Reaction of Lecithin Cholesterol Acyltransferase with Water-soluble Substrates

Bonelli, Frank S.; Jonas, Ana

doi:10.1016/s0021-9258(18)63758-5

Cited by 48 publications

(5 citation statements)

References 35 publications

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“…Calculation of recoveries was impossible because the total activity increased during the gel filtration step, perhaps due to the removal of an inhibitor. The specific activity of the purified enzyme (Tables II and III) was comparable to the activity of the A. salmonicida GCAT we isolated earlier (Buckley et al, 1982) and several orders of magnitude higher than the activity of purified LCAT (Bonelli & Jonas, 1989). Upon electrophoresis, nearly all of the protein ran as a single band of 36.0 kDa.…”

Section: Resultsmentioning

confidence: 54%

“…Another determined the production of glycerol phosphate produced by the transfer of fatty acids from the sn-2 position of phospholipids in erythrocyte ghost membranes, followed by hydrolysis of the sn-1 ester linkages (MacIntyre & Buckley, 1978). Hydrolysis of the monodisperse substrate p-nitrophenyl butyrate was also measured as described (Bonelli & Jonas, 1989). CD Measurements.…”

Section: Methodsmentioning

confidence: 99%

“…The microbial enzyme does not appear to distinguish between the sn-2 and sn-1 positions for hydrolysis, whereas LCAT shows some preference for the sn-2 position (Aron et al, 1973). Both enzymes will catalyze acyl transfer from a variety of other donors, including p-nitrophenyl esters (Buckley, 1983;Bonelli & Jonas, 1989).…”

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confidence: 99%

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Purification and spectral study of a microbial fatty acyltransferase: activation by limited proteolysis

Hilton¹,

McCubbin²,

Kay³

et al. 1990

Biochemistry

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A fatty acyltransferase with a reaction mechanism similar to that of mammalian lecithin: cholesterol acyltransferase has been purified from culture supernatants of a mutant Aeromonas salmonicida containing the cloned Aeromonas hydrophila structural gene. Typically, more than 35 mg of protein were isolated from 2 L of culture supernatant. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein corresponded to predictions based on the sequence of the gene, indicating that the signal sequence had been correctly removed during export but that no further processing had occurred. Analysis of the far-UV circular dichroic (CD) spectrum of the enzyme showed that it consists of 31% alpha-helix, 21% beta-sheet, and 16% beta-turn, with 12% of aperiodic form. Treatment of the purified protein with a variety of proteases resulted in nicking near the C-terminus. This led to an increase in enzyme activity against lipids in erythrocyte membranes and increased rate of hydrolysis of p-nitrophenyl butyrate. Activation was accompanied by a change in the CD spectrum and a change in its aggregation state. The trypsin cut site was located between the two cysteines in the enzyme. Evidence is presented that the cysteines are joined by a disulfide bond and therefore cannot participate in acyl transfer. This may distinguish the microbial enzyme from lecithin:cholesterol acyltransferase. This is the second extracellular A. hydrophila protein that we have shown can be activated by proteolysis after it is released.

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Section: Resultsmentioning

confidence: 54%

Section: Methodsmentioning

confidence: 99%

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Purification and spectral study of a microbial fatty acyltransferase: activation by limited proteolysis

Hilton¹,

McCubbin²,

Kay³

et al. 1990

Biochemistry

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“…Esterase activity was determined by the hydrolysis of a watersoluble substrate, ρ-nitrophenyl butyrate (PNPB), as previously described. 68,69 The reaction mixture contained 50 μL crude enzyme extract, 10 μL PTAE (final concentrations: 0, 0.01, 0.02, 0.76, 1.14 and 1.52 mg mL −1 ), 100 μL reaction liquid, 100 μL PNPB and 790 μL buffer. Following incubation of the mixture for 20 min at 37 °C, the formation of ρ-nitrophenoxide was monitored using a UV-VIS spectrophotometer at 400 nm.…”

