LYSOSOMAL PHYSIOLOGY IN TETRAHYMENA : III. Pharmacological Studies on Acid Hydrolase Release and the Ingestion and Egestion of Dimethylbenzanthracene Particles

The ingestion of ¹⁴C-labeled 9,10-dimethyl-1,2-benzanthracene particles, the extracellular release of acid phosphatase, ribonuclease, and α-glucosidase, and the egestion of preingested dimethylbenzanthracene particles by Tetrahymena taken from logarithmically growing cultures and resuspended in a dilute salt solution were followed in the presence of several pharmacologic agents. Serotonin, caffeine, and, to a lesser extent, dibutyryl cyclic AMP increased the rate of particle ingestion, but did not alter the rate of release of the three acid hydrolases studied. Added catecholamines did not affect either particle ingestion or acid hydrolase release, but particle ingestion was inhibited by the catecholamine antagonists, dichloroisoproterenol, desmethylimipramine, reserpine, and phenoxybenzamine. These drugs also increased the release of acid phosphatase and ribonuclease in 5-h incubations. Desmethylimipramine acted within 1 h to increase acid hydrolase release, but the effect of dichloroisoproterenol developed more slowly and was secondary to a change in cellular content of the hydrolases. Desmethylimipramine increased the energy of activation for the release of acid phosphatase, while dichloroisoproterenol did not. Both of these drugs enhanced the egestion of preingested dimethylbenzanthracene particles, supporting the view that acid hydrolase release occurs through a cytoproct egestion mechanism. Particle ingestion was also inhibited by colchicine, vinblastine, and cytochalasin B, but these agents had no effect on acid hydrolase release, thus further differentiating the properties of the ingestion mechanism from those of the egestion mechanism. It appears that both microtubules and microfilaments play a role in the ingestion process and that this process may be controlled in part by a cyclic AMP-mediated serotoninergic and adrenergic system.     

Giardia lamblia: localization of hydrolase activities in lysosome-like organelles of trophozoites

Homogenates of Giardia lamblia trophozoites exhibited the following hydrolase activities: acid phosphatase (EC 3.1.3.2), proteinase (EC 3.1.4) with urea-denatured hemoglobin and N-benzoyl-DL-arginine-2-naphthylamide as substrates, deoxyribonuclease (EC 3.1.4.5), and ribonuclease (EC 2.7.7.16). beta-N-Acetylglucosaminidase (EC 3.2.1.30), beta-galactosidase (EC 3.2.1.23), beta-glucuronidase (EC 3.2.1.31), alpha-D-glucosidase (EC 3.2.1.20), beta-D-glucosidase (EC 3.2.1.21), and beta-D-xylosidase (EC 3.2.1.37) activities were below the level of detection. Differential and isopycnic centrifugation of homogenates demonstrated that giardial hydrolases were localized in a single-particle population sedimenting at 7200g for 30 min. The particles had a buoyant density in sucrose of 1.15 and exhibited latency. Latency was completely destroyed by Triton X-100 or 15 cycles of freezing and thawing. After centrifugation of Triton- or freeze-thaw-treated particle fractions, the hydrolase activities, though no longer latent, were still sedimentable suggesting tight binding to the organelle membrane. Latency was destroyed simultaneously for all hydrolases, in direct proportion to the amount of Triton added to a particle preparation or to the number of times a particle preparation was subjected to freezing and thawing. These results support the suggestion that the hydrolases of G. lamblia trophozoites are localized in a single-particle population of lysosome-like organelles.     

