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.     

Calf adrenocortical fasciculata cells secrete aldosterone when placed in primary culture

Aldosterone production occurs in the outer area of the adrenal cortex, the zona glomerulosa. The glucocortocoids cortisol and corticosterone, depending upon the species, are synthesized in the inner cortex, the zona fasciculata. Calf zona glomerulosa cells rapidly lose the ability to synthesize aldosterone when placed in primary culture unless they are incubated in the presence of the antioxidants butylated hydroxyanisol and selenous acid, the radioprotectant DMSO, and the cytochrome P-450 inhibitor metyrapone. In the presence of these additives, calf zona fasciculata cells in primary culture synthesize aldosterone at rates which can approach those from cells isolated from the zona glomerulosa. Calf zona glomerulosa and fasciculata cells both responded well to ACTH and angiotensin II, but the zona fasciculata cells respond very poorly compared to glomerulosa cells to increased potassium in the media. Rat zona fasciculata cells in primary culture under similar conditions did not synthesize aldesterone, suggesting that the regulation of the expression of the enzymes responsible for the biosynthesis of aldosterone in the two species is different. Two distinct cytochrome P-450 cDNAs which hydroxylate deoxycorticosterone at the 11β position have been described in the rat, human and mouse. Both cytochrome P-450 cDNAs have been cloned and expressed in non-steroidogenic cells, but only one is expressed in the zona glomerulosa and only this glomerulosa cytochrome P450 can further hydroxylate deoxycorticosterone to generate aldosterone. Two bovine adrenal cDNAs have been described with 11β-hydroxylase activity and their expression products in transiently transfected COS cells can convert deoxycorticosterone into aldosterone. Both enzymes are expressed in all zones of the adrenal cortex. Zonal regulation of aldosterone synthesis in the bovine adrenal gland may be due to an 11β-hydroxylase with aldosterone synthesizing capacity which has not yet been isolated. Alternatively, a single enzyme might be responsible for the several hydroxylations in the pathway between deoxycorticosterone and aldosterone and zonal synthesis might be controlled by unknown factors regulating the expression of C-18 hydroxylation. The incubation of zona fasciculata with antioxidants and metyrapone results in atypical expression of this activity by an unclear mechanism.     

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.     

Morphological evidence for a close interaction of chromaffin cells with cortical cells within the adrenal gland

Summary The adrenal medulla appears to exert a regulatory influence on adrenocortical steroidogenesis. We have therefore studied the morphology of rat, porcine and bovine adrenals in order to characterize the contact zones of adrenomedullary and adrenocortical tissues. The distribution of chromaffin cells located within the adrenal cortex and of cortical cells located within the adrenal medulla was investigated. Chromaffin cells were characterized by immunostaining for synaptophysin and chromogranin A, both being considered specific for neuroendocrine cells. Cortical cells were characterized by immunostaining for 17-hydroxylase, an enzyme of the steroid pathway. Cellular contacts of chromaffin cells and cortical cells were examined at the electron microscopical level. In rat and porcine adrenals, rays of chromaffin cells, small cell clusters and single chromaffin cells or small invaginations from the medulla could be detected in all three zones of the cortex. Chromaffin cells often spread in the subcapsular space of the zona glomerulosa. In porcine and bovine adrenals, 17-hydroxylase immunoreactive cells were localized within the medulla. Single cortical cells and small accumulations of cells were spread throughout this region. At the ultrastructural level, the chromaffin cells located within the cortex in pig and rat adrenals formed close cellular contacts with cortical cells in all three zones. Our morphological data provide evidence for a possible paracrine role of chromaffin cells; this may be important for the neuroregulation of the adrenal cortex.     

