Assay and purification of a solubilized membrane receptor that binds the lysosomal enzyme alpha-L-iduronidase.
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Adsorption↗
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The synthesis of mannose 1-phosphate in brain.
The interconversion of mannose-6-P and mannose-1-P in brain has been shown to be catalyzed by a distinct enzyme. The enzyme has been separated from most of the phosphoglucomutase activity of the brain. The residual phosphoglucomutase activity (less than 1%) may be associated with phosphomannomutase itself. Mannose-1,6-P2 or glucose-1,6-P2 is required for the reaction as well as a divalent cation (Mg2+ greater than Co2+ greater than Ni2+ greater than Mn2+). Glucose-1-P, glucose-6-P, and 2-deoxyglucose-6-P are also substrates or inhibitors. Other phosphorylated sugars tested, glucosamine-6-P, N-acetylglucosamine-6-P, galactose-6-P, fructose-6-P, ribose-5-P, and arabinose-5-P, do not affect the rate of the reaction when assayed in the presence of mannose-6-32P.
Animals↗
Tunicamycin inhibits proteoglycan synthesis in rat ovarian granulosa cells in culture.
The effects of tunicamycin, an inhibitor of N-linked oligosaccharide biosynthesis, on the synthesis and turnover of proteoglycans were investigated in rat ovarian granulosa cell cultures. The synthesis of proteoglycans was inhibited (40% of the control at 1.6 micrograms/ml tunicamycin) disproportionately to that of general protein synthesis measured by [3H]serine incorporation (80% of control). Proteoglycans synthesized in the presence of tunicamycin lacked N-linked oligosaccharides but contained apparently normal O-linked oligosaccharides. The dermatan sulfate and heparan sulfate chains of the proteoglycans had the same hydrodynamic size as control when analyzed by Sepharose 6B chromatography. However, the disulfated disaccharide content of the dermatan sulfate chains was reduced by tunicamycin in a dose-dependent manner, implying that the N-linked oligosaccharides may be involved in the function of a sulfotransferase which is responsible for sulfation of the iduronic acid residues. When [35S]sulfate and [3H]glucosamine were used as labeling precursors, the ratio of 35S/3H in chondroitin 4-sulfate was reduced to approximately 50% of the control by tunicamycin, indicating that the drug reduced the supply of endogenous sugar to the UDP-N-acetylhexosamine pool. Neither transport of proteoglycans from Golgi to the cell surface nor their turnover from the cell surface (release into the medium, or internalization and subsequent intracellular degradation) was affected by the drug. Addition of mannose 6-phosphate to the culture medium did not alter the proteoglycan turnover. When granulosa cells were treated with cycloheximide, completion of proteoglycan diminished with a t1/2 of approximately 12 min, indicating the time required for depleting the core protein precursor pool. The glycosaminoglycan synthesizing capacity measured by the addition of p-nitrophenyl-beta-xyloside, however, lasted longer (t1/2 of approximately 40 min). Tunicamycin decreased the core protein precursor pool size in parallel to decreased proteoglycan synthesis, both of which were significantly greater than the inhibition of general protein synthesis. This suggests two possibilities: tunicamycin specifically inhibited the synthesis of proteoglycan core protein, or more likely a proportion of the synthesized core protein precursor (approximately 50%) did not become accessible for post-translational modifications, and was possibly routed for premature degradation.
Animals↗
Characterization of polyisoprenyl phosphate phosphatase activity in rat liver.
The polyisoprenyl phosphate dephosphorylating activity of rat liver has been investigated with regard to substrate specificity, subcellular distribution, and transmembrane orientation. Total liver microsomes were employed as a source of enzymatic activity against a variety of 32P-labeled substrates. Susceptibility to dephosphorylation followed the order solanesyl phosphate greater than alpha-cis-polyprenyl 19-phosphate = alpha-trans-polyprenyl 19-phosphate = dihydrosolanesyl phosphate greater than (S)-dolichyl 19-phosphate = (R)-dolichyl 19-phosphate = (R,S)-dolichyl 11-phosphate. There appeared to be no major effect of chain length from 11 to 20 isoprenes. Data obtained from inhibition studies using solanesyl [32P]phosphate as substrate were consistent with the substrate specificity studies and suggested that a single activity is responsible. With dolichyl [32P]phosphate as substrate, the phosphatase specific activity of the subcellular fractions prepared from rat liver was found to follow the sequence Golgi = smooth endoplasmic reticulum greater than plasma membrane greater than lysosomes = rough endoplasmic reticulum greater than nuclei greater than mitochondria. Transmembrane topography studies, using enzyme latency as a criterion, were consistent with an orientation of the active site facing the cytoplasm.
