Rat Liver Lysosomal Membranes Research Articles

The Arf-family protein, Arl8b, is involved in the spatial distribution of lysosomes

Lysosomes are late-endocytic organelles which primarily contribute to degradation and recycling of cellular material. From a previous proteomics study of purified rat liver lysosomal membranes we identified a protein from the Arf-family of small GTPases, Arl8b. Although proteins of the Arf-family have roles in a wide range of cellular functions, most notably roles in protein/vesicular trafficking, Arl8b represents the first from this protein family to be associated with a late-endocytic organelle. We demonstrate the co-localization of this protein with various lysosomal markers in different cell lines by confocal-immunofluorescence microscopy. We also show that GTP-restricted mutant Arl8b localizes to lysosomes and causes their redistribution to the periphery of the cell and into membrane projections. This indicates that Arl8b is involved in trafficking processes for lysosomes.

Effects of wine on cellular and endocellular membranes <em>in vitro</em>

<p style="text-align: justify;">Several observations suggest that wine consumption could have beneficial effects on health by preventing cardiovascular diseases. Apparently, ethanol is not the only component responsible for these effects, phenolic substances: tanins, flavonoids may have an important role. As these compounds are able to affect biological membranes, we investigated the effect of wine <em>in vitro</em> on two model membranes: the lysosomal membrane and the red cell membrane. The integrity of rat liver lysosomal membrane was assessed by measuring the latency of Nacetylglucosaminidase, a lysosomal enzyme, when the organelles are subjected to free radicals of oxygen in the presence of different concentrations of wine. The state of red cell membrane was followed by determining hemolysis caused by phospholipase C. Our results show that low concentrations of red wine prevent the deterioration of the membrane of lysosomes induced by oxygen free radicals generated by the xanthine-xanthine oxidase system and oppose hemolysis induced by treating red cells with <em>Clostridium welchii</em> phospholipase C. White wine is considerably less efficient. As similar effects can be obtained with some phenolic compounds, it is probable that the membrane protective effects of red wine that we describe, originate from its content in these substances.</p>

Dexamethasone megadoses stabilize rat liver lysosomal membranes by non-genomic and genomic effects.

Membrane-stabilizing effects may be part of glucocorticoid action during high-dose glucocorticoid therapy. The present study investigates the mode of action of dexamethasone megadoses on rat liver lysosomal membranes. Following intravenous administration of dexamethasone in rats, the release of beta-glucuronidase from liver lysosomes was assessed ex vivo as a marker for lysosomal membrane integrity. Dexamethasone megadoses significantly inhibited beta-glucuronidase release 10 min post-administration by 38% (3 mg/kg dexamethasone) and 33% (10 mg/kg dexamethasone) at corresponding dexamethasone liver concentrations of 3.9 x 10(-5) mol/kg and 15.1 x 10(-5) mol/kg, respectively. Comparable inhibition of beta-glucuronidase release (34% for 3 mg/kg and 38% for 10 mg/kg) was observed 24 h after administration of dexamethasone, although dexamethasone liver concentrations had already declined to 0.09 x 10(-5) mol/kg and 0.19 x 10(-5) mol/kg, respectively. A 2-h oral pretreatment of rats with the glucocorticoid receptor antagonist RU 486 (10 mg/kg) did not alter immediate (10 min) stabilization by dexamethasone (3 mg/kg). but almost completely prevented lysosomal membrane protection 24 h after dexamethasone injection. Dexamethasone megadoses may preserve lysosomal membrane integrity by a dual action involving both rapid nongenomic effects occurring instantaneously after administration and long-term receptor-dependent genomic events.

