Biomedical subjects

T Olivecrona

Publications and source records attributed to T Olivecrona.

At least 109 records · Page 6Linked to original sources

PubMed1986

Regulation of lipoprotein lipase synthesis by recombinant tumor necrosis factor--the primary regulatory role of the hormone in 3T3-L1 adipocytes.

Tumor necrosis factor (TNF), a protein homologous to cachectin, has been implicated in mediating cachexia. This effect at least in part has been suggested to occur through the influence of the hormone on adipose tissue metabolism. Using fully differentiated 3T3-L1 adipocytes as a model system, we have been investigating the effects of recombinant TNF (rTNF) on key features of adipocyte metabolism. Exposure of fully differentiated 3T3-L1 adipocytes to recombinant tumor necrosis factor resulted in a dose and time-dependent suppression of the activity of lipoprotein lipase. The loss in activity results from an effect on the synthesis of the enzyme, as determined by a decreased incorporation of [35S]methionine into immunoprecipitable lipoprotein lipase. No effect of rTNF on the half-life of the enzyme was observed. General protein synthesis, as judged by [35S]methionine incorporation into acid-insoluble protein, was minimally affected by exposure of the cells to rTNF; this was further confirmed by sodium dodecyl sulfate-polyacrylamide gel analysis of total cellular protein. As opposed to our previously reported results with crude preparations of TNF, no effect on either the ability of the adipocytes to synthesize and store or mobilize triacylglycerol was observed. Our results are consistent with the hypothesis that other hormones present in crude preparations of TNF acting either alone or synergistically with TNF play a major role in the further metabolic derangements associated with adipose tissue during cachexia.

Adipose Tissue↗

PubMed1985

Distribution of lipoprotein lipase and hepatic lipase between plasma and tissues: effect of hypertriglyceridemia.

Lipoprotein lipase and hepatic lipase were measured in rat plasma using specific antisera. Mean values for lipoprotein lipase in adult rats were 1.8-3.6 mU/ml, depending on sex and nutritional state. Values for hepatic lipase were about three times higher. Lipoprotein lipase activity in plasma of newborn rats was 2-4-times higher than in adults. In contrast, hepatic lipase activity was lower in newborn than in adult rats. Following functional hepatectomy there was a progressive increase in lipoprotein lipase activity in plasma, indicating that transport of the enzyme from peripheral tissues to the liver normally takes place. Lipoprotein lipase, but not hepatic lipase, increased in plasma after a fat meal. An even more marked increase, up to 30 mU/ml, was seen after intravenous injection of Intralipid. Plasma lipase activity decreased in parallel with clearing of the injected triacylglycerol. 125I-labeled lipoprotein lipase injected intravenously during the hyperlipemia disappeared somewhat slower from the circulation than in fasted rats, but the uptake was still primarily in the liver. Hyperlipemia, or injection of heparin, led to increased lipoprotein lipase activity in the liver. This was seen even when the animals had been pretreated with cycloheximide to inhibit synthesis of new enzyme protein. These results suggest that during hypertriglyceridemia lipoprotein lipase binds to circulating lipoproteins/lipid droplets which results in increased plasma levels of the enzyme and increased transport to the liver.

Adipose Tissue↗

PubMed1985

Studies on inactivation of lipoprotein lipase: role of the dimer to monomer dissociation.

Sedimentation equilibrium analysis demonstrated that preparations of bovine lipoprotein lipase contain a complex mixture of dimers and higher oligomers of enzyme protein. Enzyme activity profiles from sedimentation equilibrium as well as from gel filtration indicated that activity is associated almost exclusively with the dimer fraction. To explore if the enzyme could be dissociated into active monomers, 0.75 M guanidinium chloride was used. Sedimentation velocity measurements demonstrated that this treatment led to dissociation of the lipase protein into monomers. Concomitant with dissociation, there was an irreversible loss of catalytic activity and a moderate change in secondary structure as detected by circular dichroism. The rate of inactivation increased with decreasing concentrations of active lipase, but addition of inactive lipase protein did not slow down the inactivation. This indicates that reversible interactions between active species precede the irreversible loss of activity. The implication is that dissociation initially leads to a monomer form which is in reversible equilibrium with the active dimer, but which decays rapidly into an inactive form, and is therefore not detected as a stable component in the system.

