Physicochemical characterization of soluble complexes of human serum low density lipoprotein with heparin

Soluble complexes of low density lipoproteins (LDL) with heparin (HEP) and chondroitin sulphate (CS) in the absence of divalent cations have been studied by means of a micro-rolling-ball ciscometer to obtain information about molecular size and structure of the aggregates. The rheological results were supplied and corroborated by light scattering measurements, electrophoresis and analytical ultracentrifugation. Molar binding ratios were measured using gel filtration assays and ultracentrifugation. At a certain weight ratio of LD To HEP the solutions showed a significant viscosity maximum. At this weight ratio 2–3 LDL particles are held together 1–2 HEP chains. The hydrodynamic radius R_H of this complex is about 16.3 ± 0.90 nm and the rotational diffusion constant is > 7.1 × 10³ s^?1. With excess HEP the radius of the aggregates is almost the same as that of free LDL (R_H = 11.9 ± 0.70 nm). Quantitative binding studies revealed that in this case 1–2 HEP molecules are bound to a single LDL particle. An interaction was also found with CS and LDL but complex formation in this case showed different characteristics. Very low density lipoproteins (VLDL) and high density lipoproteins (HDL) gave no rheologically effective aggregates with HEP.     

Follicular fluid glycosaminoglycans inhibit degradation of low-density lipoproteins and progesterone production by porcine granulosa cells

Porcine granulosa cells were incubated with commercially available glycosaminoglycans (GAGs) or GAGs purified from porcine follicular fluid to evaluate the effects of GAGs on degradation of low-density lipoproteins (LDL) and progesterone production. Commercially available heparin and chondroitin sulfates (CS) as well as follicular CS and heparan sulfate (HS) inhibited degradation of LDL in dose-dependent manners. Doses of follicular CS and HS required to inhibit 50% of the LDL degradation corresponded to concentrations found in follicular fluid (less than 1 mg/ml). Progesterone production was also inhibited in a dose-dependent fashion by follicular GAGs at concentrations found in follicles. The ability of the follicular GAGs to inhibit degradation of LDL could represent a mechanism by which the utilization of LDL-derived sterol is temporarily restricted following permeabilization of the ovulatory follicle. Follicular GAGs may also modulate utilization of apoprotein E-containing high-density lipoproteins in unruptured follicles.     

High-field 1H and 31P NMR studies on the binding of the anticancer agent mitoxantrone to d[CpGpApTpCpG]2

A high-field 1H and 31P-NMR study of the oligomer d[CpGpApTpCpG]2 was carried out in H2O and water signal suppression was employed in all 1H NMR acquisitions. Particular attention was given to imino proton and 31P assignments. Two dimensional 31P-1H shift correlation contours were particularly useful in 31P assignments and confirming previous 1H assignments. Titrimetric addition of aliquots of the anticancer agent mitoxantrone resulted in selective and progressive chemical shifts with critical changes at stoichiometries of 1:1 and 2:1 drug to DNA ratios. The results indicate ultimate intercalative binding of the drug at both C.G. termini of the oligomer in accord with the previously determined C.G. preference and with non-nearest neighbor intercalation.     

Sulfation of chondroitin sulfate secreted by baby hamster kidney cells and their polyoma virus-transformed counterparts

Sulfation of glycosaminoglycans (GAGs) secreted by baby hamster kidney (BHK) cells and the polyoma virus-transformants (PY-BHK) was investigated. It has been reported that chondroitin sulfate (CS) of cell membranes from PY-BHK cells is undersulfated compared to that from BHK cells (Cancer Res. 43, 2712-2717, 1983). In the first series of experiments of the present study, cells were incubated with [3H]glucosamine and [35S]sulfate, and GAGs isolated from the culture medium were examined. GAG composition was comparable between the BHK and PY-BHK cultures. Disaccharide analysis of the chondroitinase ACII digests of the hyaluronate lyase-resistant materials showed a high proportion (68% for BHK and 47% for PY-BHK) of delta Di-0S, with delta Di-4S (32% for BHK and 53% for PY-BHK) as the major sulfated disaccharide on the basis of 3H-radioactivities. The beta-D-xyloside treatment did not alter the degree of undersulfation of the CS of either culture. In the second series of experiments, disaccharide analysis of the chondroitinase ABC digests of unlabeled GAGs demonstrated similar disaccharide composition for the two cell types. The BHK and PY-BHK preparations showed 28 and 17% (mol percent) of delta Di-0S, 58 and 72% of delta Di-4S, and 14 and 11% of delta Di-6S, respectively. These results indicate a considerable degree of undersulfation of secretory CS from both cells, and a slightly higher degree, if any, of under-sulfation of secretory CS from BHK cells if compared between the two cell types, which is in contrast to the results reported for membrane CS.     