EFFECTS OF SOME BIOLOGICALLY ACTIVE COMPOUNDS ON PHAGOSOME–LYSOSOME FUSION IN PERITONEAL MACROPHAGES OF MICE

Effects of biologically active compounds bilirubin (BR), farmorubicin (FR), and chelerythrine (CR) on phagosome-lysome (P–L) fusion in mouse peritoneal macrophages were studied using fluorescent dye acridine orange as lysosomal labelling and yeast cells as target. It was found that all three compounds tested enhanced P–L fusion. To investigate mechanisms of these effects, changes in fluidity of rat liver lysosomal membranes under influence of BR, FR and CR were studied by measuring fluorescence intensity, lifetime, and polarization of DPH or TMA-DPH incorporated in isolated rat liver lysosomes. In order to characterize the cytoskeleton changes under the action of these biologically active compounds F-actin content in peritoneal macrophages of mice was determined. Our results demonstrate that BR action induces a decrease in DPH and TMA-DPH polarization, FR increases DPH and TMA-DPH polarization, and CR causes only an increase in TMA-DPH polarization in lysosomal membranes. All three compounds tested increase F-actin content in peritoneal macrophages. Thus, the effect of BR on P–L fusion is connected with increasing fluidity of lysosomal membranes and the cytoskeleton changes. The enhancement of P–L fusion under the action of FR and CR can most likely be explained by changes of the cytoskeleton state.

Cationic lipids destabilize lysosomal membrane in vitro

Addition of cationic lipids to plasmid DNA considerably increases the efficiency of transfection. The mechanism has not yet been elucidated. A possibility is that these compounds destabilize biological membranes (plasma, endosomal, lysosomal), facilitating the transfer of nucleic molecules through these membranes. We have investigated the problem by determining if a cationic lipid N-(1-(2,3-dioleoxy)propyl)- N, N, N,-trimethylammonium methyl-sulfate (DOTAP, Boehringer, Mannheim, Germany) affects the integrity of rat liver lysosomal membrane. We have measured the latency of β-galactosidase, a lysosomal enzyme, and found that incubation of lysosomes with low concentrations of DOTAP causes a striking increase in free activity of the hydrolase and even a release of the enzyme into the medium. This indicates that lysosomal membrane is deeply destabilized by the lipid. The phenomenon depends on pH, it is less pronounced at pH 5 than at pH 7.4. Anionic compounds, particularly anionic amphipathic lipids, can to some extent prevent this phenomenon. It can be observed with various cationic lipids. A possible explanation is that cationic liposomes interact with anionic lipids of lysosomal membrane, allowing a fusion between the lipid bilayers which results in a destabilization of the organelle membrane.

Functional reconstitution of the lysosomal sialic acid carrier into proteoliposomes.

The lysosomal carrier for the acidic monosaccharides sialic acid and glucuronic acid was solubilized from rat liver lysosomal membranes and reconstituted into phospholipid vesicles. Membrane proteins were extracted from lysosomal membranes with Triton X-100. Upon removal of the detergent by absorption on Amberlite XAD-2 beads, the solubilized proteins were incorporated in egg yolk phospholipids. The reconstituted proteoliposomes show proton-driven carrier-mediated uptake of acidic monosaccharides. The reconstituted carrier was compared in several characteristics with the transporter as present in the native lysosomal membrane. Transporter substrate affinity (Kt for glucuronic acid = 0.4 mM) and specificity for acidic monosaccharides are completely retained. The proteoliposomes also demonstrate trans-stimulation properties with both substrates sialic acid and glucuronic acid. The transporter is inhibited, both in its native and in the reconstituted state, by the sulfhydryl-modifying agents p-chloromercuribenzoic acid, N-ethylmaleimide, and phenyl isothiocyanate. In native membrane vesicles, arginine and histidine modifiers phenylglyoxal and diethyl pyrocarbonate inactivated transport in a substrate-protectable manner. In reconstituted proteoliposomes, similar inhibition was observed. However, protection by substrates was achieved only after treatment with phenylglyoxal. These data suggest that arginine or histidine residues or both are present at or near the substrate binding site of the carrier. Possibly, other essential histidines become exposed in the reconstituted state. The successful functional reconstitution of the lysosomal sialic acid carrier represents an important step towards its purification and its detailed molecular characterization.

Purification and characterization of (Ca2+-Mg2+)-ATPase in rat liver lysosomal membranes.