Animals↗

PubMed1985

Molecular size of bovine lipoprotein lipase as determined by radiation inactivation.

We have determined the size of the functional unit of bovine lipoprotein lipase by radiation inactivation. This was done in five different situations: 1) in a buffer with high salt concentration. In this situation the enzyme is relatively soluble and stable. 2) For an enzyme-heparin complex. This may reflect the physiological state of the enzyme at the vascular endothelium, where it is believed to be bound to a heparin-like molecule. 3) In the presence of lipid substrate and 4) with lipid substrate and activator protein. Here most of the enzyme is adsorbed to the substrate droplets. 5) For an enzyme-detergent complex; another model for enzyme-lipid interaction. In all five situations the enzyme activity decayed as an exponential function of radiation dose, and the target sizes were similar. The target size did not vary with the concentration of lipase protein. The combined data for bovine lipoprotein lipase yield a functional size of 72 kDa which is close to that expected for a dimer, 77 kDa.

Animals↗

PubMed1985

Putative role of cholesteryl ester transfer protein in removal of cholesteryl ester from vascular interstitium, studied in a model system in cell culture.

A model system to study the putative role of cholesteryl ester transfer protein in the egress of interstitial cholesteryl ester is described. Confluent cultures of bovine aortic smooth muscle cells were labeled for 24 h with [3H]cholesteryl linoleyl ether and [14C]cholesteryl linoleate by incubation with bovine milk lipoprotein lipase. This method of labeling results in the transfer of cholesteryl linoleyl ether and cholesteryl ester to three compartments: a trypsin-releasable, trypsin-resistant and catabolic compartment (Stein, O., Halperin, G., Leitersdorf, E., Olivecrona, T. and Stein, Y. (1984) Biochim. Biophys. Acta 795, 47-59). The efflux of labeled cholesteryl linoleyl ether and cholesteryl ester from the extracellular and cell-surface related compartments into a serum-free culture medium containing 1% bovine serum albumin was studied during 24 h of postincubation. The efflux was expressed as a percentage of pulse value, i.e., radioactivity retained by the cell culture at the end of the labeling period. The efflux of [3H]cholesteryl linoleyl ether, [14C]cholesteryl ester and 14C-labeled free cholesterol (formed by cellular hydrolysis of cholesterol ester) into the culture medium with 1% bovine serum albumin was about 5% of the pulse value. Addition of human lipoprotein-deficient serum resulted in a 3-10-fold increase in the efflux of [3H]cholesteryl linoleyl ether and [14C]cholesteryl ester, but did not change markedly the efflux of 14C-labeled free cholesterol. Rat lipoprotein-deficient serum which does not contain cholesteryl ester transfer protein did not increase the efflux of [3H]cholesteryl linoleyl ether or [14C]cholesteryl ester. The rate of cholesteryl ester efflux in the presence of human lipoprotein-deficient serum was linear for about 6 h and increased further up to 24 h. Addition of Intralipid to medium containing human lipoprotein-deficient serum further enhanced the efflux of [3H]cholesteryl linoleyl ether and, to a lesser extent, that of cholesteryl ester. A similar effect was observed also by addition of rat VLDL to medium containing human lipoprotein-deficient serum. Inhibition of cholesteryl linoleyl ether and cholesteryl ester efflux and marked enhancement of free cholesterol efflux occurred when rat HDL was added to medium containing human lipoprotein-deficient serum, while human HDL was only slightly inhibitory. The results obtained with human lipoprotein-deficient serum were reproduced with partially purified cholesteryl ester transfer protein. Using the partially purified cholesteryl ester transfer protein, the efflux of cholesteryl linoleate was compared to that of cholesteryl oleate and was found to be the same.

Animals↗

PubMed1985

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

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.

Enzyme Activation↗

PubMed1985

Combined lipase deficiency (cld/cld) in mice. Demonstration that an inactive form of lipoprotein lipase is synthesized.

