I O O O O O O O O O O O O O O O O O O O O O O O O O O O O 80 LITERATURE CITED 0 O O O O O O O O O O O O O O O O I O O O O O O O O O 8 2 vi 1.131911111911335. Figure l 10 ll 12 13 14 Calibration curve for glucose obtained by reduction of 3,5-dini- trosalicylic acid with 1.0 ml aliquots of various concentrations Of glucose 0 O O O O O O O O O O O O I O O O O O O O O O O O O 0 Standard curve for protein determination obtained with crystal- line bovine serum albumin. . . . . . . . . . . . . . . . . . . . Relationship of amount of protein in various aliquots of a 10% homogenate of Tetrahymena to the quantity of homogenate used in eaCh determination o e e o o e o o o e e o e e o e 0.. o o o o o Standard curve for estimation of inorganic phosphate . . . . . . Standard curve for estimation of beta-glucuronidase activity . . Growth of Tetrahymena pyriformis W in standard trypton medium. Series A. e o o o o e o o o o o o o e o e o o o o o o o o e o o Relationship between age of culture following inoculation and concentration of glucose in the culture medium. Series A. . . . Acid phosphatase activity of Tetrahymena pyriformis W cultured in standard tryptone medium. Series A. . . . . . . . . . . . . Growth of Tetrahymena pyriformis W in 3 types of culture media. Series B. o e e o e e o o o o o o e o e e o o e o o e o o o o 0 Acid phosphatase activity of Tetrahymena pyziformis W cultured in 3 types Of media. Series Bo o o o o e e o e o o e o o e o 0 Growth of Tetrahymena pyriformis W in various culture media. series C. O O O I O O O O O O O O C O O O O O O O O O O O O O O 0 Acid phosphatase activity of Tetrahymena pyriformis W in various culture media. Series C.. . . . . . . . . . . . . . . . . . . . Growth of Tetrahymena pyriformis W in various culture media. series Do a o o e O O o e o o o o o o o O I o o o O O O O O O I Ackiphosphatase activity of Tetrahymena pyriformis W cultured in various media. Series D. . . . . . . . . . . . . . . . . . . vii 21 23 26 28 30 34 36 38 40 42 45 47 50 52 LIST OF FIGURES (Cont.) Figgre Page 15 16 Growth of Tetrahymena pyriformis W in standard tryptone media with various initial pH levels . . . . . . . . . . . . . . . . 56 Acid phosphatase activity of Tetrahymena pyriformis W cultured in standard tryptone media at various initial pH levels. . . . 59 viii LIST 9; TABLES Table Page 1 Stability of azo dye patterns resulting from enzyme activity . 18 2 Acid phosphatase activity of'I. pyriformis during synchronous diViSion O O O O C O O O G O O O O O O O O O O O O O C C O O O 32 3 Changes in pH of culture media during 30-day culture period. (Series D) . . . . . . . . . . . . . . . . . . . . . . . . . . 54 4 Changes in pH of culture media during 30-day culture period. (series E) 0 O O O O O O O O O O O I O O O O O O O O C O O 0 O 58 ix Plate .LIST OF PLATES Page Apparatus for zone electrophoresis . . . . . . . . . . . . . . 11 Specimens of Tetrahymena pyriformis W fixed in cold absolute acetone for 24 hours. Acid phosphatase activity localized ac- cording to the procedure of Gomori (1952). . . . . . . . . . . 89 Figure 17 Cells from l-day culture. Localization with glycerophosphate. Figure 18 Cells from 3-day culture. Localization with glycerophosphate. Figure 19 Cells from 3-day culture. Localization with glucose-l-phosphate. Figure 20 Control cells from 6-day culture exhibiting false-positive reaction. Specimens of Tetrahymena pyriformis W fixed in cold absolute acetone for 24 hours. Localization of acid phosphatase acti- vity by procedures of Gomori (1952) and Seligman and Manheimer (1949) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Figure 21 Cells from 12-day culture. Localization with glycerophosphate. Figure 22 Cells from 27-day culture. Localization with glycerophosphate. Figure 23 Cells from 15-day culture. Localization with beta-naphthyl phosphate and anthraquinone-l- diazonium chloride. Figure 24 Cells from 18-day culture. Localization with alpha-naphthyl phosphate and anthraquinone-l- diazonium chloride. Specimens of Tetrahymena pyriformis W fixed in cold acetone and electrophoretic pattern of components possessing acid phos- phatase activity. Localization by method of Seligman and Man- heimer (1949). . . . . . . . . . . . . . . . . . . . . . . . . . 93 Figure 25 Cells from 3-day culture. Localization with alpha-naphthyl phosphate and diazotized 3,3'- dichloro-4,4'-diamino~bephenyl.. X Plate 4 (Cont.) Figure 26 Figure 27 LIST 9;: PLATES (Cont. ) Cells from 15-day culture. Localization with alpha-naphthyl phosphate and diorthoanisidine. Acid phosphatase pattern following zone electro- phoresis. Localization with alpha-naphthyl phos- phate and Fast Garnet GBC.. xi INTRODUCTION The fundamental processes of all living material can be thought of in terms of an orderly functioning of enzymes. Todd (1959) points out that our knowledge concerning the role of organic phosphate and poly- phosphate esters in biological processes has increased greatly during the past 15 to 20 years. Numerous enzymes involved in the metabolism of these compounds have been studied and it is reasonable to expect that further elucidation of enzymes