Immunogold cytochemistry of cytochromes P-450 in porcine adrenal cortex. Two enzymes (side-chain cleavage and 11 beta-hydroxylase) are co-localized in the same mitochondria

In order to study the distribution of mitochondrial cytochromes P-450 in porcine adrenal glands, the glands of anesthetized pigs were fixed in situ. Polyclonal antibodies against two cytochromes P-450, i.e., C27 side-chain cleavage enzyme and 11 beta-hydroxylase, were used to study the distribution of these enzymes in cryosections of the adrenal cortex. Ultrathin cryosections were evaluated by both protein-A/gold/silver immunocytochemistry and immunoelectron microscopy using double labeling with protein-A/colloidal-gold. At light microscopy, the two cytochrome P-450 enzymes were found to be broadly distributed in both the fasciculata and glomerulosa zones of the adrenal cortex. Quantitative immunoelectron microscopy revealed that both enzymes were localized only in mitochondria, in which they were present on the inner aspects of the inner mitochondrial membrane. Both cytochromes P-450 were demonstrable in all of the mitochondria examined, and statistical evaluation of the ratios of the two enzymes present in individual mitochondria yielded a normal distribution curve. Since no evidence was found for the preferential localization of either enzyme in a special population of mitochondria, we conclude that all mitochondria of the adrenal cortex contain both enzymes. We discuss implications of these findings with respect to the regulation of steroidogenesis.     

Density gradient separation of two populations of lysosomes from rat parotid acinar cells

Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.     

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)     

Immunogold cytochemistry of cytochromes P-450 in porcine adrenal cortex

Summary In order to study the distribution of mitochondrial cytochromes P-450 in porcine adrenal glands, the glands of anesthetized pigs were fixed in situ. Polyclonal antibodies against two cytochromes P-450, i.e., C²⁷ sidechain cleavage enzyme and 11 beta-hydroxylase, were used to study the distribution of these enzymes in cryosections of the adrenal cortex. Ultrathin cryosections were evaluated by both protein-A/gold/silver immunocytochemistry and immunielectron microscopy using double labeling with protein-A/colloidal-gold. At light microscopy, the two cytochrome P-450 enzymes were found to be broadly distributed in both the fasciculata and glomerulosa zones of the adrenal cortex. Quantitative immunoelectron microscopy revealed that both enzymes were localized only in mitochondria, in which they were present on the inner aspects of the inner mitochondrial membrane. Both cytochromes P-450 were demonstrable in all of the mitochondria examined, and statistical evaluation of the ratios of the two enzymes present in individual mitochondria yielded a normal distribution curve. Since no evidence was found for the preferential localization of either enzyme in a special population of mitochiondria, we conclude that all mitochondria of the adrenal cortex contain both enzymes. We discuss implications of these findings with respect to the regulation of steroidogenesis.This work was supported by grants from the National Institutes of Health (AM28113, AM32236, CA29497, NSF-INT-8317418, and NATO 818/83)     

Aldosterone biosynthesis in mitochondria of isolated zones of adrenal cortex

An assumption that the aldosterone-synthesizing enzyme exists only in zona glomerulosa cells apparently contradicts our recent findings that a purified bovine adrenocortical cytochrome P-45011 beta catalyzes the aldosterone formation and the enzyme exists in both zones of the adrenal cortex. To gain more insight into the zone specificity of aldosterone production, the aldosterone-synthesizing activity of mitochondria prepared from the isolated zones of adrenal cortex of various animal species was investigated. The intact mitochondria from the bovine or porcine zonae fasciculata-reticularis could not produce aldosterone whereas those from the zona glomerulosa produced it at a significant rate. When the mitochondria from the zonae fasciculata-reticularis were solubilized by the addition of cholate, they produced aldosterone from corticosterone at a rate comparable to that of those from the zona glomerulosa. The presence of specific factor(s) in the zonae fasciculata-reticularis mitochondria inhibiting expression of the aldosterone synthetic activity is discussed. The mitochondria of the rat zonae fasciculata-reticularis could hardly catalyze aldosterone synthesis under the detergent-solubilized conditions, whereas those of the zona glomerulosa could. Immunoblot analysis revealed that the mitochondria of the zonae fasciculata-reticularis contained a protein of Mr 51,000 which was immunocrossreactive with a monoclonal antibody directed against P-45011 beta, whereas those of the zona glomerulosa contained two immunocrossreactive proteins of Mr 51,000 and 49,000. These results suggest that in the case of rat adrenal cortex, a specific aldosterone-synthesizing enzyme exists in the zona glomerulosa.     

