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, β-galactosidase and N-acetyl-βglucosaminidase. Realimentation of starved animals for 10 days was found to restore the enzyme levels and the sedimentation characteristics to normal profiles.     

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.     

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.     

Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient

A preparation, similar to the light mitochondrial fraction of rat liver (L fraction of de Duve et al, (1955, Biochem. J. 60: 604-617), was subfractionated by isopycnic centrifugation in a metrizamide gradient and the distribution of several marker enzymes was established. The granules were layered at the top or bottom of the gradient. In both cases, as ascertained by the enzyme distributions, the lysosomes are well separated from the peroxisomes. A good separation from mitochondria is obtained only when the L fraction if set down underneath the gradient. Taking into account the analytical centrifugation results, a procedure was devised to purify lysosomes from several grams of liver by centrifugation of an L fraction in a discontinuous metrizamide gradient. By this method, a fraction containing 10--12% of the whole liver lysosomes can be prepared. As inferred from the relative specific activity of marker enzymes, it can be estimated that lysosomes are purified between 66 and 80 times in this fraction. As ascertained by plasma membrane marker enzyme activity, the main contaminant could be the plasma membrane components. However, cytochemical tests for 5'AMPase and for acid phosphatase suggest that a large part of the plasma membrane marker enzyme activity present in the purified lysosome preparation could be associated with the lysosomal membrane. The procedure for the isolation of rat liver lysosomes described in this paper is compared with the already existing methods.     

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.     

Improved recovery of rat liver fractions enriched in lysosomes by specific alteration of the sedimentation properties of mitochondria

A method for the preparation of lysosomes from rat liver is presented. The procedure requires only standard equipment and is completed within less than 3 h. Homogenization and differential centrifugation were performed at pH 7.4 in isotonic potassium phosphate-buffered sucrose medium. The addition of potassium phosphate, at the concentration used (10 mM), accelerated the sedimentation rate of mitochondria without altering that of lysosomes resulting in the decrease in the mitochondrial contamination of the final pellet. Further purification was achieved by isopycnic centrifugation in 45% isotonic Percoll performed in an angle rotor. Lysosomal fractions representing 51.5% of the original population were recovered over a density range of 1.09 to 1.15 g/ml. The most purified fraction (37-fold purified) contained 25.3% of lysosomal beta-N-acetylglucosaminidase, and only 0.9% of mitochondrial monoamine oxidase and 0.6% of peroxisomal urate oxidase original activities. It was practically devoid to endoplasmic reticulum contamination.     

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.     

Morphologic and biochemical heterogeneity of lysosomes]

By means of isopycnic centrifugation in the continuous density gradient of sucrose two subfractions of lysosomes were isolated from rat liver homogenates: a "light" one (with the floating density p=1.13) and a "heavy" one (p=1.24). Electron microscopic, enzymatic and electron microscope enzymatic analysis of the isolated subfractions showed that the "light" subfraction consisted mainly of newly-formed primary lysosomes, while the "heavy" one was presented by secondary lysosomes. Parallel biochemical investigations demonstrated a considerable enzymatic heterogeneity of the two lysosomal subfractions: the "light" subfraction was characterized by a high specific activity of acid DNase, acid RNase and beta-galactosidase, and by almost total absence of beta-glucosidase activity, while the "heavy" one was characterized by a high specific activity of beta-glucosidase, beta-glucuronidase and beta-N-acetylglucosaminidase. Possible causes of enzyme heterogeneity of rat liver lysosomes are discussed.     

EVIDENCE FOR REGULATION OF PROTEIN SYNTHESIS AT THE TRANSLATION LEVEL IN RESPONSE TO DIETARY ALTERATIONS

Rats were maintained for several days on a protein-free diet which, approximately 14 hr before decapitation, was changed to a protein-rich diet. Microsomal subfractions of liver were obtained by sucrose density gradient centrifugation. The sedimentation patterns of the microsomal subfractions and of the polysomes were found to be unchanged after the alteration of the diet whereas the ability to incorporate amino acid was markedly enhanced. No significant differences in the RNA/protein ratio of the microsomal subfractions were observed. The changes in amino acid incorporation which were unrelated to concurrent changes in the amount of polysomes are discussed.     

The activator of cerebroside sulphatase. Lysosomal localization.

1) An activator protein necessary for the enzymic hydrolysis of cerebroside sulphate could be partially purified from unfractionated rat liver. This activator, which is similar to that of human origin, proved to be a heat-stable, non-dialyzable, low molecular weight protein with an isoelectric point of 4.1. Its activity could be destroyed by pronase. 2) For elucidation of the subcellular localization of the activator, rat liver was fractionated by differential centrifugation. The intracellular distribution of the cerebroside sulphatase activator was compared to the distribution patterns of marker enzymes for different cell organelles and found to coincide with the lysosomal arylsulphatase, thus indicating a lysosomal localization. 3) This was confirmed using highly purified secondary, i.e. iron-loaded, lysosomes. After disruption by osmotic shock, these organelles hydrolyzed cerebroside sulphate when incubations were performed under physiological conditions with endogenous as well as exogenous sulphatase A as enzyme. 4) After subfractionation of the disrupted secondary lysosomes into membrane and lysosol fractions by high speed centrifugation, it was found that the activator protein was exclusively associated with the lysosol, whereas the acid hydrolases were distributed differently between the two fractions. 5) The lysosol was further fractionated by semi-preparative electrophoresis on polyacrylamide gels. Two protein fractions were obtained: a high molecular weight fraction, containing the activator-free acid hydrolases, and a low molecular weight fraction, containing the enzyme-free activator of cerebroside sulphatase. 6) The significance of these findings for the hydrolysis of sphingolipids in the lysosomes is discussed.     

