Heterogeneity of lysosomes in human fibroblasts

Summary Lysosomes are defined traditionally with the marker enzyme acid phosphatase. We showed recently that lysosomes from human fibroblasts can be separated into a light and dense fraction as well as prelysosomal population. We now provide evidence that although acid phosphatase is enriched in all three fractions, the marker enzyme in the prelysosomal compartment is qualitatively distinct from that of the lysosomes. Ultrastructural analysis showed that the acid phosphatase in the prelysosomal vesicles deposited an extremely electron-dense reaction product, entirely obliterating the lumen of the vesicle, in contrast to that of the light and dense lysosomes which deposited a fine and diffuse product scattered throughout the luminal space. Biochemical analysis showed that only 51% of the acid phosphatase in the prelysosomes was inhibited by tartrate, while 80% of that in the lysosomes was tartrate-inhibitable. Immunoprecipitation with antibodies specific for various isozymes of acid phosphatase showed that 39% of the acid phosphatase in the prelysosomes was of the lysosomal type whereas over 5007o of the acid phosphatase in the lysosomes was of this type. These results showed that acid phosphatase in the prelysosomes of human cultured fibroblasts can be distinguished from that of the lysosomes cytochemically, biochemically, and immunologically and that lysosomes, as marked by acid phosphatase, are a heterogeneous organelle.     

A quantitative immunoelectronmicroscopic study on soluble, membrane-associated and membrane-bound lysosomal enzymes in human intestinal epithelial cells

Summary We have used quantitative immunoelectronmicroscopy to compare thein situ localization of acid -glucosidase, lysosomal acid phosphatase, -hexosaminidase and glucocerebrosidase in intestinal epithelial cells of the human duodenum. Differences between these four lysosomal enzymes were observed with respect to their presence at the apical cell surface. Transport to the apical membrane seems to be a more important intracellular route for lysosomal acid phosphatase and acid -glucosidase than it is for -hexosaminidase. The membrane associated lysosomal enzyme glucocerebrosidase is not transported to the microvilli. The studies emphasize that lysosomal enzyme transport pathways are enzyme and cell type specific.     

Dual localization of β-glucuronidase and acid phosphatase in lysosomes and in microsomes

Summary The dual localization of certain hydrolases in lysosomes and in endoplasmic reticulum as studied in enzyme staining reactions is now supported by cytobiochemical studies on mouse liver and kidney -glucuronidase and acid phosphatase. Use was made of the renal -glucuronidase response to endogenous androgen for both studies. Accordingly, sucrose homogenates were prepared of liver and kidney of male BALB/C mice previously injected with gonadotrophin along with control animals receiving saline instead. The homogenates were subjected to differential ultracentrifugation yielding six fractions. These were characterized as to their organelle composition by measurements of marker enzymes and by observations with the electron microscope. In all subcellular fractions, -glucuronidase was uniformly increased 5 to 8 times over the corresponding control value and, in fractions rich in lysosomes, this enzyme was easily released by alternate freezing and thawing. On the other hand, the microsomal -glucuronidase and acid phosphatase enzymes were not liberated by freezing and thawing nor were they after treatment with 0.1 % Triton X-100 and by employing other reagents and conditions which are known to release lysosomal enzymes. In contrast to microsomal acid phosphatase, microsomal -glucuronidase activity could be liberated by treatment with hyaluronidase. This soluble -glucuronidase showed the same optimum pH, Michaelis Constant and heat inactivation behavior as the lysosomal -glucuronidase prepared by freezing and thawing treatment. These observations define two populations of microsomal vesicles each identifiable by an individual membrane-associated acid hydrolase. One of these -glucuronidase, increases in specific activity in the animal on androgens and is released by hyaluronidase and the other, acid phosphatase, does not respond to androgen and is not released by hyaluronidase. There would appear to be a variety of mechanisms by which hydrolases enter into association with the membranes of the endoplasmic reticulum and from there, a variety of routes to the lysosomes. A comment is made concerning the question of acid phosphatases and -glucuronidase as enzyme markers for lysosomes in mouse kidney.Aided in part by Research Grant, P-106, of the American Cancer Society, Inc., New York, and by U.S.P.H.S. Grant CA-07538 and by a Research Career Award, CA-K6-18453 to William H. Fishman.     

