Response of heterogenous rat liver lysosome populations to starvation and refeeding.

Starvation-induced alterations in liver lysosomes and their recovery pattern following refeeding were investigated. Fasting of adult rats for five days caused an increase in 'free' activities of acid hydrolyses in liver homogenates and loss in sedimentation of one of the heterogenous populations of lysosomes that could be isolated by differential centrifugation. Isopycnic sucrose gradient centrifugation revealed a decrease in the median and modal equilibration densities of all the forms of lysosomes in response to the dietary deprivation. Further, starvation also evoked a distinct bimodal distribution in a population that was rich in acid phosphatases, beta-galactosidase and N-acetyl-beta-glucosaminidase. Realimentation of starved animals for 10 days was found to restore the enzyme levels and the sedimentation characteristics to normal profiles.     

SUBCELLULAR DISTRIBUTION OF d-AMINO ACID OXIDASE AND CATALASE IN RAT BRAIN

—Rat brain d -amino acid oxidase was found to be confined to the hindbrain, distributed more or less equally between cerebellum and medulla. Histochemical staining showed the enzyme to be localized largely in the molecular layer of the cerebellum. After fractionation by means of several distinct density gradient centrifugation procedures exploiting differences in sedimentation coefficient or in density or in both, the enzyme was found to be entirely or almost entirely associated with cytoplasmic particles with a median diameter of the order of 0·2 μm, and a median equilibrium density in aqueous sucrose of 1·18. Comparison with the behavior of cytochrome oxidase and of N-acetyl-β-glucosaminidase indicates that these particles are not mitochondria and are unlikely to be lysosomes. They also differ significantly from the bulk of the catalase-containing particles, which on an average appear to be somewhat smaller. The possibility that they might contain some catalase activity, and thereby qualify as ‘peroxisomes’, can however not be excluded. In any case, they differ profoundly from the peroxisomes of liver or kidney.     

The early post-natal development of the neuronal lysosome

Abstract— The hydrolysis of p-nitrophenyl-2-acetamido-2-deoxy-β-d -gluco-(I) and β-d -galacto-pyranoside (II) and of p-nitrophenyl-α-d -mannopyranoside (III) by neuronal cell bodies and glial cells isolated from the cerebral cortex of 18-day-old or adult rats was found to be equally efficient, with relative ratios of hydrolysis for I, II and III of approximately 10:1:0.5 in both cell types and at both ages. Homogenates of the neuronal cell bodies obtained from cerebral cortices of 3-, 8-, 12-, 18- and 32-day-old rats were subjected to differential centrifugation and the subcellular localization of N-acetyl-β-d -glucosaminidase (EC 3.2.1.30) hydrolysing (I)] was compared to that of the mitochondrial marker, succinate-INT- oxidoreductase (EC 1.3.99.1). A fraction in which N-acetyl-β-d -glucosaminidase exhibited maximal specific activity could be isolated at all ages, an observation indicating that the potential for active hydrolytic performance is incorporated into the neuronal lysosome very early post-natally. The specific activities of N-acetyl-β-d -glucosaminidase and succinate- INT-oxidoreductase reached their respective maxima at widely different times postnatally: at 10–12 days for the mitochondrial enzyme and at about 18 days for the glycosidase, a difference suggesting that in the cortical neuron lysosomes and mitochondria develop out of step. The mitochondrial, lysosomal and microsomal fractions obtained by differential centrifugation were subjected to equilibrium density centrifugation and the presence of two populations of N-acetyl-β-d -glucosaminidase-bearing particles was demonstrated. Although their presence was readily apparent in the neurons from 8- and 12-day old brains, it was difficult to discern their presence in the neurons from the 3- and the 18-day-old brains. In 8-day-old brains gradient fractions obtained from neurons containing N-acetyl-β-d -glucosaminidase of a specific activity up to 8-fold higher than that of the enzyme in the original neuronal homogenate were examined by electron microscopy and the concentration of numerous lysosomes and derivative bodies in these fractions was verified. Our present study demonstrates the capability of the immature and developing neuron to tightly couple the pace of its degradative processes to that of its highly efficient and highly selective synthetic activities.     