Effects of wheat germ agglutinin and concanavalin A on the accumulation of glycosaminoglycans in pericellular matrix of human dermal fibroblasts. A comparison with insulin.

The effect of insulin, wheat germ agglutinin (WGA), peanut agglutinin (PNA) and concanavalin A (ConA) on [3H]glucosamine incorporation into pericellular glycosaminoglycans (GAGs) was investigated in two lines of cultured human dermal fibroblasts. Insulin and WGA stimulated [3H]glucosamine incorporation into hyaluronic acid (HA) and heparan sulphate (HS) without any alteration of chondroitin sulphate (CS) and dermatan sulphate (DS) contents. ConA increased [3H]glucosamine incorporation into HS, CS and DS, but had no effect on [3Hglucosamine incorporation into HA. PNA affected neither the content, nor the composition of GAGs. In contrast to PNA, ConA and WGA stimulated glycolysis and demonstrated an evident antiproliferative effect on dermal fibroblasts. Thus, both the insulin-like action of WGA and ConA on cultured dermal fibroblasts and the differences between the effects of lectins on modulation of GAGs synthesis appear to be determined by their chemical structure.     

Self-association and unique DNA binding properties of the anti-cancer agent TAS-103, a dual inhibitor of topoisomerases I and II

The objective of our study was to investigate the self-association and DNA-binding properties of the DNA topoisomerases I (Topo I) and II (Topo II) dual inhibitor: 6-[[2-(dimethylamino)ethyl]amino]-3-hydroxy-7H-indeno[2,1-c]quinoline-7-one dihydrochloride (TAS-103), by means of 1H-NMR and 31P-NMR spectroscopy, structure computation techniques, thermal melting study, and UV-Visible spectroscopy. In aqueous solution, all chemical shifts of TAS-103 underwent upfield shifts depending with an increase in concentration. The two-dimensional (2D)-NMR spectra and structure computations indicated that TAS-103 self-associated through pi-pi stacking and hydrophobic interactions of the aromatic chromophores. Thermal melting indicated that the binding of TAS-103 to DNA with a potency equal to that of ethidium bromide (EtBr). The UV-Visible spectra of TAS-103 titrated by several DNA exhibited hypochromic and hypsochromic effects. The 31P-NMR spectrum of the 6:1 TAS-103/d(CGCGAATTCGCG)(2) complex showed two broadening signals. 2D-NMR spectra of the 1:1 TAS-103/d(CGCGAATTCGCG)(2) complex indicated that the chemical shift differences of the DNA are very small. However, those of the terminal region are relatively large. The chemical shift differences of TAS-103 showed that the proton resonances except H2 underwent downfield shifts. From these observations, we conclude that TAS-103 binds to DNA by two modes. The major binding mode is on the surface (outside binding) and the minor binding mode by intercalation.     