involved in phosphate metabolism will appear in the future. Acid phosphatases have been identified in microorganisms and a variety of plant and animal tissues. Elliot and Hunter (1951), who used adenylic acid as a substrate, demonstrated that phosphatases of Tetrahymena exhibit maximum activity at two pH levels, i.e., 5.3 and 9.2. These ' authors suggested that this enzyme system may play a role in the release of inorganic phosphate from the decomposing organic material present in the natural environment of the organism. The cytochemical procedures employed by Fennell and Marzke (1954) demonstrated a discrete localiza- tion and a general increase in acid phosphatase activity in aging cultures of Tetrahymena. Hunter (1959) studied acid phosphatase in Paramecium and maintained that enzyme activity is confined to the mitochondria. Rothstein and Meier (1948, 1949) identified phosphatases on the sur- face of living yeast cells. The hydrolysis of phosphorylated compounds, i.e., glycerophosphate, phenylphosphate, adenosine diphosphate, adenosine triphosphate, and pyrophosphate, occurred at an optimum rate in an acid solution. 0n the basis of enzyme activity at various pH levels and com- petition experiments, it was suggested that several enzymes might be invol- ved in hydrolysis of these phosphate esters. Gomori (1952) maintained that there is an overlapping of acid and alkaline phosphatase activities. Brandes and Elston (1956) indicated that alkaline phosphatase exists in the cell wall of Chlorella vulgaris. However, these authors did not rule out the possibility that the reaction was due in part to acid phosphatase. The observations of Taylor (1960) indicate that maximum uptake of glucose by Scenedesmus occurs at pH 4.5 to 5.5. The inhibitory effects of phloridzin and 2,4-dinitrophenol lend support to the hypothesis of Lundsgaard (1933) that a phosphorylation-dephosphorylation mechanism may be involved in active absorption of glucose. The localization studies of Wachstein (1955) demonstrated acid phos- phatase in the loops of Henle, collecting tubules, and glomeruli of the kidney. Beck (1942), who investigated the effect of 0.01 M phlorizin ont' acid phosphatase and hexokinase activities in kidney extracts, noted that the rate of inorganic phosphate released from glycerophosphate at pH 5.0 was markedly decreased at this concentration. At pH levels approaching 7.0, phlorizin had little inhibitory effect on dephosphorylation. Beck maintained that acidnphosphatase was probably not involved in phlorizin- inhibition of glucose re-absorption, since phlorizin did not inhibit dephos- phorylation at the normal intracellular pH of 6.9. Marsh and Drabkin (1947) demonstrated _i_._r_t_ 31.33 and it; 1&9. inhibition of phosphatases of the kidney by phlorizin. These results should possibly be re-evaluated in the light of the recent contributions of Morton (1958) and Nigam and Fishman (1959), who demonstrated that acid phosphatases may also function as transferases. The experiments by Brachet (1955) on Amoeba proteus indicate that phosphatase activity decreases when the nucleus is removed. This suggests that the maintenance of cytoplasmic acid phosphatase is highly dependent on nuclear control. 3 A correlation between morphogenetic pattern and acid phosphatase activity of root epidermis from festucoid and panicoid grasses was noted by Avers and Grimm (1959). The fact that trichoblasts exhibit greatest enzyme activity prior to root hair formation, while hairless cells possess little or no acti- vity, suggests that acid phosphatases may be functional in morphogenesis. Moog (1943) studied the development and distribution of phosphatases in the spinal cord of chick embryos of various ages. The typical localization pattern shifted, as the age of the embryo increased, from the dorsal half to the ventral half of the cord, particularly to the motor groups of cells. This author suggested that phosphatases may function in morphogenesis in young embryos whereas in older embryos they may take part in metabolism of such sub- strates as carbohydrates. Vorbrodt (1958) believes that acid phosphatases are involved in protein synthesis, since increased enzyme activity was observed in reticulocytes, tumor cells, fibroblasts, regenerating liver, and pancreatic tissue. On the other hand, acid phosphatase was lower in cells such as erythrocytes. Sulkin and Gardner (1948) compared the distribution of acid and alkaline phosphatases in normal and regenerating liver. They noted that the cytoplasmic acid phosphatase activity was variable in normal liver while nuclear acid phos- phatase was more constant. Increased nuclear phosphatase activity in regen- erating liver was cited as additional evidence for support of the view that phosphatases may be important in nucleoprotein metabolism. The prostate gland is one of the richest sources of acid phosphatase. Abul-Fadl and King (1948) demonstrated that the prostatic enzyme differs from acid phosphatases from other sources, i.e., plasma, erythrocytes, in that the former is selectively inhibited by tartrate. Further, this enzyme is also inhibited by