Deuterium magnetic resonance study of the interaction between chondroitin sulfate and human plasma low density lipoproteins

[²H]Chondroitin sulfate was prepared by partial $\text{N}$-deacetylation of chondroitin sulfate ($\text{via}$ hydrazinolysis) followed by treatment with [²H₆]acetic anhydride. ²H NMR spectra of [²H]chondroitin sulfate in the presence of human plasma low density lipoprotein provide evidence for a soluble complex stoichiometry of 3 (and possibly 2) lipoproteins per polysaccharide molecule, and allow a rough estimation of the dissociation constant K_d.     

Interaction of plasma apolipoproteins with lipid monolayers

The monolayer technique has been used to study the interaction of lipids with plasma apolipoproteins. Apolipoprotein C-II and C-III from human very low density lipoproteins, apolipoprotein A-I from human high density lipoproteins and arginine-rich protein from swine very low density lipoproteins were studied. The injection of each apoprotein underneath a monolayer of egg phosphatidyl[¹⁴C]choline at 20 mN/m caused an increase in surface pressure to approximately 30 mN/m. With apolipoprotein C-II and apolipoprotein C-III there was a decrease in surface radioactivity indicating that the apoproteins were removing phospholipid from the interface; the removal of phospholipid was specific for apolipoprotein C-II and apolipoprotein C-III. Although there was a removal of phospholipid from the monolayer, the surface pressure remained constant and was due to the accumulation of apoprotein at the interface. The rate of surface radioactivity decrease was a function of protein concentration, required lipid in a fluid state and, of the lipids tested, was specific for phosphatidylcholine. Cholesterol and phosphatidylinositol were not removed from the interface. The addition of 33 mol% cholesterol to the phosphatidylcholine monolayer did not affect the removal of phospholipid by apolipoprotein C-III.The addition of phospholipid liposomes to the subphase greatly facilitated the apolipoprotein C-II-mediated removal of phospholipid from the interface.     

Effects of microtubule-disruptive and membrane-stabilizing agents on low density lipoprotein metabolism by cultured human fibroblasts

The cellular mechanisms involved in the uptake and metabolism of low density lipoprotein (LDL) by cultured normal human fibroblasts have been investigated with the aid of drugs known to disrupt cytoplasmic microtubules or to inhibit membrane fusion.Two drugs which disrupt microtubules by differing mechanisms, colchicine and vinblastine, each reduced the high affinity surface binding of ¹²⁵I-labelled LDL by fibroblasts. Associated reductions of the endocytosis and degradation of the lipoprotein could be attributed almost entirely to this effect. In contrast, lumicolchicine, an analogue of colchicine without microtubule-disruptive activity, had little or no effect on ¹²⁵I-labelled LDL metabolism.Each of two groups of membrane-stabilizing agents, the phenothiazines and the tertiary amine local anaesthetics, directly inhibited both the internalization of ¹²⁵I-labelled LDL following high affinity binding to cell surface receptors and the catabolism of the lipoprotein subsequent to endocytosis, supporting previous morphological evidence for the importance of membrane fusion in these processes.     

Hepatic and extra-hepatic glutathione-S-transferase activity in wild pigeons (Columba livia)

Glutathione-S-transferase (EC 2.5.1.18) activity was assayed in hepatic and extra-hepatic tissues of pigeons using l-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Gluthathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene in pigeon was in the order: kidney > liver > testes > brain > lung> heart. The enzyme activity with 1-chloro-2,4-dinitrobenzene as substrate was 40–44 times higher in pigeon liver and kidney than that observed with 1,2-dichloro-4-dinitrobenzene as substrate.K _m values of hepatic and renal glutathione transferase with l-chloro-2,4-dinitrobenzene as substrate were 2.5 and 3 mM respectively. Double reciprocal plots with varying reduced gluthathione concentrations resulted in biphasic curves with twoK _m values (liver 0.31 mM and 4mM; kidney 0.36 mM and 1.3 mM). The enzyme activity was inhibited by oxidized gluthathione in a dose-dependent pattern. 3-Methylcholanthrene elicited about 50% induction of hepatic glutathione transferase activity whereas phénobarbital was ineffective.     

