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Structure of heparin fragments with high affinity for lipoprotein lipase and inhibition of lipoprotein lipase binding to alpha 2-macroglobulin-receptor/low-density-lipoprotein-receptor-related protein by heparin fragments.

ABSTRACT: Heparin-derived deca- and octa-saccharides were subjected to affinity chromatography on lipoprotein lipase-Sepharose and the fractions eluted at high salt concentration were analysed by strong-anion-exchange chromatography. Two high-affinity decasaccharides were isolated and the structure determined by one- and two-dimensional 1H-n.m.r. spectroscopy. The affinities of 3H-labelled low-molecular-mass heparin and size-fractionated deca-, octa-, and hexa-saccharides for lipoprotein lipase immobilized on microtitre plates were determined from saturation curves. From competition experiments the affinities of unlabelled heparins and pure deca- and hexa-saccharide fragments were determined. The binding was size- and charge-dependent, but structural dependency was also indicated. Thus substitution of a 2-O-sulphated L-iduronic acid with D-glucuronic acid was less important than the sulphation pattern of the D-glucosamine residue for affinity for lipoprotein lipase. Heparin inhibits binding of lipoprotein lipase to alpha 2-macroglobulin-receptor/low-density-lipoprotein receptor-related protein. The effects of size, charge and structure for this inhibition were studied. The ability of the heparin fragments to inhibit binding correlated with their affinity for lipoprotein lipase. This indicates that the inhibition of the binding of lipoprotein lipase to alpha 2-macroglobulin-receptor/low-density-lipoprotein receptor-related protein by heparin is exclusively mediated by binding of heparin to lipoprotein lipase.

SUBMITTER: Larnkjaer A

PROVIDER: S-EPMC1136764 | biostudies-other | 1995 Apr

REPOSITORIES: biostudies-other

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Project description:Periportal and perivenous parenchymal cells were isolated by the digitonin-pulse perfusion method. The digitonin-pulse perfusion was shown to lead to selective lysis of the correct zone with a straight and sharp border of two to three cells. The mean ratios of alanine aminotransferase activity (a marker for periportal parenchymal cells) and glutamine synthetase activity (a perivenous marker) of periportal to perivenous parenchymal cells were 1.76 and 0.025 respectively. Cells were incubated in vitro with 125I-asialo-orosomucoid (ASOR), 125I-trypsin-activated alpha 2-macroglobulin (alpha 2M-T) or 125I-beta-migrating very-low-density lipoprotein (beta-VLDL), in order to determine the zonal distribution of the asialoglycoprotein receptor (ASGPr), the alpha 2-macroglobulin receptor/low-density-lipoprotein receptor-related protein (alpha 2Mr/LRP) and the lipoprotein-remnant receptor, respectively. Maximum binding capacity for 125I-ASOR on parenchymal cells showed a periportal/perivenous ratio of 0.70. The periportal/perivenous ratio of Bmax. values of binding of 125I-alpha 2M-T to parenchymal cells was 1.51. The Bmax. values of binding of 125I-beta-VLDL, however, were about equal for both cell populations. It is concluded that the maximum binding capacity of the ASGPr on isolated periportal parenchymal cells is 0.70 times that of perivenous parenchymal cells. The 1.51-fold higher expression of the alpha 2Mr/LRP on periportal cells, compared with perivenous parenchymal cells, indicates a zonal specialization for the uptake of the suggested multiple ligands. In contrast, the observed homogeneous distribution of the lipoprotein-remnant receptor is in accordance with the suggestion that lipoprotein remnants bind to a specific receptor, which is different from the alpha 2Mr/LRP. The zonal heterogeneity in the expression of receptors suggests that receptor-dependent uptake pathways are under zonal control, leading to intrahepatic heterogeneity in the removal of ligands from the blood circulation.

Project description:Lipoprotein lipases from a variety of sources have been shown previously to bind to heparin and some related polysaccharides. For the present studies lipoprotein lipase purified from bovine milk was used. 1. In batch experiments binding of the enzyme activity to heparin-Sepharose occurred relatively slowly, so that 30min was required for the system to come to near-equilibrium. In contrast, release of the enzyme activity from heparin-Sepharose by addition of salt to the liquid phase occurred rapidly. 2. Some binding was observed also with unsubstituted Sepharose, but this binding had a low capacity compared with that observed with heparin-Sepharose. High salt concentrations, heparin or deoxycholate decreased the binding to unsubstituted Sepharose. These factors also increase the solubility of the enzyme, which is low. 3. Addition of heparin to the liquid phase caused a concentration-dependent release of enzyme activity from the gel. These results suggested that the binding of the enzyme to heparin-Sepharose was mainly through interaction with heparin. 4. The enzyme activity was also quantitatively displaced to the liquid phase at increased concentrations of salt. Among the positive ions tested the following order of effectiveness was noted: Cs(+) approximately K(+)>Na(+)>Li(+); and among the negative the following: SCN(-)>I(-)> NO(3) (-)>Br(-) approximately Cl(-). The differences were quite large. Thus addition of 0.16m-KSCN (in addition to the 0.32m-NaCl originally present) displaced one-half of the enzyme activity to the supernatant, whereas 0.8m-LiCl only displaced one-quarter. 5. The distribution of heparin in the gel also profoundly influenced the binding. Two series of gels were studied. One series was made by mixing heparin-Sepharose with unsubstituted Sepharose. Results obtained with these gels were those expected from a series of decreasing volumes of heparin-Sepharose. In contrast, a series of heparin-Sepharoses made with different degrees of substitution gave quite different results. With these gels the amount of enzyme activity bound per amount of heparin increased markedly, whereas the salt concentration needed to displace the enzyme activity from the gel decreased markedly with decreased concentration of heparin in the gel. 6. On stepwise elution of small columns of heparin-Sepharose the enzyme activity was eluted over a remarkably wide range of salt concentrations. When enzyme eluted at one salt concentration was re-applied, it gave the same elution profile as enzyme previously eluted at other salt concentrations or the entire enzyme preparation. These and other results suggested that, whereas the enzyme preparation was rather homogeneous in its binding to heparin, the heparin preparation was polydisperse in binding of lipoprotein lipase.

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Structure of heparin fragments with high affinity for lipoprotein lipase and inhibition of lipoprotein lipase binding to alpha 2-macroglobulin-receptor/low-density-lipoprotein-receptor-related protein by heparin fragments.

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