polyxenylphosphate (Humel,lg£.gl., 1958), polyestradiol phosphate, and the polymeric phosphates of phloretin, phlorrhizin, and hesperidin (Beling,.gg‘§l.,l959);. Gutman and Gutman (1938) noted that the prostate of children con- tained little acid phosphatase whereas the gland of adult males exhibited higher enzyme activity than other organs. Under normal conditions, pro- static phosphatase does not enter the circulation but following metastasis or rupture of the prostatic capsule total serum acid phosphatase is ele- vated significantly. Andersen (1959) states, ”this phenomenon is patho- gnomonic to metastasizing carcinoma of the prostate." The recent work of Kurtz and Valk (1960) demonstrated considerable variability in acid phos- phatase levels in cases of either bony metastases or benign prostatic hypertrophy. Contradictory reports in regard to phosphatase levels have appeared in the literature during the past 2 decades and the evidence to date indicates that the full significance of elevated levels of acid phosphatase activity is incompletely understood at the present time. A different approach to the study of acid phosphatases was under- taken by deDuve and his co-workers (Berthet and deDuve, 1951). They noted that when rat liver was fractionated in 0.25 M sucrose solution that acid phosphatase was restricted primarily to the mitochondrial fraction. When the classical mitochondrial fraction was further analyzed, greatest activity was found in the ”lysosomal" fraction (deDuve g£_§l,,1955)}- Identification of the enzyme in the soluble fraction following homogeni- zation of tissues in the Waring blender or after exposure to detergents led these workers to postulate that the soluble enzyme was normally con- fined to the lysosomes by an external limiting membrane. Evidence sup- porting the hypothesis that the lysosomal membrane is of a lipoprotein nature was obtained when lecithin, trypsin, and chymotrypsin Were found to change the enzyme from a sedimentable to a non-sedimentable phase (Beaufay and deDuve, 1959). Additional studies demonstrated that beta-glucuronidase, cathepsin, acid deoxyribonuclease and acid ribonuclease were also associated with acid phosphatase in the lysosomes (deDuve, g£.;§l.,l955,, Wattiaux £5.3l3—1956);- Recent studies suggest that the release of the soluble lysosomal hydrolases of liver may be induced in yigg by ischaemia and various hepato- toxic treatments.(Beaufay g£_§l,,1959)§. These results have been inter- preted as evidence for the hypothesis that the enzymes may play a role in the initiation of autolytic and necrotic processes (deDuve, 1959). It is evident from the literature that there is a great diversity of opinion as to the nature and role of acid phosphatases in normal or pathological systems. There is also a considerable difference of Opinion as to the validity of the cytochemical techniques employed for the locali- zation of activity of these enzymes (Lassek, 1947; Newman, 35 £13,1950;, Bourne, 1951). It would appear from the evidence to date, however, that acid phosphatases may be involved in one, or a combination, of the follow- ing roles: (l) cellular proliferation, (2) the absorption or utilization of metabolites, such as carbohydrates, and (3) autolytic and necrotic processes. The purposes Of this investigation are to evaluate the various methods for the cytochemical localization of acid phosphatase activity and to ascer- tain whether increased levels of enzyme activity are correlated with cellu- lar proliferation, carbohydrate uptake, or autolytic processes. MATERIALS AND METHODS Tetrahymena pyriformis W was used exclusively as a test organism in this study. Cultures of organisms were established for experimentation by transfer of 1.0 ml portions of a culture solution from a bacteria-free stock culture to 125 m1 Erlenmeyer flasks containing 75 ml of sterile cul- ture medium. The medium consisted of 10.0 gm Bacto-tryptone (Difco Labora- tories, Detroit, Michigan), 1.0 gm glucose, 1.0 gm sodium acetate, 1.0 gm potassium monobasic phosphate, 1.0 gm potassium dibasic phosphate, 0.25 microgram thiamine hydrochloride, and 1.0 liter glass-distilled water. In succeeding pages, this medium will be designated as standard tryptone medium. All media were autoclaved at 1219C and 15 pounds pressure for 20 minutes. Cultures were maintained in a constant temperature room at 23° to 25°C.. Localization gxperiments. For localization studies, samples of or- ganisms were taken at 3-day intervals from bacteria-free cultures and centrifuged in 15 m1 centrifuge tubes at 386 X g for 5 minutes. The super- natant fluid was decanted, the organisms were suspended in distilled water, and concentrated by centrifuging. The washed cells were deposited on albumin-coated slides and were air-dried. After adherence of organisms to the slide surface, they were quickly immersed in either cold acetone or chilled 10% formalin and fixed for 18 to 24 hourss. The procedures of Gomori (1952) and Seligman and.Manheimer (1949) were used for the localization of acid phosphatase activity. With the Gomori method, slides were incubated for 1 hour at 37°C in a buffered