Combined lipase deficiency, cld, is a recessive mutation within the T/t complex of mouse chromosome 17. Mice homozygous for this defect display severe functional deficiencies of lipoprotein lipase and the related hepatic lipase. They develop massive hyperchylomicronemia and die within 3 days when allowed to suckle. Heart, diaphragm muscle, and brown adipose tissue of 1-day-old cld/cld and unaffected mice incorporated in vivo [35S]methionine into a protein that could be immunoprecipitated by antilipoprotein lipase serum. The immunoprecipitated protein in all tissues had the same Mr as bovine lipoprotein lipase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proportion of radioactivity in the lipoprotein lipase band to that in total protein was 0.02% in tissues of cld/cld mice and 0.01% in tissues of unaffected mice. There was 2-6 times more lipoprotein lipase-like protein (determined by immunoassay) in tissues of defective mice than in those of unaffected mice. These findings indicate that the cld mutation did not cause deletion of the structural gene for lipoprotein lipase. Lipoprotein lipase activity in heart, diaphragm muscle, brown adipose tissue, and lung of cld/cld mice was less than 5% of that in tissues of unaffected mice. This low activity could be inhibited more than 85% by antilipoprotein lipase serum, but not by nonimmune serum. It is concluded that tissues in cld/cld mice synthesize a lipoprotein lipase-like protein which has subnormal catalytic activity.

Adipose Tissue, Brown↗

PubMed1985

Bovine milk lipoprotein lipase transfers tocopherol to human fibroblasts during triglyceride hydrolysis in vitro.

Lipoprotein lipase appears to function as the mechanism by which dietary vitamin E (tocopherol) is transferred from chylomicrons to tissues. In patients with lipoprotein lipase deficiency, more than 85% of both the circulating triglyceride and tocopherol is contained in the chylomicron fraction. The studies presented here show that the in vitro addition of bovine milk lipoprotein lipase (lipase) to chylomicrons in the presence of human erythrocytes or fibroblasts (and bovine serum albumin [BSA]) resulted in the hydrolysis of the triglyceride and the transfer of both fatty acids and tocopherol to the cells; in the absence of lipase, no increase in cellular tocopherol was detectable. The incubation system was simplified to include only fibroblasts, BSA, and Intralipid (an artificial lipid emulsion containing 10% soybean oil, which has gamma but not alpha tocopherol). The addition of lipase to this system also resulted in the transfer of tocopherol (gamma) to the fibroblasts. Addition of both lipase and its activator, apolipoprotein CII, resulted in a further increase in the cellular tocopherol content, but apolipoprotein CII alone had no effect. Heparin, which is known to prevent the binding of lipoprotein lipase to the cell surface membrane, abrogated the transfer of tocopherol to fibroblasts without altering the rate of triglyceride hydrolysis. Thus, in vitro tocopherol is transferred to cells during hydrolysis of triglyceride by the action of lipase, and for this transfer of tocopherol to occur, the lipase itself must bind to the cell membrane.

Animals↗

PubMed1985

Binding of diamine oxidase activity to rat and guinea pig microvascular endothelial cells. Comparisons with lipoprotein lipase binding.

Microvascular endothelial cells from rat and guinea pig fat pads were shown to bind diamine oxidase (DAO) activity when incubated with soluble extracts of placenta (33 DAO U/mg of placenta) and a purified placental enzyme preparation (94 U/micrograms of protein). The extent of binding was dependent on the concentration of enzyme activity and tissue. Saturation of binding sites with 5,000 U of DAO/ml resulted in levels of bound activity (up to 11-13 U/mg of endothelial cells) in excess of that observed in all tissues except placenta. Scatchard plots suggested that there were at least two DAO binding sites (apparent Km 92 and 2,450 U/ml). Although the same cell preparations bound 125I-labeled lipoprotein lipase (LPL), the presence of LPL on the endothelial cell surface did not interfere with the binding of DAO activity except when cells were exposed to high concentrations of LPL. Alternatively, bound DAO activity was partially displaced (up to 33%) only with high concentrations (30 micrograms/ml) of LPL. DAO activity may thus be bound to at least two populations of sites, one of which may bind LPL. Both enzymes, however, were displaced by heparin (0.05-5 U/ml) and DAO binding was impaired by prior treatment of cells with proteolytic and glycosaminoglycandegrading enzymes. The demonstration of DAO binding to vascular endothelial cells provides a further example of the ability of these cells to bind enzymes at their surface and thereby act on biologically active substances in the circulation.

Adipose Tissue↗

PubMed1985

Is hypertriglyceridemic very low density lipoprotein a precursor of normal low density lipoprotein?