Section: Lcat Activitymentioning

confidence: 99%

Effects of Pu-erh tea aqueous extract (PTAE) on blood lipid metabolism enzymes

et al. 2015

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Disorders of blood lipid metabolism are the primary risk factors for many diseases. Recently, the effect of Pu-erh tea on blood lipid metabolism has received increasing attention. However, the mechanism underlying its ability to regulate blood lipid metabolism is unclear. We set out to study this through assessing the effects of Pu-erh tea aqueous extract (PTAE) on the central enzymes of blood lipid metabolism, including lipoprotein-associated phospholipase A2 (Lp-PLA2), lecithin: cholesterol acyltransferase (LCAT), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and pancreatic lipase (PL). We find that the Lp-PLA2, HMRG and PL activities are inhibited by PTAE in a dose-dependent manner and that the LCAT activity tends to increase with increasing PTAE concentrations. Lineweaver-Burk plot analyses reveal that PTAE acts as a competitive inhibitor for HMGR and PL and as a noncompetitive inhibitor for Lp-PLA2. Moreover, we determine that its active ingredients include catechins, gallic acid, caffeine, free amino acids, and soluble sugar. However, the effect of each ingredient and whether any of them have synergistic effects are still unknown. The results suggest that Pu-erh tea has a potent ability to regulate blood lipid metabolism and knowledge of the mechanisms provides insights into its potential therapeutic application as an alternative hypolipidemic drug.

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“…LCAT is a 67 kDa plasma enzyme that catalyzes the transfer and esterification of sn-2 fatty acids from phosphatidylcholine to the 3-hydroxyl group of cholesterol and is then responsible for the synthesis of most of the cholesteryl esters (CE) in human plasma (review in ref 13). LCAT is also able to hydrolyze water-soluble esters (14) and plateletactivating factor (15) as well as oxidized polar phosphatidylcholine (PC) generated during lipoprotein oxidation (16). LCAT may play an important role in preventing the oxidation of LDL (7).…”

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confidence: 99%

A Novel Lecithin-Cholesterol Acyltransferase Antioxidant Activity Prevents the Formation of Oxidized Lipids during Lipoprotein Oxidation

et al. 1999

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Recent investigations suggest that high-density lipoprotein (HDL) may play an anti-atherogenic role as an antioxidant and inhibit the oxidative modification of low-density lipoprotein (LDL). The antioxidant activity of HDL has been proposed to be associated with several HDL-bound proteins. We have purified one HDL-associated protein, lecithin:cholesterol acyltransferase (LCAT), to apparent homogeneity and have found that LCAT is not only capable of esterifying cholesterol in the plasma, but can also prevent the accumulation of oxidized lipids in LDL. Addition of pure human LCAT to LDL or palmitoyl-linoleoyl phosphatidylcholine/sodium cholate (PLPC) micelles inhibits the oxidation-dependent accumulation of both conjugated dienes and lipid hydroperoxides. LCAT also inhibits the increase of net negative charge that occurs during oxidation of LDL. LCAT has the ability to prevent spontaneous oxidation and Cu2+ and soybean lipoxygenase-catalyzed oxidation of lipids. The antioxidant activity of LCAT appears to be enzymatic, since the enzyme is active for up to 10 h in the presence of mild free-radical generators. The catalytic serine, residue 181, may mediate this activity and act as a reusable proton donor. Chemical modification of the active serine residue with diisopropylfluorophosphate completely inhibits the ability of LCAT to prevent lipid oxidation. Thus, in addition to its well-characterized phospholipase and acyltransferase activities, LCAT can also act as an antioxidant and prevent the accumulation of oxidized lipid in plasma lipoproteins.

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Reaction of Lecithin Cholesterol Acyltransferase with Water-soluble Substrates

Cited by 48 publications

References 35 publications

Purification and spectral study of a microbial fatty acyltransferase: activation by limited proteolysis

Purification and spectral study of a microbial fatty acyltransferase: activation by limited proteolysis

Effects of Pu-erh tea aqueous extract (PTAE) on blood lipid metabolism enzymes

A Novel Lecithin-Cholesterol Acyltransferase Antioxidant Activity Prevents the Formation of Oxidized Lipids during Lipoprotein Oxidation

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