Lysosomes of root tip cells in corn seedlings

Summary Nine acid hydrolases are present in lysosomes which are found in the mitochondrial fraction of a cell-free extract prepared from root tips of corn seedlings.Light and heavy lysosomes can be distinguished. The latter are sedimentable in a sucrose-medium, the former only in sorbitol-medium. The fraction of heavy lysosomes is in turn composed of at least three populations of lysosomes differing in density and enzyme content.Light lysosomes are membrane-bound particles with diameters from 0.3 to 1.5 . Electron micrographs of frozen-etched tissue and isolated particles provide evidence that light lysosomes are identical with small vacuoles. This type of lysosome is characterized by presence of transaminases in addition to that of hydrolases. Heavy lysosomes are small spheres (diameters from 0.1–0.3 ) with membranes resembling those of vacuoles and of the endoplasmic reticulum. These lysosomes are characterized by high specific activities of two oxydoreductases known to occur also in the membranes of the reticulum.The different types of particles are thought to represent stages of the development of the lysosomal apparatus; according to this hypothesis the large vacuole of parenchymatous cells represents the end product of this process.     

The postnatal development of the rat kidney,with special reference to the chemodifferentiation of the proximal tubule

Summary New nephron anlages appear in the renal cortex up to the 4th postnatal day (PD). The last anlages to be formed develop into functional nephrons by PD 10, and the cortex appears mature at PD 12 after formation of the cortex corticis. The renal medulla develops by the longitudinal growth of loops of Henle and collecting ducts. The immature medulla cannot be divided into different zones and corresponds structurally to the later inner stripe of the outer zone. The inner zone is formed by PD 8, and the outer stripe of the outer zone by PD 12. The renal medulla is mature at PD 21.From the start of its development, the renal proximal tubule consists of the pars convoluta and pars recta. In both parts the formation of the brush border is accompanied by the simultaneous appearance of brush border enzymes (alkaline phosphatase, -glutamyltranspeptidase, dipeptidylamino-peptidase IV) and lysosomal enzymes (acid phosphatase, acid -galactosidase, N-acetylglucosaminidase, dipeptidylaminopeptidase II) over the full length of the proximal tubule. During the course of proximal tubule maturation, however, the lysosomal enzyme activities decline in the pars convoluta (with constant brush border enzyme activities), while the brush border enzyme activities increase in the pars recta (with constant lysosomal enzyme activities). The two parts further differ in that they exhibit different lysosomal patterns from the outset, the pars convoluta containing numerous large, highly enzyme-active lysosomes arranged in groups, and the pars recta containing only a few very small lysosomes with low enzyme activity. Thus, even in the newborn rat, the lysosomal pattern of the pars recta already corresponds to that of the mature S₃ segment. The S₁ and S₂ segments of the pars convoluta first differentiate between PD 10 and 21, as the groups of large lysosomes are progressively broken up and the extent of the lysosomal apparatus is diminished, this proceeding in a retrograde direction from the end of the immature pars convoluta.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Cytochemistry and biochemistry of acid phosphatases

Summary Acid phosphatases of the rat ventral prostate were studied cytochemically using different substrates. The results were compared to findings on isoelectric focussing gels stained for acid phosphatase activity. This is a highly specific and reproducible method which allows the distinction between secretory androgen-dependent and lysosomal acid phosphatases. Activity of lysosomal acid phosphatase was increased after castration, while the activity of the secretory enzyme gradually decreased after androgen deprivation. None of the substrates tested was selectively hydrolyzed by either secretory or lysosomal acid phosphatase. Phenylphosphate, creatine phosphate and choline phosphate were found to be inappropriate substrates for histochemical purposes, however, reproducible results were obtained with -naphthylphosphate, -glycerophosphate and p-nitrophenylphosphate. The method of isoelectric focussing (pH range 4.0–8.0) of enzymes with subsequent histochemical staining demonstrated lysosomal enzymes at pH 7.9 and 8.2 respectively. Small amounts of identical enzymes were found in liver, kidney, blood or epididymis. Secretory acid phosphatases were focussed at pH 5.5, 5.6, 5.65 and 7.15. Similar enzymes have been identified in epididymis, kidney, liver and pancreas. These results indicate that 1) at present no specific substrate for prostatic secretory or lysosomal acid phosphatases is available and 2) that no prostate-specific prostatic acid phosphatase (PAP) exists in the rat ventral prostate.Supported by the Deutsche Forschungsgemeinschaft (Au 48/6)     