RESOLUTION OF GRANULES FROM RABBIT HETEROPHIL LEUKOCYTES INTO DISTINCT POPULATIONS BY ZONAL SEDIMENTATION

Postnuclear supernates from homogenates of essentially pure rabbit heterophil leukocytes were fractionated by means of zonal differential centrifugation through a discontinuous sucrose gradient at various speeds. Three distinct groups of granules were characterized biochemically and morphologically. They were, in order of decreasing sedimentation coefficient: (a) Large, relatively dense granules, identified morphologically as the azurophil or primary granules, and containing essentially all of the myeloperoxidase activity of the preparations, about one-third of their lysozyme activity, and between 50 and 80% of their content in five acid hydrolases typically associated with lysosomes in other cells; (b) smaller, less dense granules, with the morphological appearance of the specific or secondary granules, and carrying most of the alkaline phosphatase and the remainder of the lysozyme activity of the preparations; (c) a second group of lysosome-like particles, associated with a morphologically heterogeneous fraction, and containing the remainder of the acid hydrolases, but little or no myeloperoxidase. When p-nitrophenyl phosphate was used instead of β-glycerophosphate for the assay of acid phosphatase, only small proportions of the total activity accompanied the two main lysosomal bands, and considerable activity was found in a zone slightly retarded with respect to the slowly moving band of acid hydrolases.     

Fusion of Lysosomes with Late Endosomes Produces a Hybrid Organelle of Intermediate Density and Is NSF Dependent

Using a cell-free content mixing assay containing rat liver endosomes and lysosomes in the presence of pig brain cytosol, we demonstrated that after incubation at 37°C, late endosome–lysosome hybrid organelles were formed, which could be isolated by density gradient centrifugation. ImmunoEM showed that the hybrids contained both an endocytosed marker and a lysosomal enzyme. Formation of the hybrid organelles appeared not to require vesicular transport between late endosomes and lysosomes but occurred as a result of direct fusion. Hybrid organelles with similar properties were isolated directly from rat liver homogenates and thus were not an artifact of cell-free incubations. Direct fusion between late endosomes and lysosomes was an N-ethylmaleimide–sensitive factor– dependent event and was inhibited by GDP-dissociation inhibitor, indicating a requirement for a rab protein. We suggest that in cells, delivery of endocytosed ligands to an organelle where proteolytic digestion occurs is mediated by direct fusion of late endosomes with lysosomes. The consequences of this fusion to the maintenance and function of lysosomes are discussed.     

Etude comparative d'activites enzymatiques microsomiques dans les hepatocytes de singe adulte et foetal: Comparative study of microsomal enzymic activities in adult and foetal monkey hepatocytes

Differential centrifugation was applied to adult and foetal liver of monkey. Obtained fractions were: F₁ (800 × g); F₂ (12 500 × g); F₃ (200 000 × g); and cell sap. Analysis of chemical compounds of these fractions shows that: (1) adult and foetal nucleic acids levels are similar; (2) there are more proteins in adult than in foetal hepatocytes; (3) most of the glycogen is located in F₃; the foetal level is twenty times higher than the adult level.Plasma membrane enzymes (5′-nucleotidase, adenylate cyclase) show a nucleomicrosomic distribution. The distribution of alkaline phosphatase is not significant.Mitochondrial enzymes (monoamine oxydase, succinate cytochrome c reductase, cytochrome oxydase) are enriched in F₂ without any sedimentation in F₃ There is more malate dehydrogenase liberated in cell sap during foetal liver fractionation. This indicates the foetal mitochondria are more sensitive to the homogenisation method.Lysosomal enzymes (acid phosphatase, N-acetylglucosaminidase) are enriched in F₂. The same observation for N-acetylglucosaminidase as for malate dehydrogenase leads to the same conclusion for foetal lysosomes.Endoplasmic reticulum and Golgi enzymes (glucose-6-phosphatase and related phosphotransferase activity, NADPH-cytochrome c reductase and sialyltransferase) are much enriched in F₃. Thus this fraction F₃ is pure enough to allow the observation of the modification produced on endoplasmic reticulum and Golgi apparatus during foetal and neonatal development.     

