I“ ! M ”I I ‘ U } { I! l l ‘ | I N N H! l \ $ 1 l §§ IH THS SOME OBSERVATQON$ 02% THE LOCALiZATEON, SLEESTRATE SPECEFNEW; AND THE EFFECT OF LNHEB‘ITORS ON EWERASES EN TETRAHYMENA PYRIFORMIS W ”Hosts for H10 Degree of M. S MICHIGAN STATE UNEVERSI'EY Eliezer P. Pastor 3958 1 :1?‘ 3“?) SOME OBSERVATIONS ON THE LOCALIZATION, SUBSTRATE SPECIFICITY, AND THE EFFECT OF INHIBITORS ON ESTERASES IN TETRAHYMENA fiYRIFORMIfi w By Eliezer P. Pastor A THESIS Submitted to the School of Graduate Studies of Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1958 ACKNOWLEDWTS The author wishes to express her sincere thanks to Dr. Richard.A. Fennell for his sug- . gestion of the researcn problem and also for his guidance, interest and-constant encourage- ment during the course of this investigation. The author is also indebted to Mr. P. G. Coleman, Photographer, Agricultural Experiment Station, for the photomicrographs. SHRHHHHHHHHHHRH? ii SOME OBSERVATIONS ON THE LOCALIZATION, SUBSTRATE SPECIFICITY, AND THE EFFECT OF INHIBITORS ON ESTERASES IN TETRAHYMENA PYRIFORMIS W ' By Eliezer P. Pastor AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Zoology 1958 I I [Q]... i: if 11/ "-" l1\ A‘pproved “-4! LA 1111. JJK'L'J; Ile-kcf L. (Err-r THESIS ABSTRACT . Cultures of Tetrahymena pyriformis W were established and reared axenically by inoculating Erlenmeyer flasks (125 ml) containing 75 ml of sterile Bacto-tryptone culture medium with 1 ml of inoculum from‘ bacteria-free heavily populated stock cultures 3 to 25 days subsequent to seeding. ' ‘Test organisms were fixed in 10% formalin at h degrees centigrade for 10 to 15 minutes and stained for esterase activity according to Gomori‘s Tween (5h5), cholinesterase (?h8), and modified Azo dye ('52) procedures; Nachlas and Seligman‘s Azo dye technique ('h9); and Koelle and Friedenwald's modified thiocholine technique ('h9).‘ Enzymatic activities were obtained with saturated Tweens G-7596j, Tween to and Tween 60 but not with Tween 80 which is an unsaturated fatty acid ester. Pattern of localization obtained with all three saturated Tweens was essentially similar. The complete suppression of the hydrolysis of Tween 60 by Na taurocholate is interpreted as evi- dence that the enzyme responsible for its hydrolySis is a non-specifiC' esterase and not a lipase. Apparently there is no true lipase in Tetrahymena as indicated by the absence of activity towards Tween 80. Enzymatic hydrolysis of alpha naphthyl acetate, beta naphthyl acetate and naphthyl AS acetate were also observed. The localization picture with alpha naphthyl acetate was similar to that obtained with iv the Tweens. However, the inhibitory effects induced by various in- hibitors upon their hydrolysis showed considerable differences. Beta naphthyl acetate and naphthyl AS acetate gave two types of staining reactions: a purple precipitate localized at the posterior ends of the organisms and a pink precipitate localized at the anterior ends of the organisms. Cholinesterases in E, pyriformis W were found which hydrolyzed myristoylcholine, lauroylcholine, and benzoylcholine. Myristoylcholine gave more intense reaction than the latter two substrates. Eserine sulfate at 10-4 M was without inhibitory effects upon the hydrolysis of myristoylcholine. An 80-fold increase in the concentration of eserine was required to effect a heavy inhibition on the enzyme activity. Hydrolysis of Tween 60 was completely suppressed by Na taurocholate, quinine hydrochloride, and by benzaldehyde but not by Na arsanilate. Enzymatic activity with alpha naphthyl acetate was completely inhibited by quinine hydrochloride and benzethonium chloride; heavily inhibited by Na taurocholate, Na arsanilate and by 8 x 10-3 M eserine; moderately inhibited by pontocaine hydrochloride; and slightly inhibited by Na fluoride. No inhibitory effects blef4 M eserine and by benzaldehyde were noted. I Hydrolysis of myristoylcholine was completely inhibited by ponto— caine hydrochloride and heavily inhibited by'8 x 10"4 M eserine but not by 10-4 M eserine. Inhibitory effects by Na fluoride were erratic. Esterases in Tetrahymena showed considerable resistance to fixation in formalin but not in acetone. TABLE OF CONTENTS SECTION Page I INTRODUCTION 1 III RESULTS.........;......................................... 9 Iv DISCUSSION 26 I SUMMARY in VI moss A9 LIST OF TABLES TABLE Page I Histochemistry-of esterases in 2. pyriformis W.. . . . . . .. . . . . . 22 II The effect of inhibitors on the esterases of l. mifornfis W 25 vii LIST OF PLATES PLATE Page . .. I Specimens of T. iforImLS W. fixed in 10% formalin for 15 minutes and stained Tor esterase activity according to Gomori's Tween procedure (”45) and Gomorifis modification Of the AZO dye tecmq‘le ('52)........0.0.0.9...00000......00 S6 Fig. l. Localization of esterase activity with 0-7596); Fig. 2. Localization of esterase activity with Tween ho; Fig. 3. Localization of esterase activity with Tween 60; Fig. h. Localization of esterase activity with alpha naphthyl acetate . II Specimens of "_I‘_. iformis W fixed in 10% formlin for 15 minutes and stain'é'd for esterase activity with beta naphthyl acetate and naphthyl AS acetate; test organisms fixed in cold acetone and in 10% formalin for 2h hours and stained for esterase activity with alpha naphthyl aceuteOOOOOCOOCOOOOOOCOOOOOOOOOOOOOOOOOO...OOOOCOOOOOOOOOOO 58 Fig. 5. Localization of esterase activity with beta naphthyl acetate; Fig. 6. Localization of esterase activity with naphthyl AS ac state; Fig. 7 . Esterase activity after fixation in cold acetone for 2).; hours; Fig. 8. Esterase activi after fixation in 10% formalin for 2 hours. viii LIST OF PLATES ( Cont.) PLATE . Page III? Specimens of T. ormis W fixed in 10% formalin for 15 minutes anH'SEEIEEH-TEF'cholinesterase activity according to Gomori's cholinesterase procedure ('h8)....... 60, Fig. 9 and 10. Localization of cholinestenase activity with myristoylcholine chloride; Fig. 11. Localization of cholinesterase activity with benzoylcholine chloride; Fig. 12. Localization of cholinesterase activity with laurqylcholine chloride. IV Test organisms fixed in.10% formalin.for 15 minutes and incubated in substrate solutions containing inhibitors..... 62 Fig. 13. Inhibition of esterase activity with alpha naphthyl cetate by Na arsanilate (8 x 10 ); Fig. 1h. Inhibition.of esterase activity with alpha naphthyl acetate by Na taurocholate (2 x 10'3M); Fig. 15. Inhibition of esterase activity with myristqylcholine chloride by eserine sulfate (6 x 10’3M). INTRODUCTION A review of the literature on the esterases reveals that only a few detailed observations have been made concerning this group of enzymes in lower forms of animal life. Seaman and Houlihan (‘51) made a general survey of enzyme systems in Tetrahymeng_geleii S and found an acetylcholinesterase which was correlated with ciliary action. These authors maintained that eserine and diisopropylfluorophosphate(DFP) at concentrations (e.g., 3.85 x 10-3 M) reversibly inhibited ciliary activity in Titrahymena, frog esophagus, and Egggs_gills. Seaman (751) fractionated Tetrahymena and demonstrated that acetylcholinetterase was restricted to the ciliated pellicular fractions. These observations ‘were in general agreement with those of Engelman (1880) who maintained that conduction along the fibrillar structure (a system of fibers which connects the base of individual cilium in the pellicle) was similar to conduction along nerve fibers. Fennell and Marzke (35h), employing the Tween technique, demonstrated esterase activity in Specimens of Tetgghmmena galeiiiW”which.was localized and restricted to the cytoplasm iimnediately adjacent to the nuclei. I Augustinsson and Gustafson ('h9) measured esterase activity in eggs of Paracentrotus lividus at various stages of development. Cholinesterase activity was negligible in.both unfertilized eggs and fertilized eggs (for the first few hours subsequent to fertilization) but it increased sharply at the time ciliated tufts appeared in the Echinoplutei larvae. Hawkins and Mendel ('hé) demonstrated acetyl- cholinesterase activity in cellular extracts of Planaria. The enzyme differed from the true cholinesterase in mammalian red blood cells and vertebrate brain in that enzyme activity increased by increasing the concentration of acetylcholine in the substrate solution. Eserine (lo-7M), which almost completely inhibits true Cholinesterase in verte- brate tissues, was found to have no appreciable effect on esterase activity in Plating eoctracte in optimin concentrations of acetylcholine and acetyl-b-methylcholine. .Hawkins and Mendel (3529,) identified a true cholinesterase in frog brain which resembled specific Cholinesterase in mammalian tissues in its activity-acetylcholine concentration relationship but differed from it in that it was eserine resistant. Sawyer ('h3,'h3a), who studied development and distribution of Cholinesterase during the entire larval life of Amblystoma punctatum, found a close correlation between enzyme content and motility. Esterase activity was low in.premotile stages (although concentrated in nervous and muscular tissues), higher in swimming stage embryos, and highest in older embryos (for several days subsequent to the beginning of feeding). Cholinesterase activity in developing Melanoplus differentialis eggs was investigated by Tahmisian ('h3). He found that during early prediapause, i.e., from the day of laying until the seventh day of development, grasshopper eggs contained no detectable cholinesterase. Enzymogenesis, which was noted on the seventh day of prediapause development, was observed to be closely correlated with neuroblast differentiation. Esterases in higher vertebrate tissues have been extensively investigated since the establishment of the role played by acetylcholine- cholinesterase system in neural transmission (Nachmansohn, ‘39). 