Isolation and properties of lysosomes from dark-grown potato shoots

Summary A method is described for the isolation of lysosomal fractions from dark-grown potato shoots using a single stage separation on a Ficoll gradient. Peaks of acid hydrolase activity consisting of acid phosphatase, phosphodiesterase, ribonuclease, carboxylic esterase and -glycerophosphatase were well separated from peaks of mitochondrial and glyoxysomal enzymes. A heavy lysosomal fraction with particle diameters from 0.1 to 1.6 and density of 1.10 g cm^-3 containing relatively low hydrolase activity was distinguishable from a light fraction with diameters 0.025 to 0.6 and density of 1.07 g cm^-3 with a higher level of hydrolase activity. Both fractions appeared heterogeneous by electron microscopy, but the fine structure of the membranes of both heavy and light lysosomes was similar. The heavy lysosomal fraction was rich in autophagic vacuoles (secondary lysosomes) containing organelles and amorphous cytoplasmic material. Both fractions were rich in ribonucleic acid.Freezing and thawing, high speed blending and ultrasonication either singly or in combination solubilised a maximum of ca. 30% of the acid phosphatase from crude lysosomal fractions derived from dark-grown potato shoots. Treatment with Triton X-100 and deoxycholate released appreciably more enzyme activity but acetone and carbon tetrachloride failed to solubilise any acid phosphatase. Only detergent treatments gave marked overrecovery of enzyme and indicated structure-linked latency. Liberation of enzyme from lysosomes varied with pH and was almost complete at both extremes of pH. Crude snake venom was rapid and effective in solubilising acid phosphatase from lysosomal preparations, purified phospholipase A was less effective and phospholipases C and D had negligible effects. Phospholipase and venom mediated release of acid phosphatase was accompanied by the coincident release of an acid end-product. Gel filtration of acid phosphatase liberated from heavy and light lysosomal fractions by snake venom digestion revealed that each of these fractions was characterised by the presence of distinct molecular forms of the enzyme. The nature of the association of acid phosphatase with potato shoot lysosomes is discussed.     

Lysosomes in Tetrahymena pyriformis. I. Some properties and lysosomal localization of acid hydrolases

In homogenates of Tetrahymena pyriformis, five hydrolases — phosphatase, ribonuclease, deoxyribonuclease, proteinase, amylase — with acid pH optima were found. Over 75% of their activity is sedimentable with a centrifugal force of 250,000 g. min. Only 17% of the acid phosphatase and ribonuclease is active when assayed in the presence of 0.25 M sucrose at 0°. Exposure to a lowered osmotic pressure, freezing and thawing, and incubation at temperatures over 0° result in activation of the latent phosphatase and ribonuclease. After isopycnic centrifugation in a sucrose density gradient the hydrolases show a broad distribution which differs greatly from those of enzymes associated with mitochondria (succinate dehydrogenase) or with peroxisomes (catalase). The results are interpreted as evidence that the five acid hydrolases studied are localized in lysosomes which represent a distinct population of subcellular particles in Tetrahymena.     

The heterogeneous distribution of acid hydrolases within a homogeneous population of cultured mammalian cells

1. Chinese-hamster ovary fibroblasts were cultured to provide a homogeneous cell population. Homogenates obtained from these cells were fractionated by centrifugation techniques and the resulting fractions were analysed for protein and for enzymes representative of certain subcellular particles. 2. Unlike those in rat liver homogenates, the mitochondrial and lysosomal populations proved impossible to separate by differential centrifugation owing to the similarity of their sedimentation properties. Their resolution was possible by using isopycnic centrifugation in a continuous sucrose density gradient. 3. The mitochondrial population equilibrated at a density of 1.17g.cm(-3) as in rat liver homogenates. However, the lysosomal population equilibrated at a lower rather than a higher density position than the mitochondria and the probable reasons for this are discussed. 4. The lysosomal population subdivided into two groups characterized by differences in acid hydrolase content and equilibrium densities. The fraction with a density of 1.15g.cm(-3) contained the majority of arylsulphatases A and B, of cathepsin and of beta-acetylglucosaminidase activities, whereas that with a density of 1.09g.cm(-3) contained the majority of the acid phosphatase and acid ribonuclease activities. The probable division of the lysosomal population of a single cell into a number of distinguishable subgroups is suggested.     

Lysosomes in skeletal muscle tissue. Zonal centrifugation evidence for multiple cellular sources

Postnuclear supernates from homogenates of skeletal muscle from rats subjected to starvation, injections of Triton WR-1339, dextran-500, and dextran + corticosterone were fractionated by means of rate and isopycnic zonal centrifugation in sucrose—0.02 M KCl gradients. Zonal fractions were analyzed for protein, RNA, cytochrome oxidase, and up to six acid hydrolases. The results indicate the presence of two groups of lysosome-like particles. One group contributes approximately 95% of the cathepsin D and acid phosphatase activity and 75% of the acid ribonuclease, β-glucuronidase, and arylsulfatase activity in muscle. It is characterized by a modal equilibrium density of 1.18 that is decreased by starvation, but is not shifted by dextran-500 or Triton WR-1339. The second group has a higher proportion of acid ribonuclease, β-glucuronidase, and arylsulftase; the equilibrium density can be shifted by dextran-500 and Triton WR-1339. It is suggested that this group of lysosomes is derived from macrophages and other connective tissue cells, whereas the former group represents lysosome-like particles from muscle cells.     