Animals↗
Phenobarbital-induced alterations in the activities of the transport and hydrolytic components of the glucose-6-phosphatase system in smooth and rough subfractions of the rat hepatic endoplasmic reticulum.
We have examined the influence of the phenobarbital-induced proliferation of the hepatic endoplasmic reticulum (ER) on the activities of the components of the glucose-6-phosphatase system, i.e., the enzyme, the glucose-6-P translocase (T1), and the phosphate translocase (T2). Young male rats were injected ip twice daily for 4 days with 4 mg/100 g body wt of phenobarbital (PB) or an equivalent volume of saline solution. On the fifth day, the rats were killed and smooth (SER) and rough (RER) fractions of the ER were isolated from liver homogenates. Kinetic constants for glucose-6-P hydrolysis by the system and enzyme were determined and used to calculate the kinetic constants for glucose-6-P transport. T2 activity was approximated by assaying the pyrophosphatase activity at pH 6.0 in intact microsomes. Three times more SER protein was recovered from livers of PB-treated rats. PB-treatment did not alter total liver enzyme activity, but total liver T1 activity was decreased to 59% of the control value. Maximal specific activities of the system, enzyme and T1 were all reduced by PB treatment to 44% of control values in the RER and to 68% of control values in the SER. PB treatment reduced the apparent activity of T2 in RER and SER to 35 and 49% of the respective control values. In the SER from both groups of rats, T1 activity or apparent T2 activity divided by enzyme activity was about 55% of the corresponding ratio in the RER. Our analysis of these data suggests that the lower activities of T1 and T2 in the smooth ER are the results of suppression by some intrinsic component localized in the smooth membrane. Accordingly, the reduction in total liver T1 activity and, therefore, system activity in PB-treated rats reflects the redistribution of the glucose-6-P translocase from the RER to the more abundant SER membrane where it is less active. The possibility is discussed that a higher cholesterol content within the SER membrane is responsible for the lower transport activities.
Animals↗
Maturation of asparagine-linked oligosaccharides in Dictyostelium discoideum analyzed with modification-specific probes.
Lysosomal enzymes in Dictyostelium discoideum contain high mannose oligosaccharides that contain mannose 6-phosphate and several unusual structures. The synthesis and distribution of these post-translational modifications were studied using probes for different carbohydrate groups. These probes include lectin-like antibodies directed to two distinct sulfated and one nonsulfated N-linked determinants, the lectin Con A, and the mammalian 215-kDa phosphomannosyl receptor. Only Con A binds to newly synthesized alpha-mannosidase present in the rough endoplasmic reticulum. The other modifications are acquired at different rates and are first detected on protein in light density Golgi-like membranes. Mutations which prevent protein transport to Golgi membranes block synthesis of these moieties, but inhibitors which prevent later transport steps have no effect. The majority of modified proteins are in lysosomes but significant amounts are delivered to nonlysosomal destinations. Different lysosomal proteins contain unequal amounts of each modification.
Animals↗
Adsorptive endocytosis of lysosomal enzymes by human fibroblasts: presence of two different functional systems that deliver an acid hydrolase to lysosomes.