The origin of lipid accumulated in liver lysosomes after administration of triton WR-1339

The origin of the lipids accumulated in liver lysosomes after administration of Triton WR-1339 was investigated. When Triton WR-1339 was injected into rats, serum triglyceride and cholesterol increased markedly. The highest content of triglyceride was observed in the second-day serum, from which very-low-density lipoprotein (VLDL) was isolated. The VLDL was administered to normal rats, then the light mitochondrial fraction of the liver at 24 h was centrifuged in a sucrose density gradient. The activities of lysosomal enzymes, acid phosphatase, N-acetyl-beta-D-glucosaminidase and acid lipase, were all shifted to less dense fractions as compared with those of normal lysosomes. [3H]Triglyceride-labeled VLDL was injected similarly, and at 12 and 24 h after the administration, the light mitochondrial fraction of the liver was fractionated by sucrose gradient centrifugation. Protein content and radioactivity in the immunoprecipitate with anti-VLDL serum at 12 h showed almost the same distribution as acid phosphatase activity. At 24 h, though acid phosphatase activity, immunoprecipitable protein content and radioactivity were all found in less dense fractions than in the case of normal lysosomes, the former two distributions were significantly different from the latter. The anti-VLDL serum reacted in Ouchterlony tests not only with Triton-induced VLDL and normal VLDL but also with the extract from low-density lysosomes. These results suggest that the lipids accumulated in low-density lysosomes following the administration of Triton WR-1339 were probably derived from the elevated serum VLDL induced by the treatment.     

Renin substrate in granules from rat kidney cortex.

1. Subcellular fractions of rat kidney cortex generated angiotensin I continuously over 2h when incubated at 37degreesC with rat renin, indicating the presence of renin substrate within cells in the renal cortex. 2. Renin substrate was located in highest specific concentration in particulate fractions. The particles containing renin substrate had a sedimentation velocity slightly lower than mitochondria and renin granules but greater than the microsomal fraction. 3. Isopycnic gradient centrifugation indicated a density of 1.190g/ml for the particles containing renin substrate, compared with 1.201 for renin granules, 1.177 for mitochondria, and 1.170 and 1.230 for lysosomes in the heavy-granule fraction. 4. In the liver, renin substrate was also found in particles, but these had a lower sedimentation rate than those from the kidney. 5. The molecular weights of renin substrate in kidney and liver granules and rat plasma were similar, namely 61000-62000. 6. On the basis of these biochemical findings, a mechanism for the intrarenal production of angiotensin, incorporating a subcellular reaction scheme, is proposed.     

Separation of hepatocytes from suspensions of mouse liver cells using programmed gradient sedimentation in gradients of ficoll in tissue sulture medium

Liver cells were obtained in suspension using a solution of lysozyme in Joklik's modification of minimum essential medium. Hepatocytes were separated in 74.2 ± 12.9% purity from other liver cells having different densities using isopycnic centrifugation, in 97.1 ± 1.9% purity from other liver cells having different diameters using velocity or rate-zonal centrifugation. A previously reported computer integration of the differential sedimentation equation was employed in determining the gradient design and the speed and duration of centrifugation which would permit purification of hepatocytes from other liver cells. More than 98% of the hepatocytes separated by velocity sedimentation excluded trypan blue. Velocity sedimentation is superior to isopycnic centrifugation for the separation of hepatocytes from liver cell suspensions because it gives more highly purified hepatocytes and because it requires lower centrifugal forces for shorter periods of time.     

Inhibition of liver lysosomal cholesterol esterase activity in rats by dextran blue in vivo and in vitro

Dextran blue decreases the activity of lysosomal acid cholesteryl esterase of rat liver at a concentration from 0.25 M to 10 M without altering acid phosphatase, acid beta-galactosidase and beta-glucosidase activities. The dextran blue filled lysosomes with a high degree of purity prepared by centrifugation over the linear sucrose density gradient contained insignificant impurities (up to 19%) of protein from other organelles. The specific activity of acid phosphatase, beta-galactosidase and beta-glucosidase was increased 35-40-fold in this fraction, whereas the activity of acid cholesteryl esterase rose but 14.7-fold. Chromatography on a Sepharose 2B column of the digitonin-digested native and dextran-containing lysosomes attests to the formation of large dextran aggregates with lysosomal matrix proteins. Since aggregation of dextran blue with acid phosphatase, beta-galactosidase and beta-glucosidase does not affect their activities, it is concluded that to bring about hydrolysis of lipoprotein cholesterol esters, it is necessary that cholesteryl esterase be associated with hydrophobic macromolecules. Moreover, dextran blue can be used for simulation cholesterol esters deposition in lysosomes.     

