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R L Jackson

Publications and source records attributed to R L Jackson.

At least 109 records · Page 6Linked to original sources
PubMed1985

Interaction of lipoprotein lipase and apolipoprotein C-II with sonicated vesicles of 1,2-ditetradecylphosphatidylcholine: comparison of binding constants.

The interaction of lipoprotein lipase (LpL) and its activator protein, apolipoprotein C-II (apoC-II), with a nonhydrolyzable phosphatidylcholine, 1,2-ditetradecyl-rac-glycero-3-phosphocholine (C14-ether-PC), was studied by fluorescence spectroscopy. A complex of 320 molecules of C14-ether-PC per LpL was isolated by density gradient ultracentrifugation in KBr. The intrinsic tryptophan fluorescence emission spectrum of LpL was shifted from 336 nm in the absence of lipid to 330 nm in the LpL-lipid complex; the shift was associated with a 40% increase in fluorescence intensity. Addition of C14-ether-PC vesicles to apoC-II caused a 2.5-fold increase in intrinsic tryptophan fluorescence and a shift in emission maximum from 340 to 317 nm. LpL and apoC-II/C14-ether-PC stoichiometries and binding constants were determined by measuring the increase in the intrinsic tryptophan fluorescence as a function of lipid and protein concentrations; for LpL the rate and magnitude of the fluorescence increases were relatively independent of temperature in the range 4-37 degrees C. A stoichiometry of 270 PC per LpL for the LpL-lipid complex compares favorably with the value obtained in the isolated complex. The dissociation constant (Kd) of the complex is 4.3 X 10(-8) M. For apoC-II, the stoichiometry of the complex is 18 PC per apoprotein, and the Kd is 3.0 X 10(-6) M. These data suggest that LpL binds more strongly than apoC-II to phosphatidylcholine interfaces.

Animals↗
PubMed1985

Monoclonal antibodies to bovine milk lipoprotein lipase. Evidence for proteolytic degradation of the native enzyme.

Lipoprotein lipase was purified from bovine skim milk by chromatography on heparin-Sepharose. Polyacrylamide gel electrophoresis showed a single protein with an apparent molecular weight of 55,000 in the trailing edge of the elution profile; fractions in the leading edge contained additional proteins with molecular weights of 36,000 and 18,000-22,000. Nine monoclonal antibodies were prepared against the 55,000-dalton protein. By immunoblotting, we show that the Mr = 18,000-22,000 components share common antigen determinants with the 55,000-dalton protein, suggesting that they represent proteolytic degradation products. Incubation of partially purified lipoprotein lipase for 24 h at 37 degrees C results in breakdown of the 55,000-dalton protein with concomitant enrichment in lower Mr components; the proteolytic activity is prevented by incubating the milk with phenylmethane, sulfonyl fluoride prior to chromatography on heparin-Sepharose. This study shows the presence of milk proteases which co-purify and degrade lipoprotein lipase. We suggest that this degradation could account for part of the known instability of the enzyme.

Animals↗
PubMed1985

Lipoprotein lipase-catalyzed hydrolysis of phospholipid monolayers: effect of fatty acyl composition on enzyme activity.

The phospholipase A1 activity of lipoprotein lipase (LpL) was determined with monomolecular phospholipid films. Rates of phospholipid hydrolysis were dependent on apolipoprotein C-II (the activator protein for LpL) phospholipid fatty acyl composition, and lipid-packing density. In sphingomyelin: cholesterol (2:1, molar) monolayers containing 5 mol % disaturated phosphatidylcholines (PC) and at a surface pressure of 22 mNm-1, rates of LpL hydrolysis of diC14:0PC, diC16:0PC, and diC18:0PC were 74, 207, and 65 nmol h-1 mg LpL-1, respectively. At 22 mNm-1, phospholipids containing unsaturated fatty acyl chains were hydrolyzed at rates 5-10 times greater than saturated lipids. At higher lipid packing densities, the difference in hydrolysis rates between saturated and unsaturated lipids was less apparent. Comparison of molecular areas indicate no simple dependency between the rate of LpL catalysis and phospholipid fatty acyl chain length and saturation/unsaturation.

Apolipoprotein C-II↗
PubMed1985

Nanosecond motions of the single tryptophan residues in apolipoproteins C-I and C-II: a study by oxygen quenching and fluorescence depolarization.