sub- strate mixture containing 0.01 M sodium beta-glycerophosphate and 0.004 M lead nitrate in 0.2 M acetate buffer (pH 5.2), Following incubation, slides were washed in tap water, immersed in a dilute ammonium sulfide solution, counterstained with eosin, and mounted in Canada balsam. In cases where glucose-l-phosphate and fructose-6-phosphate were employed as substrates, the incubation procedure was similar to that described above except that these substrates were substituted for glycerophosphate. With the azo dye procedure, slides werue incubated for 15 minutes in 50 ml of acetate buffer (pH 5.2) containing 15 mgm sodium beta-naphthylphos- phate and 25 mgm of either anthraquinone-l-diazonium chloride or tetrazotized-O- dianisidine (Diazo Blue B). After the incubation period, the organisms were covered with cover slips using Kaiser’s medium (8 gm gelatin, 1.0 gm phenol, 42 ml water, 50 ml glycerine) as an aqueous mounting medium. Control slides, in both procedures, were immersed in: (1) 0.1 M sodium fluoride for 30 minutes prior to incubation; (2) water at 80°C for 10 minutes prior to incubation, and (3) incubation solutions in which substrates were omitted. Preparation gf coupling agents and substrates. Diazotized salts were prepared from the following organic compounds by the procedure of Vogel (1957): 2-amino-4'-nitro-bipheny1, 3,3'dichloro-4,4'-diamino-biphenyl, beta-amino-alpha-naphthalene sulfonic acid, m-nitro-p-toluidine, 4-chloro-2-nitroaniline, 2-chloro-5-nitroaniline, 4-methy1-0-sulfanilic acid, 3-methyl-4-chloro-0-su1fanilic acid, 4-methy1-5-chloro-O-su1fanilic acid. After diazotization, the pH of the solution was increased to 5.0 by the addition of ammonium hydroxide and appropriate aliquots were transferred to coplin jars containing the buffered substrate solutions. All pH deter- minations were made electrometrically with a Beckman model H2 glass elec- trode pH meter. The procedure of Burstone. GUSSSD was used for synthesis of naphthyl AS-BI and naphthyl AS-TR phosphates. Naphthols were generously supplied by Verona Dyestuffs (Union, gNew Jersey). Subsequent to phosphorylation and evaporation of the solvent, the reaction mixture was added to cold water containing an excess of sodium carbonate for conversion of the phosphates to their disodium salts. The precipitates were collected under suction and dried in a desiccator containing anhydrous calcium chloride. Substrates prepared in this manner were used for the localization of enzyme activity. Determination 2f glucose. Estimation of glucose was made by the quantitative measurement of reduction of 3,5-dinitrosa1icylic acid as given by Carter ggugl. (1957). The colorimetric readings for this procedure were taken at 540 millimicrons in a Bausch and Lomb Spectronic 20 colori- meter. The determinations were performed at 3-day intervals on 0.5 m1 and 1.0 m1 aliquots of culture medium after the cells were removed by_ centrifugation. A standard curve for this procedure was obtained by the use of graduated increments of glucose. Quantitative determination.gf acid phosphatase activity. Quantita- tive estimates of acid phosphatase activity were carried out by a procedure similar to that given by Shinowara, Jones, and Reinhart (1942). Acid phosphatase activity was expressed as micrograms of phosphate liberated per milligram of protein per 30-minute incubation period. Preparation.2£_homogenate. Samples of organisms which were taken for enzyme assay at 3-day intervals were concentrated by centrifugation, the supernatant solution was decanted, and the cells were washed twice with glass-distilled water. The organisms were disrupted by freezing in a dry ice-acetone mixture (-70°C) and then thawing. This procedure was repeated 4 additional times to assure complete disruption of all cells. The cellu- lar material was diluted with glass-distilled water to obtain a 5-10% homogenate. Substrate solution and incubgtion procedure. The substrate solution, which contained 0.5 gm sodium beta-glycerophosphate per 100 m1 of 0.2 M acetate buffer (pH 5.2), was overlayed with petroleum ether and refriger- ated. Four m1 of substrate solution was added to each test tube and all tubes were placed in a water bath at 37‘C for 5 minutes. Two-tenths m1 of homogenate was added to each tube and all tests were performed in dupli- cate. After a 30-minute incubation period, 1.0 m1 of 10% trichloroacetic acid was added to each tube. A substrate control, homogenate control, blank, and standard were used in each experiment. Determination g; inorganic phosphate and protein. After precipi- tation with trichloroacefixzacid, the proteins were separated by centrifu- gation and enzymically released phosphate in the supernatant solution was ascertained by the method of Fiske and Subbarow (1923). A standard curve for infirganic phosphate was obtained with potassium monobasic phosphate. A11 colorimetric readings for phosphate determinations were made at 660 mu with the use of a red filter. Protein determinations were made by use of the modified biuret method of Gornallig§.gl. (1949). A calibration curve for protein was made with crystalline