A (Ca(2+)-Mg2+)-ATPase associated with rat liver lysosomal membranes was purified about 300-fold over the lysosomal membranes with a 7% recovery as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included: preparation of lysosomal membranes, solubilization of the membrane with Triton X-100, WGA-Sepharose 6B, Con A-Sepharose, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. The molecular mass, estimated by gel filtration with Sephacryl S-300 HR, was approximately 340 kDa, and SDS-polyacrylamide gel electrophoresis showed the enzyme to be composed of four identical subunits with an apparent molecular mass of 85 kDa. The isoelectric point of the purified enzyme was 3.6. The enzyme had a pH optimum of 4.5, a Km value for ATP of 0.17 mM and a Vmax of 71.4 mumol/min/mg protein at 37 degrees C. This enzyme hydrolyzed nucleotide triphosphates and ADP but did not act on p-nitrophenyl phosphate and AMP. The effects of Ca2+ and Mg2+ on the ATPase were not additive, thereby indicating that both Ca2+ and Mg(2+)-ATPase activities are manifested by the same enzyme. The (Ca(2+)-Mg2+)-ATPase differed from H(+)-ATPase in lysosomal membranes, since the enzyme was not inhibited by N-ethylmaleimide but was inhibited by vanadate. The effects of some other metal ions and compounds on this enzyme were also investigated. The N-terminal 18 residues of (Ca(2+)-Mg2+)-ATPase were determined.

Purification and characterization of dipeptidyl peptidase IV in rat liver lysosomal membranes.

Dipeptidyl peptidase IV (m-DPP IV) in rat liver lysosomal membranes was purified about 50-fold over the lysosomal membranes with 38% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The enzyme amounts to about 3% of lysosomal membrane protein constituents. The purification procedures included: extraction of lysosomal membranes by Triton X-100, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, ion exchange chromatography, and preparative polyacrylamide gel electrophoresis. The enzyme (M(r) 240,000) is composed of two identical subunits with an apparent molecular weight of 110,000. The enzyme contains about 12.4% carbohydrate and the carbohydrate moiety was composed of mannose, galactose, fucose, N-acetylglucosamine, and neuraminic acid in a molar ratio of 14:17:2:24:11. Susceptibility to neuraminidase and immunoreactivity of the enzyme in intact tritosomes were examined to study the topology of the enzyme in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the enzyme were not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock. This result indicated that both the oligosaccharide chains and the main protein portion of the enzyme are on the inside surface of the tritosomal membranes. Subcellular localization of DPP IV was determined by means of enzyme immunoassay, which indicated that bile canalicular membranes and lysosomal membranes are the major sites of localization, and DPP IV activity in lysosomes was separated into a membrane bound form (60%) and a soluble form (40%). Immunoelectron microscopy clearly confirmed that DPP IV occurs not only in the bile canalicular domain but also in the lysosomes of rat liver.

Purification and characterization of an 85 kDa sialoglycoprotein in rat liver lysosomal membranes.

Sialoglycoprotein with a molecular mass of 85 kDa (LGP85) was purified from rat liver lysosomal membranes with a 0.9% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence and in the absence of SDS. The purification procedures included: preparation of lysosomal membranes, elimination of LGP107 and LGP96 with immunoaffinity columns, WGA-Sepharose affinity chromatography, hydroxylapatite chromatography, and preparative polyacrylamide gel electrophoresis. LGP85 contains about 22.8% carbohydrate and the carbohydrate moiety is composed of mannose, galactose, fucose, glucosamine, galactosamine, and neuraminic acid, in a molar ratio of 40:20:2:23:3:13. Susceptibility to neuraminidase and immunoreactivity of the protein in intact tritosomes were examined to study the topology of the protein in tritosomal membranes. Neuraminidase susceptibility and immunoreactivity of the protein were not observed in intact tritosomes until the tritosomes had been disrupted by osmotic shock. These observations suggest that both oligosaccharide chains and the main protein portion of the protein are located on the interior surface of the tritosomal membranes. Subcellular localization of LGP85 was determined using enzyme immunoassay. The lysosomes seem to be the major location. LGP85 in the lysosomes was divided into the membrane bound form (90%) and the soluble form (10%). Immunoelectron microscopy clearly confirmed that the localization of LGP85 is mainly confined to lysosomes.

Isolation and sequencing of a cDNA clone encoding 85kDa sialoglycoprotein in rat liver lysosomal membranes