0rd and Thompson ('50) found that in tissues where acetylcholine exerted a nicotine-like action (in which it is concerned with transmission to another neurone or to a striated muscle cell) the enzyme present was predominantly true Cholinesterase. .In those tissues where acetylcholine exerted a muscarine-like action both true and pseudocholinesterases were demonstrated. ‘ Hellmann (‘52), and Benz ('5h), employing Koelle's method for the histochemical localization of'cholinesterase, demonstrated activity at the myoneural junctions in cat and rat diaphragm. Denz, who localized both.non-Specific and specific cholinesterase, maintained that non- specific cholinesterase was widely spread throughout muscles of the diaphragm whereas Specific cholinesterase was, for the most part, localized at the myoneural junctions.' Sawyer and Everett (3h9) demonstrated a high concentration of non- specific cholinesterase in salivary and Harderian glands, brown fat, ovary, uterus, liver and blood serum of albino rat. Specific choline- esterase was the predominant esterase in the brain stem, red bone marrow, spleen, thymus, lymph node, skeletal muscle, adrenal cortex and.peri~ pheral nerves. Both acetylcholinesterase and non-choline-splitting esterases were found by Mendel and Rudney (‘h3) in brain tissues of mouse and dog. Ravin, Zacks and Seligman ('53) localized acetylcholine- esterase in neurocytes of the brain, multipolar anterior horn cells of the spinal cord, and ganglion cells of the sympathetic nervous system and myenteric and submucosal plexuses. . .Antopol and Glick (‘hO) demonstrated that Cholinesterase activity is relatively lOW'in cortical cells of the adrenal gland in contrast to great activity in medullary cells. They maintained that the choline- esterase in the medulla may.serve as a control mechanism in.preventing an accumulation of an excess of acetylcholine. Hagen ('56) showed that in the bovine adrenal medulla, the bulk of the acetylcholinesterase activity was located in the microsomal fraction. Gomori ('hS), using Tweens as substrates, demonstrated lipase- esterase activity in many organs such as liver, pancreas, lung, kidney, testicles, epididymis, adrenal, adipose tissue, stomach and small intestine, Nachlas and Seligman ('h9), employing three chromogenic substrates (beta naphthyl acetate, beta naphthyl laurate, and beta naphthyl palmitateestearate), obServed differential rates of hydrolysis of the substrates by kidney, liver and pancreas. Kidney and liver . esterase hydrolyzed acetate and laurate while pancreas enZyme hydrolyzed all three substrates. -lhese observations were interpreted as evidence- for the existence ofxtwo distinct enzymes, 1.6.,.esterase and lipase in»the pancreas. . . A ‘,;ele. t . . Barrett ('52), employing indoxyl acetate as substrate, demonstrated esterase activity in tissues of albino rat. Glick and Biskind ('35) found that in beef adrenal most of the esterase activity was localized in the medulla and glomerulosa. Doyle and Liebelt ('Sh) studied the distribution of esterase in the epithelial and lymphatic tissues of rat, appendix and found maximal concentration in the epithelium. Buno and Merino ('52) observed that early in development of the chick embryo, prior to differentiation of the gut and also of hepatic and panoreatic rudiments, lipase activity was limited to the endoderm of the yolk. sac. Zacks ('Sh) confirmed this observation and also localized cholinesterase in the embryonic axis. Nonsspecific esterase activity was identified, on the basis of beta naphthyl acetate hydrolysis, in the striated border of the duedena of chick embryos and hatched chicks (Richardson, berkowitz, and MOOg, '55). The authors observed that esterase content in the duodenal epithelium of starved chicks was consistently higher than in that of the fed chicks. Since the location, time of appearance,' and rate of accumulation of either non-specific esterase or alkaline phosphomonoesterase in late embryonic stages were essentially the same the authors suggested that both enzymes were concerned with transfer of molecules through the intestinal epithelium. The classification of esterases is still open to question since there is considerable overlapping in substrate specificity patterns and also in activation and inhibition effects induced by various chemical compounds (Gomori and Chessick, '53; Seligman ét.al,, '50; Huggins and Moulton, i33). Hewever, three general classes are recognized in the literature at the present time, namely, lipases, non-specific esterases, and cholinesterases. The lipases are identified on the basis of their predilection for triglycerides of higher fatty acids (Cherry and Crandall, '32; Balls and Matlack, '38), and activation of enzyme activity with taurocholate(Gomori, m5; Nachlas and Seligman, no; Seligman 33 £13, '50). The non-specific esterases rapidly hydrolyze short-chain fatty acid esters (Balls and Matlack, '38; Cherry and Crandall, '32) and are inhibited by taurocholate (Seligman and Nachlas, 'h9; Gomori, 'hS; Barrett and Seligman, '51). The cholinesterases have predilection for choline esters (Stedman, Stedman, and Easson, '32). True cholinesterase has a higher specificity for acetylcholine and displays greater activity in low concentrations of substrate (Richter and Croft, ‘hZ; Mendel and Rudney, 'hh; Mendel 22.3l3’ 'hB). The nonrspecific or pseudocholine- esterase hydrolyze both choline and non-choline esters and exhibit maximum activity in relatively high concentrations of acetylcholine mmmlmdmmW,mD. As very little information is available about esterases in proto- zoans, this investigation is designed to determine the esterases present in Tetrgm ena mamas w by histochemical localization, substrate specificity and behavior of protozoan esterases in the presence of activators and inhibitors. MATERIALS AND METHODS Specimens of Tetrahmneg pyriformis W used in the following experi- ments were cultured axenically at 23-25 degrees centigrade in a culture medium containing: (1) 10. gm‘Bacto-tryptonewifco Laboratories, Detroit, Michigan); (2) 1 gm each of glucose, MgClz, KH2°04, K2HP04, and C2H303Na; (3)) 0.1 9n yeast extract and (100.0025 mg thiamine hydrochloride in . 1 liter of double glass-distilled water. ' Erlenmeyer flasks (125 ml) containing 75 ml of culture medium were sterilized at 15 lbs. pressure for 20 minutes. Cultures of organisms were established by inoculating each flask with 1 ml of culture medium from bacteria-free heavily populated stock cultures 3' to 21; days subse- quent to seeding. ' Test organisms were transferred into a 15 ml centrifuge tube and centrifuged lightly at 1500 r.p.m. for not more than five minutes. The supernatant fluid was removed and the organisms resuspended and fixed in 10% formalin buffered at pH 7.0 with b.02 gm NaH2P04.H30 and 16M gm NaZHP04.12 H20 for 10 to 15 mimltes at )4 degrees centigrade. The organisms were washed three times with 0.75% physiological saline centrifuging lightly each time. The organisms were affixed to albumin:- coated slides and allowed to dry in air. ' Lipase activity was demonstrated by Gomori's Tween technique ('15), non-Specific esterase by Nachlas and Selignan‘s Azo dye procedure ('19) and Gomori's modification of the Azo dye procedure ("52), and choline- esterase activity by Gomori's cholinesterase procedure ('h8), and Koelle and Friedenwald's modified thiocholine technique (“149). In control experiments, test organisms were immersed in Lugolis solution for five minutes prior to incubation in the substrate medium or orgnisms were incubated in medium where the substrate was excluded. To obtain nmdmtm effects from. inhibitors, test organisms were preincubated in aqueous solutions of the inhibitors for one hour prior to transfer to substrate solutions which contained essentially the same concentrations of the inhibitors. To detemine the effects of acetone fixation on esterase activity in L'miformis, organisms were fixed in cold acetone for l, 2, 12 ,. and 214 hours. The organisms were then stained for esterase activity and. the intensity of the reactions obtained were compared with that obtained with organisms fixed in 10% formalin for 10 to 15 minutes. RESULTS A. EBTERASE ACTIVITY IN ACETONE AND FORMALTN-FDCED SPECD’ENS 0F TETRAHYMENA PYRIFORMIS w l 1. Acetone fixation. In order to demonstrate intracellular enzymatic activity with histochemical procedures, selection of a satis- factory fixing agent is of major importance. The majority of enzymes are too labile to resist nanipulations in histochemical techniques and only a few enzymes, i.e., hydrolytic enzymes are fairly resistant to acetone (Gomori, ‘52). I Gomori (i_b_i_d_.) maintained that the best fixative for lipase and esterase is acetone. Relatively short (12-2h hours) fixation in 10% formalin (neutralized and at ice box temperature) is permissible but enzyme activity is inhibited from 50-75%. Nachlas and Seligman ('h9a) demonstrated that esterase activity, subsequent to fixation in.acetone for 2h hours, was reduced to about 60%. Specimens of Tetrahymena were fixed in cold acetone (h degrees centigrade) for varying lengths of time (1-2h hours) and stained for lipase with the Tween.procedure (Gomori,"h5), for esterase with Gomoriis modification of the.Azo dye technique ('52), and for cholinesterase with Gomori ('hB) procedure. Tween.60, alpha naphthyl acetate, and myristoylcholine chloride were used as subatrates. - .A complete loss of enzymatic activities with Tween 60 and myristoyl- choline were observed in test organisms fixed in acetone for 12 and 2h hours at h degrees centigrade. Enzymatic activity with alpha naphthyl 10 acetate was completely inhibited in majority of the organisms. Occasional cells were observed which contained small enzyme active areas in the cytoplasm.