Endocytosis of human spleen beta-glucuronidase by human fibroblasts can be completely impaired by the competitive inhibitor mannose 6-phosphate or by pretreatment with acid phosphatase or endoglycosidases H or F. However, endocytosis of bovine spleen and liver beta-glucuronidase is partially impaired by the same treatments, suggesting that the bovine enzyme contains two endocytosis recognition markers located in separate enzyme domains. The mannose 6-phosphate recognition marker seems to be responsible for approximately 23% of the bovine enzyme endocytosis. The existence of two lysosomal endocytosis systems in human fibroblasts is supported by the following facts: (a) the rate of endocytosis of mannose 6-phosphate-containing human beta-glucuronidase was not affected by the presence of high levels of the bovine enzyme (which has only the other marker). (b) Anti-215K mannose 6-phosphate receptor antibodies selectively impair the endocytosis of the beta-glucuronidase containing mannose 6-phosphate. (c) Weak bases exert a differential effect on human and bovine endocytosis. beta-Glucuronidase internalized by either system is targeted to secondary lysosomes of human beta-glucuronidase-deficient fibroblasts, where it is able to degrade accumulated glycosaminoglycans. These results suggest that human fibroblasts have two different and independent endocytic systems for targeting of acid hydrolases to lysosomes.
Ammonium Chloride↗
The aggregation and dissociation properties of a low molecular weight mannose 6-phosphate receptor from bovine testis.
The aggregation state of low molecular weight mannose 6-phosphate receptor from bovine testis was determined in membrane preparations and in purified soluble preparations. The effect of aggregation on binding of the receptor to immobilized pentamannose 6-phosphate was also examined. Nonreducing SDS-PAGE followed by immunoblotting revealed that interchain disulfide bonds exist in detergent-solubilized and purified receptor preparations, but not in membrane-associated receptor. Reduction of the receptor with dithiothreitol abolished its ligand binding activity and drastically altered its ability to bind antibodies. The results of receptor crosslinking and molecular sieving chromatography studies suggest that the receptor exists in membranes as a noncovalently linked dimer and in solution as oligomeric forms, largely as a tetramer. The formation of the tetramer is affected by the concentration of the receptor, but not by its solubilization from membranes with detergent, nor by the presence of mannose 6-phosphate. Mono-, di-, and tetramer forms of 125I-labeled receptor were separated by molecular sieving chromatography and examined for their ability to bind to immobilized ligand, agarose-pentamannose-phosphate. The order of binding observed was tetramer greater than dimer greater than monomer. Binding of the monomer and dimer to immobilized ligand was dependent on the presence of divalent cations while the tetramer had little requirement for divalent cations.
Animals↗
Specific lipids enhance the activity of UDP-GlcNAc: dolichol phosphate GlcNAc-1-phosphate transferase in rat liver endoplasmic reticulum membrane vesicles.
The rate of the reaction catalyzed by UDP-N-acetylglucosamine (GlcNAc):dolichol phosphate GlcNAc-1-phosphate transferase in rat liver endoplasmic reticulum vesicles was shown to be influenced by particular lipids. Utilizing in vitro assay conditions where the membrane vesicles retained latency of glucose-6-phosphatase activity, the addition of phosphatidylethanolamine, cardiolipin, or monogalactosyldiglyceride resulted in severalfold increases in the rate of dolichol pyrophosphate N-acetylglucosamine synthesis. Other phospholipids were not stimulatory. These rates were dependent on the concentrations of the exogenous lipids and of the substrate dolichol phosphate. In the presence of cardiolipin, the membrane-bound enzyme became more susceptible to inactivation by protease K and to inhibition by tunicamycin. Titration of cardiolipin-containing endoplasmic reticulum vesicles with adriamycin indicated that the majority of the cardiolipin was exposed on the outer surface. These results suggest that the particular lipids altered membrane structure in a way that allowed further access of the enzyme to substrate, inhibitor, and other molecules. Lipids observed in these studies to be stimulatory are known to exist in the macromolecular hexagonal phase and may therefore be affecting the GlcNAc-1-phosphate transferase by locally disrupting the bilayer structure of the membrane. As other dolichol-utilizing enzymes have been previously observed by other investigators to be similarly influenced by such lipids, the effects may be common to enzymes of the dolichol cycle.
Animals↗
Biosynthesis and endocytosis of lysosomal enzymes in human colon carcinoma SW 1116 cells: impaired internalization of plasma membrane-associated cation-independent mannose 6-phosphate receptor.
