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Binding of active and inactive forms of lipoprotein lipase to heparin. Effects of pH.

ABSTRACT: Lipoprotein lipase has been shown to bind to, be internalized by, and perhaps be transferred through, a variety of cells. These processes may involve a heparin-like cell-surface receptor and passage through acidified cell compartments. We have therefore studied effects of low pH on the binding of the lipase to heparin and on its catalytic activity. The rate of inactivation of the lipase in solution was found to increase as the pH was lowered. Addition of heparin stabilized the enzyme. Binding of active lipoprotein lipase to heparin-Sepharose could be demonstrated at pH down to 6.5. At pH below 6, binding could not be studied directly because the lipase was too unstable in solution. Lipase bound to heparin-Sepharose could, however, be exposed to pH 4.5 at 10 degrees C with little loss of activity. Binding to heparin-Sepharose also stabilized under physiological conditions (37 degrees C, 0.15 M-NaCl, pH 5.5-7.4). Catalytically inactive lipoprotein lipase retained the ability to bind to heparin-Sepharose. Higher concentrations of salt were needed to displace both active and inactive lipase from heparin-Sepharose at lower pH, indicating that the affinity increased as pH was lowered. The inactive lipase was, however, displaced by lower concentrations of salt than was active lipase.

SUBMITTER: Bengtsson-Olivecrona G

PROVIDER: S-EPMC1144727 | biostudies-other | 1985 Mar

REPOSITORIES: biostudies-other

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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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Binding of active and inactive forms of lipoprotein lipase to heparin. Effects of pH.

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