bovine serum albumin (Armour Laboratories, Kankakee, Illinois) as a standard. The colorimetric readings werermadec at 540 mp. Zone electrophoresis. The method used for the electrophoretic characterization of acid phosphatases was that described by Smithies (1955). A starch-gel suspension was prepared from 13.0 gm hydrolyzed starch (Con- naught Mbdical Laboratories, Toronto, Canada) and 100 m1 borate buffer (pH 8.9, 0.153 M). The suspension was heated in a flask over an open flame until it became slightly less viscous. Air bubbles were removed under a vacuum and the suspension was poured into plastic trays (0.75 cm X 2.4 cm X 22.5 cm). The gels were usually prepared 12 hours prior to electrophoretic 10 experiments. Trays were covered with a plastic cover and stored in the refrigerator until use. The samples were inserted by either of two methods: (1) a transverse section was made in the gel with a razor blade and a piece of filter paper with the same cross-sectional dimensions as the gel was impregnated with the homogenate and inserted between the transected surfaces, (2) a trans- verse section of 2 mm in width was removed from the gel. The homogenate was suspended in crude potato starch and introduced into the transected area by means of a pipette. The exposed surface of the homogenate was covered by a small piece of glass (1.0 cm X 2;5 cm). Three filter paper bridges extended from each end of the gel into the electrophoretic buf- fer (pH 8.9, 0.023 M). The electrodes of a variable voltage regulated power supply (The Heath Company, Benton Harbor, Michigan) were immersed in the electrophoretic buffers. A current of 5 ma per starch block and a 6-volt drop per centi- meter were maintained for the 6-8 hour electrophoretic period. The‘discontinuous buffer system suggested by Poulik (1959) was also used in electrophoresis experiments. In this system, the gel was made with a tris-citrate buffer (pH 8l6); a sodium hydroxide-borate buffer (pH 816) was used in the electrophoretic vessels. The identification of enzyme-active areas within the gel strips was made by a procedure similar to the method used by Hunter and Markert (1957). The strips were incubated in buffered substrate solutions containing alpha- naphthyl phosphate (pH 5.2) for 60 minutes. Fast Garnet GBC, Fast Garnet GC, and Fast Violet LB salts were employed as coupling agents (courtesy of Verona Dyestuffs, Union, New Jersey). Synchronization 2f cell division. The procedure used for the synchroni- zation of cell division was essentially the same as given by Scherbaum and ll PLATE 1- Apparatus for zone electrOphoresis. Legend: Power supply; Voltage meter; Electrophoretic buffer vessels; Starch-gel trays with inserted sample; Anode lead wire; Cathode lead wire. Zeuthen (1954). The tubes containing organisms were left in a water- bath at 34°C for 30 minutes and then transferred to a water-bath at 28°C for 30 minutes. This cycling was repeated 6 times and then all tubes were maintained at 28°C. Subsequent to the completion of shock treatments, organisms were examined at 20 minute intervals in order to ascertain the time when division of cells was at a maximum. Amicronucleate strains of Tetrahymena exhibit nuclear division without the usually recognized stages of mitotic division (McDonald, 1958). Acid phosphatase activity, therefore, was ascertained at the divisional stages comparable to those described by Sullivan (1959), namely: interphase, early division, mid—division, late division, and very late division. Cells collected at each stage were homogenized by freeze-thawing and quantitative estimates of acid phosphatase acti- vity were performed as previously described. Direct cell counts. The number of cells per cubic millimeter of culture medium was ascertained by the use of a Sedgewick-Rafter counting chamber and a Whipple ocular micrometer (Scherbaum, 1957). All cell counts given in the succeeding pages represent the arithmetic mean of counts from 3 random samples. Alteration‘gf culture conditions. In series A experiments, organisms were cultures in standard tryptone medium. In experiments described as series B, organisms were cultured in 3 types of media, i.e., standard tryptone medium, standard tryptone medium lacking glucose and sodium acetate, and standard tryptone medium with a final concentra- tion of 1.3 X 10'4 M phloridzin. 14 In experiments designated as series C, cultures were divided into 4 lots of 16 each. Six days subsequent to inoculation, malonic acid was added to lot number 1 (final concentration 10-3 M). Twelve days after inoculation, malonic acid (final concentration 10'2 M) and 2,4-dinitro- phenol (final concentration 10'5 M) were added to lots 2 and 3 respec- tively, and lot 4 was used as a control. All the cultures in series D were allowed to proceed without alter- ation until 9 days following inoculation. After 9 days, the total number of cultures was divided into 5 groups. Group 1 served as a control. Groups 2 through 5, respectively, were treated to give the desired con- centrations of the following compounds: 9 X 10"4 M di-sodium malonate, 8.8 X 10"2 M di-sodium malonate, 10'2 