Effect of freezing and thawing on the distribution of lysosomal hydrolases in leaves of Solanum tuberosum L.

Density-gradient ultracentrifugation techniques showed that freezing and thawing of potato leaves resulted in a change in the density of subcellular particles containing acid phosphatase and acid ribonuclease (RNase). Gel filtration experiments were used to characterise the molecular forms of the hydrolases associated with the various cell fractions. Freezing and thawing promoted a release of a portion of the complement of acid phosphatase and RNase from the lysosomes to the supernatant fluid fraction of cell homogenates. The freezing treatment appeared to activate latent lysosomal RNase.     

Effect on lysosomes of invertase endocytosed by rat-liver

The intracellular localization of invertase endocytosed by rat liver was investigated by analytical centrifugation in sucrose and Percoll gradients of mitochondrial fractions originating from rats killed 15 h after injection. After isopycnic centrifugation in a sucrose gradient, invertase is located in higher density zones than acid hydrolases. The difference between the distribution of invertase and that of acid hydrolases increases with the amount of invertase injected. When the invertase dose is sufficiently high, a change of lysosomal enzyme distribution is clearly visible. It consists in the shift of a proportion of these enzymes to higher density regions where invertase is located. The proportion of hydrolase activity affected by invertase is different for each enzyme measured; it is the least pronounced for acid phosphatase, and most for acid deoxyribonuclease and arylsulfatase. A pretreatment of the rat with Triton WR 1339 considerably decreases the equilibrium density of structures bearing invertase. Nevertheless invertase distribution is quite distinct from that of the bulk of lysosomal enzymes that are recovered in lower density zones of the gradient; on the other hand the invertase injection to rats treated with Triton WR 1339 causes a spreading of the acid hydrolase distribution towards higher density zones. The distribution of acid hydrolases and invertase in a Percoll gradient depends on the sucrose concentration of the solvent. It is shifted towards higher densities when the sucrose concentration increases. The phenomenon is more important for invertase. These results are best explained by supposing that invertase accumulates in a distinct population of lysosomes that can be individualized as a result of the density increase they are subjected to by the invertase they accumulate. It is proposed that these lysosomes mainly originate from non-parenchymal cells of the liver.     

Specificity and localization of the hepatitis B virus-associated protein kinase.

The nature of the protein kinase (PK) which phosphorylates the core protein of hepatitis B virus in vitro was studied. The PK copurified with the core particles during rate zonal centrifugation and gel chromatography. It showed the same size heterogeneity as the core particles, which consisted of a main fraction of 28-nm particles and a subfraction of 22- to 26-nm particles. DNA-containing heavy core particles with a density of 1.33 to 1.35 g/ml and less endogenous PK than did the light cores. The phosphorylation reaction had a rapid initial phase (several minutes) and a slow but long-lasting second phase (many hours). The PK had a high affinity for ATP (KM = 0.5 mumol/liter). Only few of the several hundred P21.9 subunits in one core particle were phosphorylated in vitro. The only amino acid which was phosphorylated in vitro was serine. The resistance of the introduced phospho group against alkaline phosphatase showed that the PK acceptor, and probably the enzyme itself, was located inside the core particle.     

Seasonal Changes in Molecular Forms of Acid Phosphatase and Ribonuclease of Potato Tubers and the Effects of Infection by Phytophthora erythroseptica

Sequential changes in total activity and molecular forms ofacid phosphatase and ribonuclease from potato tubers were studiedby seasonal assays and Sephadex gel filtration. Ribonucleaseand p-nitrophenyl phosphatase activity fluctuated during storageof tubers, while ß-glycerophosphatase declined toa low level coincident with initiation of sprout growth. Inrecently-lifted tubers acid phosphatase activity occurred ina single high molecular weight peak. Two new forms of lowermolecular weight appeared during ageing of stored tubers. Theinfluence of infection by a tuber-rotting fungus, Phytophthoraerythroseptica,on these seasonal changes was variable. No consistent effectson total hydrolase activities were observed, while post-infectionalchanges in molecular forms included a pronounced shift in themajor acid phosphatase peak. The possible significance of thismolecular weight change in infected samples is discussed inthe light of recent evidence concerning the sub-unit structureof acid phosphatase from potato tubers.     

