Evaluation of cytotoxicity, acute toxicity, genotoxicity and antioxidant and antigenotoxicity activities of the sarcotesta fraction of punica granatum L. rich in lectin (PgTel).

Punica granatum, popularly known as pomegranate, is a fruit tree with wide worldwide distribution, containing numerous phytochemicals of great medicinal value. The aim of the present study was to determine the phytochemical profile and antioxidant potential of a protein fraction (PF) derived from P. granatum sarcotesta which is rich in lectin. In addition, the acute oral toxicity, genotoxicity and antigenotoxicity of this protein fraction (PF) from P. granatum sarcotesta was measured. The phytochemical profile of PF was determined using HPLC. The in vitro antioxidant effect was assessed using the methods of total antioxidant capacity (TAC) and DPPH and ABTS+ radical scavenging. Acute oral toxicity was determined in female Swiss mice administered a single dose of 2000 mg/kg. This PF was examined for genotoxicity and antigenotoxicity at doses of 500, 1000 and 2000 mg/kg, utilizing mouse peripheral blood cells. Phytochemical characterization detected a high content of ellagic acid and antioxidant capacity similar to that of ascorbic acid (positive control). PF was not toxic (LD50 >2000 mg/kg) and did not exert a genotoxic effect in mice. PF protected the DNA of peripheral blood cells against damage induced by cyclophosphamide. In conclusion, this PF fraction exhibited significant antioxidant activity without initiating toxic or genotoxic responses in mice.

Cephalosporium curvulum lectin causes mycotic keratitis by initiating infection through MyD88 dependent cellular proliferation and apoptosis in human corneal epithelial cells.

Physiological role of a core fucose specific lectin from Cephalosporium curvulum isolated from mycotic keratitis patient in mediating pathogenesis was reported earlier. CSL has opposite effects on HCECs, at the initiation of infection when lectin concentration is low, CSL induces proinflammatory response and at higher concentration it inhibits growth as the infection progresses. Here we delineate detailed mechanism of opposing effects of CSL by confirming the binding of CSL and anti TLR 2 and 4 antibodies to TLRs 2 and 4 purified from HCECs using Galectin-3 Sepharose 4B column. Further, the expression of signaling proteins were monitored by Western blotting and apoptosis assay. At concentration of 0.3 µg/ml, CSL induced the activation of TLR-2,-4 and adapter protein MyD88. CSL also induced the expression of transcription factors NFkB, C-Jun and proinflammatory cytokines like interleukins -6 and -8 essential in maintaining cell proliferation. In contrast at higher concentrations i.e. 5 µg/ml CSL induces apoptotic effect as evidenced by increase in early and late apoptotic population as demonstrated by Annexin V-PI assay. Western blotting revealed that CSL treated HCECs at higher concentration lead to MyD88 dependent expression of apoptotic proteins like FADD, Caspase -8 and -3. All these results are in line with and substantiate our earlier results that indeed CSL is involved in mediating host pathogen interactions by interacting with cell surface TLRs, activating downstream signaling pathways leading to pathogenesis. Findings are of clinical significance in developing carbohydrate based therapeutic strategy to control infection and the disease.

Internalization of a novel, huge lectin from Ibacus novemdentatus (slipper lobster) induces apoptosis of mammalian cancer cells.

An N-acetyl sugar-binding lectin (termed iNoL) displaying cytotoxic activity against human cancer cells was isolated from the slipper lobster Ibacus novemdentatus (family Scyllaridae). iNoL recognized monosaccharides containing N-acetyl group, and glycoproteins (e.g., BSM) containing oligosaccharides with N-acetyl sugar. iNoL was composed of five subunits (330, 260, 200, 140, and 30 kDa), which in turn consisted of 70-, 40-, and 30-kDa polypeptides held together by disulfide bonds. Electron microscopic observations and gel permeation chromatography indicated that iNoL was a huge (500-kDa) molecule and had a polygonal structure under physiological conditions. iNoL displayed cytotoxic (apoptotic) effects against human cancer cell lines MCF7 and T47D (breast), HeLa (ovarian), and Caco2 (colonic), through incorporation (internalization) into cells. The lectin was transported into lysosomes via endosomes. Its cytotoxic effect and incorporation into cells were inhibited by the co-presence of N-acetyl-D-mannosamine (ManNAc). Treatment of HeLa cells with iNoL resulted in DNA fragmentation and chromatin condensation, through activation of caspase-9 and -3. In summary, the novel crustacean lectin iNoL is incorporated into mammalian cancer cells through glycoconjugate interaction, and has cytotoxic (apoptotic) effects.