Pulsatile secretion of bioactive luteinizing hormone in adult male rhesus macaques: acute and chronic effects of orchidectomy

Glycosaminoglycans (GAGs) were purified from bovine follicular fluid, and their effectiveness to compete for heparin-binding sites in granulosa cells was evaluated. The GAGs dermatan sulfate (DS) and heparan sulfate (HS) were purified by anion-exchange high-performance liquid chromatography. Approximately 5 micrograms of protein from suspensions of bovine granulosa cells were incubated with 101 pmoles of [3H]heparin and 0.01-5.0 mg/ml of HS or DS for 2 h at 37 degrees C in 40 mM tris(hydroxymethyl)aminomethane (Tris), pH 7.35. Heparan sulfate obtained from small and medium follicles displaced [3H]heparin in a dose-dependent manner from 0.1 to 5 mg/ml, but HS from large follicles did not displace [3H]heparin. The DS obtained from small, medium, and large follicles displaced [3H]heparin in a dose-dependent manner, and the potency of the DS to displace [3H]heparin increased as the size of the follicles from which the DS was purified increased. Those results were independent of the maturational state of the granulosa cells. In a separate experiment, heparin (17.1% sulfate) was N-desulfated (11.8%), and the desulfated heparin did not displace [3H]heparin. It was concluded that the effectiveness of follicular HS and DS to compete for heparin-binding sites on granulosa cells was dependent on the maturation of the follicle from which the fluid was obtained rather than on the source of granulosa cells. The binding interaction of the GAGs relies, to some extent, on the presence and positions of sulfate moieties.     

Comparable interaction of doxorubicin with various acidic phospholipids results in changes of lipid order and dynamics

We have characterized the interaction of the antitumor drug doxorubicin with model membranes of the anionic phospholipids dioleoylphosphatidic acid (DOPA), dioleoylphosphatidylserine (DOPS), cardiolipin and dioleoylphosphatidylglycerol (DOPG) as compared to the zwitterionic dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE). The saturating binding levels were: 2.4 (DOPA), 1.3 (cardiolipin), 1.5 (DOPS, DOPG) and 0.02 (DOPC) doxorubicin per lipid phosphorus (mol/mol). The half-saturating free drug concentrations were comparable for DOPA, cardiolipin, DOPS and DOPG: 20, 16, 35 and 18 microM, respectively. Doxorubicin fluorescence revealed the simultaneous existence of at least two populations of bound drug in the various anionic phospholipids: (1) fluorescent molecules with chromophores that reside between the lipid molecules and (2) above 0.01-0.02 doxorubicin bound per lipid phosphorus: non-fluorescent drug-stacks that are closer to the aqueous phase than the fluorescent molecules. Small-angle X-ray scattering indicated that doxorubicin can reorganize anionic phospholipid dispersions into closely-packed multilamellar structures. Addition of the drug caused leakage of entrapped 6-carboxyfluorescein. Neither 2H-NMR on [2-2H]serine-labelled DOPS nor 31P-NMR revealed any significant effect of doxorubicin on headgroup conformation, but 2H-NMR on di[11,11-2H2]oleoyl-labelled phospholipids showed that the drug had a strong acyl chain-disordering effect on anionic phospholipids. 2H-NMR relaxation measurements indicated that the drug immobilized the headgroups and acyl chains of anionic phospholipids. The implications of these observations for the cellular activity of the drug are indicated.     

Involvement of Ni protein in the functional coupling of the atrial natriuretic factor (ANF) receptor to adenylate cyclase in rat lung plasma membranes

In the presence of 1 microM atrial natriuretic factor (ANF) and low (0.1 mM) Mg2+ concentrations, the initial rate of binding of [3H]guanosine 5'-[beta, gamma-imido)triphosphate [( 3H]p[NH]ppG) to rat lung plasma membranes was increased twofold to threefold. ANF-dependent stimulation of the initial rate of [3H]p[NH]ppG binding was reduced at high (5 mM) Mg2+ concentrations. Preincubation of membranes with p[NH]ppG (5 min at 37 degrees C) eliminated the ANF-dependent effect on [3H]p[NH]ppG binding whereas ANF-dependent [3H]p[NH]ppG binding was unaffected by similar pretreatment with guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). An increase in ANF concentration from 10 pM to 1 microM caused a 40% decrease in forskolin-stimulated or isoproterenol-stimulated adenylate cyclase activities (IC50 5 nM) in rat lung plasma membranes. GTP (100 microM) was obligatory for the ANF-dependent inhibition of adenylate cyclase, which could be completely overcome by the presence of 100 microM GDP[beta S] or the addition of 10 mM Mn2+. Reduction of Na2+ concentration from 120 mM to 20 mM had the same effect. Pertussis toxin eliminated ANF-dependent inhibition of adenylate cyclase by catalyzing ADP-ribosylation of membrane-bound Ni protein (41-kDa alpha subunit of the inhibitory guanyl-nucleotide-binding protein of adenylate cyclase). The data support the notion that one of the ANF receptors in rat lung plasma membranes is negatively coupled to a hormone-sensitive adenylate cyclase complex via the GTP-binding Ni protein.     