Rotational freedom of the single tryptophan residue in human plasma apolipoproteins C-I (apo C-I) and C-II (apo C-II) was investigated by oxygen quenching and lifetime-resolved anisotropies. The tryptophan in both apo C-I and C-II was highly accessible to oxygen quenching. The tryptophan residue in both apo C-I and C-II and their sodium dodecyl sulfate (SDS) or dimyristoylphosphatidylcholine (DMPC) complexes displayed significant motional freedom on the nanosecond time scale. Lifetime-resolved anisotropies of tryptophan residues under conditions of oxygen quenching revealed an increase in the amplitude of the segmental motions at 40 degrees C as compared to that at 5 degrees C. It was concluded from these studies that both the apoprotein C-I and C-II are highly flexible molecules, and that the nanosecond motions of the tryptophan residue are sensitive to the fluidity of its environment in both SDS and DMPC complexes.

Apolipoprotein C-I↗
PubMed1985

Preparation of a homogeneous and stable form of bovine milk lipoprotein lipase.

Lipoprotein lipase was purified to homogeneity from bovine skim milk by a two-step procedure using chromatography on heparin-Sepharose. As determined by gradient-polyacrylamide gel electrophoresis in sodium dodecyl sulfate, purified lipoprotein lipase showed a single band with an apparent molecular weight of 55,000. The use of Triton N-101 in the washing buffers was the major improvement from previously reported purification procedures that resulted in a stable homogeneous preparation of the enzyme.

Animals↗
PubMed1985

Comparison of gradient gel electrophoresis and zonal ultracentrifugation for quantitation of high density lipoproteins.

The study was conducted to compare gradient gel electrophoresis (GGE) and zonal ultracentrifugation for quantitation of human plasma high density lipoproteins (HDL). Plasma samples were obtained from seven normal subjects consuming a high fat diet (65% total calories) followed by a high carbohydrate diet (65% total calories). HDL were fractionated into HDL2 and HDL3 by zonal ultracentrifugation and lipid and protein mass were determined. HDL were also fractionated by GGE and the results were compared to the zonal method. Zonally isolated HDL2 represented a homogeneous particle population that was equivalent to HDL2b as determined by GGE. By the zonal method, HDL2 accounted for 27 +/- 4% (mean +/- SEM) of total HDL mass in subjects on the high fat diet as compared to 16 +/- 2% in subjects fed the high carbohydrate diet; by GGE, the HDL2b values were 27 +/- 4% and 14 +/- 1%, respectively. The coefficient of correlation (n = 25) for the two methods was 0.894 (P less than 0.001).

Adult↗
PubMed1984

Specificity of cholecystokinin antibody may influence choice of tracer for radioimmunoassay.

When CCK33 was iodinated at His 20 with lactoperoxidase, the labelled hormone was less immunoreactive under radioimmunoassay conditions than CCK39 which is readily iodinated at Tyr 1. Since the difference in immunoreactivities could not be due to different degrees of oxidative damage, the regional specificity of the assay antibody (UT122, from the laboratory of J.C. Thompson, University of Texas) was re-examined. A synthetic parital sequence, CCK33 6-16, which had similar conformation to the same sequence in the intact peptide, as shown by CD analysis, was devoid of immunoreactivity. Results with sulphated and non-sulphated C-terminal octapeptides of the hormone, CCK8 (CCK33 27-33), indicate limited dependence of the C-terminal region for binding to antibody. Thus, the lower binding with 125I-CCK33 than with 125I-CCK39 indicates that His 20 is an important feature of the immunogenic site and that the iodine atom is large enough to cause steric hindrance. This explains why the method of iodination might have to be varied depending on the regional specificity of antibodies.

Amino Acid Sequence↗
PubMed1984

Lipoprotein lipase- and phospholipase A2-catalyzed hydrolysis of phospholipid vesicles with an encapsulated fluorescent dye. Effects of apolipoproteins.

The self-quenching dye, 6-carboxyfluorescein, has been encapsulated into sonicated vesicles of egg phosphatidylcholine. Porcine pancreatic phospholipase A2 and bovine milk lipoprotein lipase catalyze the hydrolysis of the phosphatidylcholine resulting in the release of the encapsulated dye and a large increase in 6-carboxyfluorescein fluorescence. The fluorescence increase occurs in parallel with the formation of lysophosphatidylcholine and is strongly dependent on Ca2+ for phospholipase A2 catalysis and on apolipoprotein C-II for hydrolysis by lipoprotein lipase. Other apolipoproteins, including apolipoproteins C-III, C-I, and A-I, do not enhance lipoprotein lipase activity towards this substrate. We conclude that the enhancement of lipoprotein lipase activity by apolipoprotein C-II is a specific property of the activator protein due to its interaction with lipoprotein lipase or an enzyme/lipid interface and not a characteristic of lipid-binding proteins in general.