Catfish rhamnose-binding lectin induces G0/1 cell cycle arrest in Burkitt's lymphoma cells via membrane surface Gb3.

Silurus asotus egg lectin (SAL), an α-galactoside-binding protein isolated from the eggs of catfish, is a member of the rhamnose-binding lectin family that binds to Gb3 glycan (Galα1-4Galß1-4Glc). We have previously demonstrated that SAL reduces the proliferation of Gb3-expressing Burkitt's lymphoma Raji cells and confirm here that it does not reduce their viability, indicating that unlike other lectins, it is not cytotoxic. The aim of this study was to determine the signal transduction mechanism(s) underlying this novel SAL/Gb3 binding-mediated effect profile. SAL/Gb3 interaction arrested the cell cycle through increasing the G0/1 phase population of Raji cells. SAL suppressed the transcription of cell cycle-related factors such as c-MYC, cyclin D3, and cyclin-dependent protein kinase (CDK)-4. Conversely, the CDK inhibitors p21 and p27 were elevated by treatment with SAL. In particular, the production of p27 in response to SAL treatment increased steadily, whereas p21 production was maximal at 12 h and lower at 24 h. Activation of Ras-MEK-ERK pathway led to an increase in expression of p21. Notably, treatment of Raji cells with anti-Gb3 mAb alone did not produce the above effects. Taken together, our findings suggest that Gb3 on the Raji cell surface interacts with SAL to trigger sequential GDP-Ras phosphorylation, Ras-MEK-ERK pathway activation, p21 production, and cell cycle arrest at the G0/1 phase.

Cytotoxic effects of Aeromonas hydrophila culture supernatant on peripheral blood leukocytes of Nile tilapia (Oreochromis niloticus): Possible presence of a secreted cytotoxic lectin.

Number of exotoxins like haemolysin, leukocidin, aerolysin etc. were reported from Aeromonas hydrophila. In this study, we report the haemolytic and cytotoxic effect of A. hydrophila culture supernatant (CS) that is specifically inhibited by lactose and also by serum and mucus of Nile tilapia (Oreochromis niloticus). Hence, we assume the presence of a secreted lectin in the CS. CS is toxic to peripheral blood leukocytes (PBL) of O. niloticus as revealed by MTT assay and by flow cytometry. DNA laddering assay indicates that CS causes necrosis to PBL. As a result of necrosis, CS treated PBL showed increased production of reactive oxygen species as indicated by nitroblue tetrazolium and 2',7' -dichlorofluorescin diacetate assays. CS treated PBL showed reduced mRNA expression of TNF-α and IFN-γ genes. When CS was subjected to polyacrylamide gel electrophoresis, it showed a single band corresponding to the molecular weight of 45 kDa. However, upon concentrating the CS by ultrafiltration, many bands were visualized. Further studies at molecular level are required to unravel this macromolecular-leukocyte interaction which would ultimately benefit the aquaculture industry.

[Antagonistic effect of gingerols against TNF-α release, ROS overproduction and RIP3 expression increase induced by lectin from Pinellia ternata].

To explore the antagonistic effect of gingerols against the inflammation induced by lectin from Pinellia ternata. In this study, ELISA method was used to determine the effect of different extracts from gingerols on the release of inflammatory factor TNF-α from macrophages induced by lectin from P. ternata. The fluorescence probe was used to determine the effect of gingerols on the changes in ROS of macrophages induced by lectin from P. ternata. The western-blot method was applied to study the effect of gingerols on the increase in expression of cell receptor interacting protein RIP3 in macrophages induced by lectin from P. ternata. The scanning electron microscope (SEM) was used to study the effect of gingerols on morphological changes in macrophages induced by lectin from P. ternata. According to the results, gingerols can significantly inhibit the release of inflammatory factor from macrophages induced by lectin from P. ternata, ROS overproduction and increase in RIP3 expression. SEM results showed that gingerols can inhibit the cytomorphosis and necrocytosis induced by lectin from P. ternata. Fresh ginger's detoxication may be related to gingerols' effects in inhibiing release of inflammatory factor, ROS overproduction and increase in RIP3 expression caused by macrophages induced by lectin from P. ternata, which are mainly inflammatory development.