Binding of [3H]heparin to human plasma low density lipoprotein.

The binding of [³H]heparin to human plasma lipoproteins was measured using a gel filtration assay on columns of Ultrogel AcA 54. [³H]Heparin formed a soluble complex with low density lipoprotein (LDL) as evidenced by the appearance of a new radioactive peak emerging at the void volume where the lipoproteins elute. Free heparin on the other hand was retarded on this column and eluted at a later volume. Heparin binding to LDL could also be demonstrated on columns of Sepharose 4B, in which case two included peaks of ³H were observed to elute in the area of LDL and of heparin. [³H]Heparin did not bind to either high or very low density lipoproteins as determined by the gel filtration assay. The binding of the [³H]heparin to LDL was proportional to both the concentration of LDL and of heparin and both showed saturation kinetics. Cations were not necessary for binding, nor was binding inhibited by EDTA. LDL showed a marked specificity for heparin. Thus, the binding of [³H]heparin to LDL was strongly inhibited by the addition of unlabeled heparin, while other glycosaminoglycans such as chondroitin sulfate, heparan sulfate, keratan sulfate, and dermatan sulfate were not effective inhibitors except at very high concentrations. Salts, especially K₂HPO₄ and (NH₄)₂SO₄, also inhibited binding when added at concentrations of 10 mm or higher suggesting an ionic interaction between heparin and LDL. The pH optimum for binding was between 7.5 and 8.5 but binding fell off markedly above pH 9.0. The [³H]heparin was heterogeneous and could be separated into four fractions on columns of Sephadex G-75. When these fractions were tested for binding to LDL, only the high molecular weight fraction bound to any significant extent. LDL was treated with reagents used to selectively modify basic amino acid residues, and the effect of these treatments on heparin binding was examined. Thus, ethoxyformic anhydride was used for histidine modification, acetic anhydride and succinic anhydride for lysines and cyclohexanedione for arginine residues. In each case there was a significant loss in heparin binding suggesting that various basic amino acids are involved in binding and/or that basic amino acids are necessary to maintain the proper conformation of LDL.     

Interactions of La2+ with phosphatidylserine vesicles: binding, phase transition, leakage, 31P-NMR and fusion

The interaction of La2+ with phosphatidylserine vesicles is studied by differential scanning calorimetry, 140La binding, 31P-NMR chemical shifts and relaxation rates, carboxyfluorescein and [14C]sucrose release, X-ray diffraction and freeze-fracture electron microscopy. In the presence of La3+ concentrations above 1 mM and an incubation temperature of 38 degrees C, i.e., at the phase transition temperature of the complex La/phosphatidylserine, the binding ratio of La/lipid exceeds a 1/3 ratio, reaching saturation at a 1/2 ratio. Analysis, employing a modified Gouy-Chapman equation, indicates a significant increase in the intrinsic binding constant of La/phosphatidylserine when the La3+ concentrations exceeds the threshold concentration for leakage. The analysis illustrates that at the molecular level the binding of La3+ can be comparable to or even weaker than that of Ca2+, but that even when present at smaller concentrations La3+ competes with and partially displaces Ca2+ from membranes or other negatively charged surfaces. The results suggest that the sequence La3+ greater than Ca2+ greater than Mg2+ reflects both the binding strength of these cations to phosphatidylserine as well as their ability to induce leakage, enhancement of 31P spin-lattice relaxation rates, fusion and other structural changes. The leakage, fusion, and other structural changes are more pronounced at the phase transition temperature of the La/lipid complex.     

