Isolation of hepatocyte-derived rat liver lysosomal fractions

Colloidal gold is accumulated in much smaller concentrations in lysosomes of hepatocytes of the ‘dense body’ type than in the larger lysosomes of sinus endothelial cells. These differences were used for labeling of lysosomal subpopulations as well as for the isolation of a hepatocytederived lysosomal subpopulation by combined differential and density gradient centrifugation. The origin of isolated lysosomes was determined by measurements of the gold-grain content and by cytomorphometric analyses giving size distribution curves of lysosomal profiles before and after isolation. The specific activities of β-galactosidase, acid phosphatase and especially of β-N-acetyl-glucosaminidase and β-glucuronidase were lower in lysosomal subfractions derived mainly from hepatocytes than in a mixed lysosomal fraction containing both lysosomes from hepatocytes and sinus endothelial cells.     

The effect of environmental pH upon acid hydrolase activities of cultured human diploid fibroblasts

Cultured human diploid fibroblasts were grown for 4 days in media at five different pH values between pH 6.8 and 8.0. The specific activities of α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, β-glucuronidase and hexosaminidase were determined. Increasing environmental pH levels produced increased levels of β-glucosidase activity and decreased levels of activity for the other five enzymes.     

Identification of membrane proteins mediating the interaction of human platelets

Mild acid hydrolysis of phosphomannan secreted by the yeast hansenula holstii (NRRL Y- 2448) produces two phosphomannyl fragments which differ strikingly in their potency as inhibitors of pinocytosis of human β-glucuronidase by human fibroblasts. The larger molecular weight polyphosphomonoester fragment is 100,000-fold more potent an inhibitor of enzyme uptake than the smaller penta-mannosyl-monophosphate fragment. Binding to attached fibroblasts at 3 degrees C was much greater with the polyphosphomonoester fragment than with the pentamannosyl-monophosphate. The larger molecular weight fragment was also subject to adsorptive pinocytosis and was taken up by fibroblasts at a rate 30- fold greater than the rate of uptake of pentamannosyl-monophosphate. Evidence that the polyphosphomonoester fragment is taken up by the phosphomannosyl-recognition system that mediates uptake of lysosomal enzymes includes: (a) its pinocytosis is inhibited by the same compounds that competitively inhibit enzyme pinocytosis (mannose-6-phosphate and phosphomannan from saccharomyces cerevisiae mutant mnn-1); (b) alkaline phosphatase treatment greatly reduces its susceptibility to pinocytosis; (c) its pinocytosis is competitively inhibited by high-uptake human β-glucuronidase; and (d) this inhibition by high-uptake enzyme is dramatically reduced by prior treatment of the enzyme with alkaline phosphatase or endoglycosidase-H. Endoglycosidase-H treatment human β-glucuronidase dramatically reduced its susceptibility to pinocytosis by fibroblasts. The phosphomannosyl components of high- uptake enzyme released by endoglycosidase-H treatment were much less effective inhibitors of polyphosphomonoester pinocytosis than when present on the phosphomannyl-enzyme. These results suggest that high-uptake acid hydrolases may be polyvalent ligands analogous to the polyphosphomonoester mannan fragment whose pinocytosis depends on interaction of more than one phospho-mannosyl recognition marker with pinocytosis receptors on fibroblasts.     

Concanavalin a promotes the uptake of lysosomal hydrolases by human fibroblasts

Human placental hexosaminidase B and β-galactosidase are taken up very poorly by human fibroblasts in culture. However, if fibroblasts manifesting genetically determined deficiencies of these lysosomal hydrolases are first treated with concanavalin A, then enzyme uptake is markedly increased. Enzyme activity which becomes associated with concanavalin A-treated fibroblasts maintained at 4°C can be greatly removed by treatment with haptene sugar, while enzyme activity which becomes associated with cells maintained at 37°C is refractory to haptene treatment. These results are interpreted as an initial binding of enzyme to concanavalin A molecules located at the cell surface, followed by an active cellular process leading to internalization of the lectin-enzyme complexes.     