The fungal chimerolectin MOA inhibits protein and DNA synthesis in NIH/3T3 cells and may induce BAX-mediated apoptosis.

The Marasmius oreades mushroom agglutinin (MOA) is a blood group B-specific lectin carrying an active proteolytic domain. Its enzymatic activity has recently been shown to be critical for toxicity of MOA toward the fungivorous soil nematode Caenorhabditis elegans. Here we present evidence that MOA also induces cytotoxicity in a cellular model system (murine NIH/3T3 cells), by inhibiting protein synthesis, and that cytotoxicity correlates, at least in part, with proteolytic activity. A peptide-array screen identified the apoptosis mediator BAX as a potential proteolytic substrate and further suggests a variety of bacterial and fungal peptides as potential substrates. These findings are in line with the suggestion that MOA and related proteases may play a role for host defense.

The chitinase-like protein YKL-40 increases mucin5AC production in human bronchial epithelial cells.

Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. However, the regulatory mechanisms that mediate excessive mucin production remain elusive. Recently, the level of YKL-40, a chitinase-like protein, has been found to be significantly increased in chronic inflammatory airway diseases and has been shown to be associated with the severity of these diseases. In this study, we sought to explore the effect of YKL-40 on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in this process. We found that elevated YKL-40 levels increased the mRNA and protein expression of MUC5AC in a dose- and time-dependent manner, in association with the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), reflecting their activation. These responses were significantly suppressed by the knockdown of protease-activating receptor 2 (PAR2) with specific small interfering RNA or the inhibitors of ERK and NF-κB. YKL-40-induced MUC5AC overproduction was also effectively attenuated by the inhibitor of focal adhesion kinase (FAK). Taken together, these results imply that YKL-40 can stimulate excessive MUC5AC production through PAR2- and FAK-mediated mechanisms.

In vitro antiproliferative effects of Vatairea macrocarpa (Benth.) Ducke lectin on human tumor cell lines and in vivo evaluation of its toxicity in Drosophila melanogaster.

Tumor cells may develop alterations in glycosylation patterns during the initial phase of carcinogenesis. These alterations may be important therapeutic targets for lectins with antitumor action. This work aimed to evaluate the in vitro cytotoxicity of VML on tumor and non-tumor cells (concentration of 25 µg/mL and then microdiluted) and evaluate its in vivo toxicity at different concentrations (1.8, 3.5 and 7.0 µg/mL), using Drosophila melanogaster. Toxicity in D. melanogaster evaluated mortality rate, as well as oxidative stress markers (TBARS, iron levels, nitric oxide levels, protein and non-protein thiols). The cytotoxicity assay showed that VML had cytotoxic effect on leukemic lines HL-60 (IC50 = 3.5 µg/mL), KG1 (IC50 = 18.6 µg/mL) and K562 (102.0 µg/mL). In the toxicity assay, VML showed no reduction in survival at concentrations of 3.5 and 7.0 µg/mL and did not alter oxidative stress markers at any concentrations tested. Cytotoxicity of VML from HL-60, KG1 and K562 cells could arise from the interaction between the lectin and specific carbohydrates of tumor cells. In contrast, effective concentrations of VML against no-tumor cells human keratinocyte - HaCat and in the D. melanogaster model did not show toxicity, suggesting that VML is a promising molecule in vivo studies involving leukemic cells.

A lectin from the mussel Mytilus galloprovincialis has a highly novel primary structure and induces glycan-mediated cytotoxicity of globotriaosylceramide-expressing lymphoma cells.

A novel lectin structure was found for a 17-kDa α-D-galactose-binding lectin (termed "MytiLec") isolated from the Mediterranean mussel, Mytilus galloprovincialis. The complete primary structure of the lectin was determined by Edman degradation and mass spectrometric analysis. MytiLec was found to consist of 149 amino acids with a total molecular mass of 16,812.59 Da by Fourier transform-ion cyclotron resonance mass spectrometry, in good agreement with the calculated value of 16,823.22 Da. MytiLec had an N terminus of acetylthreonine and a primary structure that was highly novel in comparison with those of all known lectins in the structure database. The polypeptide structure consisted of three tandem-repeat domains of ∼50 amino acids each having 45-52% homology with each other. Frontal affinity chromatography technology indicated that MytiLec bound specifically to globotriose (Gb3; Galα1-4Galß1-4Glc), the epitope of globotriaosylceramide. MytiLec showed a dose-dependent cytotoxic effect on human Burkitt lymphoma Raji cells (which have high surface expression of Gb3) but had no such effect on erythroleukemia K562 cells (which do not express Gb3). The cytotoxic effect of MytiLec was specifically blocked by the co-presence of an α-galactoside. MytiLec treatment of Raji cells caused increased binding of anti-annexin V antibody and incorporation of propidium iodide, which are indicators of cell membrane inversion and perforation. MytiLec is the first reported lectin having a primary structure with the highly novel triple tandem-repeat domain and showing transduction of apoptotic signaling against Burkitt lymphoma cells by interaction with a glycosphingolipid-enriched microdomain containing Gb3.