The precursor-product relationship of very low density (VLDL) and low density lipoproteins (LDL) was studied. VLDL obtained from normal (NTG) and hypertriglyceridemic (HTG) subjects was fractionated by zonal ultracentrifugation and subjected to in vitro lipolysis. The individual subfractions and their isolated lipolysis products, as well as IDL and LDL, were rigorously characterized. A striking difference in the contribution of cholesteryl ester to VLDL is noted. In NTG subfractions, the cholesteryl ester to protein ratio increases with decreasing density (VLDL-I----VLDL-III). This is the expected result of triglyceride loss through lipolysis and cholesteryl ester gain through core-lipid transfer protein action. In HTG subfractions there is an abnormal enrichment of cholesteryl esters that is most marked in VLDL-I and nearly absent in VLDL-III. Thus, the trend of the cholesteryl ester to protein ratios is reversed, being highest in HTG-VLDL-I and lowest in VLDL-III. This is incompatible with the precursor-product relationship described by the VLDL----IDL----LDL cascade. In vitro lipolysis studies support the conclusion that not all HTG-VLDL can be metabolized to LDL. While all NTG subfractions yield products that are LDL-like in size, density, and composition, only HTG-VLDL-III, whose composition is most similar to normal, does so. HTG VLDL-I and VLDL-II products are large and light populations that are highly enriched in cholesteryl ester. We suggest that this abnormal enrichment of HTG-VLDL with cholesteryl ester results from the prolonged action of core-lipid transfer protein on the slowly metabolized VLDL mass. This excess cholesteryl ester load, unaffected by the process of VLDL catabolism, remains entrapped within the abnormal particle. Therefore, lipolysis yields an abnormal, cholesteryl ester-rich product that can never become LDL.

Adult↗

PubMed1985

Plasma phospholipid transfer protein enhances transfer and exchange of phospholipids between very low density lipoproteins and high density lipoproteins during lipolysis.

In order to determine the effects of a plasma phospholipid transfer protein on the transfer of phospholipids from very low density lipoproteins (VLDL) to high density lipoproteins (HDL) during lipolysis, biosynthetically labeled rat 32P-labeled VLDL was incubated with human HDL3 and bovine milk lipoprotein lipase (LPL) in the presence of the plasma d greater than 1.21 g/ml fraction or a partially purified human plasma phospholipid transfer protein (PTP). The addition of either the PTP or the d greater than 1.21 g/ml fraction resulted in a 2- to 3-fold stimulation of the transfer of phospholipid radioactivity from VLDL into HDL during lipolysis. In the absence of LPL, the PTP caused a less marked stimulation of transfer of phospholipid radioactivity. Both the d greater than 1.21 g/ml fraction and the PTP enhanced the transfer of VLDL phospholipid mass into HDL, but the percentage transfer of phospholipid radioactivity was greater than that of phospholipid mass, suggesting stimulation of both transfer and exchange processes. Stimulation of phospholipid exchange was confirmed in experiments where PTP was found to augment transfer of [14C]phosphatidylcholine radioactivity from HDL to VLDL during lipolysis. In experiments performed with human VLDL and human HDL3, both the d greater than 1.21 g/ml fraction and the PTP were found to stimulate phospholipid mass transfer from VLDL into HDL during lipolysis. Analysis of HDL by non-denaturing polyacrylamide gradient gel electrophoresis showed that enhanced lipid transfer was associated with only a slight increase in particle size, suggesting incorporation of lipid by formation of new HDL particles. In conclusion, the plasma d greater than 1.21 g/ml fraction and a plasma PTP enhance the net transfer of VLDL phospholipids into HDL and also exchange of the phospholipids of VLDL and HDL. Both the transfer and exchange activities of PTP are stimulated by lipolysis.

Animals↗

PubMed1985

Lipoprotein lipase: an attempt to correlate its molecular properties to its function.

Hydrolysis by lipoprotein lipase in capillaries of the adipose tissue is the step that determines the rate at which fatty acids from lipoprotein triacylglycerols are transported into the tissue for storage. Like many other key enzymes, adipose tissue lipoprotein lipase can be regulated both in amount and in activity. The amount of active lipoprotein lipase at the capillary endothelium is under hormonal control. The enzyme molecule itself carries several functional sites. Three of these sites, for interaction with lipid interfaces, with activator protein, and with fatty acids, regulate the action of the enzyme's active site. Another, independent, site on the molecule anchors it to cell surface heparan sulfate chains and thus holds it in place at the endothelium. Together these properties allow for a fine-tuned regulation of the lipoprotein lipase reaction so that lipids are transferred into the adipose tissue at the right rate, at the right time, and in the right place.