The effects of sucrose loading on lysosomal hydrolases

Summary The addition of 88 mM sucrose to the culture medium of human skin fibroblasts from normal subjects caused remarkable increase in the intracellular lysosomal hydrolase activities. The mechanism of this induction by sucrose loading was carefully studied with several fibroblast strains of different inherited lysosomal storage disorders. In single lysosomal hydrolase defect such as GM₁-gangliosidosis, mannosidosis and Sandhoff disease, no induction of the deficient hydrolase was found with 88 mM sucrose loading. In contrast, sucrose loading caused normalization of intracellular lysosomal hydrolase activities in I-cell disease fibroblasts and cytoplasmic inclusion materials disappeared. Subsequent investigations reveal that I-cell disease cells are classified into three subgroups by the degree of hydrolase induction by sucrose loading; a high responding, an intermediate responding and a no-response group. The heterogeneity may be based upon different induction by sucrose loading of the enzyme, probably the residual phosphotransferase which is involved in the processing steps of lysosomal enzyme molecules. With the addition of mannose-6-phosphate and 10 mM NH₄Cl to cultured skin fibroblasts, it was shown that sucrose loading caused increased synthesis of lysosomal enzyme proteins. The result of the test with 2,4-dinitrophenol suggests that sucrose is indeed pinocytosed by cultured human skin fibroblasts and localized in lysosomes and that this event is the essential factor to trigger the induction of lysosomal hydrolases. Simultaneous loading of both invertase and sucrose in cultured cells caused no induction of -mannosidase activity. This result indicates that invertase is also pinocytosed, reaches the lysosomes and hydrolyzes sucrose in the lysosomes. Lysosomal overloading with sucrose resulted in induction of lysosomal hydrolases and invertase blocked the induction of -mannosidase activity. However, some induction still exists in -galactosidase and -fucosidase activity. Thus it is very likely that the induction of lysosomal hydrolases demands a complicated process.In this article, we investigated the effects of sucrose on the lysosomal hydrolases in cultured human skin fibroblasts of several inherited lysosomal storage disorders and normal subjects and discuss the possible mechanism. of the induction of lysosomal hydrolase activities by sucrose loading.     

Lysosomal enzymes in experimental allergic encephalomyelitis: Time course and evidence of the source

The lysosomal enzymes acid proteinase and -glucuronidase, were assayed in spinal cords of rats during the course of experimental allergic encephalomyelitis (EAE). Histological and histochemical examination was carried out versus controls, in selected areas of the same cords biochemically assayed, to look at the distribution of the lysosomal enzyme acid phosphatase. The biochemical assay showed a significant increase of the enzyme activities during the disease and the increase was significantly correlated with the intensity of the disease. The distribution in the nervous tissue of the increase in acid phosphatase activity observed in animals with EAE, suggests that endogenous nervous cells may contribute to the lysosomal enzyme increase in EAE.     

The effects of lethal procedures on the histochemical localization of acid hydrolases

Summary The distribution and activities, as revealed by histochemical techniques, of acid phosphatase, -glucuronidase, arylsulphatase and -galactosidase in the kidney, liver and skeletal muscle of rats and dystrophic hamsters killed by each of the following methods were studied: overdose of ether, chloroform and nembutal anaesthesia, immersion in liquid air, stunning followed by decapitation, and carbon dioxide asphyxiation.In rats killed with overdoses of ether or chloroform, the activity of acid phosphatase in sections of formaldehyde-fixed kidney was, it was found, less than that in decapitated ones. Sometimes the activity was abolished completely. The intensity of colour, the particulate granularity and the distribution of final reaction product normally obtained for other acid hydrolases and tissues were also affected, e.g. the distribution was more diffuse. Often the minimum time of incubation for the final reaction product to appear was shortened. One example where this happened was the activity of arylsulphatase against 6-benzoyl-2-naphthyl sulphate. The activities of the three other enzymes studied were affected in the same way but to a different degree. However, killing animals with nembutal or liquid air or by decapitation seemed to have little effect on the activities of any of lysosomal enzymes investigated.In order to obtain a reproduceable localization of strong acid hydrolase activity in a particulate form (presumably indicative of lysosomes), it is necessary to bring experimental animals to a basal physiological condition by isolating them overnight away from other animals before killing them (with nembutal or by stunning and decapitation) after a substantial period of rest.     