Effects of food lectins on the transport system of human intestinal Caco-2 cell monolayers.

The effects of 16 lectins isolated from foodstuff on the transport system across human intestinal Caco-2 cell monolayers were investigated by using four fluorescent markers: lucifer yellow (LY) for the paracellular pathway, fluorescein (FL) for the monocarboxylic acid transporter-mediated pathway, rhodamine 123 for the P-glycoprotein-mediated efflux pathway, and calcein for the multidrug resistance associated protein-related efflux pathway. The transepithelial electrical resistance (TER) values for the monolayers were also measured. WGA from wheat germ, ABA from white mushroom, AOL from Aspergillus oryzae, and CSL3 from chum salmon eggs (each at 100 µg/mL) decreased the TER value by 20-40% which resulted in increased LY transport. These lectins, as well as such other lectins as SBA from soybean, RBA from rice bran, and Con A from jack bean, affected other transport pathways too. These results indicate that the lectins modulated the transepithelial transport system in different ways, probably because of their specific binding characteristics toward Caco-2 cell monolayers.

Disparate proteins use similar architectures to damage membranes.

Membrane disruption can efficiently alter cellular function; indeed, pore-forming toxins (PFTs) are well known as important bacterial virulence factors. However, recent data have revealed that structures similar to those found in PFTs are found in membrane active proteins across disparate phyla. Many similarities can be identified only at the 3D-structural level. Of note, domains found in membrane-attack complex proteins of complement and perforin (MACPF) resemble cholesterol-dependent cytolysins from Gram-positive bacteria, and the Bcl family of apoptosis regulators share similar architectures with Escherichia coli pore-forming colicins. These and other correlations provide considerable help in understanding the structural requirements for membrane binding and pore formation.

A lectin-mediated resistance of higher fungi against predators and parasites.

Fruiting body lectins are ubiquitous in higher fungi and characterized by being synthesized in the cytoplasm and up-regulated during sexual development. The function of these lectins is unclear. A lack of phenotype in sexual development upon inactivation of the respective genes argues against a function in this process. We tested a series of characterized fruiting body lectins from different fungi for toxicity towards the nematode Caenorhabditis elegans, the mosquito Aedes aegypti and the amoeba Acanthamoeba castellanii. Most of the fungal lectins were found to be toxic towards at least one of the three target organisms. By altering either the fungal lectin or the glycans of the target organisms, or by including soluble carbohydrate ligands as competitors, we demonstrate that the observed toxicity is dependent on the interaction between the fungal lectins and specific glycans in the target organisms. The toxicity was found to be dose-dependent such that low levels of lectin were no longer toxic but still led to food avoidance by C. elegans. Finally, we show, in an ecologically more relevant scenario, that challenging the vegetative mycelium of Coprinopsis cinerea with the fungal-feeding nematode Aphelenchus avenae induces the expression of the nematotoxic fruiting body lectins CGL1 and CGL2. Based on these findings, we propose that filamentous fungi possess an inducible resistance against predators and parasites mediated by lectins that are specific for glycans of these antagonists.

Cyclic neoglycodecapeptides: how to increase their inhibitory activity and selectivity on lectin/toxin binding to a glycoprotein and cells.