Glycosaminoglycan-lipoprotein interaction

Glycosaminoglycans (GAGs) bound to various proteoglycans (PGs) present in the cardiovascular system have been proposed to perform a wide range of functions. These include conferring viscoelastic properties; interacting with and modulating growth factors and enzymes; and as receptors and co-receptors in lipoprotein metabolism. Binding of apoB-100 lipoproteins, particularly low density lipoproteins (LDL), to GAGs of extracellular matrix PGs in arteries has been proposed to be an initiating event in development of atherosclerosis. This study was initiated with the aim of getting an overview of the binding patterns of different lipoprotein subclasses with individual GAG categories. We thus evaluated the interaction of lipoproteins with GAGs commonly found in the cardiovascular system using a gel mobility-shift assay developed for this purpose. The same procedure was used to measure lipoproteins binding to metabolically [(35)S]-labeled whole PGs prepared from three cell types, arterial smooth muscle cells, THP-1 macrophages and from HepG2 cells. The effect of GAG composition on PGs on lipoprotein binding was evaluated by enzymatic degradation of the carbohydrate chains. Heparan sulfate was found to bind beta very low density lipoproteins (beta-VLDL) and a chylomicron remnant model (beta-VLDL+apoE), but not LDL. Dermatan sulfate was found to bind LDL, but not beta-VLDL or the chylomicron remnant model. Chondroitin sulfate and heparin were found to bind all lipoproteins tested (LDL, beta-VLDL and beta-VLDL+apoE) although with different affinities. We can conclude that each lipoprotein subclass tested binds a specific assortment of the GAGs tested. The observations made contribute to the understanding of new and complex mechanisms by which carbohydrate and lipid metabolism may be linked.     

Biological evaluation of a series of 2-acetamido-2-deoxy-D-glucose analogs towards cellular glycosaminoglycan and protein synthesis in vitro

Using primary hepatocytes in culture, various 2-acetamido-2-deoxy-D-glucose (GlcNAc) analogs were examined for their effects on the incorporation of D-[³H]glucosamine, [³⁵S]sulfate, and L-[¹⁴C]leucine into cellular glycoconjugates. A series of acetylated GlcNAc analogs, namely methyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-(3) and β-D-glucopyranoside (4) and 2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (5), exhibited a concentration-dependent reduction of D-[³H]glucosamine, but not of [³⁵S]sulfate incorporation into isolated glycosaminoglycans (GAGs), without affecting L-[¹⁴C]leucine incorporation into total protein synthesis. These results suggest that analogs 3–5 exhibit an inhibitory effect on D-[³H]glucosamine incorporation into isolated GAGs by diluting the specific activity of cellular D-[³H]glucosamine and by competing for the same metabolic pathways. In the case of the corresponding series of 4-deoxy-GlcNAc analogs, namely methyl 2-acetamido-3,6-di-O-acetyl-2,4-dideoxy-α-(6) and β-D-xylo-hexopyranoside (7) and 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-D-xylo-hexopyranose (8), compound 8 at 1.0 mM exhibited the greatest reduction of D-[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs, namely to ∼7% of controls, and a moderate inhibition of total protein synthesis, namely to 60% of controls. Exogenous uridine was able to restore the inhibition of total protein synthesis by compound 8 at 1.0 mM. Isolated GAGs from cultures treated with compound 8 were shown to be smaller in size (∼40 kDa) than for control cultures (∼77 kDa). These results suggest that the inhibitory effects of compound 8 on cellular GAG synthesis may be mediated by the incorporation of a 4-deoxy moiety into GAGs resulting in premature chain termination and/or by its serving as an enzymatic inhibitor of the normal sugar metabolites. The inhibition of total protein synthesis from cultures treated with compound 8 suggests a uridine trapping mechanism which would result in the depletion of UTP pools and cause the inhibition of total protein synthesis. A 1-deoxy-GlcNAc analog, namely 2-acetamido-3,4,6-tri-O-acetyl-1,5-anhydro-2-deoxy-D-glucitol (9), also exhibited a reduction in both D -[³H]glucosamine and [³⁵S]sulfate incorporation into isolated GAGs by 19 and 57%, of the control cells, respectively, at 1.0 mM without affecting total protein synthesis. The inability of compound 9 to form a UDP-sugar and, hence, be incorporated into GAGs presents another metabolic route for the inhibition of cellular GAG synthesis. Potential metabolic routes for each analog's effects are presented.     