We used the oligonucleotide probe corresponding to the internal amino acid sequence of a lysosomal membrane glycoprotein with a molecular weight of 85 K (LGP85) and isolated and characterized cDNA clones containing the entire coding region. The isolated cDNA comprised 2065 nucleotides. The predicted amino acid sequences of LGP85 consisted of 478 amino acid residues (Mr.54,090) and the protein has 11 potential N-glycosylation sites. Since the NH 2 terminal sequence determined from purified LGP85 was identical to the NH 2 terminal sequence deduced from the nucleotide sequence of the cDNA, except for the lack of initiator methionine which is likely to be cleaved off posttranslationally, it is likely that LGP85 has an uncleavable signal peptide at the NH 2 terminus. Hydropathy plots show that LGP85 possesses two strong hydrophobic regions at the NH 2 terminus (residues 4–26) and near the COOH terminus (residues 433–457), respectively. Either one or both of the domains might be used for membrane anchoring. A comparison of the sequences of the other lysosomal membrane glycoproteins with that of LGP85 revealed no homology. Glycine-tyrosine residues (so-called GY motif) which are thought an important signal for delivery of lysosomal membrane glycoproteins to lysosomes were not contained in the cytoplasmic tail of LGP85 (residues 458–478). LGP85 appears to be an unique lysosomal membrane glycoprotein that does not require tyrosine residues for targeting to lysosomes. Tyrosine residue may not be an essential signal for delivering newly synthesized lysosomal membrane glycoproteins to lysosomes.

Release of Acid Phosphatase from Lysosomal Membranes by Cathepsin D1

We examined the mechanism of release of acid phosphatase (APase) from lysosomal membranes into the lysosomal matrix. When rat liver lysosomal membranes were incubated at various pH values with APase-free tritosomal contents prepared by the treatment of tritosomal contents with anti-APase IgG Sepharose, 86% of the APase activity in the lysosomal membranes became soluble at pH 5.0. Immunoblots revealed that the membrane-bound APase (67 kDa) was released in a 64 kDa form, and the 67 and 64 kDa forms were converted to 45 and 41 kDa forms by Endo F treatment, respectively, thereby indicating that the release of APase from the lysosomal membranes was accompanied by a limited proteolysis involving loss of a 4 kDa fragment. The release of APase was strongly inhibited by pepstatin A, a potent inhibitor of aspartyl protease, but other inhibitors such as leupeptin, antipain, Ep-475 and 1,10-phenanthroline showed no effect. The release of APase did not occur when the lysosomal membranes were incubated with the tritosomal contents free of APase and cathepsin D, prepared by treatment of the APase-free tritosomal contents with anti-cathepsin D IgG Sepharose. The purified lysosomal cathepsin D released 71% of the APase activity from the lysosomal membranes and the released APase had a molecular mass of 65 kDa, that is, larger than the enzyme released by using the APase-free tritosomal contents. Endo F converted the 65 kDa form to the 43 kDa form.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of a major glycoprotein in rat liver lysosomal membrane.

A major lysosomal membrane glycoprotein (LGP107) which has an apparent molecular weight (Mr) of 107 kilodaltons (kDa) was purified from rat liver by a simple method with a yield of 1 mg/87 g wet weight of liver. The purification procedures include; preparation of tritosomal membranes of triton-filled lysosomes (tritosomes), extraction of tritosomal membranes by Lubrol PX, wheat germ agglutinin (WGA)-Sepharose affinity chromatography, and monoclonal antibody-Sepharose affinity chromatography. The quantitative immunoblot analysis indicated that LGP107 represents 6.2% of the total protein of tritosomal membranes. The isoelectric point of the purified glycoprotein was 2.7, and it moved toward neutral pH after sialidase treatment, with its molecular weight decreased by about 10 kDa. LGP107 contained 52% carbohydrates, and the carbohydrate moiety was compared of Fuc, Man, Gal, GlcNAc and sialic acid in a molar ratio of 7.2:68.2:40.6:63.0:32.3, respectively, indicating that LGP107 was highly glycosylated with N-linked complex-type olgosaccharide chains. Out of the N-linked glycans released from the glycoprotein by hydrazinolysis/N-reacetylation, about 70% was sialylated. Anion exchange and reverse-phase high performance liquid chromatography analysis on the structure of N-glycans revealed that a disialyl biantennary form is a major component in the oligosaccharide chains of LGP107.

