Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism最新文献

In plasma, the bulk of apoSAA, a positive acute phase reactant protein, is transported in high density lipoproteins (HDL), especially HDL_H (apoA₁-rich HDL). In this study we tested whether apoA₁ deficiency would adversely affect apoSAA concentration and lipid distribution in mouse plasma lipoproteins. Acute phase response (APR) was induced in C57BL/6J (apoA₁^+/+) and apoA₁-knockout mice (apoA₁^−/−) by a subcutaneous injection of silver nitrate. The APR increased cholesterol concentrations in LDL of apoA₁^−/− mice and apoA₁^+/+ mice in a like manner. In contrast to apoA₁^+/+ mice, concentrations of cholesterol, phospholipids and proteins in both HDL_L (1.063<d<1.103 g/ml) and HDL_H (1.103<d<1.21 g/ml) were significantly increased by the APR in apoA₁^−/− mice. Total concentration of plasma apoSAA and its distribution in lipoprotein fractions was similar in both APR groups. The bulk of plasma apoSAA was contained in HDL and not in VLDL or LDL even when the HDL concentration was low. In apoA₁^−/− mice, HDL_L and HDL_H contained more apoSAA than in apoA₁^+/+ mice. These results indicate that apoA₁^−/− mice are not deterred from mounting an apoSAA response similar to apoA₁^+/+ mice and that apoA₁-rich HDL particles are not necessary for apoSAA transport in the plasma.

Fatty acid α-oxidation in cucumber (Cucumis sativus) involves enzymatic conversion of long-chain C_n-fatty acids to the C_(n−1)-aldehyde and CO₂. However, the mechanism of this process is not well understood. In this study, the α-oxidation of the fatty acid analogues tetradecylthioacetic acid (TTA) and tetradecylthiopropionic acid (TTP) with a sulphur atom substituting the methylene group in positions 3 and 4, respectively, was investigated and compared to palmitic acid. Both [1-¹⁴C]TTA and [1-¹⁴C]TTP could be α-oxidised in the cucumber subcellular 150 000×g_max fraction. [1-¹⁴C]TTP was an even better substrate compared to the natural palmitic acid, while [1-¹⁴C]TTA was α-oxidised to a lower extent. [2-¹⁴C]TTA revealed no ¹⁴CO₂, indicating that only one cycle of α-oxidation occurred. TTA was an inhibitor of the palmitic acid α-oxidation, and the inhibitory effects were examined.

Triacylglycerol hydrolase activities were characterised in homogenates, cytosol, and microvillous membranes (MVM) of human placenta. Homogenates of placenta exhibited three distinct triacylyglycerol hydrolase activities with pH optima 4.5, 6.0 and 8.0. On further fractionation, placental cytosol exhibited both acid cholesterol ester hydrolase (pH 4.5) and hormone sensitive lipase (pH 6.0) activities, whereas purified placental MVM exhibited two distinct triacylyglycerol hydrolase activities; a minor activity at pH 8.0 and a second major activity at pH 6.0. Triacylglycerol hydrolase activity at pH 8.0 of MVM appeared to be lipoprotein lipase (consistent with criteria such as serum stimulation and salt inhibition), whereas at pH 6.0 the activity was unique in that it was almost abolished by serum, but was not affected by high NaCl concentrations. Our data, for the first time, demonstrate that human placental MVM, in addition to lipoprotein lipase, contain a newly identified triacylglycerol hydrolase activity at pH 6.0.

We explored the possibility that the biliary protein fraction may support part of the variation in the nucleating activity previously measured in gallbladder biles of pigs. Eighteen gallbladder aspirates freshly obtained from three dietary groups (0, 5, or 10% β-cyclodextrin) of six pigs were chromatographed to purify their total protein fraction. Proteins were quantified, and analysed through electrophoresis and immunoblotting or enzyme-linked immunosorbent assay for albumin, and five putative effectors of cholesterol crystallisation, mucins, immunoglobulin A, 130 kDa, apolipoprotein A-I, and anionic polypeptide fraction. Each total protein fraction was also assayed for its ability to influence cholesterol precipitation, when added to supersaturated model bile. The current data provided evidence that the cholesterol crystallisation-promoting activity of biliary proteins in model biles increased with the β-cyclodextrin dietary content. This occurred without any significant change in the total biliary protein content, but was associated with a significant decrease in the concentration of albumin and apolipoprotein A-I, resulting in changes in the overall balance of proteins in bile. Comparison of these results with the crystallisation figures previously obtained from the corresponding native biles led us to conclude that biliary proteins might influence the outcome of the crystallisation process, namely the final crystal concentration at equilibrium, but would not systematically represent a major driving force for determining the velocity of crystal formation in native bile of pigs.