Adipose Tissue↗

PubMed1984

Hepatic and extrahepatic uptake of intravenously injected lipoprotein lipase.

Rats were injected intravenously with 125I-labeled bovine lipoprotein lipase. The lipase disappeared within minutes from the blood due to uptake both in the liver (about 50% of the injected dose) and in extrahepatic tissues. Lipase enzyme activity disappeared in parallel to the 125I radioactivity. Thus, there was no inactivation of lipase in the circulating blood. Similar results were obtained when lipoprotein lipase purified from guinea pigs was injected into guinea pigs. Using supradiphragmatic rats we could show that the extrahepatic uptake was saturable and that the amounts of lipase that could be bound far exceeded the amounts of endogenous lipase expected to be present on the endothelium. When the lipase was denatured before injection, its removal in supradiaphragmatic rats became slower, and in intact rats the fraction of the uptake that occurred in extrahepatic tissues was much decreased. It is concluded that recognition by the extrahepatic receptors depends on the native conformation of the lipase. The extrahepatic uptake was strongly impeded by injection of heparin prior to injection of the lipase, and the uptake could to a large extent be reversed by injection of heparin after the lipase. Even after 1 h lipase that had been taken up by extrahepatic tissues reappeared immediately in the blood on injection of heparin. This was true both for enzyme activity and for enzyme radioactivity. Thus, internalization-inactivation-degradation occur only slowly in extrahepatic tissues. It is possible that the extrahepatic binding occurs to the enzyme's physiological receptors. The hepatic uptake was not dependent on the native conformation of the lipase, was less sensitive to heparin, could not be reversed by heparin and was not saturable. The enzyme was not rapidly inactivated after uptake; its activity could be detected in liver homogenates even after 1 h. Degradation to acid-soluble products in the liver was relatively slow; the t1/2 for native lipase was about 1 h. In comparison, in parallel experiments asialofetuin was degraded with a t1/2 of about 15 min.

Animals↗

PubMed1984

Lipoprotein lipase mediated uptake of non-degradable ether analogues of phosphatidylcholine and cholesteryl ester by cultured cells.

Lipoprotein lipase mediated transfer of cholesteryl ester and its ether analog, cholesteryl linoleyl ether, from unilamellar liposomes, prepared from a nonhydrolyzable ether analog of 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleyl ether-sn-glycero-3-phosphocholine (DOEPC), was studied in various cells in culture. It was found that lipoprotein lipase enhanced the uptake of cholesteryl linoleyl ether and of DOEPC. These findings provided a definitive proof that hydrolysis of liposomal PC is not needed for the lipoprotein lipase catalyzed transfer of cholesteryl linoleyl ether and cholesteryl ester to cells. The lipids transferred by lipoprotein lipase to cells were localized in three compartments, trypsin-releasable, resistant and metabolic; the latter was a chloroquine-sensitive pool as evidenced by inhibition of cholesteryl ester hydrolysis. Labeled PC and, to a lesser extent DOEPC, in the trypsin-releasable pool was able to return to the medium, while cholesteryl linoleyl ether and cholesteryl ester required cholesteryl ester transfer protein for release. The transfer of cholesteryl linoleyl ether and cholesteryl ester into a trypsin-resistant compartment did not require metabolic energy and occurred also in formaldehyde-fixed cells. Metabolic energy was needed for the translocation of cholesteryl linoleyl ether and cholesteryl ester into the lysosomal compartment, presumably by a process of endocytosis. The physiological relevance of the present findings is that as intravascular hydrolysis of triacylglycerol-rich lipoproteins is mediated by lipoprotein lipase attached to endothelial cells, the latter can provide a very extensive surface for removal and metabolism of phospholipids and cholesteryl ester by a mechanism mediated by lipoprotein lipase.

Animals↗

PubMed1984

Lipoprotein lipase enhances the cholesteryl ester transfer protein-mediated transfer of cholesteryl esters from high density lipoproteins to very low density lipoproteins.

