Degradation of pyrene-labelled phospholipids by lysosomal phospholipases in vitro. Dependence of degradation on the length and position of the labelled and unlabelled acyl chains.
ABSTRACT: The hydrolysis of pyrenylacyl phosphatidylcholines (PyrnPCs)(n indicates the number of aliphatic carbons in the pyrene-chain) by crude lysosomal phospholipases in vitro was investigated. PyrnPCs consist of several sets in which the length of the pyrene-labelled or the unlabelled acyl chain, linked to the sn-1 or sn-2 position, was systematically varied. Lysophosphatidylcholine and fatty acid were the only fluorescent breakdown products detected, thus indicating that PyrnPCs were degraded by A-type phospholipases and lysophospholipases. Of these, mainly A1-type phospholipases appear to be involved, as determined from the relative amounts of labelled fatty acid and lysolipid released from the positional isomers. Based on the effects of the length and position of the pyrene-labelled and unlabelled chains it is suggested that (1) the lysosomal A-type phospholipases acting on PyrnPCs recognize the carboxy-terminal part of the lipid acyl chains and (2) the relevant part of the binding site is relatively narrow. Thus phospholipids with added bulk in the corresponding region, such as those that are peroxidized and polymerized, may not be good substrates for the lysosomal phospholipases mentioned. The impaired hydrolysis of the most hydrophobic PyrnPCs indicates that lysosomal phospholipases may not be able to penetrate significantly into the substrate interphase, but upward movement of the lipid may be required for efficient hydrolysis. Finally, the rate of hydrolysis of many pyrenyl derivatives was found to be comparable to that of a natural phosphatidylcholine species, both in micelles and in lipoprotein particles, indicating that these derivatives can be used as faithful reporters of lysosomal degradation of natural lipids in vivo and in vitro.
Project description:Selective hydrolysis of phosphatidylcholine species, which are selectively radioactively labelled in vivo, does not appear to interfere with a radiochemical assay for hydrolysis of microsomal phosphatidylcholine by C-type phospholipases from Bacillus cereus or Clostridium perfringens. Both phospholipases substantially hydrolysed phosphatidylcholine over the pH range 4.0-10.0.
Project description:Recently, oxidized phospholipid species have emerged as important signaling lipids in activated immune cells and platelets. The canonical pathway for the synthesis of oxidized phospholipids is through the release of arachidonic acid by cytosolic phospholipase A2? (cPLA2?) followed by its enzymatic oxidation, activation of the carboxylate anion by acyl-CoA synthetase(s), and re-esterification to the sn-2 position by sn-2 acyltransferase activity (i.e. the Lands cycle). However, recent studies have demonstrated the unanticipated significance of sn-1 hydrolysis of arachidonoyl-containing choline and ethanolamine glycerophospholipids by other phospholipases to generate the corresponding 2-arachidonoyl-lysolipids. Herein, we identified a pathway for oxidized phospholipid synthesis comprising sequential sn-1 hydrolysis by a phospholipase A1 (e.g. by patatin-like phospholipase domain-containing 8 (PNPLA8)), direct enzymatic oxidation of the resultant 2-arachidonoyl-lysophospholipids, and the esterification of oxidized 2-arachidonoyl-lysophospholipids by acyl-CoA-dependent sn-1 acyltransferase(s). To circumvent ambiguities associated with acyl migration or hydrolysis, we developed a synthesis for optically active (d- and l-enantiomers) nonhydrolyzable analogs of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC). sn-1 acyltransferase activity in murine liver microsomes stereospecifically and preferentially utilized the naturally occurring l-enantiomer of the ether analog of lysophosphatidylcholine. Next, we demonstrated the high selectivity of the sn-1 acyltransferase activity for saturated acyl-CoA species. Importantly, we established that 2-15-hydroxyeicosatetraenoic acid (HETE) ether-LPC sn-1 esterification is markedly activated by thrombin treatment of murine platelets to generate oxidized PC. Collectively, these findings demonstrate the enantiomeric specificity and saturated acyl-CoA selectivity of microsomal sn-1 acyltransferase(s) and reveal its participation in a previously uncharacterized pathway for the synthesis of oxidized phospholipids with cell-signaling properties.
Project description:The A-type phospholipases (PLAs) are key players in glycerophospholipid (GPL) homeostasis and in mammalian cells; Ca(2+)-independent PLA-? (iPLA?) in particular has been implicated in this essential process. However, the regulation of this enzyme, which is necessary to avoid futile competition between synthesis and degradation, is not understood. Recently, we provided evidence that the efflux of the substrate molecules from the bilayer is the rate-limiting step in the hydrolysis of GPLs by some secretory (nonhomeostatic) PLAs. To study whether this is the case with iPLA? as well, a mass spectrometric assay was employed to determine the rate of hydrolysis of multiple saturated and unsaturated GPL species in parallel using micelles or vesicle bilayers as the macrosubstrate. With micelles, the hydrolysis decreased with increasing acyl chain length independent of unsaturation, and modest discrimination between acyl positional isomers was observed, presumably due to the differences in the structure of the sn-1 and sn-2 acyl-binding sites of the protein. In striking contrast, no significant discrimination between positional isomers was observed with bilayers, and the rate of hydrolysis decreased with the acyl chain length logarithmically and far more than with micelles. These data provide compelling evidence that efflux of the substrate molecule from the bilayer, which also decreases monotonously with acyl chain length, is the rate-determining step in iPLA?-mediated hydrolysis of GPLs in membranes. This finding is intriguing as it may help to understand how homeostatic PLAs are regulated and how degradation and biosynthesis are coordinated.