Enhanced channelling of sulphate through a rapidly exchangeable sulphate pool in response to stimulated glycosaminoglycan synthesis in pancreatic epithelial cells.

The ability of cells to decorate glycosaminoglycans (GAGs) with sulphate in highly specific patterns is important to extracellular matrix biogenesis and placing appropriate glycosulphated ligands on the cell surface. We have examined sulphate metabolism in two pancreatic duct epithelial cell lines - PANC-1 and CFPAC-1 (derived from a cystic fibrosis patient) with a view to understanding how pancreatic cells utilise intracellular sulphate. [35S]Sulphate uptake was rapid and reached near steady state levels within 10 min. However, the intracellular specific activity of [35S]sulphate for PANC-1 and CFPAC-1 reached only 35 and 10%, respectively, of the medium specific activity at 10 min. Therefore, sulphate appears to reside within two compartments; a rapidly exchangeable sulphate pool (RESP) and a slowly exchangeable sulphate pool (SESP). Reducing chloride in the medium, increased the specific activity of [35S]sulphate within cells and increased the size of the inorganic sulphate pool, suggesting that the RESP was enlarged. Sulphate pools were not different in size between the two cell lines in physiological NaCl. Increasing the size of the sulphate pool had no effect on [35S]sulphate:[3H]glucosamine ratios incorporated into glycosaminoglycans (GAGs); however, stimulating the synthesis of GAGs with 4-methylumbelliferyl-beta-d-xyloside, stably elevated [35S]:[3H] ratios. This was due to higher [35S]sulphate incorporation. [35S]Cysteine contributed less than 0.1% of the cells' sulphate requirements. We conclude that in the face of elevated demand for sulphate, pancreatic cells appear to channel a greater proportion through the RESP.     

Transformation of calf uterine progesterone receptor: analysis of the process when receptor is bound to progesterone and RU38486

Effects of different transforming agents were examined on the sedimentation characteristics of calf uterine progesterone receptor (PR) bound to the synthetic progestin [3H]R5020 or the known progesterone antagonist [3H]RU38486 (RU486). [3H]R5020-receptor complexes [progesterone-receptor complexes (PRc)] sedimented as fast migrating 8S moieties in 8-30% linear glycerol gradients containing 0.15 M KCl and 20 mM Na2MoO4. Incubation of cytosol containing [3H]PRc at 23 degrees C for 10-60 min, or at 0 degrees C with 0.15-0.3 M KCl or 1-10 mM ATP, caused a gradual transformation of PRc to a slow sedimenting 4S form. This 8S to 4S transformation was molybdate sensitive. In contrast, the [3H]RU486-receptor complex exhibited only the 8S form. Treatment with all three activation agents caused a decrease in the 8S form but no concomitant transformation of the [3H]RU486-receptor complex into the 4S form. PR in the calf uterine cytosol incubated at 23 or at 0 degrees C with 0.3 M KCl or 10 mM ATP could be subsequently complexed with [3H]R5020 to yield the 4S form of PR. However, the cytosol PR transformed in the absence of any added ligand failed to bind [3H]RU486. Heat treatment of both [3H]R5020- and [3H]RU486-receptor complexes caused an increase in DNA-cellulose binding, although the extent of this binding was lower when RU486 was bound to receptors. An aqueous two-phase partitioning analysis revealed a significant change in the surface properties of PR following both binding to ligand and subsequent transformation. The partition coefficient (Kobsd) of the heat-transformed [3H]R5020-receptor complex increased about 5-fold over that observed with PR at 0 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of synthesis of lactosylceramide and long chain bases in normal and familial hypercholesterolemic cultured proximal tubular cells