Studies on a HCO3 minus-stimulated ATPase and carbonic anhydrase system in rat liver lysosomes

Lysosome-rich fractions were prepared by discontinuous sucrose gradient centrifugation of liver homogenates from rats pretreated with Triton-WR-1339. The lysosome-rich fraction contained 48% of the crude homogenate hexosaminidase applied to the centrifuge tube and its specific activity was 10-fold greater than the original homogenate. A Mg²⁺-requiring ATPase that was stimulated by 20 mm HCO₃^? was associated with the lysosomal-enriched fraction. Its specific activity was 50–65% of that compared with the mitochondrial-rich fractions.The properties of the HCO₃^?-stimulated ATPase from rat liver lysosomes were similar to those previously reported from gastric mucosa, submaxillary gland, and pancreas with respect to substrate specificity, anion stimulators, and inhibitors. Double-reciprocal plots were nonlinear with respect to ATP (n_H = 2.23) and HCO₃^? (n_H = 1.84), and the corresponding K_m values were estimated to be 0.33 and 7.25 mm.Carbonic anhydrase activity was also found associated with the lysosomes at activities comparable to those of the mitochondrial-rich fractions. The lysosomal carbonic anhydrase was inhibited 33% by 100–200 μm acetazolamide, whereas that in mitochondrial fractions was inhibited by 68–71% by 100-μm levels of the drug. The ATPase and carbonic anhydrase system of rat liver lysosomes represents a possible mechanism for the maintenance of intralysosomal proton gradients.     

LYSOSOMAL FRACTIONS FROM TRANSITIONAL EPITHELIUM

Histochemical data suggested that the so called lipoid granules of transitional epithelium in some species are equivalent to lysosomes. Scrapings of bovine and canine transitional epithelium were subjected to differential centrifugation to confirm this identification biochemically. Fractions of rat liver, the classic source of lysosomes, were also prepared by the same methods to compare with the fractions obtained from urinary epithelium. In contrast to rat liver, uroepithelial fractions with a high relative specific activity for hydrolases were sedimented before the heavy mitochondria. Microscopically, these fractions contained the highest proportion of lipoid granules. The size and sedimentation characteristics of lysosomes from transitional epithelium more closely resembled those of lysosomes derived from rat kidney than those isolated from liver.     

Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats

1.The content of non-heme iron and the degree of lipid peroxidation were measured in liver mitochondria isolated from rats injected with either Jectofer (an iron-sorbitol-citric acid complex) or iron-nitrilotriacetate. 2. The sedimentation profiles of the mitochondria from controls and iron-treated rats as revealed by analytical differential centrifugation, indicated single population of mitochondria with $\text{s}{}_{\mathrm{4,B}}$ values of 13200± 560 S and 14200±590 S for controls and iron-loaded animals, respectively. In contrast, the sedimentation profiles of the acid phosphatase activity and the non-heme iron revealed marked polydispersities with at least three populations of particles for both controls and iron-loaded animals. 3. The mitochondria and iron-rich lysosomes were separated by density-gradient centrifugation in an isotonic medium of Percoll and sucrose. With this technique, the amount of non-heme iron in a mitochondrial fraction by differential centrifugation decreased from 69±28 nmol/mg protein to 5.6±1.1 nmol/mg protein and from 19.3±5.6 nmol/mg protein to 3.3±0.6 nmol/mg protein for Jectofer and iron-nitrilotriacetate injected rats, respectively. For control rats the amount of mitochondrial non-heme iron was about 2.7 nmol/mg protein both before and following density gradient centrifugation. The extra amount of non-heme iron still present in the purified mitochondrial fraction from iron-loaded rats, as compared to controls, was further characterized by the reactivity towards bathophenanthroline sulfonate. The results suggest that the extra iron was due to a small amount of either ferritin or hemosiderin still contaminaning the mitochondrial fraction. The amount of mitochondrial heme iron was the same in iron-loaded rats and controls. 4. The degree of lipid peroxidation in the mitochondria was estimated from the amount of malondialdehyde. The thiobarbituric acid method used for the quantitation of malondialdehyde was modified so that it was insensitive to variable amounts of iron present in the samples. No difference in the degree of lipid peroxidation was observed between the mitochondria from iron-loaded rats and controls. 5. In contrast to recent proposals (Hanstein, E.G. et al. (1981) Biochim. Biophys. Acta 678, 293–299), the present study showed that the amounts of non-heme iron and the degrees of lipid peroxidation are the same in mitochondria isolated from iron-loaded and control animals.     