Cell surface-associated lysosomal enzymes in cultured human skin fibroblasts

Trypsin released from the surface of intact human skin fibroblasts β-N-acetylglucosaminidase. The amount of trypsin removable β-N-acetylglucosaminidase in 4 control and 14 mucopolysaccharidosis cell lines was equivalent to 1.5% (range 0.5–4.3%) of the intracellular activity. Cell surface-associated β-N-acetylglucosaminidase was absent in mucolipidosis II and III fibroblasts that form lysosomal enzymes defective in binding to the cell surface receptors of fibroblasts and in β-N-acetylglucosaminidase deficient fibroblasts (Sandhoff's disease). Indirect immunofiuorescence with monospecific antisera allowed the demonstration of β-N-acetylglucosaminidase, α-N-acetylglucosaminidase, α-mannosidase and β-glucuronidase on the cell surface of fibroblasts, whereas these enzymes were absent on the cell surface of mucolipidosis II and III fibroblasts. Simultaneous staining for β-glucuronidase and β-N-acetylglucosaminidase showed presence of both enzymes in almost identical areas of the same cell. Cross-reacting material was present on the cell surface of fibroblasts with a deficiency of β-N-acetylglycosaminidase, α-N-acetylglucosaminidase (mucopolysaccharidosis III B), α-mannosidase (mannosidosis) and β-glucuronidase (mucopolysaccharidosis VII). The demonstration of lysosomal enzymes on the cell surface is in agreement with the hypothesis that in fibroblasts transport of lysosomal enzymes to the lysosomal apparatus involves cycling of lysosomal enzymes via the cell surface.     

Isolation of autofluorescent ‘aged’ human fibroblasts by flow sorting : Morphology, enzyme activity and proliferative capacity

In cultures of human fibroblasts the percentage of bright autofluorescent (AF) cells increases with increasing passage number. These autofluorescent cells were isolated using a FACS II cell sorter and compared with sorted non-fluorescent (NF) cells. The AF cells showed an increase in population doubling time (2.3-fold), cell protein (1.9-fold), and in specific activities of the lysosomal enzymes: β-hexosaminidase (4.2-fold), β-galactosidase (3.8-fold) and acid phosphatase (2.5-fold). The specific activities of two non-lysosomal enzymes glucose-6-phosphate dehydrogenase and lactate dehydrogenase had increased only slightly (1.1-fold) respectively (1.5-fold).The autofluorescence in the AF cells was restricted to small round organelles. The distribution and size of these autofluorescence granules were similar to the acid phosphatase-containing granules in the cytochemically stained cells. Electronmicroscopical examination showed that these AF cells contained a large amount of small electron-dense granules containing amorphosmophilic material. These granules which were positive for the acid phosphatase reaction, were classified as secondary lysosomes. The low percentage of the sorted AF cells which incorporate [³H]thymidine during a 24 h test period (19%) as compared with the labelling percentage of sorted NF cells (73%) from the same culture, indicate that the autofluorescent cells in a ‘young’ culture have a very limited remaining proliferative capacity. The results imply, that by flow sorting it is possible to isolate ‘aged’ cells with characteristics of ‘phase III’ cells out of non-aged fibroblast cultures.     

Correction of I-cell defect by hybridization with lysosomal enzyme deficient human fibroblasts

I-cell fibroblasts with a multiple intracellular lysosomal enzyme deficiency were hybridized with cells from patients with different types of single lysosomal enzyme defects. Fusion with G_M2 gangliosidosis, type 2, (Sandhoff disease) fibroblasts resulted in a restoration of the hexosaminidase activity, in a normalization of the electrophoretic mobility of the isoenzymes, and in a decreased activity in the medium. Fusion of I-cells with fibroblasts from G_M1 gangliosidosis, type 1, led to enhancement of β-galactosidase (β-gal) activity. This complementation must be the result of the presence of normal polypeptide chains in I-cells, whereas the other cell types provide a factor that causes the intracellular retention of the enzymes. Restoration of β-gal was also observed in heterokaryons after fusion of I-cells with β-galactosidase/neuraminidase-deficient (β-gal⁻/neur⁻) variants, indicating that the neuraminidase(s) and the posttranslational modification of β-gal are affected in a different way in I-cell disease and in β-gal⁻/neur⁻ variants. Fusion of I-cells with mannosidosis fibroblasts resulted in a restoration of the acidic form of α-mannosidase and in a decrease of the extracellular activity of both this enzyme and the hexosaminidase enzyme, indicating that fusion of I-cells with different types of fibroblasts with a single lysosomal enzyme deficiency not only leads to complementation for one particular enzyme but also to a correction of the basic defect in I-cells.     