Lysosomal Physiology in Tetrahymena. II. Effect of Culture Age and Temperature on the Extracellular Release of 3 Acid Hydrolases*

Tetrahymena cultures were grown from a single inoculum and collected on 3 successive days corresponding to the log, transitional, and early stationary phases of growth. Cells were washed and incubated for 5 hr in a dilute salt solution. Intra- and extracellular activities of acid phosphatase, α-glucosidase, and ribonuclease were assayed, and extracellular activities corrected for proteolytic degradation. A marked increase in the cellular content of acid phosphatase and significant decreases in α-glucosidase and ribonuclease occurred with advancing culture age. The intracellular changes in enzyme activities during incubation were roughly similar for cells of all ages. Protein content did not change appreciably during incubation. Extracellular A₂₅₅ release, monitored as an indication of the loss of RNA breakdown products, was at a minimum during incubation of transition cells. Significant quantities of all 3 acid hydrolases were released from cells of all ages except for ribonuclease from transition cells. The release of acid phosphatase and α-glucosidase was approximately proportional to the initial cellular content of these enzymes for cells of different ages and in log cells the effect of temperature on the rates of release was described by the Arrhenius equation. Release of ribonuclease, however, was not proportional to its intracellular content nor did it vary with temperature according to the Arrhenius equation. The results suggest that acid phosphatase and α-glucosidase are released via a first-order process.     

Calcium transport in isolated bone cells. I. Bone cell isolation procedures

Differential centrifugation of homogenates of Harding-Passey melanoma demonstrated that aryl sulfatase A and β-glucuronidase sediment with particles (i.e., lysosomes) distinct from those particles bearing tyrosinase (i.e., melanosomes). The sedimentation curves for the lysosomal enzymes and tyrosinase, however, demonstrated that an adequate separation of these particle types could not be obtained by differential centrifugation. Isopycnic density gradient centrifugation was used to obtain the necessary resolution. The results of the density gradient studies demonstrated that lysosomes and melanosomes could be separated by this technique, as judged by enzyme distribution among the fractions recovered from the gradients and from electron microscopic examination of the melanosome fractions. It was further evident that the purified and washed melanosomes contained significant amounts of both acid hydrolase activities. Indeed 24% to 27% of the total acid hydrolase activities recovered from the density gradients were associated with the melanosome fractions. The acid hydrolases associated with the melanosomes could not be solubilized by treatment with 0.1% (v/v) Triton X-100, nor by exposure to hypo-osmotic shock. The melanoma lysosomes, however, did release most of both their hydrolase activities into soluble form after treatment with the same percentage of detergent. The lysosomes were, however, very resistant to rupture by exposure to hypo-osmotic conditions.     

Acid hydrolases in the suckling rat small intestine. II. On the importance of alkaline phosphatase inhibition in the histochemical localization of acid phosphatase activity.

Phosphatase activities against beta-glycerophosphate, I-naphthyl phosphate and naphthol AS-TR phosphate were investigated, at acid and aldaline pH levels, using unfixed and fixed cryostat sections of suckling rat jejunum. The use of 10 mm EDTA and 10 mm NaF as inhibitors indicated that alkaline phosphate is predominantly located in the microvillous region of the adsorptive cells, while acid phosphatase is located in small particles distributed between the brush borders and the nuclei of these cells. Alkaline phosphatase activity was found to interfere with the localization of acid phosphatase unless EDTA was included in the incubation medium. A modified Gomori medium, containing 10 mm EDTA and additional lead nitrate, is described. Latency experiemtns using this medium, with unfixed sections, indicated the lysosomal nature of particulate acid phosphatase. The discussion stresses the importance of including an aldaline phosphatase inhibitor in incubation media designed to localize extralysosomal acid phosphatase activity.     