The human colon adenocarcinoma cell lines SW 948, SW 1116, and SW 1222 were tested for their ability to sort and internalize lysosomal enzymes. The biosynthesis of the lysosomal enzymes cathepsin B, arylsulfatase A, and beta-hexosaminidase in these cell lines exhibits no significant differences to that in human fibroblasts. The intracellular targeting of newly synthesized hydrolases to the lysosomes relies in colon carcinoma cells on the mannose 6-phosphate receptor system. Both the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor are expressed in all colon carcinoma cell lines investigated. Endocytosis of lysosomal enzymes via mannose 6-phosphate receptors is reduced in colon carcinoma cells as compared with human fibroblasts. SW 1116 cells were shown to be deficient in receptor-mediated endocytosis of mannose 6-phosphate containing ligands. Ligands of other endocytic receptors as well as the fluid-phase marker horseradish peroxidase were internalized at normal rates. While antibodies against CI-MPR bind to the surface of SW 1116 cells, these antibodies cannot be internalized. These data suggest that the cycling of CI-MPR is specifically impaired in SW 1116 cells.
Carcinoma↗
Biosynthesis of lipophosphoglycan from Leishmania donovani: characterization of mannosylphosphate transfer in vitro.
Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes and is composed of a capped polymer of repeating PO4-6Gal(beta 1,4)Man alpha 1 disaccharide units linked via a phosphosaccharide core to a lyso-1-O-alkylphosphatidylinositol anchor. An exogenous acceptor composed of the glycolipid anchor portion of LPG was shown to stimulate the enzymatic synthesis of the repeating phosphorylated disaccharide units of LPG in a cell-free system. Using the exogenous acceptor, GDP-[3H]Man, [beta-32P]GDP-Man, and unlabeled UDP-Gal as substrates, membrane preparations from an LPG-defective mutant of L. donovani that lacks endogenous acceptors catalyzed the incorporation of the doubly labeled mannosylphosphate unit into a product that exhibited the chemical and chromatographic characteristics of LPG. Analysis of fragments generated by mild acid hydrolysis of the radiolabeled product indicated that [3H]mannose-1-[32P]PO4 had been transferred from the dual-labeled sugar nucleotide. These results are consistent with the proposal that the repeating units of the L. donovani LPG are synthesized by the alternating transfer of mannose 1-phosphate and galactose from their respective nucleotide donors.
Animals↗
Phosphatidylcholine mobility in liver microsomal membranes.
Purified phosphatidylcholine exchange protein from bovine liver was used to exchange rat liver microsomal phosphatidylcholine for egg phosphatidylcholine. It was found that at 25 and 37 degrees C rat liver microsomal phosphatidylcholine was completely and rapidly available for replacement by egg phosphatidylcholine. In contrast, phosphatidylcholine in vesicles prepared from total microsomal lipids could only be exchanged for about 60%. At 8 and 0 degrees C complex exchange kinetics were observed for phosphatidylcholine in rat liver microsomes. The exchange process had neither effect on the permeability of the microsomal membrane to mannose 6-phosphate, nor on the permeability of the phosphatidylcholine vesicles to neodymium (III) cations. Purified phospholipase A2 from Naja naja could hydrolyze some 55-60% of microsomal phosphatidylcholine at 0 degrees C, but 70-80% at 37 degrees C. Microsomal phosphatidylcholine, remaining after phospholipase treatment at 37 degrees C, could be exchanged for egg phosphatidylcholine at 37 degrees C, but at a slower rate than with intact microsomes. Microsomal phosphatidylcholine remaining after phospholipase treatment at 0 and 37 degrees C had a lower content of arachidonic acid than the original phosphatidylcholine. These results are discussed with respect to the localization and transmembrane movement of phosphatidylcholine in liver microsomes.
Animals↗
Enzyme asymmetry in hepatic microsomal vesicles. Criteria for localization of lumenal enzymes with proteases.
Chymotrypsin inactivation of lysophosphatidic acid acyltransferase activity in detergent-disrupted rat liver microsomes, but not in intact microsomes, falsely indicated a lumenal location for the enzyme. Inhibition by several other proteases in the absence of detergent showed that lysophosphatidic acid acyltransferase activity is located on the cytoplasmic surface of microsomes. Chymotrypsin inactivation did not occur in vesicles disrupted by nitrogen cavitation unless deoxycholate was present, suggesting that deoxycholate exposes a cryptic chymotrypsin cleavage site. Criteria for localization of lumenal microsomal enzymes should include studies using several proteases and/or employ more than one method of microsomal disruption.