(fig. 7). Further, fixation artifacts were noted, i.e., cells ‘were shrunken and irregular in outline (fig. 7). To minimize shrinkage, organisms were suspended in culture medium maintained at about h degrees centigrade prior to fixation in cold acetone (Gomori, ‘52). Shrinkage artifacts were essentially the same as those observed when pretreatment was omitted. The fixation.period was shortened from 2h hours to 1-2 hours and enzymatic activities towards the same substrates were again observed. Slight hydrolysis of alpha naphthyl acetate was demonstrated. Small aggregates of the black azo dye precipitate could be seen irregularly scattered.within the cytoplasm of some cells. Tween 60 and myristoyl- choline chloride remained unhydrolyzed and shrinkage was still evident notwithstanding the shorter fixation period. 2. Fonualin fixation. Seligman, Chauncey, and Nachlas ('51) observed that rat liver esterase activity was considerably lost with prolonged fixation (i.e., 2h and h8 hours at h degrees centigrade) in 10% formalin or even with brief fixation at higher temperatures (1 and 2 hours at 25 degrees centigrade; l and 2 hours at 37 degrees centi- grade). Mbderate inactivation of the enzyme was noted with formalin fixation for 2 hours at h degrees centigrade. Residual esterase activity was reported to be 82% compared to 23% obtained with fixation for the same length of time at 37 degrees centigrade. Organisms fixed in 10% formalin buffered at pH 7.0 at )4 degrees centigrade for 2).; hours hydrolyzed alpha naphthyl acetate, Tween 60, and myristoylcholine chloride. Shrinkage artifacts were greatly reduced but diffusion artifacts were abundant (fig. 8). The golden brown pre- cipitate obtained with the Tween procedure was usually concentrated near or at the posterior ends of the cells but there .was a tendency for the enzymatic products to diffuse anteriorly and into the surrounding medium. Patches of the precipitate were frequently found in an inter- cellular position and on the surfaces of individual cells. Strong staining reactions were obtained with alpha naphthyl acetate and mistoylcholine chloride. Black azo dye precipitate was formed with the hydrolysis of alpha naphthyl acetate and black finely granular precipitate, which was usually localized at the anterior ends of the cells, was formed with the hydrolysis of myristoylcholine chloride. In a few cells, the precipitate was randomly or uniformly distributed in the cytoplasm. By shortening the fixation period in 10% formalin to fifteen minutes enzyme active areas were usually sharply localized . Diffusion artifacts were eliminated with the A20 dye procedure, reduced to a minimum with the cholinesterase technique and slightly reduced with the Tween technique. The above observations are indicative of the relative resistance of esterases in Tetrahyidena to fixation in 10% formalin and a consider- able inactivation of enzyme activity with acetone. 12 B. HISTOCI-EMICAL LOCALIZATION OF ESTERASES l. m technige. Gomori ('15) developed a technique for the histochemical demonstration of lipase activity using Tweens (Atlas Powder 00., Wilmington, Del.) as substrates. These are water-soluble long- chain fatty acid esters of polymer glycols and hexitans. The histo- chendCal procedure consists of the incubation of the organisms in the substrate medium in the presence of CaClz. The Ca ions form insoluble soap with the liberated fatty acid (one of the split products of hydrolysis) whiCh is then precipitated in £31.33. The Ca soap is con- verted to Pb soap by treatment with 3% lead nitrate solution. The colorless Pb soap is finally transformed into colored precipitate by immersion in dilute ammonium sulfide for 5 minutes. Sites of lipase- esterase activity were in golden-brown shades. For the demonstration of esterase activity, three saturated 'I‘txeens, polymcyethylene sorbitan monolmmate, polyoxyethylene sorbitan mono- palmitate, and polyoxyethylene sorbitan monostearate, with trade-names 6-75963, Tween to, and Tween 60 respectively, were used. To determine esterase activity towards an unsaturated fatty acid ester, polyox'yetlwlene sorbitan monooleate (Tween 80) was employed. It'is evident from figures 1, 2, and '3 that enzymatic activities were obtained with (Er-75963, Tween LLO and 60. No hydrolysis was observed with Tween 80. Three fairly distinct types of reactions were obtained I .with the saturated Tweens: a strong reaction, a moderate reaction, and a weak reaction. 13 In organisms which exhibited a strong reaction, the reactive area frequently covered as much as three-fourths of the cell. The precipitate which filled the reactive area was compact and formed a continuous mass throughout. ‘ In most.of the moderately and weakly reacting cells, the precipitate was usually localized in vacuole-like structures (fig. 2). In some moderately reacting cells, the precipitate occurred as a homogeneous and continuous mass similar to the compacted precipitates observed in the strongly reacting cells. In both moderately and strongly reacting cells, the compacted precipitates probably owe their development to the diffusion of either enzyme or hydrolytic products or both from the vacuole-like structures to the cytoplasm where the precipitate coalesced to form a compact mass. The localization.of the precipitate in these vacuole-like structures was observed in highly reactive cells. The enzyme was also observed to diffuse out into the incubating medium as evidenced by the intercellular position of some precipitated masses and by the precipitates on cell surfaces. The foregoing observations are essentially the same for all three saturated Tweens. 2. Egg dye technigu . The azo dye technique for esterases (Nachlas and Selignan, '14?) is based on the enzymatic hydrolysis of naphthol esters in the presence of a stabilized salt. The'diazonium salt couples with the liberated naphthol and forms an insoluble dye which is pre— cipitated in 32.132 . For accurate localization of the enzyme activity, - sufficient amount of the diazoniluu salt should be present in the 1h incubating medium in order to insure binding and immediate precipitation of the naphthol as it is liberated. Conditions should also be favorable for prompt coupling. In this investigation, tetrazotized diortho- anizidine Diazo Blue B was used as a coupler. Beta naphthyl acetate, alpha naphthyl acetate and naphthyl AS acetate were employed as sub- strates. . The localization of enzymatic activity with alpha naphthyl acetate .was observed to be very similar to that seen with the Tweens (fig. )4). The dye was usually seen to occupy the. entire posterior two-thirds of the heavily reacting cells (fig. 1;). In the moderately and weakly reacting cells, the dye would clearly be seen restricted in rounded Spots which were randomly arranged at the posterior ends of the cells, frequently extending anteriorly (fig. h) . Some cells display small reactive areas at the anterior part of the cytoplasm. To determine the effect of pH upon the localization picture, test organisms were incubated in alpha naphthyl acetate substrate solutions at pH 7.7 and 6.5. A sharper localization picture was obtained in those organisms incubated at pH 7.7 than in those incubated at pH 6.5. The precipitates occurred as distinct" rounded masses in the cells incubated at the higher pH. The azo dye precipitate was irregularly scattered at the posterior ends of the cells incubated at the lower pH. Gomori ( ' S 2) maintained that coupling was sooner at higher pH than at lower pH. In this study, coupling at pH 6.5 was apparently slower than at pH 7.7 resulting in the diffusion of the enzyme or hydrolytic products or both 15 to neighboring areas in the cytoplasm. Consequently, less precise localization pictures were obtained. Two types of staining reactions were obtained with the hydrolysis of beta naphthyl acetate and naphthyl AS acetate. Purple precipitates, which had the tendency to diffuse, were observed at the posterior ends of the cells :(fig. 5). In addition to the purple precipitates bright pink precipitates were found in vacuole-like structures of varying sizes and shapes usually restricted to the anterior ends of the cells (figs. h and 5). Small rounded masses of the pink precipitate were observed irregularly scattered throughout the cytoplasm (figs. 14 and 5). Both precipitates were found soluble in alcohol and xylene, hence, the organisms were mounted in glycerol jelly. In aqueous mounts, the pink precipitates were observed to be more stable than the purple precipitates as evidenced by their persistence while the latter faded and disappeared after several days. 3. Cholinesterase procedure. The Gomori (91(8) procedure was used for the localization of cholinesterases. Myristoylcholine chloride, benzoylcholine chloride, and lauroylcholine chloride were employed as substrates. No enzymatic activity was observed with myristoylcholine chloride at 0.0214. A weak staining reaction or no staining reaction at all were observed where the concentration of mistoylcholine was. either 0.00m or 0.00214. In weakly staining cells sparse amounts of granules were usually localized at the anterior ends of the cells. In other cells, the granules were randomly scattered in the cytOplasm or arranged in 16 the form of a halo on or near the nuclear surface. ' It is evident from figures 9 and 10, and table I that a very strong reaction was obtained with myristoylcholine chloride at 0.000h- M. The black granular precipitate in most of the cells was restricted to