Coexistence of acid phosphatase and acid mucosubstance in the nucleoid of human blood platelet granules

Summary Fine structural localization of acid phosphatase and acid mucosubstance in the human blood platelet has shown that these two components coexist in the platelet granules. Within the granules acid phosphatase activity and acid mucosubstance both appear confined to the nucleoid region. Additionally, heavy acid phosphatase activity filled occasional small cytoplasmic bodies as well as elongated irregular cell organelles which latter appeared to correspond with atypical granules in morphologic preparations. A complex of mucosubstance with nonenzymatic protein, sequestering the lysosomal enzyme, or a complex of mucosubstance with the enzyme reversibly inactivating it, might explain the cytochemical latency of acid phosphatase in certain lysosomes and the masked dialyzed iron affinity in these sites after glutaraldehyde fixation.     

Multivesicular bodies isolated from rat hepatocytes

Summary Plama lipoproteins (and other ligands) are endocytosed by hepatocytes and appear in multivesicular bodies (MVBs) in the Golgi-lysosome region of the cell prior to their degradation. We have isolated MVB fractions from livers of estradiol-treated rats, permitting studies of their properties (Hornick et al. 1985). Here we report our cytochemical studies of lysosomal enzyme activity in partially and highly purified MVB fractions and in MVBs in hepatocytes in situ.Only about 15% of partially or highly purified MVBs were positive for acid phosphatase and arylsulfatase, consistent with the prelysosomal nature of this compartment. Partially purified MVB fractions contained small round vesicles, 70–120 nm in diameter, which, stained intensely for these enzymes; occasionally these vesicles appeared to fuse with MVBs, suggesting that these structures are primary lysosomes. Such stained vesicles were rarely seen in highly purified MVB preparations. Acid phosphatase reaction product with cerium as capture reagent appeared as uniform precipitates surrounding endocytosed plasma lipoproteins in positively stained MVBs. Arylsulfatase reaction product, however, appeared as distinctive are or plaque-like deposits just inside the MVB-limiting membrane, often in continuity with intense reaction product contained in a fusing primary lysosome. Similar putative primary lysosomes were occasionally observed in isolated, intact Golgi fractions from the same livers. Similar histochemical reactivities of MVBs and putative primary lysosomes were observed in thin sections of hepatoyctes in situ.These observations support the conclusion that, in hepatocytes, MVBs represent the immediate prelysosomal compartment in the endocytic pathway of macromolecular catabolism, and suggest that MVBs are converted to secondary lysosomes by direct fusion with primary lysosomes arising from closely adjacent Golgi compartment.A portion of this work was published in abstract form in the Journal of Histochemistry and Cytochemistry, Vol. 34, page 120, 1986.This work was supported by U.S. Public Health Service Grant HL 14237 (Arteriosclerosis SCOR)     

The demonstration of acid phosphatase in cultured 3T3 mouse cells

Summary Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium -glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.     

Oxygen free radicals induced release of lysosomal enzymes in vitro

Summary The effect of oxygen free radicals, generated by xanthine and xanthine oxidase, was studied on the release of lysosomal hydrolase from rat liver lysosomes in vitro. A lysosomal enriched subcellular fraction was prepared, using differential centrifugation technique, from the homogenate of rat liver. The biochemical purity of the lysosomal fraction was established by using the markers of different cellular organelles. Oxygen free radicals were generated in vitro by the addition of xanthine and xanthine oxidase. The release of lysosomal hydrolase (-glucuronidase) from the lysosomal fraction was measured. There was a 3 to 4 fold increase in the release of -glucuronidase activity in the presence of xanthine and xanthine oxidase when compared to that in the absence of xanthine and xanthine oxidase. In the presence of superoxide dismutase (SOD), a scavenger of oxygen free radicals, the xanthine and xanthine oxidase system was unable to induce the release of -glucuronidase activity from the lysosomes. Sonication (2 bursts for 15 sec each) and Lubrol (2 mg/10 mg lysosomal protein) treatment, which are known to cause membrane disruption, also induced the release of -glucuronidase from lysosomal fraction. This release of -glucuronidase by sonication and lubrol treatment was not prevented by SOD. These data indicate that lysosomal disruption is a consequence of oxygen free radicals, generated by xanthine and xanthine oxidase.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - EGTA Ethylene Glycol Bis-(-aminoethyl ether)N,N,-N,N-tetracetic acid - Tris Tris (hydroxymethyl) aminomethane - SOD Superoxide Dismutase     