Adriamycin-induced leakage of lysosomal enzymes in vitro

Chronic treatment of rats with adriamycin has been shown to affect myocardial lysosomes as well as enzyme activities in the serum fraction. In this study, we examined in vitro effects of adriamycin (10^–6 to 10^–3 M) on the lysosomal fraction isolated from rat ventricular tissue. Morphological examination revealed that the isolated fraction was mainly vesicular in nature. Higher concentrations of adriamycin (10^–3 M) caused a significant loss of acid phosphatase and N-acetyl-B-d-glucosaminidase activity from the lyosomal vesicles. The enzyme leakage was not accompanied by any intravesicular localization of lanthanum, an extravesicular electron dense tracer. Preincubation of lysosomal vesicles with 10 g/ml superoxide dismutase did not protect against adriamycin-induced loss of lysosomal enzymes. The study shows that adriamycin induces loss of lysosomal enzymes in vitro and the superoxide radical may not be involved in this change.     

Immunohistochemical localization of adrenal ferredoxin in bovine adrenal cortex.

Adrenal ferredoxin, the iron-sulfur protein associated with cytochrome P-450 in adrenocortical mitochondria, has been localized at the light microscopic level in bovine adrenal cortex. Localization was achieved through the use of rabbit antiserum to bovine adrenal ferrodoxin in an unlabeled antibody peroxidase-antiperoxidase method. When sections of bovine adrenal glands were exposed to the adrenal ferredoxin antiserum, intense staining was observed in parenchymal cells of the three cortical zones. Staining for adrenal ferredoxin was not detected in the medullary chromaffin cells. The presence of adrenal ferredoxin in the three cortical zones was verified by electron paramagnetic resonance spectrometry. These determinations also revealed that while the zona fasciculata and the zona reticularis contained approximately equal concentrations of adrenal ferredoxin, the concentration of the iron-sulfur protein in the zona glomerulosa was considerably lower. Similar results were obtained when the levels of cytochrome P-450 were determined in the three cortical zones. These results represent the first immunohistochemical localization within an intact tissue or cell of any component of an NADPH-dependent electron transport sequence which is responsible for the reduction of cytochrome P-450.     

The synthesis of aldosterone by the adrenal cortex. Two zones (fasciculata and glomerulosa) possess one enzyme for 11 beta-, 18-hydroxylation, and aldehyde synthesis

In order to establish the nature of the aldosterone synthetase activity in the adrenal cortex, we have used porcine adrenal, bovine adrenal cortex, highly purified bovine and porcine 11 beta-/18-hydroxylase, and antibodies raised against the latter enzyme. Mitochondria from two zones (glomerulosa and fasciculata) of the bovine cortex synthesize aldosterone, but those from glomerulosa are much more active than those from fasciculata. Partially purified (cholate-extracted plus ammonium sulfate-precipitated) extracts of mitochondria from the two zones are equally active in catalyzing all three steps in the conversion of 11-deoxycorticosterone to aldosterone. 18-Hydroxylase and aldehyde synthetase activities (18-hydroxycorticosterone----aldosterone) were completely precipitated from cholate extracts of mitochondria from bovine adrenal by antibodies to the pure porcine enzyme. No activity corresponding to any of the three steps in the conversion of 11-deoxycorticosterone to aldosterone was found in extramitochondrial fractions of the bovine cortex. Synthesis of aldosterone by the pure porcine enzyme was inhibited by antibodies to this enzyme and by metyrapone (an inhibitor of 11 beta-/18-hydroxylase). When fractions of porcine adrenal, resulting from purification of the enzyme from mitochondria, were exhaustively tested for any of the enzyme activities required for the synthesis of aldosterone, activity was found only in those fractions containing the 11 beta-/18-hydroxylase, i.e. no additional enzyme was discarded during the purification procedure. It is concluded that the only adrenocortical enzyme capable of synthesizing aldosterone in bovine and porcine adrenal is the well known 11 beta-hydroxylase, that the conversion of 18-hydroxycorticosterone to aldosterone is catalyzed by this cytochrome P-450, and that this step (aldehyde synthetase) requires the heme of the P-450 as demonstrated by the photochemical action spectrum.     