the anterior ends and along the surface of the cells (figs. 9 and 10). In the heavily staining cells, the precipitate frequently extended to the cmtrally located cytoplasm. Interspersed among the cells exhibit- ing a strong reaction were few weakly reacting cells with the reactive areas located in essentially the same place in the cytoplasm as described above. Benzoylcholine chloride was observed to be weakly hydrolyzed at 0.0021! and weakly to moderately hydrolyzed at 0.00514 (fig. 11). The black granular precipitate obtained was found localized anteriorly or centrally in most of the cells but posteriorly situated precipitates were occasionally observed. In cells where the reactive areas were anteriorly located, precipitates were frequently arranged to form a reticulum in the vicinity of the buccal aperture. When the precipitate was located at the center of the cyt0plasm, it was usually found adjacent to the cell nuclei. Lwroylcholine chloride gave a weak reaction in 0.000hM concen- tration (fig. 12). The precipitates were usually found near the central areas of the cytoplasm adjacent to the nuclei. Occasional cells were seen to show anterior localization of the precipitate. In smaller number of cells, the precipitate was randomly dispersed in the posterior one—fourth of the cytoplasm . l7 )4. @ocholine procedure £93; cholinesterase. The thiocholine technique of Koelle and Freidemrald (”49) utilizes thio-analogues of choline esters, i.e., acetylthiocholine and butyrylthiocholine, as substrates. Processing of tissues is a two step process. Firstly, tissues or organisms are incubated in media containing thiocholine substrate and copper glycinate. Theoretically, the enzymatically liberated thiocholine forms a.white precipitate of copper mercaptide at sites of enzyme activity. Secondly, copper mercaptide is converted to a brown amorphous deposit of copper sulfide by treatment with dilute ammonium sulfide solution. ' Koelle ('50) found that acetylthiocholine was hydrolyzed by both specific and non-specific cholinesterases but at an accelerated rate by the former. Butyrylthiocholine, on the other hand, was hydrolyzed exclusively by the non-specific cholinesterase. Using the above criteria in addition to the use of specific inhibitors, specific cholinesterase could then be distinguished from non-Specific cholinesterase. In this study, no reaction was obtained in both fixed and unfixed organisms incubated in either acetylthiocholine iodide or buty‘rylthio- choline iodide although the incubation periods were extended from 2 to 2).; hours . (3. EFFECT OF VARIOUS mHIBITORS ON ESTERASE ACTIVITY p‘ Effect of inhibitors on the mticmgwis of Tween 60, alpha naughty. l acetate:L and Eistoylcholwine chloride. Differentiation of esterases on the basis of substrate specificity alone was found ' 18 inadequate due to considerable overlapping in specificity patterns with various substrate solutions. For this reason, investigations aimed at identification of esterases were supplemented with inhibitor studies. Various compounds have been found to have specific inhibitory or accel- erating effects on esterase activity, i.e., Nu 683 was found to specific- ally inhibit pseudocholinesterase activity and Nu 1250 was found to specifically inhibit true cholinesterase activity (Hawkins and Mendel, 11493 Hawkins and Gunter, '146) . Cholinesterases in vertebrate tissues, regardless of the substrate employed were found to be completely inhibited by 10'"‘5 M eserine while simple esterases were insensitive to 100 to 3.000 times this concentration (Richter and Croft, 'h2). Test organisms were preincubated in aqueous solutions of the inhibitors for one hour at room temperature prior to incubation in the mbstrate solutions which contained essentially the same final concen— tration of the inhibitors. Esterase activity was ascertained with Gomori's Ween (U45) and cholinesterase ('hB) procedures, and Gomori's modification of the A20 dye technique ('52). The extent of inhibition was determined by comparing the intensity of staining obtained in test organisms incubated with the inhibitors with the intensity of staining in orynisms of the same age incubated. simultaneously in substrate solu- tions without inhibitors. The following values have beal used to desig- nate the various degrees of inhibition observed; )4, complete inhibition; 3, heavy inhibition; 2, moderate inhibition; 1, slight inhibition; and 0, no inhibition. 19 The inhibitors added to Tween 60 substrate solutions were Na tauro- cholate (2 x 10-214), Na arsanilate (8 x lo’zn), quinine hydrochloride (10-114), and benzaldehyde (1.6 x 10-311). The results obtained as sumrized in Table II show that Na taurocholate completely suppressed the enzyrmtic hydrolysis of Tween 60. No inhibitory effects by Na arsanilate were demonstrated. On the other hand, complete abolition of enzymatic activities towards Ween 60 was obtained with benzaldehyde and. quinine hydrochloride. Enzymatic hydrolysis of alpha naphthyl acetate was heavily inhibited by Na taurocholate (2 x 10-214) and Na arsanilate (8 x lO-2M), slightly inhibited by Na fluoride (10-11“!) and completely inhibited by quinine hydrochloride (lo-1M) . Eserine sulfate (10-41!) and benzaldehyde (1.6 x 10314) were noted to have no inhibitory effects towards the hydrolysis of alpha naphthyl acetate. However, benzethonium chloride (1.2 x 10.311) completely inhibited it. Eserine (8 x 10-311) produced a heavy inhibition on the esterase activity towards alpha naphthyl acetate while pontocaine hydrochloride (8 x 10-314) moderately inhibited it. The inhibitory effects of eserine sulfate at two different concen- trations (10‘414 and 8 x lO-BM) were tested with nyrietoylcholine chloride. At 10-411, eserine was observed to have no inbitory effects on the enzyme activity, whereas by "increasing the concentration 80 times, a heavy inhibition on the hydrolysis of myristoylcholine was noted. Pontocaine hydroChloride (8 x 10-314) completely inhibited enzymatic activity. Inhibitory effects obtained with Na fluoride were -2 erratic. At h.2 x 10 M, Na fluoride was without inhibitory effects '20 -1 while at 10 M, it displayed no inhibition to complete inhibition of esterase activity. 21 TABLE I Test organisms were stained according to Gomori's Tween ('15), cholinesterase (MB) and modified Azo dye ('52) pro- cedures; Nachlas and Seligmn‘s Azo dye technique ('19); and Koelle and Friedenwald's modified thiocholine technique for cholinesterase (U49 ) . acplanation of the table number values is as follows: 0, no reaction; 1, weak reaction; 2, moderate reaction; 3, strong reaction. 22 TABLE I HISTOCHEHIBTRY OF FSTERA SE8 IN 11;. PYRIFOFMIS W _-__ '— substrate Tweens: (saturated) l. 0-75963 2. Tween to 3. Tween 60 (unsaturated) h. Tween 80 Naphthol esters: S . Alpha naphtl'wl acetate 6. Beta naphthyl acetate Intensity of A— Location of Enzwue Active Centers Small aggregates of golden.brown precipitate usually localized posteriorly or irregularly scat- tered throughout the cytoplasm of the cells. Golden-brown.precipitate in vacuole-like structures restricted to posterior ends of most cells, at center or at anterior ends of some cells; precipitate compacted into a continuous mass.in heavily reacting cells. Localization essentially the same as with Tween to. No reaction Observed. Black finely granular precipitate occurring as rounded masses ran! domly distributed posteriorly or throughout the cells; precipitate, a compacted continuous mass occupy- ing almost three-fourths of the cell in the heavily staining organisms. Purplevprecipitate at posterior ends of the cells; a tendency to diffuse; a.pink precipitate in vacuole-like structures localized anteriorly or found as small aggre- gates randomly distributed through- out the cytoplasm in some cells F.— v __—"---__-— Substrate Intensity of Reaction TABLE I (cont.) .-- .-——--.—--.—-_—.- _--—.-—--..-~~_---—c—._- Location of Enzyme Active Centers Naphthol esters: 7. Naphthol AS acetate Choline esters: 8 . Hyristoylcholine chloride 9. Lauroylcholine chloride 10. Benzoylcholine chloride Thiocholine esters: 11a Acetylthiocholine iodide 12. Butyrylthiocholine iodide ‘7 Localization essentially the same as preceding. Black granular'precipitate local- ized anteriorly in majority of the cells, extending centraILy in the heavily reacting cells; few cells with.precipitate local- ized centrally or irregularly throughout the cytoplasm. Precipitate more frequently localized at the center of the cells adjacent to nuclei; local- ization at the anterior ends of some cells. Precipitate localized anteriorly or centrally in majority of the cells, posteriorly in some cells. No reaction observed. No reaction observed. TABLEII Organisms were tested for esterase activity with Gomori's Tween procedure (‘15), Gomori's cholinesterase procedure (”48), and Gomori's modification of the Azo dye technique ('52) . Organisms incubated in substrate solutions without the inhibitors were simultaneously run to serve as controls. The degree of inhibition was given values ranging from O to h: 0, no inhibition; 1, slight inhibition; 2, moderate inhibition; 3, heavy inhibition; )4, complete inhibition. 25 TABLE II THE EFFECT OF INHIBITORS ON THE ESTERASES OF I, PYRIECRMIS'W mfg: Final Concentration Extent of 10 o-h subatrate Inhibit°r of Inhibitor Inhibition W ._—V— W— -2 ._+_ w Tween 60 Na taurocholate 2 x 10 M h -2 Na arsanilate 8 x lo M O -1 Quinine hydro— lO M h chloride _3 Benzaldehyde 1.6 x 10 M h _2 Alpha naphthyl Na taurocholate 2 x 10 M 3 ‘cetate -2 Na arsanilate 8 x 10 M 3 Na fluoride 10.1 M l -1 Quinine hydro- 10 M h chloride _4 Eserine sulfate 10 M O -3 Eserine sulfate 8 x 10 M 3 -3 Benzaldehyde 1.6 x 10 M O -3 Benzethonium 1.2 x 10 M h chloride -3 Pontocaine hydro- . 