Animals↗
PubMed1984

Comparison of acyl-oxyester and acyl-thioester lipids as substrates for bovine milk lipoprotein lipase.

The dithioester analog of dihexanoylphosphatidylcholine, rac-1,2-S,S-dihexanoyl-3-phosphocholine-1,2-dimercapto-3-propanol was synthesized and compared to the corresponding acyl-oxyester lipid as a substrate for bovine milk lipoprotein lipase. The apparent maximal reaction velocity (Vmax) for dihexanoyldithiophosphatidylcholine was considerably lower than that for dihexanoylphosphatidylcholine (0.12 vs. 5.0 mumol product released/min per mg lipoprotein lipase, respectively). The apparent Km values were 1.9 and 4.0 mM, respectively. 3-Butyrylthio-1,2-dibutyryloxypropane was also compared to tributyrylglycerol as a substrate for lipoprotein lipase; hydrolysis of the acyl-thioester bond was insignificant when compared to the corresponding oxyester derivative. Apolipoprotein C-II, the activator protein of lipoprotein lipase for long-chain fatty acyl substrates, had no effect on the hydrolysis of either the thio- or oxyester short-chain substrates. The low lipoprotein lipase activity for the thioester substrates is discussed in relation to the structure of the lipid and the active site of the enzyme.

Animals↗
PubMed1984

Effect of calcium-channel-blocking drugs on lysosomal function in human skin fibroblasts.

Recent reports suggest that certain Ca2+-channel-blocking drugs reduce the severity of atherosclerosis in cholesterol-fed animals. To determine whether the suppression of atherogenesis is related to altered lipoprotein metabolism, we have assessed the effects of these drugs on the catabolism of plasma low density lipoproteins (LDL) by human skin fibroblasts. The Ca2+-channel-blocking drugs verapamil and diltiazem inhibit the lysosomal degradation of LDL by these cells; degradation of epidermal growth factor was also inhibited by the same drugs, suggesting a general effect of these drugs on lysosomal function. In contrast, nifedipine did not affect the degradation of LDL or epidermal growth factor. None of the drugs affected phospholipid or protein synthesis. Entry of LDL into the lysosomes also was not affected. [3H]Diltiazem, which inhibited LDL degradation, accumulated in the lysosome-rich fraction, whereas [3H]nimodipine, a drug structurally and functionally similar to nifedipine, did not accumulate. We suggest that the inhibitory effect of some of the Ca2+-channel-blocking drugs on lysosomal function is due to their basic nature, causing them to accumulate in lysosomes, thereby increasing intralysosomal pH.

Calcium Channel Blockers↗
PubMed1984

Modification of apolipoprotein C-II with 1,2-cyclohexanedione and 2,3-butanedione. Role of arginine in the activation of lipoprotein lipase.

Apolipoprotein C-II, the activator protein of lipoprotein lipase, contains 78 amino acids with a single residue of arginine at position 49. Chemical modification of apolipoprotein C-II with 1,2-cyclohexanedione or 2,3-butanedione results in a loss of both the arginine residue and the ability of the protein to enhance the activity of bovine milk lipoprotein lipase toward a trioleoylglycerol substrate; removal of the modifying group restores arginine and more than 70% of the activating property of the apolipoprotein. Arginine modification of apolipoprotein C-II does not effect its lipid-binding properties as assessed by its association to sonicated vesicles of dimyristoylphosphatidylcholine. Furthermore, secondary structure associated with complex formation with dimyristoylphosphatidylcholine are nearly identical for the unmodified, 1,2-cyclohexanedione-modified or modified-reversed proteins. These results suggest that arginine-49 of apolipoprotein C-II is situated at or near an amino acid sequence domain involved in the activation of lipoprotein lipase. However, a guanidinium group is not required for lipid binding.

Animals↗
PubMed1984

Effect of apolipoprotein C-II on the temperature dependence of lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholines. A hydrophobic model for the mechanism.