Protein (lectin/toxin)-glycan interaction can be clinically harmful so that the design of inhibitors has become an aim. Cyclic decapeptides are suited as rigid carriers for carbohydrate derivatives. We herein document the bioactivity of sugar headgroups covalently attached to this carrier for the cases of five proteins, i.e. a potent biohazardous plant agglutinin, a leguminous model lectin and three adhesion/growth-regulatory human lectins. They represent the different types of topological organization within the galectin family. The relative inhibitory activities of glycoclusters with the three ligands (galactose, lactose and the disaccharide of the Thomsen-Friedenreich antigen) reflected the affinity of free carbohydrates, hereby excluding an impairment of binding activity by chemical derivatization and conjugation. Headgroup tailoring is thus one route to optimize activity and selectivity of cyclopeptide-based glycoclusters. The increase of ligand density from tetra- to hexadecavalency added a second route. The plant toxin and tandem-repeat-type galectin-4 were especially sensitive to this parameter change. Strategically combining solid-phase assays for screening with analysis of lectin binding to cells in different systems revealed efficient inhibition by distinct glycoclusters, thereby protecting cells from lectin association. Cyclic neoglycodecapeptides thus warrant further study as lectin-directed pharmaceuticals.

Isolation, functional, and partial biochemical characterization of galatrox, an acidic lectin from Bothrops atrox snake venom.

Snake venom lectins have been studied in regard to their chemical structure and biological functions. However, little is known about lectins isolated from Bothrops atrox snake venom. We report here the isolation and partial functional and biochemical characterization of an acidic glycan-binding protein called galatrox from this venom. This lectin was purified by affinity chromatography using a lactosyl-sepharose column, and its homogeneity and molecular mass were evaluated by high-performance liquid chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. The purified galatrox was homogeneous and characterized as an acidic protein (pI 5.2) with a monomeric and dimeric molecular mass of 16.2 and 32.5 kDa, respectively. Alignment of N-terminal and internal amino acid sequences of galatrox indicated that this protein exhibits high homology to other C-type snake venom lectins. Galatrox showed optimal hemagglutinating activity at a concentration of 100 µg/ml and this effect was drastically inhibited by lactose, ethylenediaminetetraacetic acid, and heating, which confirmed galatrox's lectin activity. While galatrox failed to induce the same level of paw edema or mast cell degranulation as B. atrox crude venom, galatrox did alter cellular viability, which suggested that galatrox might contribute to venom toxicity by directly inducing cell death.

Correlated expression of T cell growth factor dependence, sensitivity to Vicia villosa lectin, and cytolytic activity in hybrids between cytolytic T cells and T lymphomas.

Somatic cell fusion between cytolytically active, T cell growth factor- (TCGF) dependent murine T cell lines (CTL lines) and noncytolytic, TCGF-independent murine T lymphoma lines has yielded two types of somatic cell hybrids (5): cytolytic hybrids, growth of which is dependent on TCGF, and hybrids with very weak or undetectable cytolytic activity which grow at the same rate with or without TCGF. Here we report that the former can produce stable variants that resemble the latter type. Some of these TCGF-independent variants still have TCGF receptors. High susceptibility to the cytotoxic effects of Vicia villosa lectin, a marker distinguishing the parental CTL lines from T lymphomas, is expressed by the TCGF-dependent hybrids but not by the TCGF-independent variants. The two types of hybrids also differ in the expression of surface glycoproteins. We propose that there exists a genetic element in the CTL line that represses the TCGF-independent replication mechanism of the T lymphoma parent in the TCGF-dependent hybrids and that this genetic element is lost or switched off in the TCGF-independent variants.

Increased cytotoxicity of normal rabbit serum for lectin-resistant mutants of animal cells.

Plant lectins are cytotoxic and can be used to select for mutants of animal cells that exhibit structural changes in cell surface carbohydrates reflecting glycosylation defects. We isolated eight lectin mutants of Chinese hamster ovary (CHO) cells that appear to represent three different phenotype classes. These lectin mutants were much more sensitive to the cytotoxic action of normal rabbit serum (NRS) than were the parental cells. This increased cytotoxicity was heat sensitive, specifically absorbed, and inhibited by simple and complex carbohydrates. No killing was observed under conditions in which only the alternate complement pathway was active. An NRS-resistant subclone that was isolated from one lectin mutant was shown to have also regained wild type behavior when tested with the lectins. The possibility that naturally occurring antibodies in rabbit serum are reacting with incomplete carbohydrate chains on the surface of the lectin mutants is discussed.

Green tea polyphenols mitigate the plant lectins-induced liver inflammation and immunological reaction in C57BL/6 mice via NLRP3 and Nrf2 signaling pathways.