8 x lO M 2 chloride -4 Wristoylcholine Eserine sulfate 10 M O chloride -3 Eserine sulfate 8 x lO M 3 -3 Pontocaine hydro- 8 x 10 M )1 chloride _2 Na fluoride h.2 x 10 M O -1 Na fluoride ’ M 26 DISCUSS ION ‘1. Effects of fixation. The prime requisite for valid histo- chemical staining reactions for enzymes is immobilization of the enzyme (Doyle and Liebelt, 'Sh). Pearse ('53) maintained that theoretically the best way of showing enzyme activity by histochemical means would be by the use of fresh frozen sections. He observed, however, that loss of the enzyme and of other protein and non-protein materials into the incubating medium was much greater than in fixed material . This was confimed by Doyle and Liebelt ('94) who demonstrated considerable diffusion of esterase activity into the incubating medium in fresh frozen rabbit appendix. Majority of enzyme works has then been carried out on materials fixed in acetone, alcohol, or formlin (Pearse, '53). The relative resistance of esterases to the inactivating effects of fixatives has been found by various investigators to vary depending upon the source of the enzyme under study. Chessick (' Sh) demonstrated that after fixation in neutral formlin at )4 degrees centigrade there was a complete loss of enzymatic activity in lobster nerve and con- siderable reduction at end plates of the fro g' , whereas the end plates of other species studied, i.e., human, dog, rat and mouse, resisted the fixation without noticeable loss of activity. D . Taxi (' 52) found considerable difference in residual cholinesterase activity in tissues from various species after fixation in formalin for one hour at 18 degrees centigrade . Pseudocholinesterase from dog 2? pancreas was observed to be less readily inactivated in comparison to pseudocholinesterase from horse serum. _True cholinesterase from hemolyzed beef blood corpuscles was noted to be more resistant to formalin than true cholinesterase from beef, rat, and dog caudate nucleus, whereas all enzymatic activity was abolished in the Torpedo electric organ. The author also found that increasing the temperature to 37 degrees centigrade. or the time of fixation to 21; hours further reduced the residual pseudocholinesterase activity and completely inactivated. true cholinesterase. A comparison of the degree of inactivation by formalin on specific and non-specific cholinesterase showed that the former was more heavily inactivated than the latter (1.233.) . Gomori ('55) demonstrated that hum tissues fixed in cold acetone gave very intense and extensive reactions with various naphthol esters. He noted a reduction of enzymatic activity to 50% after fixation in fonmJJn. Nachlas and Seligmn (th9) maintained that fixation of tissues- in cold acetone produced less destruction of esterase activity than other fixing agents. They observed complete inactivation in 10% formalin, ethyl alcohol, or methyl alcohol for 2).; hours. Ravin, Tsou and Selignan ('53) demonstrated a loss of 75 to 80% of serum cholinesterase activity from horse serum in cold acetone for 2 hours or in 10% formalin for 30 minutes while non-specific esterase activity remained unaffected . Seligman, Chauncey and Nachlas ('51) observed that esterases in rat liver tissue were less inactivated when fixed in 10% formalin for 2 hours at )4 degrees centigrade than when fixed for the same period of time at 37 degrees centigrade. Hermann ('52) maintained that cat 28 diaphragn fixed in 10% formalin for .148 hours at room temperature gave clear and precise localization pictures of cholinesterase activity. Utilizing indoxyl acetate as substrate; Barrett (‘53) found that brief fixation of tissues in cold acetone significantly reduced the activity of non-specific esterases and lipase. and almost completely abolished the activity of cholinesterase. . In contrast to the observations of Gomori ('52) and of Nachlas and Seligmn (”49), esterases in Timon: were found to resist fixation in-forrflin but not in acetone. Test organisms fixed in cold acetone for 12 and 21; hours and stained for non-Specific esterase and choline- ssterase activity gave no reaction at all. When the fixation period was shortened from 2h hours to l and 2 hours weak hydrolysis of alpha naphthyl acetate but. not of mristoylcholine was noted . {This is in agreement with the observations of Barrett ('53) that non-specific esterases and lipases display some resistance to the inactivating effects of acetone while cholinesterases exhibit almost none at all. Fixation in 10% formalin (especially when fixation was brief) gave excellent results both for non-specific esterases and cholinesterase activity. It was demonstrated in this investigation that prolonged fixation in formalin, i.e., 214 hours did not appreciably reduce esterase activity although the localization picture was not as precise as when fixation was short. This is in agreement with Chessick's observation (fSh) that esterase activity persisted apparently undiminished in tissues kept in formalin for extended periods of time. 29 It is evident from the results obtained in this study, in addition to those cited, that the resistance of esterases to the inactivating effects of fixation depends upon the type and source of the enzyme. This suggests that esterases are not the same everywhere but vary from species to species and from tissue to tissue. Gomori and Chessick ('53) maintained that esterases form a large family of closely related enzymes .which differ froln each other, in more or less significant details, by one or several of their prosthetic groups thus producing the effect of overlapping similarities and divergencies. No fixative could therefore be prescribed as ideal for esterases in general. The inactivating effects of the various fixing agents on esterases are variable and unpredictable. It is then of utmost import- ance to discover the relative resistance of any given enzyme under investigation to different fixatives before histochemical studies are made. This is to avoid misinterpretation of negative results for absence of the enzyme when the case might be that enzyme activity was only suppressed by the fixative employed. 2. glagsification of esterases. Esterases, in the broadest sense of the word, is used to refer to those enzymes which catalyze the hydroly- sis of carboxylic acid esters. Their classification into lipase, non- specific esta‘ases, and cholinesterases is based upon the efficiency with which various substrates are hydrolyzed, their distribution in tissues, and the effect of inhibitors upon their activity. Hence, lipases are those enzymes which have a predilection for long-chain fatty acid esters and which are found most abundant in the pancreas, while the 30 nonpspecific esterases are those enzymes which have a substrate prefer- ence for short-chain.fatty acid esters and abundant in the liver, kidney, spleen.and other organs of the body. Cholinesterases, on the other hand, have a predilection for choline esters and are most abundant in the nervous system, red blood cells and in the electric organ of the Torpedo. However, even at the present time, the classification of esterases is still open to question due to the discovery of considerable overlapping in substrate Specificity and distribution patterns and variability in inhibition effects. Cherry and Crandall ('32) demenstrated, following obstruction of the dog pancreas, an increase in the hydrolysis of olive oil in the blood while the hydrolysis of ethyl butyrate or tributyrin remained unchanged. This was interpreted as evidence for the Specificity of lipase which is most abundant in the pancreas. It was then suggested that the term "lipase" be reserved for enzymes capable of splitting true fats and oils and the term "esterase" be used for the enzymes acting upon simple esters. Balls and Matlack ('38) compared the mates of hydrolysis of horse pancreatic and liver extracts on tributyrin, tripropionin and on.butyrate and stearate esters and found that horse liver which is rich in ethyl butyrase and tributyrinase had no hydrolytic power on steanate esters in contrast to the pancreatic extract which hydrolyzed them with facility. The most important differentiating factor, therefore, between lipase and non-specific esterases is the length of the fatty acid moiety of the substrate hydrolyzed (ibid.). This was confirmed by Nachlas and 31 Seligman ('h9b) who demonstrated that kidney and liver readily hydro- lyzed acetic (Ca) and lauric (013) esters of beta naphthol with neglig- ible effects on.palmitic-stearic acid esters while pancreas hydrolyzed from 50 to 100 times of the palmitic-stearic acid ester (Clg-CIB) as' did the kidney and the liver. . The first histochemical procedure for the demonstration_of lipase was developed by Gomori ('hS). He employed the "Tweens" which are long- chain fatty acid esters of glycols and hexitans. Gomori (ééigs) main— .tained that with the Tween.procedure he was able to demonstrate lipase in.many organs such as liver, pancreas, lung, kidney, testicles, epididymis, adrenals, adipose tissue, stomach and small intestine. No lipase activity was found in the spleen, lymph nodes, brain and muscle. 