We have investigated the effects of lipoproteins on sphingolipid metabolism in proximal renal tubular cells from normal subjects and low density lipoprotein (LDL) receptor-negative homozygous familial hypercholesterolemic subjects employing radioactive precursors, e.g. [3H]serine, [3H]glucose, and [14C]galactose. Compared to cells incubated with lipoprotein-deficient serum, maximum suppression (70-80%) of incorporation of [3H]glucose and [3H]serine into ceramide and LacCer occurred when the LDL concentration in the medium was 25 micrograms/ml medium, and addition of higher amounts of LDL (up to 500 micrograms/ml medium) to normal cells did not produce further suppression. In contrast, high density lipoproteins did not suppress the incorporation of [3H]glucose into lactosylceramide (LacCer) in normal cells. The incorporation of [14C] galactose into LacCer was also suppressed by LDL (50% suppression at a concentration of 100 micrograms/ml medium). In contrast, LDL modified by reductive methylation of lysine residues did not suppress the incorporation of [3H]glucose into LacCer and the incorporation of [3H]serine into ceramide, whereas, native LDL exerted a concentration-dependent suppression of [3H]serine incorporation into ceramide and sphingomyelin in normal cells. At high concentrations of LDL (50-500 micrograms/ml medium), the incorporation of [3H]glucose and [14C]galactose into LacCer in homozygous FH cells was stimulated approximately 2-fold. Maximum stimulation of [3H]serine incorporation into ceramides, LacCer, and sphingomyelin occurred at 100 micrograms LDL/ml medium. Our studies indicate that the endogenous synthesis of sphingolipids in normal renal cells is regulated by the LDL receptor. Modification of the lysine residues in LDL by reductive methylation results in the inability to suppress sphingolipid synthesis in normal cells. Lack of LDL receptors, as in the case of homozygous FH cells, results in the lack of suppression of endogenous sphingolipid synthesis.     

Binding of [99mTc]chondroitin sulfate to scavenger receptors on human chondrocytes as compared to binding of oxidized [125I]LDL on human macrophages

Chondroitin sulfate (CS) used for treatment of osteoarthritis exerts distinct effects on human articular chondrocytes in vitro. We performed a binding analysis with 99mTc-labeled CS (Condrosulf, a commercial CS preparation containing calcium stearate) and cultured human chondrocytes in order to evaluate the presence of specific receptors. Saturation binding at 37 degrees C for 2 h revealed the presence of high-affinity binding sites for CS with a Kd of 2.3 x 10(-9) mol/L and a Bmax of 5.0 x 10(8). Extensive dialysis of Chondrosulf led to a decrease of the binding affinity by 52.5 +/- 19.5% and of the number of CS binding sites/cell by 62.0 +/- 14.0%, demonstrating that the additive present in the Condrosulf preparation enhances CS binding. The nature of the binding site is not yet known but evidence exists in the literature that the scavenger receptor CD36, thoroughly investigated on macrophages, is also found on chondrocytes and might be involved in CS binding. Therefore, we undertook a comparative binding study with human monocytes and labelled LDL and oxidized LDL, the latter being a postulated atherogenic agent in atherosclerosis. For [125I]-LDL binding we found a Kd of 0.45 x 10(-8) mol/L and a Bmax of 0.14 x 10(6) on quiescent monocytes and for [125I]-(ox)LDL binding a Kd of 1.8 x 10(-8) mol/L and a Bmax of 1.3 x 10(6) using LPS-activated monocytes. These data are comparable to the binding affinity found for lipoprotein-proteoglycan-complexes and hence are an indication but not a proof that CD36 is involved in CS binding to human chondrocytes.     