A dual enzyme amperometric biosensor for monitoring organophosphorous pesticides

An amperometric biosensor consisting of two enzyme membranes, one a potato layer rich in acid phosphatase and the other immobilized glucose oxidase membrane, when operated in conjunction with a Clark type dissolved O 2 elec-trode, detected the pesticide, Paraoxon, at 1 g/ml. The advantage of this biosensor is that the inhibition of acid phosphatase by the pesticide is reversible and thereby eliminates the problem of enzyme inactivation and the necessity for its reactivation which is not efficient.     

Cytochemical evidence for the leakage of acid phosphatase through ultrastructurally intact lysosomal membranes

Synopsis Fixation under improper conditions ofin vitro cultivated cells results in an extensive diffusion of the lysosomal enzyme acid phosphatase because of the influence of a low effective osmotic pressure. In the present investigation, advantage was taken of this predictable diffusion in order to establish whether or not leakage of acid phosphatase could take place through ultrastructurally intact lysosomal membranes.In order to reveal small holes in the lysosomal membranes, secondary lysosomes were labelled with thorium dioxide particles, which were presumed to appear free in the cell sap if ruptures in the membranes larger than about Ioo Å were created.The experiments revealed that following the fixation ofin vitro cultivated human glia cells under improper conditions, mitochondria and ground cytoplasm show considerable swelling artifacts, while secondary lysosomes appear to be essentially unaffected. The lysosomes, nevertheless, apparently lost most of their content of acid phosphatase, as judged from enzyme cytochemical studies. These findings indicate that leakage of acid phosphatase from ultrastructurally intact lysosomes is possible.     

Effect of triparanol (Mer 29) on rat liver lysosomes: cytochemical and biochemical study

Summary Rats treated with triparanol (MER-29) develop numerous membranous inclusions-myeloid bodies in the cytoplasm of liver cells. The myeloid bodies did not show cytochemically demonstrable acid phosphatase. Instead diffuse activity was observed throughout the cytoplasm. Biochemically, acid phosphatase was found in the liver lysosomal fraction obtained from triparanol treated rats. This fraction, however, did not show the structure-linked latency of acid phosphatase which is characteristic of normal lysosomes. It is suggested that myeloid bodies are lysosomes with altered membranes.     

Evaluation of histochemical observations of activity of acid hydrolases obtained with semipermeable membrane techniques: A combined histochemical and biochemical investigation

Summary The reliability of enzyme histochemical observations of activities of acid hydrolases was investigated with a combined histochemical and biochemical study. Specimens of m. soleus, m. plantaris, m. gastrocnemius and diaphragm of normal and of vitamin E deficient rabbits were used. For the histochemical investigation, activity and localization of acid phosphatase, -glucuronidase, leucine aminopeptidase and E₆₀₀ resistant non-specific aryl-esterase were examined with semipermeable membrane techniques. For the biochemical investigation, activity of acid phosphatase, -glucuronidase, cathepsin D, acid maltase and neutral maltase was determined.By means of statistical calculations the enzyme activities demonstrated with histochemical techniques were compared with the enzyme activities determined with biochemical techniques.In the present communication the histochemical findings are reported and discussed. From the histochemical findings it appeared that activity of the acid hydrolases investigated is strongly increased in both a granular and a diffuse pattern in skeletal muscle of vitamin E deficient rabbits. The statistical calculations of the histochemical findings clearly reveal that the increased activity of one acid hydrolase was highly significantly paralleled by an increased activity of a second acid hydrolase. Moreover the probability that the activity of all other histochemically studied acid hydrolases was significantly increased was rather high.The increase in activity of the acid hydrolases studied was the same in muscles with an aerobic or an anaerobic metabolism. Moreover there was no difference in activity and localization of the acid hydrolases in aerobic type I and anaerobic type II fibres.The localization of acid phosphatase and -glucuronidase activity in muscle fibres mostly coincided. In cases where these enzymes were localized both centrally and in the subsarcolemnal areas of the muscle fibres, the activity of E₆₀₀ resistant naphtholesterase was usually, and the activity of leucine aminopeptidase was exclusively located in the subsarcolemnal areas. All of the examined acid hydrolases were found to be present in the inflammatory exudate and in the connective tissue.This study was partly extracted from the Ph. D. thesis of D.E. Israël (1977).     