Purification and characterization of human lysosomes from EB-virus transformed lymphoblasts

A method was developed for the isolation of unmodified lysosomes of human origin using cultured EB-virus transformed lymphoblasts. The cells were lysed carefully by repeated resuspension in buffered isotonic sucrose. A crude granular fraction derived from this lysate was further purified by isopyknic centrifugation in an isotonic colloidal silica gel gradient and by free-flow electrophoresis. The following relative specific activities (mean ± S.D.) of lysosomal marker enzymes were measured in a pooled lysosomal fraction obtained from the final electrophoresis step (representing less than 0.1% of the initial protein): β-N-acetylglucosaminidase 85.6 ± 15.5; β-galactosidase 87.6 ± 13.4; acid β-glycerophosphatase 41.7 ± 3.5; β-glucuronidase 36.6 ± 6.1. With respect to the final two enzymes the recovery within this pooled fraction was 5–6% of the initial lysate. The great differences in relative specific activities achievable may be due mainly to different extralysosomal portions of the lysosomal marker enzymes, as was found for acid β-glycerophosphatase which was largely distributed within non-lysosomal structures in lymphoblasts when studied by histochemical staining. The final fraction consisted almost exclusively of lysosomes when examined by electron microscopy. Most lysosomes appeared club-shaped immediately after cell lysis and throughout the preparation procedure. Examination by electron microscopy and measurement of the latency of lysosomal enzyme activity revealed an exceptional integrity of the lysosomal membrane. This method provides the opportunity to study highly purified lysosomes from patients with lysosomal disorders.     

Concanavalin A promotes the uptake of lysosomal hydrolases by human fibroblasts.

Human placental hexosaminidase B and beta-galactosidase are taken up very poorly by human fibroblasts in culture. However, if fibroblasts manifesting genetically determined deficiencies of these lysosomal hydrolases are first treated with concanavalin A, then enzyme uptake is markedly increased. Enzyme activity which becomes associated with concanavalin A-treated fibroblasts maintained at 4 degrees C can be greatly removed by treatment with haptene sugar, while enzyme activity which becomes associated with cells maintained at 37 degrees C is refractory to haptens treatment. These results are interpreted as an initial binding of enzyme to concanvalin A molecules located at the cell surface, followed by an active cellular process leading to internalization of the lectin-enzyme complexes.     

Lysosomal hydrolases of human vascular cells: response to agonists of endothelial function

Endothelial injury has been proposed as a feature of a wide variety of vascular diseases, and release of endothelial lysosomal hydrolases could contribute to the pathological changes seen. We have determined the relative activities of 14 glycosidases, two esterases and four peptide hydrolases in human umbilical vein endothelial cells and investigated whether known agonists of endothelial function, or materials known to modulate hydrolase secretion in other phagocytic cells, influenced the activity or secretion of these enzymes by human umbilical vein endothelial cells. Hexosaminidase, beta-galactosidase, beta-glucuronidase and alpha-iduronidase accounted for most of the measured glycosidase activity. Acid phosphatase activity greatly exceeded arylsulphatase activity, and most of the measured peptidase activity was due to acid peptidases. Optimum pH and apparent Km values were determined for the most abundant hydrolases. Exposure of human umbilical vein endothelial cells to bradykinin, thrombin or interleukin-1 resulted in negligible release of either hexosaminidase or lactate dehydrogenase (LDH), in contrast to phorbol myristate acetate, which caused a parallel, dose-dependent release of both enzymes. Treatment of these cells with calcium ionophore A23187, trypsin or platelet-activating factor, caused less than 10% release of either hexosaminidase or LDH. Agents known to modulate lysosomal enzyme secretion by other phagocytic cells failed to induce selective secretion of lysosomal enzymes by human umbilical vein endothelial cells.