M urethan (ethyl carbamate), and 10'1 M urethan. The standard medium in series E was divided into 3 groups. The pH of the medium of the control group was 7.2, the pH of group 2 was adjusted to 6.0 with N HCl, and the pH of group 3 was adjusted to 7.7 with tris (hydroxymethylaminomethane). Quantitative estimation‘gf beta-glucuronidase activity. The procedure of Talalay 3;.gl., (1946) was employed for estimation of beta- glucuronidase activity. The substrate was prepared by addition of an excess of 2 N HCl to 100 mgm of the cinchonidine salt of phenolphthalein monoglucuronide and the mixture was extracted with two S-ml portions of ethyl acetate. The combined ethyl acetate extracts were evaporated under vacuum. Five m1 of glass-distilled water was added to the residue. The latter was neutralized with N sodium hydroxide and the volume was made up to 10 ml (0.013 M). 15‘ Four ml of buffer was put into each test tube. Acetate buffer (0.1 M) was used for obtaining pH values of 3.8, 4.0, 5.0, 5.6 and Tris buffer (0.1 M) was employed for obtaining pH values of 6.9, 7.4, and 7.8. Five-tenths m1 of 0.013 M sodium phenolphthalein was added to each tube with the exception of the control tube. A 10% homogenate of Tetrahymena (0.5 ml) was added to each tube, the contents were mixed, and they were incubated at 37°C for 60 minutes. After incubation, 5.0 m1 of 0.4 M glycine buffer (pH 10.4) was added to each tube and the intensity of color was read at 540 mp. A phenolphthalein calibration curve was prepared with each tube containing 4.0 ml of 0.1 M acetate buffer (pH 4.5), 5.0 ml of 0.4 M glycine buffer (pH 10.4), and 1.0 ml of various dilutions of phenol- phthalein. 15 Four m1 of buffer was put into each test tube. Acetate buffer (0.1 M) was used for obtaining pH values of 3.8, 4.0, 5.0, 5.6 and Tris buffer (0.1 M) was employed for obtaining pH values of 6.9, 7.4, and 7.8. Five-tenths ml of 0.013 M sodium phenolphthalein was added to each tube with the exception of the control tube. A 10% homogenate of Tetrahymena (0.5 ml) was added to each tube, the contents were mixed, and they were incubated at 37°C for 60 minutes. After incubation, 5.0 ml of 0.4 M glycine buffer (pH 10.4) was added to each tube and the intensity of color was read at 540 mp. A phenolphthalein calibration curve was prepared with each tube containing 4.0 m1 of 0.1 M acetate buffer (pH 4.5), 5.0 m1 of 0.4 M glycine buffer (pH 10.4), and 1.0 m1 of various dilutions of phenol- phthalein. 15 Four ml of buffer was put into each test tube. Acetate buffer (0.1 M) was used for obtaining pH values of 3.8, 4.0, 5.0, 5.6 and Tris buffer (0.1 M) was employed for obtaining pH values of 6.9, 7.4, and 7.8. Five-tenths ml of 0.013 M sodium phenolphthalein was added to each tube with the exception of the control tube. A 10% homogenate of Tetrahymena (0.5 ml) was added to each tube, the contents were mixed, and they were incubated at 37°C for 60 minutes. After incubation, 5.0 m1 of 0.4 M glycine buffer (pH 10.4) was added to each tube and the intensity of color was read at 540 mp. A phenolphthalein calibration curve was prepared with each tube containing 4.0 m1 of 0.1 M acetate buffer (pH 4.5), 5.0 ml of 0.4 M glycine buffer (pH 10.4), and 1.0 ml of various dilutions of phenol- phthalein. 16 RESULTS Localization Experiments Gomori method. The Gomori procedure, in which beta-glycerophosphate was used as a substrate, resulted in localization patterns which were con- fined to a definite cytoplasmic location. Cells from 1- to 3-day cultures exhibited black precipitates of lead sulfide which appeared to be confined to small, spherical cytoplasmic entities (Figures 17, 18). When glucose-l- phosphate and fructose-6-phosphate were used as substrates, essentially the same localization patterns were observed. In fact, no differences in intensity of precipitate or localization were distinquishable between these substrates (Figures 18, 19). Cells from 6-, 9-, and 12-day cultures appeared to increase in size and exhibited a larger number of enzyme- active centers within the cytoplasm. A gradual change in shape of the cells was noted as the age of the cultures increased from 15 to 30 days. The cells changed from a pyriform to a more spherical shape. Cellular debris, from autolyzed cells, began to accumulate on the bottom of the culture flasks. Changes in the pat- tern of acid phosphatase activity appeared to accompany the morphological changes. In older cultures, enzyme activity appeared to shift from the vesicular cytoplasmic pattern to a pattern that was more diffuse and general throughout the entire cytoplasm (Figures 21, 22). Occasionally, diffuse black precipitates were observed on the outer pellicular surface of the cells. Cells on control slides were invariably devoid of the typical cytoplasmic precipitates of Lead sulfide.lvoccasidnally,uhowever, they exhibited a very Small black precipitate which was Localized in the pOSterior portion ofochefcehlsc(Figure“20)3 17 A20 dye method. The localization patterns obtained by use of the azo dye procedures differed considerably from those obtained with the Gomori procedure. When