Fine structural localization of acid phosphomonoesterase in the brush border region of osteoclasts

Summary The fine structural localization of acid phosphomonoesterase was studied in osteoclasts of fracture callus in the rat. In addition to being present in the Golgi apparatus and the lysosomes, reaction product was precipitated over tubular elements, vesicles, and vacuoles located subjacent to the brush border. The findings are compatible with the idea that the calcified matrix can be destroyed by the combined effects of absorption with subsequent intracellular digestions in osteoclasts, and actual secretion of lysosomal enzyme to the extracellular space.This study was supported by grants from the Swedish Medical Research Council (Project no. B72-12X-1006-07A) and Konung Gustav V:s åttioårsfond". The assistance of Miss Silwa Mengarelli and Mrs. Britt-Marie Åkerman is gratefully acknowledged.     

Lysosomal hydrolase secretion by Tetrahymena: a comparison of several intralysosomal enzymes with the isoenzymes released into the medium.

Tetrahymena pyriformis were grown in proteose-peptone medium and then washed and incubated in a dilute salt solution for one hour. The cells were then discarded and the lysosomal hydrolases that had been secreted were subjected to DEAE cellulose column chromatography. At least three isoenzymes of acid phosphatase, three of acid protease, and two of beta-N-acetylhexoseaminidase were found, as well as single peaks of alpha-mannosidase, beta-galactosidase, and beta-fucosidase. The latter two activities were not resolved by the DEAE column and could not be separated in a second chromatographic step on CM-cellulose. Cells were also grown under identical conditions and homogenized in 0.25 M sucrose in order to allow comparison of some of the intracellular lysosomal hydrolases with their secreted counterparts. Two lysosomal populations were resolved by sucrose density gradient sedimentation, a heavy lysosomal fraction, contered at a density of about 1.25 gm/cm3, and a light lysosomal fraction, centered at a density of about 1.16 gm/cm3. These two populations differed in that the light lysosomes did not appear to contain significant amounts of beta-fucosidase, beta-galactosidase, or acid protease, whereas all six of the hydrolase activities studied were present in the heavy lysosomes. The light lysosomal peak occurred in cells grown to transition phase, but was markedly reduced in cells from cultures grown to stationary phase. In addition to these two fractions a third very light particle, containing only alpha-mannosidase activity, was detected just inside the gradient. Measurements were made of the effect of heat (10 minutes at 66 degrees) and of a change in pH from 4.5 (standard assay condition) to 6.0 on the three acid phosphatases and two beta-N-acetylhexoseaminidase isoenzymes resolved by DEAE column chromatography of the secreted hydrolases and on these hydrolyases in the heavy and light lysosomal fractions on the sucrose gradient. Use of the thermostability and pH criteria permitted computation of the expected properties of the intralysosomal acid phosphatase and hexoseaminidase activities if these consisted of the respective isoenzymes in the proportions secreted. It was found that neither the intralysosomal acid phosphatase nor the intralysosomal hexoseaminidase had the properties expected if they consisted of the secreted mixture of the respective isoenzymes, indicating that modification of some of these isoenzymes may have occurred during the 1-hour starvation period or after secretion.     

Subcellular distribution of polysaccharide depolymerase and glycoside hydrolase enzymes in rumen ciliate protozoa

The distribution of polysaccharide depolymerase and glycoside (acid) hydrolase activity in nine genera of rumen entodiniomorphid and holotrich ciliate protozoa was examined by differential centrifugation. Sedimentable activity was detected in all of the protozoa examined and occurred principally in fractions that were prepared by centrifugation at 1000g for 10 min, 10,000g for 10 min, and 20,000g for 20 min (fractions F1, F2, and F3). Acid phosphatase was present in these subcellular fractions which contained membrane-bound vesicles 0.1–0.8 m in size. The enzyme location profile of the subcellular fractions differed within the genera examined. The distribution of the enzyme activity in the subcellular fractions indicated the presence of distinct populations of hydrolase-containing organelles and other functional vesicles in the rumen ciliates.