In vivo suppression of coding associated with bacteriophage-induced S-adenosylmethionine hydrolase

The methylation of ribosomal and transfer ribonucleic acid (RNA) synthesized after the induction of a hydrolase for S-adenosylmethionine by phage T3 infection is reducible to 50% of the methylation of RNA in uninfected cells. Hypomethylated ribosomal RNA is found in 70S particles that dissociate in 100 mum Mg(++) to yield only 30S and 50S subunits. By this criterion, the omitted methyl groups apparently are not required for ribosomal maturation or stability. The rate of production of alkaline phosphatase in a phosphatase amber mutant was examined after phage infection in the presence and in the absence of streptomycin to determine the effect on the translation process consequent to S-adenosyl-l-methionine (SAM) hydrolase induction. Significant increases in the rates of phosphatase production were found when ultraviolet-inactivated T3 or streptomycin was added. The effects were cumulative when the cells were treated with both bacteriophage and the drug. Ultraviolet-inactivated T7, a phage closely related to T3 but which does not produce the SAM hydrolase, did not enhance the rate of alkaline phosphatase production. We suggest that the production of SAM hydrolase affects the stability of the translation process by the observed hypomethylation or by mechanisms concerning polyamine metabolism.     

Analog modeling of glucagon-induced autophagy in rat liver. II. Evaluation of iron labeling as a means for identifying telolysosome, autophagosome and autolysosome populations.

To evaluate the potential usefulness of iron labeling as a means for identifying the telolysome, autophagosome and autolysosome populations of rat liver, animals treated with Jectofer (iron-citric acid-sorbitol complex), or with Jectofer followed by glucagon, have been studied with a variety of biochemical and morphological methods. Differential centrifugation studies of liver homogenates revealed that the sedimentation velocity and mechanical fragility of acid phosphatase bearing particles increase with the duration of Jectofer treatment and that iron accumulates in the mitochondrial and nuclear fractions. Rate sedimentation studies confirmed the change in sedimentation velocity, which was shown to be due in part to a marked increase in particle density. Quantitative morphological analysis of liver M + L and N + M + L fractions revealed a nearly complete absence of pericanalicular dense bodies after 6–7 days of Jectofer treatment. In these fractions a new type of particle containing fine electron dense granules was seen. The mean volume of these particles was decreased and their number increased when compared to dense bodies but the general morphology and overall size distribution of the two particle classes were similar. In animals given both Jectofer and glucagon, autophagic vacuole formation was similar to that found in animals receiving only glucagon. However, the increase in osmotic fragility of acid phosphatase bearing particles usually seen after glucagon administration occurred at a significantly slower rate. Examination of paniculate fractions revealed the presence of autophagic vacuoles with (autolysosomes) and without (autophagosomes) fine dense granules. The number of autolysosomes and their relative proportion in the autophagic vacuole population were correlated with an increase in the osmotic fragility of the acid phosphatase bearing particles in the same fraction. Organelle degeneration was observed more frequently in autolysosome profiles. These results support the contention that iron labeling can be used to separate the principal particle populations participating in the autophagic response induced by glucagon.     

Quantitative immunogold localization of Na, K-ATPase along rat nephron.

Ultrastructural localization of Na, K-ATPase alpha-subunit along rat nephron segments was investigated quantitatively by immunogold electron microscopy on LR-White ultrathin sections using affinity-purified antibody against alpha-subunit of the enzyme. Ultrathin sections were incubated with the antibody at a saturation level and the number of gold particles bound per micron of the plasma membrane (particle density) of the tubular epithelial cells from the proximal tubule to the collecting duct was determined. In all the tubular epithelial cells, gold particles were located exclusively on the basolateral surface, and no significant binding of gold particles to the apical surface was observed. Distribution of gold particles on the basolateral membranes was quite heterogeneous; lateral membranes and infolded basal membranes were highly labeled, whereas the basal membranes which are in direct contact with the basal lamina were scarcely labeled. The average particle density on the basal surface was highest in the distal straight tubule cells (11.4 units), very high in the distal convoluted tubule cells (9.8 units), intermediate in the proximal tubule cells (3.3 units), in the connecting tubule cells (4.3 units), and in the principal cells of the collecting duct (5.6-3.8 units), low in the thin limb of Henle's loop (1.0 unit), and at the control level in the intercalated cells in the connecting and collecting duct. The relative number of gold particles/mm nephron segment and the relative number of gold particles in the various nephron segments were calculated using quantitative morphological data. The estimated distribution profile of the former was in good agreement with the Na, K-ATPase activity profile in rat nephron, which was determined biochemically with a microenzymatic method.     

The subcellular distribution and properties of Crithidia sp. hydrolases with particular reference to pyrophosphate and orthophosphate monoester phosphohydrolases.