anthraquinone-l-diazonium chloride was used as a coupling agent with either alpha- or beta-naphthyl phosphates, the resultant orange-brown precipitates displayed a very diffuse cytoplasmic pattern (Figure 23). The dye was readily soluble in alcohol and diffused throughout the entire cell, extending into extracellular areas. Dif- fusion of the dye was somewhat retarded by immersion of slides in alka- line solutions of barium carbonate and mounting in an aqueous mounting medium; however, diffusion readily occurred within an hour after the localization procedure was completed. (Figure 24). The localization pattern obtained when diazotized-O-dianisidine was used as a coupler was superior to that described in the preceding paragraph. Nevertheless, this pattern was tuat as definite as that observed with the Gomori procedure. (Figure 26). A summary of the stability of the azo dye pattern obtained with each of the diazotized couplers when alpha-naphthyl phosphate was used as substrate is shown in Table 1. It is evident from Table l as well as from Figure 25 that less diffusion occurred when 3,3'-dichloro-4,4'- diamino-biphenyl and alpha-naphthyl phosphate were used than when other combinations were employed. Further, enzyme-active areas obtained by this procedure were localized in essentially the same regions of the cytoplasm as those obtained by the Gomori procedure. Enzyme-active areas were never observed in control organisms. Enzyme-active centers were never observed when naphthol-AS deri- vatives were employed as substrates. The latter compounds were highly soluble in N,N-dimethylformamide but, on the addition of acetate buffer TABLE I Stability of azo dye patterns resulting from enzyme activity Structure of Stability of Amine , , Localization Diazonium Ion Pattern O l-amino-anthra- poor quinone O / \ diorthoanisidine N+ ' N+ fair 2 Z (SI-I30 OCH3 - ' - t- ' _ 2 antino 4 nitro O N fair biphenyl Z + N2 3, 3' -dichloro-4,4'- + ood diamino- N2 N g biphenyl C1 C1 SO3H beta-amino-alpha— N naphthalene- poor sulfonic acid m-nitro-p- poor toluidine TABLE 1 (cont.) Structure of Stablhty 0f Amine ' , , Localization Diazonium Ion Pattern 4- chloro- 2 - nitroaniline poor 02 Z-chloro-S- N+ nitroaniline 2 poor Cl SO3H 4-methyl-0- + sulfanilic CH N poor . 3 2 ac1d SO3H + 3-methyl-4-Chloro- C1 ‘ N2 O-sulfanilic acid \ / POOI‘ CH 3 4-methyl- 5-chloro- poor 0- sulfanilic acid 20 (pH 5.2) to the substrate solutions, a chalky-pink precipitate was formed. Specimens of Tetrahymena, when incubated in the latter, did not exhibit localization patterns even though the incubation time was ex- tended several hours. Calibration Curves A calibration curve for glucose was obtained by reduction of 3,5-dinitrosalicylic acid with 1.0 m1 aliquots of various concentrations of glucose. It is evident in Figure 1 that the optical density read- ings obtained following the addition of fixed increments of glucose are essentially linear. Figure 2 shows that when fixed increments of standard protein are added to the biuret reagent, the optical density readings are directly related to the concentration of protein. Since homogenates of Tetrahymena are slightly turbid, an experiment was designed to deter- mine whether turbidity would introduce errors into protein determination. The amount of protein (in milligrams) in various aliquots of a 10% homo- genate of Tetrahymena (from 0.1 ml to 0.5 ml), as interpreted from the standard curve, was plotted against the quantity of homogenate used in each determination. It is evident from Figure 3 that the concentration of protein in various aliquots of cellular homogenate is directly propor- tional to the amount of homogenate used. A calibration curve for inorganic phosphate (Figure 4) was ascer- tained by the method of Fiske-Subbarow (1925). It is evident that the concentration of inorganic phosphate exhibits a linear relationship to the optical density readings. 21 FIGURE 1 Calibration curve for glucose obtained by reduction of 3,5-dinitrosa1icylic acid with 1.0 m1 aliquots of various concentrations of glucose. Ordinate, optical density at 540 millimicrons; abscissa, micrograms glucose. 0.9 0.8 i L . i p 7 IO 5 4 O. 0. 0 0. d2 cam wfimzmo qan o.~m o.wm o.ow mousafia Qua puma kuo> H.mNH «.mmH m.oHH sausage oNH cowmw>wm mama o.moH w.~m m.¢HH nouoawa ooH oowmw>fiunu«a H.mm ¢.ww w.mw mouoafia om oonH>Hv waned a.aw N.mw n.bm sauna afinnnannasa annaannnta c.