'With the addition of 0.2% Na taurocholate to the substrate solutions, Gomori (3229,) noted intensification of enzymatic reaction only in the pancreas while the enzymatic activity in all other organs was greatly inhibited. Cherry and Crandall ('32) employing olive oil as substrate demonstrated lipase in significant amounts only in.the pancreas, in» testinal mucosa, liver and spleen, while all other tissues investigated hydrolyzed tributyrin which is split most rapidly by non-Specific esterases. ' Huggins and Moulton (*h8)_observed that paranitrophenyl.propionate was hydrolyzed by essentially the same tissues which hydrolyzed the Tweens. Nachlas and Seligman ('h?) noted the pattern of distribution of esterase activity with the.Azo dye procedure to be essentially 32 similar to that obtained with the Ween procedure. Esterase activity was found to be highest in the liver, pancreas, kidney and lung. ' Activity was also demonstrated in the gastrointestinal tract, testis, adrenal, and prostate while no activity was observed in the heart, skeletal mscle, spleen, lymph nodes, thyroid and’brain. The specificity of the Tween procedure for lipase activity was questioned by Huggins and Moulton ('h8) and by Nachlas and Selig-an (U49). They suggested that Tweens are not hydrolyzed exclusively by lipases but also by non-specific esterases. Gomori's ('h5,'h6) observations with Na taurocholate would lend support to this suggestion. Na tauro— cholate has been found to be a potent activator of lipase activity and an inhibitor of non-specific esterase activity. If the hydrolysis of the Tweens as observed by Gomori (1.92.3 .) was specific for lipase, the reaction in all the positive orgns should have been accelerated. Only the pancreatic enzyme, however, was activated . The enzyme activity in all the rest of the organs was inhibited. Ehridently, both lipase and non-specific esterase were responsible for the hydrolysis of the Tweens. In the present investigation, test organisms of Tetraigwm'evn‘a; stained intensely with G—75963, Tween to, and Tween 60, which are saturated long-chain fatty acid esters . Intracellular localization of enzyme active areas obtained with the above-smbstrates was noted to be essen- tially similar. This is in agreement with the observations of Gomori ('LtS ,‘h6) . He demonstrated that the localization and intensity of reaction in the various organs investigated with the saturated Tweens as substrates remained essentially the same . The observation made in 33 the present study is interpreted as evidence that only one type of esterase is responsible for the hydrolysis of G-7S96j, Tween ho, and Tween 60. To determine whether it was a lipase or a non-specific esterase which was responsible for the hydrolysis of the Tweens, inhibition or activation effects by Na taurocholate was tested . The result obtained was that of complete inhibition of enzyme activity on Tween 60 by 0.02M . Nataurocholate. It is concluded, therefore, that the enzyme respons- ible for the hydrolysis of the Tweens is a non-specific esterase. Gomori (”49) extending his investigation on the Specificity of lipases discovered a new distinctive feature for the characterization of true lipases. He demonstrated the unique ability of true lipases to hydrolyze unsaturated fatty acid esters. Evidently, no true lipases : are present in Tetral‘fl I ena as indicated by the absence of activity .of test organisms on Tween 80 which is an unsaturated fatty acid ester. 'Ille histochemical procedure devised by Nachlas and Selignan ('li9a) for the demonstration of esterases was found by Gomori ('50a) to be unsatisfactory for precise localization of enzyme activity due to the solubility of the azo dye formed.‘ He demonstrated that part of the azo dye reaction-product remitted in solution for mxy minutes, especially in the presence of more than 1% acetone. This resulted in diffusion - artifacts which made accurate localization impossible. Substituting alpha naphthyl acetate for beta naphthyl acetate Gomori (_i_b_i_c1.) noted instant precipitation of the reaction products at pH 7.0.‘ He, therefore, suggested a modification of the technique with the use of alpha naphthyl 3h acetate and a minimum of acetone. Pearse ('53) compared the original ,method by Nachlas and Seligman ('h9a) with Gomori's ('52) modification and confirmed the latter author's findings that localization pictures ‘ obtained with alpha naphthyl acetate were far more precise than with beta naphthyl acetate. He demonstrated that the azo dye formed with the latter substrate was soluble not only in alcohol and xylene but also in water. - Results obtained in this investigation are in general agreement with the foregoing observations. It was demonstrated in Tetrahymgna that the purple azo dye formed by coupling beta naphthyl acetate with Diazo Blue B dissolved in alcohol and xylene. In aqueous mounts, the dye diffused resulting in blurring of enzyme active areas. Hewever, the pink dye restricted at the anterior ends of the cells was noted to be more stable. On the other hand, precise localization pictures were consistently obtained with alpha naphthyl acetate as substrates. 'With the naphtholic substrates, i.e., alpha naphthyl acetate, Gomori (‘52) found the distribution.pattern quite similar to, but more 'wideSpread than that obtained with the Tweens., Many structures comp pletely negative by the Tween method were found to stain.more or less intensely by the naphthol methods. Conversely, some Tweenépositive tissues showed no naphthol reaction.at all. In addition, Gomori ' observed different inhibitory effects by various compounds on the hydroly- sis of Tweens and naphthol esters. This was interpreted to suggest the existence of separate enzymes responsible for the reactions with the naphthol esters. Hence, Gomori ('52) designated the alpha naphthyl acetate-splitting enzyme "alpha esterase." 35 .Alpha naphthyl acetate was readily hydrolyzed by Specimens of Tetrahymena_and the localization.of the enzyme active areas was noted to be very similar to that observed with the Tweens. However, inhibitory effects by various compounds on the hydrolysis of alpha naphthyl acetate and Tween 60 were found to vary considerably; While Na taurocholate completely inhibited the reaction with Tween 60, it only heavily . inhibited the hydrolysis of alpha naphthyl acetate. Na arsanilate was found to have no inhibitory effects on.Tween 60 while it heavily inhibited enzyme activity towards alpha naphthyl acetate. Quinine completely inhibited reactions with both substrates . On the other hand, benzaldehyde, which is an inhibitor of lipase (west and Todd, '57) inhibited enzyme activity with Tween 60 leaving unaffected the hydrolysis of alpha naphthyl acetate. These observations suggest the presence of two, possibly closely related nonpspecific esterases which are response ible for the separate activities towards Tween 60 and alpha naphthyl acetate. Gomori {‘52) maintained that localization.pictures obtained with beta naphthyl acetate and alpha.naphthyl acetate appeared identical. In variance with the foregoing observation, the pattern of localization with alpha naphthyl acetate differed considerably from that obtained ‘with beta naphthyl acetate in test organisms of Tetrahymena. On the other hand, the pattern of localization with beta naphthyl acetate was found to be identical with that obtained with naphthol AS acetate. ‘With the latter substrates the precipitates were found in.two regions in the organisms, namely, the diffuse purple precipitate at the posterior 36 ends of the organisms and the pink precipitate localized at the anterior endsbf the organisms.- The black azo dye formed with the hydrolysis of alpha naphthyl acetate, on the other hand, was predominantly localized at the posterior half of the cells. Gomori ('52) demonstrated that the topographic patterns of activity as shown by alpha naphthyl acetate and naphthyl AS acetate were closely similar but not identical. There were .SOme structures, i.e., duodenum of the rat, certain unidentified cells in the heart of the .rat and seme cells of the mouse lung which were strongly positive with alpha _ naphthyl acetate and only faintly positive or entirely negative with naphthyl AS acetate. Conversely, Brunner's glands of the rat, Imman mast cells and large motor cells of the brain of mm and rat stained intensely with naphthyl AS acetate but not with alpha naphthyl acetate. Conseqicntly, he referred to the enzyme hydrolyzing naphthyl AS acetate as "As esterase." Pearse ('53) , however, maintained that he did not find sufficient differences in tissue distribution of localization of esterases when naphthyl AS acetate was used as substrate instead .of alpha naphthyl . acetate to warrant the desiglation of the enzyme hydrolyzing the former substrate "AS esterase" as proposed by Gomori ('52). Chessick ('53) mintained to have observed numerous instances of overlapping of locali- zation in some tissues with alpha naphthyl acetate and naphthyl AS acetate although there were wide differences in distribution patterns in other tissues varying with the species employed. 