The lipoprotein lipase-catalyzed hydrolysis of diacylphosphatidylcholines (PC) in mixed micelles of Triton X-100/PC was studied as a function of temperature in the presence and absence of apolipoprotein C-II (apo-C-II), the activator protein for lipoprotein lipase. Dilauroyl-, dimyristoyl-, dipalmitoyl-, and distearoyl-phosphatidylcholine (di-C12-PC, di-C14-PC, di-C16-PC, and di-C18-PC, respectively) were used as substrates. No systematic relationship between substrate fatty acyl chain length and either the rates of the activation energies for hydrolysis in the presence or absence of apo-C-II was observed. However, there was a linear relationship between fatty acyl chain length and both the logarithm of the activation factor (the ratio of enzyme activity with apo-C-II to that without apo-C-II) and the difference in activation energy in the presence and absence of apo-C-II. These relationships were not the result of an alteration in the physical form of the substrate, since a mixture of di-C14-PC and di-C16-PC gave activation factors for each PC which were the same as those obtained for each individual lipid. From the temperature dependence of the activation factor, thermodynamic functions of the apo-C-II-induced change in the reaction pathway were calculated. The free energy of activation decreased linearly with increasing chain length as the result of a linear increase in activation entropy which more than offset the unfavorable increase in activation enthalpy. We propose that the apo-C-II-mediated increase in the rate of the lipoprotein lipase-catalyzed hydrolysis of phosphatidylcholine is associated with transfer of a fatty acyl chain of the substrate or product to a more hydrophobic environment within the transition state complex.

Animals↗
PubMed1984

Degradation of apolipoprotein B-100 of human plasma low density lipoproteins by tissue and plasma kallikreins.

Human plasma low density lipoproteins (LDL) contain one major apoprotein of apparent Mr = 550,000 designated apolipoprotein B-100 (apo-B-100) and in some LDL preparations, minor components termed apo-B-74 (Mr = 410,000) and apo-B-26 (Mr = 145,000). The structural and metabolic relationships among these LDL apoproteins remain obscure. In the present study, we show that the mixing of proteolytic inhibitors with blood at the moment of collection prevents the appearance of apo-B-74 and -26 in plasma LDL indicating that these peptides are derived by proteolytic degradation of apo-B-100. In order to simulate the degradation in vitro, LDL were digested with plasmin, trypsin, chymotrypsin, thrombin, and tissue and plasma kallikreins and the degradation products analyzed by polyacrylamide gradient gel electrophoresis. While plasmin, trypsin, and chymotrypsin caused extensive degradation of apo-B-100, thrombin, and tissue and plasma kallikreins generated limited cleavage patterns. LDL digested with thrombin contained stoichiometric amounts of two peptides with apparent Mr = 385,000 and 170,000. Mixing experiments showed that the thrombin-derived peptides of apo-B-100 did not co-migrate with apo-B-74 and B-26 during electrophoresis indicating that these peptides were different. In contrast, LDL digested with kallikrein contained stoichiometric amounts of two peptides with apparent molecular weights identical to apo-B-74 and -26. Together, the above results indicate that apo-B-74 and -26 are degradation products of apo-B-100 and are not produced by the action of thrombin. Whether the expression of a kallikrein-like activity in vivo accounts for the specific degradation of LDL B-100 to yield LDL B-74 and -26 remains to be determined.

Apolipoproteins↗
PubMed1984

Lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine. Effect of lipid organization and apolipoprotein C-II on enzyme activity.

The effect of phospholipid organization on the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine was examined with sonicated vesicles and Triton X-100 or lysomyristoylphosphatidylcholine solubilized lipid. Triton X-100-dimyristoylphosphatidylcholine substrates were prepared at various ratios of detergent to phospholipid so as to produce lipid structures varying from bilayers to micelles. Apolipoprotein C-II, the activator protein for lipoprotein lipase, enhanced the rate of the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine for each substrate tested. Although the absolute rate of lipoprotein lipase catalysis was different for each, the factor (the ratio of lipoprotein lipase activity with apolipoprotein C-II to that without the activator protein) was nearly constant, with a value of approximately 16. We conclude that the enhancement of lipoprotein lipase activity by apolipoprotein C-II is independent of the physical form of the phospholipid substrate.

Animals↗
PubMed1984

Reactivity of heparin with the human plasma heparin-binding proteins thrombin, antithrombin III, and apolipoproteins E and B-100.