A cell fractionation scheme was developed for studying the distribution of certain hydrolases, especially phosphohydrolases in a Crithidia sp. (Trypanosomatidae). Whilst between 26-56% of the total cellular hydrolase activities were soluble (probably of flagellar pocket origin), a certain percentage, 5-40%, was sedimentable. A particulate fraction obtained after isopycnic density gradient centrifugation (p = 1.187-1.241), designated fraction FA/FB, was enriched in various acid hydrolases (relative specific activities 1.33-6.24) and displayed latent phosphohydrolase activities. The density gradient distributions of this hydrolytic enzymes were compared with reference to one another and malate dehydrogenase (mitochondrial marker). From the results obtained it appears that the sedimentable acid hydrolases of Crithidia are associated with a heterogeneous population of subcellular particles. Cytochemical observations on the FA/FB fraction supported this finding and revealed the association of acid phosphatase reaction product with subcellular elements resembling multivesicular bodies.     

Selective Release of Proteins from Spirillum itersonii by Tris(hydroxymethyl)aminomethane and Ethylenediaminetetraacetate

Treatment of Spirillum itersonii with tris(hydroxymethyl)aminomethane (Tris)-ethylenediaminetetraacetate (EDTA) results in the quantitative release of alkaline phosphatase and ribonuclease into the surrounding medium. At the same time, about 90% of the total cellular soluble cytochrome c is liberated. This process occurs within 1 min of treatment at both 24 and 4 C. Release of these proteins by Tris-EDTA treatment is highly selective, since only 9% of the total cell protein is liberated, concomitantly with less than 5% ribonucleic acid, deoxyribonucleic acid, and malate dehydrogenase. Different sigmoidal curves are obtained for release of proteins as a function of EDTA concentration. The order of liberation with increasing EDTA is as follows: alkaline phosphatase, protein, soluble cytochrome c, and ribonuclease. Treatment of cells with Tris-EDTA under conditions which cause extensive loss of alkaline phosphatase, soluble cytochrome c, and ribonuclease results in cell death, with cessation of protein and ribonucleic acid synthesis. Cells treated with EDTA in phosphate buffer (in the absence of Tris) liberate a large portion of their soluble cytochrome c, but negligible amounts of alkaline phosphatase and ribonuclease. Addition of Tris to cells pretreated with phosphate-buffered EDTA releases high levels of alkaline phosphatase, but not ribonuclease. These results suggest that a common surface alteration is not solely responsible for release of periplasmic proteins. More likely, each protein of the periplasm is bound in an independent and specific manner.     

The development of ribonuclease and acid phosphatase during germination of Pisum arvense

1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.     

Structural characterization of polysomal poly(A)-protein particles in rat liver

Poly(A)-protein particles were prepared from rat liver polyribosomes, washed with 0.5 M KCl or unwashed, after digestion with pancreatic ribonuclease and ribonuclease T1 by two successive rounds of sucrose gradient centrifugation. The particles were sedimented in a range of 5--13 S with a peak at about 9 S. The KCl wash of polysomes had no effect on the sedimentation properties of the particles. The particles isolated in this manner were 99% resistant to further pancreatic ribonuclease treatment and contained about 96% adenylic acid. The length of the poly(A) molecules prepared from the poly(A)-protein particles showed a broad distribution of about 70--290 nucleotides with a peak around 130 nucleotides, as measured by polyacrylamide gel electrophoresis. In CsCl density gradient the poly(A)-protein particles banded in a density range of 1.30--1.42 g/cm3 with a peak at 1.36 g/cm3, which amounts to about 80% of the protein content. Sodium dodecyl sulfate/polyacrylamide and urea/sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated six polypeptides with molecular weights of 50 000, 54 000, 58 000, 63 000, 76 000 and 90 000 in the poly(A)-protein particles, but the main components were dependent on the method. The treatment of polysomes with KCl resulted in a loss of the 90 000-molecular-weight component. Amino acid analysis of the polypeptides bound to poly(A) revealed that they contained a relatively large amount of aspartic plus glutamic acid (21.6%) as well as hydrophobic amino acids (41.4%). Digestion of glutaraldehyde-fixed particles with ribonuclease T2 showed that about 50% of poly(A) was accessible to the enzyme, thus this part of poly(A) was located on the surface of the particles. In the electron micrographs the shadowed poly(A)-protein particles appeared in a globular, somewhat elongated form and were mostly 14-18 nm in diameter. On the basis of the results a model for the 'average' 9-S particles was constructed.