An assessment of some molecular parameters of mevalonate kinase from plant and animal sources

The molecular weights, diffusion coefficients, and sedimentation coefficients of mevalonate kinase in partially purified preparations from Hevea brasiliensis latex, Cucumis melo cotyledons, Phaseolus vulgaris cotyledons, bakers yeast, chicken liver, and rabbit liver have been determined by gel filtration in Sephadex G-100 and G-200 and by sucrose density gradient centrifugation. The enzyme had similar molecular weights (94800–103500), diffusion coefficients (5.39–5.62 × 10^?7 cm²/sec), and sedimentation coefficients (5.85–6.00 S) in the six preparations.     

Further biochemical and morphological studies of granule fractions from rabbit heterophil leukocytes

Fractionation of rabbit heterophil leukocyte homogenates by isopycnic centrifugation as well as by zonal sedimentation has helped to characterize further the particulate components of these cells. Four classes have been identified: (A) Large (0.5–0.8 µm) and dense (1.26) azurophil or primary granules, containing all the myeloperoxidase, one-third of the lysozyme, and a major proportion of the lysosomal acid hydrolase activities of the cells. (B) Smaller (0.25–0.40 µm) and less dense (1.23) specific or secondary granules, containing 90% of the alkaline phosphatase and the remainder of the lysozyme activities, but very little if any acid hydrolases. (C) Particles of low density (1.20), containing the remainder of the lysosomal acid hydrolases. This fraction was heterogeneous, but showed abundant small rod- or dumbbell-shaped particles of moderate electron opacity, surrounded by a single membrane (tertiary granules?). The possible origin of these lysosomes from contaminating macrophages could not be ruled out but appeared unlikely. (D) Slowly sedimenting material of very low density (1.14), made up of large, empty vesicular membrane structures, and containing 10% of the alkaline phosphatase, and all of a thiol-dependent acid p-nitrophenyl phosphatase, an enzyme clearly different from the lysosomal acid phosphatase.     

Pressure effects on the association of the and 2 subunits of tryptophan synthetase from Escherichia coli and Salmonella typhimurium

Sucrose density gradient centrifugation was employed to study the association of the α and β₂ subunits of the enzyme tryptophan synthetase from Escherichia coli and Salmonella typhimurium. In both cases, the fully associated enzyme (α₂β₂) showed a sedimentation coefficient of 6.4 S, in agreement with the values reported by other workers for the E. coli enzyme. The substrate, l-serine, and the cofactor, pyridoxal phosphate, were required for complex formation in both cases. Generation of moderately high pressures by increasing the centrifuge speed from 39,000 rpm to 50,000 rpm was found to interfere with complex formation in both species at 5 °C. This effect was reversed by a temperature increase from 5 °C to 20 °C or by low concentrations of a nonpolar solvent, ethanol, at 5 °C. These results indicate that hydrophobic bonding plays an important role in the formation of the active tryptophan synthetase α₂β₂ complex. Monovalent and divalent cations also interfered with the formation of the α₂β₂ complex, indicating the possibility that ionic bonds are also involved.     

Isolation of lysosomes from brain tissue. A separation method by means of alteration of mitochondrial and synaptosomal sedimentation properties