37 In the present investigation, the difference in the localization patterns between alpha naphthyl acetate, on the one hand, and beta naphthyl acetate. and naphthyl AS acetate, on the other hand suggests that the enzyme or enzymes responsible for the reaction with the two latter substrates are distinct from that hydrolyzing the former. Denz (‘53) demonstrated that with beta naphthyl acetate as sub- strates, fresh frozen sections of muscle fibers stained uniformly crimson while the line of the myoneural Junctions purple. In acetone- fixed tissues the general crimson staining persisted but the purple staining at the woneural junctions was lost. In addition, the crimson stain was found to be much more resistant to inhibition, i.e., to lO'BM eserine, than the purple staining. He further demonstrated that crude preparations of Specific cholinesterase from rat brain and diaphragm hydrolyzed beta naphthyl acetate at rates similar to acetylcholine . These observations were interpreted to suggest that the purple staining was due to specific cholinesterase and the diffuse crimson staining due to a non-specific esterase. Results obtained in specimens of TetrahEnena are in general agree— 'ment with ‘Denz's 051;) observations with respect to the two types of staining produced. The difference in localization of the two types of staining suggests that they are formed by activities of two different, spatially separated enzymes in the cells. It could not be determined at the present time, however, whether the reactions obtained were both due to non~specific esterase activity or whether the purple or the pink staining was due to the activity of a cholinesterase. Further studies 38 with selective inhibitors would be necessary to clarify this question. gtedmn, Stedman, -and Easson ('32) discovered an enzyme in horse serum which differed from liver esterase in that it attacked choline esters more rapidly than non-choline esters. Stedmsn and Stedman (‘35) also demonstrated an enzyme in red blood cells and brain tissue which exhibited a predilection for choline esters. However, the foregoing authors were unable to distinguish between the serum enzyme from the. enzyme present in red blood cells and brain tissue. It was between 19140 and 191:3 when it was definitely established that more than one kind of cholinesterase occurred in the animal body (Whittaker, '51). The first workers to prove this were Alles and Hawes ('h2) who compared the specificity of the cholinesterase in red blood cells and in the serum. They demonstrated that the hydrolysis of acetylcholine by the taro enzymes had different kinetic characteristics and that while both hydrolyzed acetyl-a-methylcholine, only the red cell cholinesterase hydrolyzed acetyl-b-methylcholine . Mendel and Rudney (”43) suggested that the serum enzyme be called psaldocholinesterase since it had a predilection not only for choline esters but also for. non-choline esters. The enzyme in red blood cells and brain tissue, on the other hand, was suggested to be designated true cholinesterase for its greater specificity for acetylcholine. ‘ In addition to the difference in substrate specificity patterns between the two types of cholinesteraSes it has been found that true cholinesterase displayed umdmum activity with low substrate concen- trations and inhibited with high concentrations of acetylcholine , while 39 pseudocholinesterase activity increased with increasing concentration of the substrate (Alles and Hawes, 'h23 Mendel and Rudney, ”43). However, Mendel and Rudney ('hh) demonstrated that the substrate- activity curve of true cholinesterase could be altered to resemble the substrate-activity curve of pseudocholinesterase by protamines. In addition, true cholinesterases have been found in Planaria which dis- played a substrate-activity curve characteristic of pseudocholinesterase in vertebrates (Mendel and Rudney, U414) . It was concluded by Mendel and Rudney gm.) that the substrate-activity relationship displayed by the taro types of cholinesterases is but a secondary property which is determined by their environments. Gomori (U48) maintained that long-chain fatty acid esters of choline were hydrolyzed satisfactorily by cholinesterases. Using lauroyl, wristoyl, palmitoyl and stearoylcholine he demonstrated marked dif- ferences in species specificity towards these substrates. Some Species hydrolyzed these esters readily while others only very slowly or not at all. Purified bovine red blood cell cholinesterase and purified acetylcholinesterase from electric organ of Togedo failed to hydrolyze nyristoyl, palmitoyl, or stearoyl esters. In view of the failure of the Gomori method to demonstrate activity in red blood cells and in electric organ of Togedo, Koelle and Friedemtald (U49) concluded that the mistoylcholine method (Gomori, ”48) localized only non-specific cholinesterase. Hard and Peterson ('50) demonstrated a mistoylcholine-wand lauroylcholine-splitting enzyme localized at 1111ch areas and points of synapse in both central and to peripheral nervous system of the dog. The enzyme was inhibited by both prostignine (lo-5M) and excess of choline. The authors, however, were unable to determine the particular type of cholinesterase responsible for the observed reaction. I A mristoylcholine—splitting enzyme was localized at the myoneural junctions of rat diaphragm which reacted with selective inhibitors in a way characteristic of specific cholinesterase (Benz, '53-). Using (the Warburg method Denz (3221') showed hydrolysis. of nyristoylcholine by highly active preparations of specific cholinesterase from rat brain and sheep red blood cells. These observations were interpreted as evidence that the activity obtained with myristoylcholine at the myoneural junctions of rat diaphragn was due to specific cholinesterase. Pearse ('53) maintained that the Gomori method is capable of demonstrating both specific and non-specific cholinesterase. It was demonstrated in the present investigation that choline- esterases are present in letrahymfiena which can hydrolyze myristoyl- choline and lauroylcholine . It was noted that more intense reactions were obtained with the former substrate than with the latter. The pattern of localization observed with the above substrates did not exactly coincide but a certain degree of overlap was evident in most of the test organisms. The reactive. areas with mistoylcholine were predominantly localized at the anterior ends of the cells frequently extending towards the central and posterior regions of the cytoplasm. With lauroylcholine the precipitate was usually localized (at the central regions of the cytoplasm and extending anteriorly in some of the test hl organisms. The evidence at hand do not warrant interpretation that two separate enzymes are responsible for the activities towards the above substrates. It is evident, however, that greater activity is. exhibited towards mistoylcholine than towards lauroylcholine. Benzoylcholine, which. is a substrate hydrolyzed exclusively by. pseudocholinesterase (Mendel and Rudney, ”43), was demonstrated to be moderately hydrolyzed at 0.002M and 0.00514 concentration. by test organisms of EEWE‘E- Localization of the granular reaction products were essentially similar to that obtained with mistoylcholine. The proper assessment of the foregoing observations is limited for the very reason that there still remains considerable uncertainty as to the type of cholinesterase responsible for the hydrolysis of mistoyl- choline. However, the observed inhibition of enzymatic hydrolysis of wristoylcholine at substrate concentration of 0.02M and the subsequent removal of inhibition at a very much lower substrate concentration, i.e. , 0.000104, suggests that the enzyme under question has features in common with true cholinesterase in vertebrate tissues. It is well founded (Mendel and Rudney, '13) that true cholinesterases in vertebrate tissues are easily inhibited by excess substrate (acetylcholine) and the inhibition- is apparently due to choline (Roepke, l3?) . The rryristoyl- choline-Splitting enzyme in Tet-ram ens, however, d iffers from true cholinesterases in vertebrate tissues in “that its activity was not appreciably affected by 10-4M eserine. It was necessary to increase the concentration of eserine eighty times to effect a heavy inhibition on the enzymatic activity. It is also evident from the results obtained h2 in this study that there is present in Tetflymena a benzoylcholine— splitting esterase. The failure to demonstrate cholinesterase activity-by means of Koelle and Friedanwald's thiocholine technique need not be interpreted to man complete absence of enzyme activity towards acetylthiocholine. Seaman ('51) mintained that he obtained positive reactions in specimens of Tetra—m ' ‘ ena @331 S with Koelle‘s method. It' is entirely possible, therefore, that certain conditions during the processing of the test orgnisms in‘ this study were not met to permit histochemical visuali- zation of cholinesterase activity by means of this technique. 3. Effegts of inhibitors. As the amount of information on esterases increased, it became more apparent that substrate specificity is by no means an absolute one. Considerable overlapping in substrate specificity patterns have been observed (Benz, '53; Nachlas and Seligman, 'h9b; Pearse, '53; Gomori, ”45, '16; Balls and Matlack, '38). For this reason, studies aimed at identification and characterization of esterases are always supplemented with inhibitor studies. It was shown earlier in the discussion that both lipase and non- Specific esterases can be demonstrated histochemically by the Ween procedure. The problem of determining activity due to lipase from that due to non—specific esterases immediately arises. This can be resolved by Na taurocholate which specifically activates lipase activity and inhibits non-Specific esterase activity. Numerous workers have demon- strated activation of lipase and inhibition of nonpspecific esterase activity in vertebrate tissues (Gomori, ”45, MB, t1:9; Seligman, 1:3 Nachlas and Mallomo, '50; Nachlas and Seligman, 'h9b; Gomori and Chessick, '53). ‘ It was observed that the hydrolysis of Tween 60 in test organisms of Tetrahymena was completely inhibited by Na taurocholate (2 x 10-21%) . A heavy inhibition was also noted on the hydrolysis of alpha naphthyl acetate. Gomori and Chessick ('53) noted no appreciable. effect of tauro- cholate on the hydrolysis of naphthol esters, but Seligman, Nachlas, and Hallomo ('50) demonstrated increase in the hydrolysis of naphthol esters in pancreas and inhibition in liver. 