Acyltransferases↗
Short-chain aliphatic alcohols increase rat-liver microsomal membrane fluidity and affect the activities of some microsomal membrane-bound enzymes.
n-Butyl and isoamyl alcohols decrease the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and enhance the efficiency of pyrene excimer formation when these probes are incorporated in rat-liver microsomal membrane, suggesting an increase in rotational and translational mobilities. Neither alcohol modifies NADH-ferricyanide reductase activity but both increase NADH-cytochrome c reductase activity. This was interpreted as an increase in the rate of lateral diffusion of the proteins cytochrome b5 and cytochrome b5 reductase as a consequence of the enhanced membrane lipid phase fluidity. Microsomal delta 9 and delta 6 desaturase activities in the presence of isoamyl alcohol were also studied. This alcohol decreases delta 9 desaturation when it is measured at a low substrate concentration (13 microM palmitic acid), but it is not modified when it is measured at a high substrate concentration (66 microM palmitic acid). delta 6 desaturation is diminished by isoamyl alcohol when it is measured with both 13 microM and 66 microM linoleic acid. The influence of isoamyl alcohol on the glucose-6-phosphatase system activity was also studied. In non-detergent-treated microsomes, isoamyl alcohol enhances glucose-6-phosphatase activity. However, if microsomes are previously treated with 0.1% Triton X-100 isoamyl alcohol does not modify this activity. The enhancement of the glucose 6-phosphate transport rate is not due to membrane permeability barrier disruption, since isoamyl alcohol does not modify mannose-6-phosphohydrolase latency. This would suggest that an increase in membrane lipid phase fluidity specifically activates glucose 6-phosphate transport across the membrane.
Animals↗
Comparison of type I hexokinases from pig heart and kinetic evaluation of the effects of inhibitors.
Type I hexokinase (ATP:D-hexose 6-phospotransferase, EC 2.7.1.1) of porcine heart exists in two chromatographically distinct forms. These do not differ significantly in size, electrophoretic mobility at pH 8.6 or kinetic properties. Both forms obey a sequential mechanism and are potently inhibited by glucose 6-phosphate. In contrast to observations of type I hexokinase from brain, inhibition by glucose 6-phosphate is not relieved by inorganic phosphate. Under most conditions, low concentrations of phosphate (less than 10 mM) have little effect on the kinetic behaviour of the enzyme but at higher concentrations this ligand is an inhibitor. Mannose 6-phosphate inhibits in a manner analogous to glucose 6-phosphate but the Ki is much greater. In view of the similarity of the kinetic parameters governing phosphorylation of mannose and glucose, this difference in affinity for the inhibitor site is seen as consistent with the existence of a separate regulatory site on the enzyme. MgADP inhibits hexokinase but behaves as a normal product inhibitor and inhibition is competitive with respect to MgATP and non-competitive with respect to glucose.
Adenine Nucleotides↗
13-cis-retinoic acid stimulates in vitro mannose 6-phosphate hydrolysis and inhibits retinol esterification and benzo[a]pyrene hydroxylation by rat-liver microsomes.
13-cis-Retinoic acid, a drug used at high doses in the treatment of recalcitrant acne, increased the permeability of rat-liver microsomal membranes to mannose 6-phosphate in vitro, as indicated by an increase in mannose-6-phosphatase activity. At the same concentrations, four other amphiphiles, including all-trans-retinoic acid, were much less effective. 13-cis-Retinoic acid also inhibited retinol esterification and benzo[a]pyrene hydroxylation in microsome preparations in vitro. Although the molecular mechanism and the reversibility of these effects have not yet been studied, the interaction of 13-cis-retinoic acid with cell membranes may well be involved in both its therapeutic and toxic manifestations.
Animals↗
Mannose-6-p and mannose-1-p in rat brain, kidney and liver.
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Aging↗
Binding of lysosomal hydrolases by the membranes of rat liver lysosomes: evidence for phosphomannosyl recognition markers.
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Animals↗