Steroidogenesis in the zona glomerulosa of the adrenal cortex II. Distribution of cytochrome P-450 in the zona glomerulosa of the bovine adrenal cortex

Microsomes were obtained from the zona glomerulosa of the bovine adrenal cortex. Contamination of microsomes with other cellular organelles was examined using various marker enzymes and the electron microscope. Distribution of cytochrome P-450 in the zona glomerulosa was studied using various fractions including microsomes, described above, and mitochondria. The amount of cytochrome P-450 in mitochondria and in microsomes was determined to be 0.73 and 0.32 nmol/mg protein, respectively. The CO difference spectrum was affected not only by the concentration of added deoxycholate but also by the incubation time after addition. Approximately 40–50% of cytochrome P-450 in the samples was converted to cytochrome P-420 within 20–30 sec of incubation with deoxycholate.The content of RNA, phospholipids, and cytochromeb ₅ in microsomes obtained from the zona glomerulosa is also evaluated in comparison to that in microsomes obtained from the zona fasciculoreticularis.     

X-ray microanalysis of colloidal-gold-labelled lysosomes in rat liver sinusoidal cells after incubation for acid phosphatase activity

Summary The lysosomal apparatus of the Kupffer and endothelial cells of the sinusoidal lining of the rat liver was found to take up colloidal-gold particles with a mean diameter of 5 nm, prepared according to a modified method. After incubation of the glutaraldehyde-perfusion-fixed tissue in a lead-containing medium for the demonstration of acid phosphatase activity, a reaction product was observed in the gold-loaded lysosomes. By X-ray microanalysis of such lysosomes, the presence of osmium, gold and lead was detected qualitatively in the unstained sections from the tissue, which after the incubation had been post-fixed with an OsO₄-solution to which K₄Fe(CN)₆ had been added to enhance the contrast. The quantitative computer-assisted processing of the X-ray microanalytical data from such lysosomes enabled to determine the gold-to-lead ratio and the individual gold and lead peak intensities derived from both the M and L values in the spectra. On the basis of these results and those obtained similarly in control lysosomes containing either only gold or only lead phosphate precipitate, it was found that only the L values were reliable, whereas the M values from the same lysosomal spectra were unrealistic, due to deconvolution problems in the computer programs applied. Based upon the L values it was found that among the population of lysosomes in single Kupffer cells, studied after a 60-min interval between the injection of the gold colloid and fixation, three types of lysosomal contents could be quantitated by X-ray microanalysis, viz. one type with only gold, one with only lead, one with gold and lead, in various ratios. This quantitative approach might make it possible to detect variations in lysosomal composition associated with ageing.The unit for analytical electron microscopy was established by collaboration between: the Erasmus University of Rotterdam (W.C. de Bruijn), the University of Leiden, and the Organization for Health Research TNO. The analytical electron microscope was purchased with funds from the Netherlands Organization for Pure Scientific Research (ZWO)     

Morphology and ultrastructure of the adrenal gland in Bactrian camels (Camelus bactrianus)

In the present study, we examined the morphological features of the adrenal gland in Bactrian camel by means of digital anatomy, light and electron microscopy. Our findings testified that the gland was divided into three parts, capsule, cortex and medulla from outside to inside as other mammals, and the cortex itself was further distinguished into four zones: zona glomerulosa, zona intermedia, zona fasciculate and zona reticularis. Notably, the zona intermedia could be seen clearly in the glands from females and castrated males, whereas it was not morphologically clear in male. There was a great deal of lipid droplets in the zona fasciculate, while it was fewer in the zona glomerulosa and zona reticularis. The cytoplasm of adrenocortical cell contained rich mitochondria and endoplasmic reticulum. The adrenal medulla was well-developed with two separations of external and internal zones. The most obvious histological property of adrenal medulla cells were that they contained a huge number of electron-dense granules enveloped by the membrane, and so medulla cells could be divided into norepinephrine cells and epinephrine cells. Moreover, the cortical cuffs were frequently present in adrenal gland. Results of this study provides a theoretical basis necessary for ongoing investigations on Bactrian camels and their good adaptability in arid and semi-arid circumstances.     

Discrepancy between the histochemically and biochemically determined 3β-hydroxysteroiddehydrogenase activity in the calf adrenal cortex

Summary In sections of the calf adrenal cortex the histochemically determined3-hydroxysteroiddehydrogenaae activity is lower in the zona glomerulosa than in the zona fasciculata. Biochemically the activity of this enzyme was found in mitochondrial as well as in microsomal fractions. The mitochondrial, respectively the microsomal fractions of the two zones showed identical enzyme activities. So there is a discrepancy between the histochemical results on tissue sections and biochemical results obtained with isolated subcellular fractions.However, if the histochemical determination of 3-hydroxysteroiddehydrogenase activity is carried out on sections of mitochondrial and microsomal pellets of the two zones the results are in agreement with the biochemical findings. Therefore the observed discrepancy rather seems to be related with the state of tissue—intact or cell fractions—than with the used histochemical method.     