¢mH w.wm~ ¢.omH comm up vooHMuofioa. Houuooo oHuwMMWHum N H ..oHS on Noaououm Ema \maws uooaumouu nouwo mafia ammum .oowma>fim wooaousoahm magnum mwahomaumm .H.mo hua>uuoo omoumnmmono ufio uGM P/ MGM PROTEIN/ 30 MIN. 49 of control cultures from 12 to 18 days and then appeared to remain relatively low. Table 3 shows the relationship between hydrogenéion concentration of the medium and age of control cultures and cultures to which malonate and urethan were added. The initial pH of all culture media was 7.2. It can be seen that the pH of the culture media in the control group increased from 7.2 in 3-day cultures to 8.2 in 30-day cultures, whereas those from malonate (9 X 10-4) and urethan (10"2) increased to pH 8.3. The media of cultures with lowest acid phosphatase activity, i.e., those treated with the highest concentrations of malonate and urethan, were pH 7.5 and 7.6, respectively, at 30 days. Series E. Cultures within this series were divided into 3 groups based on the initial pH of the media. Figure 15 indicates that the number of cells in control cultures (initial pH 7.20) reached a maximum at 15 days subsequent to inoculation and then decreased rather rapidly to a minimum at 30 days. The number of cells in cultures with an ini- tial pH of 6.00 reached a maximum at 9 days and was consistently higher from 18 to 30 days than in the other two groups. It is evident that the number of cells in culture media with an initial pH of 7.65 was lowest at all culture ages. Cells within the latter media appeared very small and cellular debris accumulated on the bottom of the culture flasks as the culture age increased from 15 to 30 days. Table 4 shows that the pH of the media of groups 1 and 3 decreased slightly for the first 6 days after inoculation and then gradually in- creased to pH 7.80 and 8.00, respectively, at 30 days. The pH of the media of group 2 cultures increased more rapidly from pH 6.00, at the time of inoculation, to pH 7.50 after 30 days. 50 FIGURE 13 Growth of Tetrahymena pyriformis W in various culture media. Series D. Ordinate, cells per cubic millimeter culture medium; abscissa, culture age in days. Legend: Standard tryptone medium; ----- Standard tryptone medium containing 9 X 10-4 M di-sodium malonate; . . . . Standard tryptone medium containing 8.8 x 10’2 M di-sodium malonate; -°-----'- Standard tryptone medium containing -2 10 M urethan; -...-...- Standard tryptone medium containing 10"1 M urethan. mwawfl ZH MO< HMDBADU m: Id m~ NH d 00m 00¢ 000 com CELLS PER CUBIC MILLIMETER 50 FIGURE 13 Growth of Tetrahymena pyriformis W in various culture media. Series D. Ordinate, cells per cubic millimeter culture medium; abscissa, culture age in days. Legend: Standard tryptone medium; ----- Standard tryptone medium containing 9 X 10'° M di-sodium malonate; . . . . Standard tryptone medium containing 8.8 x 10'2 M di-sodium malonate; -'------- Standard tryptone medium containing -2 10 M urethan; --------- Standard tryptone medium containing 10.1 M urethan. mw<0 ZH MU< MMDHJDU CON 00¢ 000 com CELLS PER CUBIC MILLIMETER 52 FIGURE 14 Acid phosphatase activity of Tetrahymena pyriformis W cultured in various media. Series D. Ordinate, acid phosphatase activity in micrograms phosphate liberated per milligram protein per 30-minute incubation period; abscissa, culture age in days, Legend: Standard tryptone medium; Standard tryptone medium containing 9 X 10’4 M di-sodium malonate; Standard tryptone medium containing 8.8 X 10-2 M di-sodium malonate; Standard tryptone medium containing -2 10 M urethan; Standard tryptone medium containing -1 10 M urethan. WW¢HH7nHHDZmeEPBJHKu ‘1? .1 .1 .1 1 I L SN 4 00¢ 4 8a A com 000a r i acme \ 2....1. uGM P/ MGM PROTEIN/ 30 MIN. TABLE 3.7:Changes in pH of culture media during 30-day culture period. culture age pH in days control 9 x 10'4 M 8.8 x 10'2 M 10'2 M 10'1 M malonate malonate urethan urethan 3 7.20 -- -- -- -- 6 7.20 -- -- -- -- 9 7.20 -- -- -- -- 12 7.33 7.23 7.30 7.30 7.20 15 7.32 7.35 7.35 7.30 7.22 18 7.40 7.43 7.30 7.45 7.30 21 7.50 7.60 7.30 7.60 7.35 24 7.50 7.60 7.40 7.70 7.40 27 -- -- -- -- -- 30 8.20 8.30 7.50 8.30 7.60 55 Figure 16 indicates that acid phosphatase activity of Tetrahymena was highest from 6 to 18 days in cultures with an initial pH of 7.65 and lowest in cultures with an initial pH of 6.00. Electrophoresis Studies The results of electrophoresis experiments indicate that T2332: hymena possesses at least 5 components with acid phosphatase activity (Figure 27). Good separation of enzymes was obtained when the current was applied for a period of 6 hours. Shrinkage of the starch-gel blocks occurred when the discontinuous buffer system of Poulik (1959) was employed. Consequently, the borate-buffer system of Smithies (1955) was used in all further experiments. The results of quantitative determination of acid phosphatase activity in aging cultures (previously described) indicated that enzyme activity increased significantly as the culture age increased. No dif- ferences were observed, however, between the electrophoretit pattern of acid phosphatases of organisms from young and old cultures. Inhibition studies disclosed that all 5 components are inhibited by 10"1 M sodium fluoride, 10-1 M.semicarbazide hydrochloride, and 10'3 M ammonium.molybdate. Only one component was inhibited by 10"1 M sodium arsenate. No inhibition was observed with 10"2 M sodium oxalate, 10"1 M phloridzin, or 10"2 M L-tartaric acid. Beta-Glucuronidase Assay Beta-glucuronidase activity could not be demonstrated in homogen- ates of Tetrahymena over a pH range of 3.8 to 7.8. 56 FIGURE 15 Growth of EEIIEDXEEEEHRZLILQEEIE W in standard tryptone media with various initial pH levels. Series E. Ordinate, cells per cubic millimeter culture medium; abscissa, culture age in days. Legend: Initial pH 7.20; ----- Initial pH 6.00; . . . . . Initial pH 7.65. mw