Isolation and sequencing of a cDNA clone encoding 96 kDa sialoglycoprotein in rat liver lysosomal membranes

We isolated and sequenced LGP 96, a cDNA clone corresponding to the entire coding sequence of the rat liver lysosomal membrane sialoglycoprotein with an apparent Mr of 96 K, LGP 96. The deduced amino acid sequence indicates that LGP 96 consists of 411 amino acid residues (Mr 45,163) and the 26 NH 2-terminal residues presumably constitute a cleavable signal peptide. The major portion of LGP 96 resides on the luminal side of the lysosome and bears a large number of N-linked heavily sialylated complex type carbohydrate chains, giving the mature molecule of 96 kDa. The protein has 17 potential N-glycosylation sites and 32.1 and 65.3% sequence similarities in amino acid to LGP 107 and human lamp-2,respectively. The glycosylation sites are clustered into two domains separated by a hinge-like structure enriched with proline and threonine. LGP 96 possesses one putative transmembrane domain consisting of 24 hydrophobic amino acids near the COOH-terminus and contains a short cytoplasmic segment constituting 12 amino acid residues at the COOH-terminal end. Comparison of LGP 96 and recently cloned lysosomal membrane glycoprotein sequences reveals strong similarity in the putative transmembrane domain and cytoplasmic tail. It is very likely that these portions are important for the targeting of molecules to lysosomes. A comparison of LGP 96 and LGP 107 showed numberous structural similarities.

Isolation and sequencing of a cDNA clone encoding acid phosphatase in rat liver lysosomes

A full length cDNA for acid phosphatase in rat liver lysosomes was isolated and sequenced. The predicted amino acid sequence comprises 423 residues (48,332 Da). A putative signal peptide of 30 residues is followed by the NH 2-terminal sequence of lysosomal acid phosphatase (45,096 Da). The deduced NH 2-terminal 18-residue sequence is identical with that determined directly for acid phosphatases purified from the rat liver lysosomal membranes. The primary structure deduced for acid phosphatase contains 9 potential N -glycosylation sites and a hydrophobic region which could function as a transmembrane domain. It exhibits 89% and 67% sequence similarities in amino acids and nucleic acids, respectively, to human lysosomal acid phosphatase. The amino acid sequence of the putative transmembrane segment shows a complete similarity to that of the human enzyme. Northern blot hybridization analysis identified a single species of acid phosphatase mRNA (2.2 kbp in length) in rat liver.

Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis.

A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn-78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with alpha-2-macroglobulin at the low temperature. Thus, the process of lysosomal maturation appears to involve the progressive delivery of lysosomal enzymes to various types of endosomes that may have already received some of the lysosomal membrane proteins. Although endolyn-78 would be one of the proteins added early to endosomes, other lysosomal membrane proteins may be added only to multivesicular endosomes that represent very advanced stages of maturation.

Acid phosphatase in rat liver lysosomal membranes: purification and characterization.

Acid phosphatase associated with rat liver lysosomal membranes (M-APase) was purified about 4,200-fold over the homogenate with 10% recovery to apparent homogeneity, as determined from the pattern on polyacrylamide gel electrophoresis in the presence of SDS. The purification procedure included; preparation of lysosomal membranes, solubilization of the membranes with 1% Triton X-100, immunoaffinity chromatography, and gel filtration with FPLC equipped with a Sephacryl S-300HR column. The molecular weight, estimated by gel filtration through TSK SW 3000G, was approximately 320K and SDS gel electrophoresis showed that the enzyme is composed of four identical subunits with an apparent molecular weight of 67K. The enzyme contains about 24.3% carbohydrate consisting of mannose, galactose, fucose, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylneuraminic acid in a molar ratio of 38:20:5:36:4:11, respectively. In addition, three soluble forms of acid phosphatase (C-APase I, II, and III) in lysosomal contents were separated from rat liver lysosomal contents with DEAE-Sephacel. These three enzymes were also purified using immunoaffinity chromatography followed by gel filtration. C-APase I, II, III, and M-APase have isoelectric points of 7.7-8.2, 6.6-7.0, 5.7-6.7, and 3.4-3.8, respectively. All four APases are sensitive to endo-beta-N-acetylglucosaminidase H. However, only C-APase III and M-APase are digestible with neuraminidase. Susceptibility of M-APase to neuraminidase in intact tritosomes was examined to study the topography of M-APase in tritosomal membranes. Neuraminidase susceptibility of M-APase was not observed in the intact tritosomes until the tritosomes had been disrupted by osmotic shock.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and sequencing of a cDNA clone encoding 107 kDa sialoglycoprotein in rat liver lysosomal membranes