The heparin-binding properties of human plasma apolipoproteins B-100 and E (apoB-100 and E) of low density lipoproteins (LDL), thrombin, and antithrombin-III (AT-III) were investigated. A highly reactive heparin (HRH) to apoB-100 was isolated by chromatography of crude heparin on a column of LDL immobilized to Affi-Gel 10. This HRH showed a high, Ca2+-dependent precipitating activity towards LDL; 1 microgram HRH uronic acid precipitated 50-70 micrograms LDL-protein. HRH was fractionated further by chromatography on a column of AT-III bound to concanavalin A-Sepharose. The unretained fraction of heparin (HRH1) had a low affinity for AT-III. The bound heparin (HRH2) had a high affinity for AT-III and precipitated LDL in the presence of Ca2+. To assess further their heparin-binding properties, the proteins were subjected to gradient-gel electrophoresis under denaturing conditions, transferred to nitrocellulose by electrophoresis, and then assayed for their ability to bind [125I]-labeled HRH2. Autoradiographic analysis showed that thrombin, apolipoproteins E and B-100, and the AT-III . thrombin covalent complex bound HRH2. Denatured AT-III did not bind HRH2, indicating that its heparin recognition site may depend on conformation.

Antithrombin III↗
PubMed1984

Effect of monolayer lipid structure and composition on the lipoprotein lipase-catalyzed hydrolysis of triacylglycerol.

The effect of lipid composition and structure on the lipoprotein lipase-catalyzed hydrolysis of triacylglycerols was determined in a monolayer system consisting of purified bovine milk lipoprotein lipase and fatty acid-free albumin. In a monolayer of dioleoylphosphatidylcholine containing 1-6 mol% of either tri[14C]oleoylglycerol or tri[14C] palmitoylglycerol , lipoprotein lipase catalyzed the hydrolysis of the unsaturated triacylglycerol at a higher rate than the saturated lipid and in either the presence or absence of apolipoprotein C-II, the activator protein for the enzyme. For example, with 3 mol% triacylglycerol and in the presence of apolipoprotein C-II, the rate of the lipoprotein lipase-catalyzed hydrolysis of tri[14C]oleoylglycerol was 27 mumol oleic acid produced/h per mg enzyme vs. 12 mumol for tri[14C] palmitoylglycerol . The effect of phospholipid fatty acyl chain length and unsaturation/saturation, polar head group and surface density on the lipoprotein lipase-catalyzed hydrolysis of tri[14C]oleoylglycerol was determined. The rate of enzyme hydrolysis of triacylglycerol was similar whether the phospholipid was a diester or diether lipid or the polar head group was ethanolamine or choline. In general, phospholipids with shorter and unsaturated fatty acyl chains gave higher rates of lipoprotein lipase hydrolysis of triacylglycerol than the corresponding longer and saturated lipids. However, with all phospholipids tested, the rate of enzyme hydrolysis decreased with increasing surface density. Lipoprotein lipase showed no activity toward triacylglycerol in a monolayer of sphingomyelin; addition of dioleoylphosphatidylcholine to the monolayer enhanced the rate of enzyme catalysis. Cholesterol (50 mol%) in a dipalmitoylphosphatidylcholine monolayer increased the rate of the lipoprotein lipase-catalyzed hydrolysis of tri[14C]oleoylglycerol, whereas cholesterol decreased the rate in a dioleoylphosphatidylcholine monolayer. The effect of phospholipid structure and surface density on lipoprotein lipase activity could not be accounted for by the amount of apolipoprotein C-II which was present at the interface. Based on these findings and other reports in the literature, we suggest that the catalytic activity of lipoprotein lipase toward tri[14C] oleylglycerol in various monolayers is dependent on the conformation or appropriate physical state of the triacylglycerol substrate at the lipid interface.

Animals↗
PubMed1984

Hydrolysis of a fluorescent phospholipid substrate by phospholipase A2 and lipoprotein lipase.

The fluorescent phospholipid 1-acyl-2-[6-[(7-nitro-2,1,3benzoxadiazol-4 -yl) amino]-caproyl] phosphatidylcholine (C6-NBD-PC) was used as a substrate for porcine pancreatic phospholipase A2 (PA2) and bovine milk lipoprotein lipase (LpL). Hydrolysis of C6-NBD-PC by either enzyme resulted in a greater than 50-fold fluorescence enhancement with no shift in the emission maximum at 540 nm; Ca++ was required for PA2 catalysis. Identification of the products of hydrolysis showed cleavage at the sn-1 and sn-2 positions for LpL and PA2, respectively. For PA2, but not for LpL, there was a marked enhancement of enzyme catalysis at lipid concentrations above the critical micellar concentration of the lipid. Furthermore, apolipoprotein C-II, the activator protein of LpL for long-chain fatty acyl substrates, did not enhance the rate of catalysis of the water-soluble fluorescent phospholipid for either enzyme.

Apolipoprotein C-II↗