Relationship between mitochondrial lipid peroxidation and alpha-tocopherol levels in the guinea-pig adrenal cortex

Lipid peroxidation in mitochondria from the functionally distinct inner (zona reticularis) and outer (zona fasciculata + zona glomerulosa) zones of the guinea-pig adrenal cortex was investigated. Ferrous ion (Fe2+)-induced lipid peroxidation was far greater in inner than outer zone mitochondria. Ascorbic acid similarly initiated lipid peroxidation to a greater extent in inner zone mitochondrial preparations. Differences in the unsaturated fatty acid content of inner and outer zone mitochondria could not account for the regional differences in lipid peroxidation. Total fatty acid concentrations were greater in the outer than in the inner zone, and the relative amounts of each fatty acid were similar in the two zones. However, mitochondrial concentrations of alpha-tocopherol, an antioxidant known to inhibit lipid peroxidation, were approx. 5-times greater in the outer than inner zone. The results demonstrate that there are regional differences in mitochondrial lipid peroxidation in the adrenal cortex which may be attributable to differences in alpha-tocopherol content. Thus, alpha-tocopherol may serve to protect outer zone mitochondrial enzymes from the consequences of lipid peroxidation and thereby contribute to some of the functional differences between the zones of the adrenal cortex.     

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.     

Endocytosis and the adaptive acid hydrolases in Tetrahymena pyriformis GL

Endocytosis of yeast cells by Tetrahymena pyriformis GL for a period of 2.5 h produced changes in cellular acid hydrolases. Acid phosphatase, acid deoxyribonuclease and acid proteinase activities were markedly increased, whereas there was a decrease in acid ribonuclease activity and little change in -glucosidase activity. These alterations do not appear to be due to any alteration in the rates of secretion of these enzymes into the milieu. Evidence is presented that the cellular enzyme increases found upon endocytosis of yeast reflect changes in lysosomal enzymes, because it was shown that the acid phosphatase activity increase resulted in an increased amount of latent enzyme within the cell. The results also support the idea that there are at least 3 distinct populations of lysosomes, in addition to phagolysosomes, present in Tetrahymena pyriformis GL, with different modes of formation. There appears to be a large excess of lysosomes, uncombined with phagosomes, present in these fed cells since latency averaged 66% in broken-cell preparations which contained very few intact phagolysosomes. The phagolysosomal acid phophatase activity cannot account for more than 34% of that present in the cell. The endocytosis of yeast in the presence of growth medium resulted in a marked drop in the rate of cell division as compared to cells growing in the growth medium alone. The results are discussed.     

Ultrastructural and immunocytochemical study of skin fibroblasts from normal and sialidosis patients

The objectives of this study were to analyze morphologically, morphometrically and immunocytochemically the lysosomal compartment of normal fibroblasts and of fibroblasts with neuraminidase deficiency. The immunocytochemical analyses consisted of quantifying the distribution of saposins and -galactosidase in the lysosomes of these cells to test the hypothesis that neuraminisdase deficiency is associated with an impairment in the transport of these proteins to the lysosomal compartment. To test this idea, cultured skin fibroblasts of patients with or without sialidosis were prepared for electron microscopy and probed with antibodies against lysosomal -galactosidase and lysosomal saposins. The lysosomes of the affected cells had an abnormal accumulation of incompletely digested membranes which was associated with a significant lowering in the density of antigenic sites per lysosome. However, due to a significant increase in the number of lysosomes per affected cell, the total number of antigenic sites in control and neuraminidase deficient cells was similar. This presumably compensatory effect indicates that although the rate of production of -galactosidase and saposins remains unchanged, the transport of these molecules to the lysosomes is somehow affected. Our data also indicate that in the fibroblasts, lysosomes require a normal concentration of the three enzymes to maintain neuraminidase activity and sphingolipid degradation.