0n the basis of the results obtained with Na taurocholate in the present study, it is concluded that the enzyme reaponsible for the reaction with Tween 60 is a non- sPecific esterase. The inhibited portion of the reaction obtained with alpha naphthyl acetate is interpreted as due to non-specific esterase. The partial inhibition of the reaction with alpha naphthyl acetate by eserine at a concentration which heavily inhibited cholinesterase activity towards myristoylcholine indicate that part of the activity towards alpha naphthyl acetate is due to cholinesterase . The residual activity observed with Na taurocholate is therefore interpreted as due to cholinesterase activity. . Inhibitory effects by arsaniJate have been shown by a number of investigtors. Nachlas and Selignan (Heb) demonstrated inhibition by 10~1M arsanilate on liver and kidney esterases greater than on pmcreas enzyme. Gomori ('hBa) observed inhibition of lipase-esterase activity by the same compound. Arsanilate inhibited the hydrolysis of indoxyl acetate (Barrett, '53) . In the present study, inhibition by arsanilate (8 x lO-zM) on alpha naphthyl acetate is in good agreement with the foregoing observations. Tween 60, however, remained unaffected. The general effect of, quinine on esterase activity is that of moderate to complete inhibition except in few reported cases of acceler- ation (Richter and Croft, 3142; Nachlas and Seligman, th9). Barrett ('53) demonstrated inhibition by quinine on the hydrolysis of indoxyl acetate. Gomori (the) noted. inhibition of the Tweens inliver, kidney and pancreas while Gomori and 0hessick ('53) observed no appreciable effect of quinine on the hydrolysis of naphthol esters. In the present investigation, quinine proved to be a potent inhibitor of esterase activity towards Tween. 60 and alpha naphthyl acetate. Benzaldehyde has been cited as-an inhibitor of lipase (West and Todd, '57). At 1.6 x 10-314 benzaldehyde completely inhibited mrdrolysis of Tween 60 . Hydrolysis of alpha naphthyl acetate, however, remained, unaffected. These observations suggest that non-specific esterases reSponsible for the hydrolysis of the Tweens in Tetrgmgena have features similar to lipases in vertebrate tissues. Pontocaine hydrolchloride (Tetracaine) moderately inhibited re- action with alpha naphthyl acetate in Totem A ‘ ena . This observation is in general agreement with the findings of Chessick ('Sh).- Phemerol (benzethonium chloride) was demonstrated to be a potent inhibitor of non-specific esterases in this study. Na fluoride at a relatively high concentration, i.e., lo'lh, was found to only slightly inhibit the reaction with alpha naphthyl acetate . Nachlas and Seligman (' )49) demonstrated inhibition “of liver and kidney esterases at very much 1:5 lower concentration, i.e., 3 mg/cc. Esterases in Tetrabflena seem to be more resistant to Na fluoride than esterases in vertebrate tissues. Richter and Croft (”42) were the first workers to differentiate cholinesterase activity from aliesterase activity by the selective inhibitory effects of eserine at low concentrations. Denz ( '53) demonstrated inhibition by lO-BM eserine on the hydrolysis of nyristoyl- choline and Chessick ('SLL) and 0omori and Chessick ('53) observed inhibi- tion of the hydrolysis of alpha naphthyl acetate at motor end plates. In variance with the foregoing observations, 10-41! eserine, which is sufficient to completely inhibit cholinesterase activity in vertebrate tissues was found to have no noticeable effect on the hydrolysis of alpha naphthyl acetate and myristoylcholine. It required an 80-fold increase in the molar concentration of eserine to inhibit enzyme activity. At 8 x 10-314, eserine heavily inhibited the reaction with alpha naphthyl acetate and mistoylcholine . In this respect, the cholinesterases in Tetrahymena resemble the true cholinesterase found by Hawkins and Mendel (mo) in Planaria extracts and frog brain which displayed pro- nounced resistance to eserine. Divers ('53) also demonstrated in the Harderian gland of the rat and hamster an eserine—insensitive enzyme capable of splitting butyrylcholine but with very little activity towards benzoylcholine and acetylcholine . ' Pontocaine hydrochloride (8 x 10-31!) completely suppressed choline- esterase activity in Tetm (ena. Na fluoride was found to have no inhibitory effects on the central and peripheral nervous system (Hard and Fox , ‘51). Results obtained in this study were erratic. 146 _2 At h.2 x 10 M, Na fluoride displayed no effect at all upon the hydroly- sis of myristoylcholine. By increasing the concentration of the .1 inhibitor to 10 M no inhibition to complete suppression of enzyme activity was observed . h? SUMRY 1. Specimens of 11;. pyriformis W reared axenically were stained for esterase activity by means of Gomori's Tween method (”45) , Nachlas and - Seligman's Azo dye technique (”49), Gomori's modified Azo dye technique ('52), Gomori's cholinesterase technique ('hB), and Koelle ’snd Friedenwald' 8 modified thiocholine technique ('19) . 2. Staining reactions were obtained with 6—75963, Tween ho, and 60, alpha naphthyl acetate, beta naphthyl acetate, naphthyl AS acetate, myristoylcholine chloride, lauroylcholine chloride, and benzoylcholine chloride but not with Tween 80, acetylthiocholine and butyrylthiocholine. 3. It was found that non-specific esterases were reSponsible for the hydrolysis of the saturated Tweens and part of the reactions obtained with alpha naphthyl acetate, beta naphthyl acetate and naphthyl AS acetate. )4. The presence of cholinesterases capable of hydrolyzing mistoyl— choline, lauroylcholine , and benzoylcholine was demonstrated in test organisms of renames. S . The activity of the myristoylcholine-Splitti-ng enzyme was found to be unaffected by 10.41! eserine but was heavily inhibited by 8' x lo'3M eserine. In this respect, it resembles the true cholinesterase demon- strated by Hawkins and Mendel (W6) in Planaria and froé brain which exhibited pronounced resistance to eserine. 6. The partial inhibition of the hydrolysis of alpha naphthyl -3 acetate by 8 x lo M eserine which markedly inhibited hydrolysis of 1L8 myristoylcholine suggests that cholinesterase activity is reaponsible for part of the reaction obtained with it. 7. Esterases in _‘I‘_etrahymena were. found to be considerably inhibited by cold acetone but. relatively resistant to 10% formalin. . 8. No staining reactions were observed with, Koelle and Friedenwald's modified thiocholine technique ('51). q , 9. Hydrolysis of Tween 60 was found completely inhibited by Na taurocholate (2 x lo’zn), quinine hydrochloride (lo-In), and by benzal- dehyde (1.6 x 10-314), but unaffected by Na arsanilate (8 x 10-214). 10 . Reaction with alpha naphthyl acetate was heavily inhibited by Na taurocholate (2 x “lo-2M), Na arsanilate (8 x 10-314), eserine (8 x lO-SMM completely inhibited by quinine hydrochloride (lo-IN) and benzethonium chloride (1.2 x 10-314); slightly inhibited by Na fluoride (lo-1M) , and moderately inhibited by pontocaine hydrochloride (8 x lO-aM) . Eserine (lo-4M) and benzaldehyde (1.6 x lO-3M) were noted to have no effects on the hydrolysis of alpha naphthyl acetate. ll. Hydrolysis of mistoylcholine chloride was heavily inhibited by 8 x 10-311 eserine but unaffected by 10-414 eserine. Reaction was completely inhibited by pontocaine hydrochloride. Results obtained with Na fluoride were erratic. 2. 3. h. 9. 10. 12. 13. 149 REFERENCES .Alles, G. A. , and Hawes, R. C. (19142). Cholinesterase in blood of m. J. B101. Chem.,1332 375‘3900 Antopol, W., and Glick, D. (1910). The histological distribution of cholinesterase in the adrenal bland. J. Biol. Chem., 132: 669- 673. Augustinsson, K. 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SS PLATE I Specimens of Tet%m ena ‘ ifornrls w fixed in 10% formalin for 15 min- utes and stained or esterase activity'according to Gomori's Tween procedure ('115) and ,Gomori's modification of the Azo dye technique ('52). Micrometer scale insert: ‘ 1 space a: 0.01 mm. ' Fig. 1. Localization of esterase activity with 0459633 Fig. 2. Localization of esteraSe activity with TweenhO; Fig. 3. Localization of esterase activity with Tween 60;. Fig. 11. Localization of esterase activity with alpha naphtmrl acetate . 57 PLATE II Specimens of W mgomis W fixed in 10% formalin and stained for esterase activity with beta naphthyl acetate and naphthyl AS acetate; figure 7 fixed in cold acetone for 2h hours, figure 8 fixed in 10% formalin for 211 hours and stained for esterase activity with alpha naphthyl acetate. Micrometer scale insert; 1 space = 0.01 mm. Fig. 5. Localization of esterase activity with beta naphthyl acetate; Fig. 6. Localization of esterase activity with naphthyl AS acetate: Fig. 7.. Esterase activity after fixation in acetone for 211 hours 5 - Fig. 8. Esterase activity after fixation in 10% formalin ' for 211 hours. 59 PLATE III Specimens of Tet ena pyriformis W'fixed in 10% formalin for 15 min— utes and stain or c o ineSterase activity according to Gomori's cholinesterase procedure (3&8). Micrometer scale insert; 1 space a 0.01 .mm. i Fig. 9 and 10. Localization of cholinesterase activity with myristoylcholine chloride; Fig. 11. Localization of cholinesterase activity with benzoylcholine chloride; Fig. 12. Localization of cholinesterase activity with lauroylcholine chloride. 61 PLATE IV Test organisms fixed in 10% formalin for 15 minutes and incubated in substrate solutions containing inhibitors. Micrometer scale insert; 1 space a 0.01 mm. Fig. 13. Inhibition of esterase activity with alpha _2 naphthyl acetate by Na.arsanilate (8 x 10 M); Fig. 1h. Inhibition,of esterase activity with alpha naphthyl acetate by Na taurocholate (2 x 10 2M); Fig. 15. Inhibition of esterase activity with myristoyl- choline chloride by eserine sulfat-e (8 x 10 M). mm ‘ E 2%.: '1‘“, U ’1' I 3 1H 1:11 ‘ .f ‘ 3.". '1): ‘ "My ~ .7 A' MICHIGAN STATE UNIVERSITY LIBRARIES 1111 11111111111 3 1293 3103 7744