1. A crude mitochondrial-lysosomal preparation from brain tissue was layered on a sucrose gradient containing 20mm-succinate, 10mm-Tris and 1mm-disodium EDTA at pH7.4. The lysosomes were separated from the mitochondria and synaptosomes by means of a twosteps centrifugation procedure. In a first low-speed step (40min at 5300g at 15 degrees C) the sedimentation rate of mitochondria and mitochondria-containing synaptosomes was enlarged due to passage of these subcellular structures through the sucrose gradient with the above-mentioned chemicals (called ;chemical field'). That part of the gradient which contained the mitochondria and synaptosomes was removed and substituted by a gradient suitable for isopycnic isolation of lysosomes in a second centrifugation step. The achieved purification for bovine brain lysosomes was 5-8-fold, for rat brain lysosomes 7-10-fold, over the homogenate. 2. The enlargement of the sedimentation rate of mitochondria and synaptosomes was brought about by the presence of succinate, but also by one of the following salts in the chemical field: malonate, fumarate, pyruvate, phosphate and chloride. 3. Comparison of the chemical-field method with other methods for the isolation of lysosomes showed that (a) with the chemical-field method a 2-3-times higher purification of the rat and bovine brain lysosomal fraction can be achieved than with the procedure described by Koenig, Gaines, McDonald, Gray & Scott [(1964) J. Neurochem.11, 729-743], and that (b) similar purification results for rat liver lysosomes were obtained when the chemical-field method and the procedure described by van Dijk, Roholl, Reijngoud & Tager [(1976) FEBS Lett.62, 177-181] were compared.     

CYTOCHEMICAL LOCALIZATION OF ACID PHOSPHATASE ACTIVITY IN GRANULE FRACTIONS FROM RABBIT POLYMORPHONUCLEAR LEUKOCYTES

When rabbit peritoneal exudates (97% polymorphonuclear [PMN] leukocytes, 2% mononuclear cells) were fractionated by zonal sedimentation or isopycnic centrifugation, four fractions (A, B, C, and D) were obtained, as reported earlier. "A" consisted largely of PMN azurophil granules, "B" of PMN specific granules, and "D" of membranous elements. The source of the more heterogeneous "C" fraction (containing acid hydrolases) was uncertain. To gain further information on the nature of this fraction, cytochemical tests for acid phosphatase (AcPase) were carried out on the starting cells and on the fractions. In intact PMN, lead phosphate reaction product was found in Golgi complexes, perinuclear cisternae, and some azurophil granules (immature forms or disrupted mature forms) of a few cells. The specifics and the intact azurophils were not reactive. Reaction product was also found within Golgi cisternae, secondary lysosomes, and some of the azurophil granules of mononuclear cells. Observations on the A and B fractions confirmed those in situ regarding the localization of reaction product in disrupted PMN azurophils, its absence from specifics, and the latency of the enzyme activity in intact azurophils. In the C fraction, AcPase was found in three structures (a) Golgi cisternae, (b) dense bodies, and (c) small pleomorphic granules Comparison with the starting cells indicates that the Golgi complexes are probably derived from both PMN leukocytes and mononuclear cells, whereas the remaining elements resemble (in size, shape, and density) secondary lysosomes and azurophil granules of mononuclear cells. The results indicate that the bulk of the cytochemically detectable AcPase present in the C fraction is derived from mononuclear cells, rather than from PMN leukocytes     

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondrai of mice with porphyria induced by griseofulvin

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 ± 0.0043, 4.11 ± 0.58 and 22.2 ± 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4–5 weeks, respectively. The energy coupling of the mitochondia was greatly diminished after 4–5 weeks of treatment, and the ferrochelatase activity was inhibited 80–90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36±0.15, 3.97±0.40 and 3.59±0.23 nmol/mg protein for control animals, 3 days- and 4–5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tangerås, A. (1985)) Biochim. Biophys. Acta 843 199–207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.     

Isolation and some properties of components of nuclear polyhedra from the cabbage looper, Trichoplusia ni

Nuclear polyhedra obtained from diseased cabbage looper, Trichoplusia ni, were digested with sodium carbonate-saline buffer, pH 11.0. The dissolved polyhedra formed 3 general zones when subjected to density gradient centrifugation. The slowest sedimenting component (Zone 1) had an ultraviolet absorption curve typical of protein and a sedimentation coefficient of 11 S. Capsids, 310 × 40 nm, were located in Zone 2. Virus particles were found in 1–3 bands (Zone 3); those with envelopes measured 300 × 72 nm, and those without envelopes measured 300 × 33 nm. Virus preparations stained with phosphotungstic acid at pH 7.0 exhibited extensive disruption whereas preparations stained at pH 3.0 did not. Virus particles in the sodium carbonate-saline-digested polyhedra had a sedimentation coefficient of 1228 S. Virus particles isolated by high speed centrifugation had a sedimentation coefficient of 1530 S.     