Plant-derived dietary lectins have been reported to be involved in the pathogenesis of several inflammatory diseases, including hepatitis, inflammatory bowel disease, diabetes, and celiac disease. In this present study, we aimed to assess whether green tea polyphenols (GTPs) exerts protective effects against plant lectins-induced liver inflammation and immunological reaction in mice. The C57BL/6 mice received intragastric GTPs (200 mg/kg b.w.) once per day for 7 consecutive days prior to plant lectins stimulation (50 mg/kg b.w., intraperitoneally). GTPs supplementation alleviated the histopathological changes of liver and the disorder of serum biochemical parameters in plant lectins-challenged mice. GTPs supplementation also alleviated plant lectins-induced oxidative stress and liver inflammation, decreasing protein contents and gene expression levels of pro-inflammatory cytokines in the plasma and hepatic tissue and increasing antioxidant capacity in the liver. GTPs decreased the protein expression levels of myeloperoxidase, F4/80 and neutrophil, as determined by immunohistochemical analysis, and T lymphocytes (CD4 and CD8) contents as determined by immunofluorescence analysis, in the liver. Moreover, we found that GTPs inhibited Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome expression and increased nuclear factor erythroid 2-related factor 2 (Nrf2) pathways in the liver tissues of plant lectins-challenged mice. Taken together, these results show that GTPs alleviates hepatic inflammatory damage and immunological reaction after plant lectins challenge, and GTPs (or green tea intake) supplements can be beneficial for people exposed to plant lectins.

Aspergillus niger lectin elicits MyD88 dependent proliferation and apoptosis at lower and higher doses in immortalized human corneal epithelial cells leading to pathogenesis.

An L-fucose lectin, ANL from the corneal smears of a mycotic keratitis patient was reported earlier. Interaction of ANL with immortalized Human Corneal Epithelial Cells (HCECs) was studied in order to assign the role of ANL in pathogenesis. ANL showed strong binding to HCECs which could be blocked by L-fucose and mucin. At concentrations below 0.6 µg/mL ANL showed proliferative effect and highest at 0.07 µg/mL leading to expression of proinflammatory cytokines IL-6 and IL-8. ANL induced proinflammatory response is mediated by TLR-2,-4, MyD88, NFkB and C-Jun dependent signaling. In contrast, ANL at concentrations above 0.6 µg/mL showed growth inhibitory effect at 48 h with an IC50 of 2.75 µg/mL. Western blot analysis revealed that HCECs treated with ANL at lower concentration induced the expression of proinflammatory signaling proteins TLR-2, 4, MyD88, NFkB and C-Jun which maintain high cell proliferating state. At higher concentration ANL induced apoptotic effect in HCECs with an increase in early apoptotic population as demonstrated by Annexin V-PI assay. ANL induced the expression of apoptotic proteins FADD, Caspase 8 and -3 mediated by MyD88. These findings demonstrate implication of ANL in pathogenesis and the findings are of clinical significance in developing strategy for controlling the infection leading to mycotic keratitis.

Impaired lysosomes in a temperature-sensitive mutant of Chinese hamster ovary cells.

We describe here the properties of a mutant of Chinese hamster ovary cells that expresses a conditional-lethal mutation affecting dense lysosomes. This mutant, termed V.24.1, is a member of the End4 complementation group of temperature-sensitive mutants selected for resistance to protein toxins (Colbaugh, P. A., C.-Y. Kao, S.-P. Shia, M. Stookey, and R. K. Draper. 1988. Somatic Cell Mol. Genet. 14:499-507). Vesicles present in postnuclear supernatants prepared from V.24.1 cells harvested at the restrictive temperature had a 50% reduction in acidification activity, assessed by the ATP-stimulated accumulation of the dye acridine orange in acidic vesicles. To investigate whether specific populations of vesicles were impaired in acidification, we measured acidification activity in three subcellular fractions prepared from Percoll gradients: one containing endosomal and Golgi markers, one containing buoyant lysosomes, and the third containing dense lysosomes. Activity in dense lysosomes was reduced by 90%, activity in the buoyant lysosome fraction was unaffected, and activity in the endosome-Golgi fraction was mildly reduced. The activity of three lysosomal enzymes--beta-hexosaminidase, beta-galactosidase, and beta-glucocerebrosidase--was also reduced in dense lysosomes but nearly normal in the buoyant lysosome fraction. However, beta-hexosaminidase and beta-glucocerebrosidase activity was increased two- to threefold in the endosome-Golgi fraction. We conclude that the lesion selectively impairs dense lysosomes but has little effect on properties of buoyant lysosomes.