These studies were undertaken to examine the effects of lipoprotein lipase (LPL) and cholesteryl ester transfer protein (CETP) on the transfer of cholesteryl esters from high density lipoproteins (HDL) to very low density lipoproteins (VLDL). Human or rat VLDL was incubated with human HDL in the presence of either partially purified CETP, bovine milk LPL or CETP plus LPL. CETP stimulated both isotopic and mass transfer of cholesteryl esters from HDL into VLDL. LPL caused only slight stimulation of cholesteryl ester transfer. However, when CETP and LPL were both present, the transfer of cholesteryl esters from HDL into VLDL remnants was enhanced 2- to 8-fold, compared to the effects of CETP alone. The synergistic effects of CETP and LPL on cholesteryl ester transfer were more pronounced at higher VLDL/HDL ratios and increased with increasing amounts of CETP. In time course studies the stimulation of cholesteryl ester transfer activity occurred during active triglyceride hydrolysis. When lipolysis was inhibited by incubating LPL with either 1 M NaCl or 2 mM diethylparanitrophenyl phosphate, the synergism of CETP and LPL was reduced or abolished, and LPL alone did not stimulate cholesteryl ester transfer. These experiments show that LPL enhances the CETP-mediated transfer of cholesteryl esters from HDL to VLDL. This property of LPL is related to lipolysis.

Carrier Proteins↗

PubMed1984

Lipoprotein lipase--the molecule and its interactions.

The lipoprotein lipase molecule carries several functional sites. Three of these sites, for interaction with lipid interfaces, with activator protein, and with fatty acids, regulate the action of the enzyme's active site. Another, independent, site on the molecule anchors it to cell surface heparan sulfate and thus holds it in place at the endothelium. Together these properties allow for a fine-tuned regulation of the lipoprotein lipase reaction so that lipids are transported from the right particles, into the right tissues at the right rate.

Binding Sites↗

PubMed1983

Immunochemical properties of lipoprotein lipase. Development of an immunoassay applicable to several mammalian species.

The reaction of bovine lipoprotein lipase with its antibodies was found to be conformation-dependent. One aspect of this was that most antisera were more reactive with denatured than with native 125I-labeled lipoprotein lipase. Another aspect was that denatured lipase did not compete effectively with native lipase for those antibodies which caused inhibition of the enzyme's activity. This latter observation leads to the conclusion that the inhibiting antibodies recognize conformation-dependent determinants on the native enzyme. Fab fragments prepared from an inhibiting antiserum blocked the binding of the lipase to triacylglycerol/phospholipid droplets. This suggests that the inhibition results from reaction of the antibodies with the enzyme as it exists in solution, either covering the lipid-binding site on the enzyme or making it impossible for the enzyme to go through the conformational transitions necessary for binding to lipid. Most rabbit antisera did not react with rat or mouse lipoprotein lipase, but some sera showed a weak cross-reaction. Antisera raised in hens showed a much stronger cross-reaction, enough to be useful for heterologous immunoassays. An immunoassay for the bovine lipase was developed. For reproducible results it was necessary to have tracer, standard and samples in denatured form. This was accomplished by heating them in SDS, and running the immunoreaction in a Triton X-100-containing medium.

Animals↗

PubMed1983

The effects of pH and salt on the lipid binding and enzyme activity of lipoprotein lipase.

This paper demonstrates a striking difference between the effects of salt and pH on the activity of lipoprotein lipase against two different substrates: Intralipid and bovine milk fat droplets. With the former substrate 1 M NaCl caused only a slight reduction in enzyme activity and the stimulation by apolipoprotein C-II was the same from 0.1 to 1.1 M NaCl. In contrast, 0.5 M or more NaCl virtually abolished the enzyme activity in the milk system. In this system the salt also abolished binding of the enzyme to the lipid droplets, whereas in the Intralipid system most of the enzyme remained bound even at 1 M NaCl. A similar picture was obtained with respect to effects of pH. In the milk system the activity decreased sharply at pH values above 8.5, whereas in the Intralipid system it continues to rise to pH 10, and the stimulation by activator protein is the same at all pH values. Correlating with this, the binding of the enzyme to the lipid droplets was highly dependent on pH values in the milk systems, with optimum binding around pH 8, whereas in the Intralipid system most of the enzyme remained bound to the lipid droplets at all pH values. These studies demonstrate that apolipoprotein C-II can activate lipoprotein lipase at a wide range of salt concentrations and of pH. They suggest that the well-known effects of high salt concentrations and of high pH to decrease lipoprotein lipase activity are exerted primarily on the enzyme itself.

Animals↗