A cDNA for 107 kDa sialoglycoprotein (LGP 107), the major protein component of rat liver lysosomal membranes, was isolated and sequenced. The 1.8 kbp cDNA contained an open reading frame encoding a polypeptide consisting of 386 amino acid residues ( M r 41914). The deduced NH 2-terminal 10-residue sequence is identical with that determined for purified LGP 107. The primary structure deduced for LGP 107 contains 20 potential N-glycosylation sites and exhibits 82.5, 43 and 60% sequence similarities to mouse LAMP-1, chicken LEP 100, and a 120-kDa human lysosomal glycoprotein, respectively. Among these lysosomal glycoproteins, the amino acid sequence of the putative transmembrane segment is highly conserved. Northern blot hybridization analysis identified a single species of LGP 107 mRNA (2.1 kbp in length) in rat liver, kidney, brain, lung, spleen, heart and pancreas, although its level in pancreas was very low.

5'-Nucleotidase in rat liver lysosomal membranes is anchored via glycosyl-phosphatidylinositol.

We have previously demonstrated that 5'-nucleotidase, known as a plasma membrane enzyme, is also distributed both in rat liver tritosomal membranes and contents (J. Biochem. 101, 1077-1085, 1987). When the lysosomal membranes isolated from rat livers were incubated with phosphatidylinositol-specific phospholipase C purified from B. thuringiensis, about 70% of 5'-nucleotidase activity was released from the membranes. Judging from the result by phase separation with Triton X-114, the enzyme solubilized by the phospholipase C digestion showed a hydrophilic nature such as that of the tritosomal contents. Immunoblot analysis showed that the molecular weight of 5'-nucleotidase released from the lysosomal membranes by the phospholipase C digestion was almost identical with that of the enzymes from the Tritosomal contents. The above results showed that the phosphatidylinositol-specific phospholipase C-like enzyme in the lysosomes may be responsible for the conversion of the lysosomal membrane-bound 5'-nucleotidase to the soluble form present in the lysosomal matrix.

A rat liver lysosomal membrane flavin-adenine dinucleotide phosphohydrolase: Purification and characterization

An enzyme hydrolyzing flavin-adenine dinucleotide (FAD) to flavin mononucleotide and AMP was identified and purified from rat liver lysosomal (Tritosomal) membranes. The purified enzyme showed a single band on silver-stained denaturing gels with an apparent M r 70,000. Periodate-Schiff staining after denaturing gel electrophoresis of whole membrane preparations revealed that this enzyme is one of the major glycoproteins in lysosomal membranes. FAD appeared to be the preferred substrate for the purified enzyme; equivalent concentrations of NAD or CoA were hydrolyzed at about one-half of the FAD rate. Negligible activity (⩽16%) was noted with ATP, TTP, ADP, AMP, FMN, pyrophosphate, or p-nitrophenylphosphate. The enzyme was inhibited by EDTA or dithiothreitol. It was stimulated by Zn, and was not affected by Ca or Mg ions, nor by p-chloromercuribenzoate. The pH optimum for FAD hydrolysis was 8.5–9 with an apparent K m of 0.125 m m. Antibodies prepared against the purified enzyme partially (50%) inhibited FAD phosphohydrolase activity in lysosomal membrane preparations but had no effect on the soluble lysosomal acid pyrophosphatase known to hydrolyze FAD. This enzyme could not be detected immunochemically in preparations of microsomes, Golgi, plasma membranes, mitochondrial membranes, or the soluble lysosomal fraction, suggesting that the enzyme is different from either soluble lysosomal acid pyrophosphatase or other FAD hydrolyzing activities in the liver cell.

Isolation of highly purified rat liver lysosomal membranes using two percoll gradients

Normal rat liver lysosomal membranes in the form of membrane vesicles have been purified using Percoll density gradient centrifugation. Lysosomes (density = 1.111) were purified approximately 63 ± 12-fold (mean ± standard deviation, n = 5) using a gradient of Percoll made isotonic with sucrose and buffered to pH 7.0. These lysosomes were then exposed to 10 m m methionine methyl ester, pH 7.0, the uptake of which resulted in swelling and breakage of the lysosomes with subsequent vesicle formation. These vesicles (density = 1.056) were further separated from residual mitochondrial and plasma membrane enzyme activities using a second Percoll density gradient. Marker enzyme analysis and electron microscopy indicated that the lysosomal membranes were essentially free of both β-hexosaminidase, a soluble lysosomal enzyme, and contaminating organelles. The specific activity of lysosomal ATPase in the lysosomal membranes was fourfold greater than in the intact lysosomes.