Structure analysis and molecular model of the selenoenzyme glutathione peroxidase at 2.8 Å resolution

The three-dimensional structure of bovine erythrocyte glutathione peroxidase, a tetrameric enzyme containing 4 gram atoms of selenium per mole (M_r = 84,000), has been determined at 2.8 Å resolution using the multiple isomorphous replacement method. By correlation calculations in Patterson space the tetramers were shown to exhibit molecular [222] symmetry, proving the monomers to be identical or at least very similar.The monomer consists of a single polypeptide chain of 178 amino acid residues. Its shape is nearly spherical with a radius of $\text{r ≈ 19}\text{A}\text{?}$. A tentative sequence corresponding to a partially refined model (R = 0.38) is given. Each subunit is built up from a central core of two parallel and two anti-parallel strands of pleated sheet surrounded by four α-helices. One of the helices runs antiparallel to the neighbouring β-strands giving rise to a βαβ substructure, an architecture that has been found in several other proteins e.g. flavodoxin, thioredoxin, rhodanese and dehydrogenases. A comparison of the glutathione peroxidase subunit structure with thioredoxin-S₂ revealed large regions of structural resemblance. The central four-stranded β structure together with two parallel α-helices resembles nearly 80% of the thioredoxin fold.The active sites of glutathione peroxidase are located in flat depressions on the molecular surface. Probably each active centre is built up by segments from two subunits. The catalytically active selenocysteines were found at the N-terminal ends of long α-helices and are surrounded by an accumulation of aromatic side-chains. A difference Fourier map between oxidized and substrate-reduced glutathione peroxidase as well as heavy-atom binding led to the conclusion that the two-electron redox-cycle involves a reversible transition of the active-site selenium from a selenenic acid (RSeOH) to a seleninic acid (RSeOOH).     

Epinephrine potentiates activation of human platelets by low density lipoproteins

Low density lipoproteins activate phosphoinositide turnover, increase free cytoplasmic calcium concentration and stimulate phosphorylation of 20- and 47-kDa proteins in blood platelets. All these effects are substantially potentiated by epinephrine.     

On the mechanism of photosynthetic electron transfer in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-(n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b₅₀ and cytochrome b_?90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q^?_I) and the secondary acceptor ubiquinone (Q_II) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b₅₀ reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b₅₀ reduction increased about 10-fold as the E_h(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with E_m(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b₅₀ reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q^?_I and Q_II. It was an effective inhibitor of cytochrome b₅₀ photoreduction at E_h(7.0) 390 mV, but not at E_h(7.0) 100 mV. At the latter E_h, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q^?_I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At E_h(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b₅₀ reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b₅₀ oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high E_h values. However, at lower redox potentials, cytochrome b₅₀ oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b_?90 in the rapid reactions of the cyclic electron transfer chain     

Synthesis of two forms of apolipoprotein B by cultured rat hepatocytes

Cultured rat hepatocytes were used to demonstrate that the liver can synthesize two forms of apolipoprotein B. Separation of apolipoprotein B by disc gel electrophoresis indicated that hepatocyte low density lipoprotein contains predominantly apolipoprotein B with an apparent molecular weight of 345,000 ± 5,055. In contrast, the major apolipoprotein B component of hepatocyte very low density lipoprotein is a variant form with a molecular weight of 242,000 ± 2,720. Hepatocyte high density lipoprotein, unlike plasma HDL, also contains apolipoprotein B with an apparent molecular weight of 244,000 ± 2,742. Incorporation of [³H] leucine into hepatocyte apolipoprotein B components suggested de novo synthesis.     

Distribution of chylomicron degradation products in the isolated,perfused rat heart

Chylomicron degradation by hearts from fed and fasted rats was studied using a perfusion technique, which allows the separate collection of coronary (Q_rv) and interstitial effluent (Q_i). Upon perfusion with [³H]-cholesterol-containing chylomicrons the tissue recovery of label was highest in the fasted state, while label recovered in Q_i was highest in the fed state. Density gradient centrifugation of Q_i indicated that the label was recovered in lipoproteins with higher densities: low density lipoproteins (1.019<d<1.050), high density lipoproteins (1.050<d<1.21) and a fraction of d>1.21. These particles probably represent chylomicron degradation products (remnants and “surface fragments”). Our results indicate that tissue cholesterol uptake during chylomicron degradation may be inhibited in the fed state. Furthermore, the role of the myocyte (or interstitial) lipoprotein lipase in chylomicron degradation is discussed.