Changes in lysosome populations in the rat kidney cortex induced by experimental proteinuria

1. Experimental proteinuria (262.9 mg protein/24 hr urine) was induced in rats by repeated intraperitoneal injections of BSA. 2. Hypertrophy of the kidney cortex was significant 8 days after the start of the BSA injections, and the activities of lysosomal enzymes in kidney cortex and urine were significantly higher in proteinuric compared to nonproteinuric rats. 3. Lysosome populations in the kidney cortex were examined by rate sedimentation of the homogenate and by rate zonal and isopycnic centrifugation of the lysosome-rich ML fraction. 4. The activity of lysosomal enzymes in the kidney cortex increased slightly, essentially in the large, fragile lysosomes mainly recovered from the proximal tubule. 5. Proteinuria induced a shift/reduction in the density of small lysosomes from 1.235 and 1.20 g/ml to 1.225 and 1.185 g/ml, respectively. 6. Proteinuria induced a new population of small lysosomes (density 1.185 g/ml) enriched in cathepsin D.     

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.     

Cestode lysosomes

By differential centrifugation method a lysosomal fraction was obtained from five species of cestodes, which possesses the highest specific activity of acidic phosphatases as compared to other subcellular fractions. By isopyknic centrifugation in the density gradient of saccharose the lysosomal fraction is divided into primary and secondary lysosomes. Lysosomes of cestodes are similar to those of vertebrate animals in the character of fractional distribution of acidic phosphatase, sedimentation abilities and sensitivity of membranes to triton X-100.     

Subcellular localization of acyl coenzyme A: dihydroxyacetone phosphate acyltransferase in rat liver peroxisomes (microbodies).

Upon differential centrifugation of rat liver homogenate, the enzyme acyl-CoA:dihydroxyacetone-phosphate acyltransferase (EC 2.3.1.42) was found to be localized in the light mitochondrial (L) fraction which is enriched with lysosomes and peroxisomes. Peroxisomes were separated from lysosomes in a density gradient centrifugation using rats which were injected with Triton WR 1339. By comparing the enzyme distribution with the distribution of different marker enzymes, it was concluded that dihydroxyacetone phosphate acyltransferase is primarily localized in rat liver peroxisomes (microbodies). Similarly, the enzyme acyl dihydroxyacetone-phosphate:NADPH oxidoreductase (EC 1.1.1.101) was shown to be enriched in the peroxisomal fraction, although a portion of this reductase is also present in the microsomal fraction.     

Isolation of peroxisomes from the dog kidney cortex.

The present study was undertaken to separate peroxisomes of the dog kidney cortex by the methods of discontinuous sucrose density gradient and zonal centrifugation. The separation of subcellular particles was evaluated by measuring the activities of reference enzymes, beta-glycerophosphatase for lysosomes, succinate dehydrogenase for mitochondria, glucose-6-phosphatase for microsomes, and catalase and D-amino acid oxidase for peroxisomes. The activities of D-amino acid oxidase and catalase were mainly observed in fractions 1 and 2 (1.6 and 1.7 M sucrose) obtained by discontinuous sucrose density-gradient centrifugation. Small amounts of acid phosphatase and succinate dehydrogenase contaminated these fractions. Considerably higher activity of catalase was determined in the supernatant, while D-amino acid oxidase showed a lower activity. By the method of zonal centrifugation, the highest specific activities of catalase and D-amino acid oxidase were found in fraction 50 (1.73 M sucrose) with no succinate dehydrogenase, acid phosphatase or glucose-6-phosphatase activity. These results suggested that peroxisomes of dog kidney cortex were clearly separated in 1.73 M sucrose from mitochondria, lysosomes and microsomes by zonal centrifugation.     

Fate of asialofetuin endocytosed by rat liver

We have investigated the endocytosis by rat liver of asialofetuin coupled to [125I] tyramine cellobiose: [125I] TCASF. Subcellular distribution of radioactive compounds was established after differential and isopycnic centrifugation and by analysing the fractions by SDS electrophoresis. Labelling secondary lysosomes was performed by injecting rats with Triton WR 1339 four days before injecting the protein. Results show that after being associated with endosomes [125I] TCASF is recovered in organelles where they are subjected to a first degradation, the density of these organelles is practically not affected by Triton WR 1339 injection. Later the degradation products are associated with lysosomes whose density is markedly lowered by Triton WR 1339 treatment. These observations suggest that the first intracellular organelles where [125I] TCASF is subjected to digestion are distinct from the secondary lysosome population. This could be in agreement with the hypothesis that supposes that endosomes acquire enzymes from primary lysosomes before fusion with secondary lysosomes.