Enzyme analysis and subcellular fractionation of human peripheral blood lymphocytes with special reference to the localization of putative plasma membrane enzymes

Human lymphocytes were isolated from defibrinated blood by Ficoll-Hypaque centrifugation with erythrocyte hypotonic lysis. Homogenates of mixed lymphocytes were subjected to analytical subcellular fractionation by sucrose gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their marker enzymes: cytosol (lactate dehydrogenase), plasma membrane (5′-nucleotidase), endoplasmic reticulum (neutral α-glucosidase), mitochondria (malate dehydrogenase), lysosomes (N-acetyl-β-glucosaminidase), peroxisomes (catalase). γ-Glutamyl transferase was exclusively localized to the plasma membrane. Leucine amino-peptidase, especially when assayed in the presence of Co²⁺, was also partially localized to the plasma membrane. Experiments with diazotized sulphanilic acid, a non-permeant enzyme inhibitor, showed that these plasma membrane enzymes are present on the cell surface. No detectable alkaline phosphatase was found in the lymphocytes. Acid phosphatase and β-glucuronidase were localized to lysosomes and there was some evidence for lysosomal heterogeneity. Leucine amino peptidase, optimal at pH 8.0, showed a partial localization to intracellular vesicles, possibly lysosomes, especially when assayed in the presence of EDTA. These studies provide a technique for determining the intracellular distribution of hitherto unassigned lymphocyte constituents and serve as a basis for investigating the cell pathology of lymphocytic disorders.     

Is there a mechanism for introducing acid hydrolases into liver lysosomes that is independent of mannose 6-phosphate recognition? evidence from I-cell disease

We have examined frozen liver tissue for N-acetylglucosamine-l-phosphotransferase, an enzyme required for the formation of the mannose 6-phosphate recognition marker of lysosomal enzymes. Using [β³²P]-UDPGlcNAc and placental β-hexosaminidase B as N-acetylglucosamine l-phosphate donor and acceptor, respectively, we were unable to find activity of the transferase in 100,000 × g membranes prepared from livers of patients with I-cell disease, whereas activity was readily observed in membranes from control livers stored under the same conditions. Yet the activity of several lysosomal enzymes (β-N-acetylglucosaminidase, β-glucuronidase, α-mannosidase and α-$\text{L}$-iduronidase) was comparable in liver tissue of I-cell patients and controls, and only β-galactosidase activity showed a marked reduction. These results suggest that in contrast to cultured skin fibroblasts, liver may be able to introduce into lysosomes acid hydrolases that lack the mannose 6-phosphate recognition marker.     

Physical Properties of Lactic Dehydrogenase-Elevating Virus and Its Ribonucleic Acid

Infectious lactic dehydrogenase-elevating virus propagated in primary cultures of mouse peritoneal macrophages in the presence of ³H-uridine and isolated by isopycnic centrifugation was found to have a density of 1.12 g/cm³. Ribonucleic acid extracted from the virus by treatment with sodium dodecyl sulfate was single stranded with a sedimentation coefficient of approximately 48S.     

Characterization of porcine adrenocortical lysosomes

Porcine adrenocortical lysosomes were characterized by differential centrifugation, acid hydrolase contents, latency of cathepsin D, release of bound acid hydrolases in soluble form, and isopycnic density gradient centrifugation. Cathepsins D and B, beta-N-acetylglucosaminidase, beta-galactosidase and arylsulphatase were found exclusively in the lysosomes, while alpha-mannosidase and beta-glucuronidase were in both the lysosomal and microsomal fractions. The activity of cathepsin D was remarkably high, amounting to more than 6 times that in porcine liver and to more than 10 times that in liver of Sprague-Dawley rats in terms of units per g wet tissue. Porcine adrenocortical lysosomes showed a modal isopycnic density value of 1.155, but mitochondria a value of 1.145. The validity of these values was studied by investigating the possibilities of agglutination of organelles, damage to lysosomal membranes, disruption of mitochondria due to the hydrostatic pressure and by applying the same procedures of isopycnic centrifugation to hog and rat livers. After these validity tests, porcine adrenocortical lysosomes were concluded to be unique in their strikingly high content of cathepsin D as well as in their low modal isopycnic density which is very close to that of porcine adrenocortical mitochondria.