CBA/J and other inbred strains of mice that express the amyloidogenic apolipoprotein serum amyloid A (apoSAA) apoSAA₂, together with apoSAA₁, are susceptible to amyloid A (AA) amyloidosis, whereas CE/J mice that express a single unique isoform, apoSAA_CEJ, are resistant. Studies indicate that CBA/J×CE/J hybrid mice that express apoSAA₂ in the presence of apoSAA_CEJ are protected from amyloidogenesis. To define a mechanism by which expression of apoSAA_CEJ may protect from AA formation in the presence of apoSAA₂, binding of recombinant apoSAA (r-apoSAA) isoforms, validated by N-terminal sequencing, to a murine macrophage cell line was investigated. Maximal specific binding occurred after incubation of radiolabeled apoSAA with IC-21 macrophages (1×10⁵ cells/ml) for 30 min at 4°C. The binding of ¹²⁵I-r-apoSAA₁, ¹²⁵I-r-apoSAA₂ and ¹²⁵I-r-apoSAA_CEJ was specific and saturable, with an affinity (K_d) of about 2.8, 3.2 and 1.3 nM, respectively, and approximately 2–4×10⁶ sites per cell. Competitive binding experiments indicate apoSAA_CEJ binds with higher affinity to macrophages than does either apoSAA₁ or apoSAA₂. We suggest that greater cellular affinity of apoSAA_CEJ compared to apoSAA₂ may contribute to protection from AA amyloid in certain CBA/J×CE/J hybrid mice by interfering with interaction of apoSAA₂ by macrophages and hence either membrane associated or intracellular degradation.

The cho1/pss mutant of Saccharomyces cerevisiae, which is auxotrophic for choline or ethanolamine because of the deficiency in phosphatidylserine synthesis, grew in the presence of 0.05 mM phosphatidylcholine (PC) with octanoic acids (diC8PC) or decanoic acids (diC10PC), but not in the presence of PC with longer acyl residues. It did not grow in the presence of the soluble hydrolytic products of PC, phosphorylcholine or glycerophosphorylcholine, at comparable concentrations. Addition of 10 mM hemicholinium-3, a choline transport inhibitor, or disruption of the CTR gene, which encodes a choline transporter, inhibited the growth of the cho1/pss mutant in the presence of choline, but not in the presence of 0.1 mM diC8PC. Under diC8PC-supported growth conditions, octanoic acid was barely detectable in the cellular phospholipid fraction, but was recovered in the culture medium as the free acid, and the phosphatidylethanolamine (PE) content was low in comparison to the choline-supported conditions. These results suggest that PCs with short acyl residues were taken up by the cho1/pss mutant and remodeled as they were used, and that PCs with short acyl residues do not inhibit conversion of PE to PC. The current results provide a new direction in the analysis of intracellular phospholipid movement and metabolism in yeast.

The protein, p100, was previously identified as a G-protein related protein that cycles on and off the cytoplasmic face of the endosome membrane (Traub et al., Biochem. J. 280 (1991) 171–178). Here we present evidence that the inositol polyphosphates, inositol 1,4,5-trisphosphate (IP₃) and inositol hexakisphosphate (IP₆), release p100 from light-density microsomal membranes and inhibit rebinding of p100 through receptors, which are specific for IP₃ or for IP₆. These receptors can be co-extracted with p100 from the microsomes by 0.5 M Tris-HCl and, in the soluble state, they exhibit similar binding activity towards the inositol polyphosphates as do untreated microsomes. Soluble p100 self-aggregates and this aggregation is blocked by both IP₃ and IP₆. Stimulation of permeabilized rat basophilic leukemia (RBL-2H3) cells with carbachol, via transfected muscarinic m1 receptors, results in increased levels of inositol polyphosphates and the quantitative release of p100 into the cytosol. This effect is reversible and cytosolic p100 rebinds to the membrane as the levels of inositol polyphosphates decline. These findings suggest that p100 may belong to a family of IP-binding proteins whose intracellular localization is determined by extracellular signals.

Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains. They are present in all groups of organisms, i.e., bacteria, fungi, plants and animals, and play a key role in the maintenance of the proper structure and functioning of biological membranes. The desaturases are characterized by the presence of three conserved histidine tracks which are presumed to compose the Fe-binding active centers of the enzymes. Recent findings on the structure and expression of different types of fatty acid desaturase in cyanobacteria, plants and animals are reviewed in this article. Roles of individual desaturases in temperature acclimation and principles of regulation of the desaturase genes are discussed.

Group V phospholipase A₂ (GV-PLA₂) has been shown to be involved in signal transduction and inflammatory processes in cellular studies, but the physical and biochemical properties of this important enzyme have been unclear. We report the over-expression and characterization of GV-PLA₂. The GV-PLA₂ cDNA was synthesized from human heart polyA⁺ mRNA by RT-PCR, and an expression construct containing the GV-PLA₂ was established. After expression in Escherichia coli cells, the protein was solubilized and purified to homogeneity in a single step using nickel affinity chromatography. The purified GV-PLA₂ protein was folded to form active enzyme. The recombinant GV-PLA₂ has an absolute requirement for Ca²⁺ for enzymatic activity. The optimum pH for this enzyme is pH 8.5 in Tris-HCl buffer with sonicated vesicles as substrate. GV-PLA₂ preferentially hydrolyzes phosphatidylethanolamine (PE) vesicles compared to phosphatidylcholine (PC) vesicles. However, hydrolysis of PC and PE is equivalent in mixed vesicles of the phospholipids. The fatty acid preference of GV-PLA₂ is linoleoyl>palmitoyl>arachidonyl with a PC head group and sonicated vesicles. 3-(3-Actamide-1-benzyl-2-ethylindolyl-5-oxy)propane phosphonic acid (LY311727), a potent inhibitor of human group IIA PLA₂, strongly inhibits GV-PLA₂ with an IC₅₀ value of about 36 nM which is comparable to its inhibition of group IIA PLA₂.