Glycosaminoglycans from bovine eye vitreous humour and interaction with collagen type II

Glycosaminoglycans (GAGs) play an important role in stabilizing the gel state of eye vitreous humour. In this study, the composition of GAGs present in bovine eye vitreous was characterized through disaccharide analysis by liquid chromatography-mass spectrometry. The interaction of GAGs with collagen type II was assessed using surface plasmon resonance (SPR). The percentage of hyaluronic acid (HA), chondroitin sulfate (CS) and heparan sulfate (HS), of total GAG, were 96.2%, 3.5% and 0.3%, respectively. The disaccharide composition of CS consisted of 4S (49%), 0S (38%) 6S (12%), 2S6S (1.5%) and 2S4S (0.3%). The disaccharide composition of HS consisted of 0S (80%), NS2S (7%), NS (7%), 6S (4%), NS6S (2%), and TriS, 2S and 4S6S (each at 0.1%). The average molecular weights of CS and HS were 148 kDa and 204 kDa, respectively. SPR reveals that collagen type II binds to heparin (primarily composed of TriS) with a binding affinity (K _D) of 755 nM and interacts with other GAGs, including CSB and CSE. Both bovine vitreous CS and HS interact with collagen type II, with vitreous HS showing a higher binding affinity.     

A multinuclear, high-resolution NMR study of bovine casein micelles and submicelles

High-resolution, natural abundance 13C[1H] (100.5 MHz), 31P[1H] (161.8 MHz) and 1H (400.0 MHz) NMR spectroscopy was used to identify the calcium-binding sites of bovine casein and to ascertain the dynamic state of amino acid residues within the casein submicelles (in 125 mM KCl, pD = 7.4) and micelles (in 15 mM CaCl2/80 mM KCl, pD = 7.2). The presence of numerous, well-resolved peaks in the tentatively assigned 13C-NMR spectra of submicelles (90 A radius) and micelles (500 A radius) suggests considerable segmental motion of both side chain and backbone carbons. The partly resolved 31P-NMR spectra concur with this. Upon Ca2+ addition, the phosphoserine beta CH2 resonance (65.8 ppm vs DSS) shifts upfield by 0.2 ppm and is broadened almost beyond detection; a general upfield shift (up to 0.3 ppm) is also observed for the 31P-NMR peaks. The T1 values of the alpha CH envelope for submicelles and micelles are essentially identical corresponding to a correlation time of 8 ns for isotropic rotation of the caseins. Significant changes in the 31P T1 values accompany micelle formation. Data are consistent with a loose and mobile casein structure, with phosphoserines being the predominant calcium-binding sites.     

Nuclear magnetic resonance and calorimetric study of the structure, dynamics, and phase behavior of uranyl ion/dipalmitoylphosphatidylcholine complexes.

The interaction of UO2(2+) with dipalmitoylphosphatidylcholine (DPPC) has been studied as a function of temperature and composition using nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC), and monolayer studies. Computer simulations of the 31P-NMR powder spectra of DPPC dispersions in the presence of various concentrations of UO2(2+) are consistent with the binding stoichiometry of [UO2(2+)]/[DPPC] = 1:4 at [UO2(2+)]/[DPPC] less than 0.3. This complex undergoes a phase transition to the liquid crystalline phase at T'm = 50 +/- 3 degrees C with a breadth delta T'm = 7 +/- 3 degrees C. This broad transition gradually disappears at higher UO2(2+) concentrations, suggesting the presence of yet another UO2(2+)/DPPC complex (or complexes) whose NMR spectra are indistinguishable from those of the 1:4 UO2(2+)/DPPC species. The temperature-dependent 13C powder spectra of 2(1-13C) DPPC dispersions in the presence of 1.2 mol ratio of UO2(2+) show that this higher order complex (complexes) also undergoes a phase transition to the liquid crystalline state at T'm +/- = 58 +/- 3 degrees C with a breadth delta T"m = 15 +/- 5 degrees C. The NMR spectra indicate that exchange among these various UO2(2+)/DPPC complexes is slow. In addition, computer simulations of the 31P-, 13C-, and 2H-NMR powder spectra show that axial diffusion of the DPPC molecules about their long axes is quenched by addition of UO2(2+) and acyl chain isomerization is the dominant motional mode. The isomerization is best described as two-site hopping of the greater than C-D bond at a rate of approximately 10(6) s-1, a motional mode which is expected for a kink diffusion.