Project description:An in vitro method based on 15N-labelled forage nitrogen (N) was developed to study ruminal N metabolism of soluble N (SN), insoluble N (ISN) and neutral detergent insoluble N (NDIN) fractions of timothy forage. Timothy grass was grown on replicated experimental plots with one plot receiving 15N-labelled and the other unlabelled N fertilizer. Harvested grass was preserved as dried grass or as formic acid treated or untreated silage. The intact forages and their corresponding N fractions were incubated in buffered rumen fluid in vitro to determine degradation parameters based on the 15N fluxes between labelled feed N and ammonia N pools. A high percentage (25-38%) of 15N-labelled ammonia disappeared from ammonia N pool during the first 15 min of incubation. Microbial uptake of dried grass SN fraction was higher than of silage SN fractions. Fractional degradation rates of SN from formic acid treated silage, untreated silage and dried grass during the first 6 hours of incubation were 0.145, 0.125 and 0.115 /h, respectively. By the end of the incubation period (28 h), 69, 66 and 43%, of the SN fraction of formic acid treated silage, untreated silage and dried grass, respectively were recovered as ammonia. The percentage of ISN fractions degraded to ammonia N were 9, 34 and 27%, respectively. Based on the changes in 15N-labelled ammonia N pool in blank incubation and appearance of 15N to ammonia N pool from 15N-labelled NDIN fractions, it was estimated that a significant portion of microbial lysis occurred when incubations were carried out for longer than 20 hours. With dried grass the contribution of ammonia N for microbial N synthesis was greater than with silages. Use of 15N-labelled forages together with this in vitro method is a promising technique for determining soluble N degradation parameters, but it requires further development to be used for determining degradation parameters of insoluble N fractions and work with whole feeds.
Project description:1. [(32)P]Lecithin and [(32)P]phosphatidylethanolamine were prepared by incubating rat liver mince with [(32)P]phosphate. With these (32)P-labelled phospholipids conditions for the quantitative assay of phospholipase A activity were established. 2. The distribution of phospholipase A activity between subcellular fractions of the bovine adrenal medulla was determined. Phospholipases A(1) and A(2), with pH optima at 4.2 and 6.5 respectively, were found in the large-granule fraction. By means of sucrose-density-gradient centrifugation it was shown that both these phospholipases were localized in lysosomes. 3. Lysosomal phospholipase A(1) catalysed the hydrolysis of [(32)P]lecithin and [(32)P]phosphatidylethanolamine at the same rate. The enzymic activity was inhibited by 70% in the presence of 10mm-calcium chloride. 4. Lysosomal phospholipase A(2) catalysed the hydrolysis of [(32)P]phosphatidylethanolamine more rapidly than it hydrolysed [(32)P]lecithin. The hydrolysis of [(32)P]phosphatidylethanolamine, but not that of [(32)P]lecithin, by phospholipase A(2) was activated by 0.8mm-calcium chloride. However, the hydrolysis of both substrates was inhibited by 8mm-calcium chloride. 5. The significance of the presence of phospholipase activity in lysosomes is discussed in relation to the functions of lysosomes in general and in the adrenal medulla.
Project description:We have investigated whether pyrene-labelled cholesterol esters (PyrnCEs) (n indicates the number of aliphatic carbons in the pyrene-chain) can be used to observe the degradation of low-density lipoprotein (LDL)-derived cholesterol esters (CEs) in the lysosomes of living cells. To select the optimal substrates, hydrolysis of the PyrnCE species by lysosomal acid lipase (LAL) in detergent/phospholipid micelles was compared. The rate of hydrolysis varied markedly depending on the length of the pyrenyl chain. Pyr10CE was clearly the best substrate, while Pyr4CE was practically unhydrolysed. Pyr10CE and [3H]cholesteryl linoleate, the major CE species in LDL, were hydrolysed equally by LAL when incorporated together into reconstituted LDL (rLDL) particles, thus indicating that Pyr10CE is a reliable reporter of the lysosomal degradation of native CEs. When rLDL particles containing Pyr4CE or Pyr10CE were incubated with fibroblasts, the accumulation of bright intracellular vesicular fluorescence was observed with the former fluorescent derivative, but not with the latter. However, when the cells were treated with chloroquine, an inhibitor of lysosomal hydrolysis, or when cells with defective LAL were employed, Pyr10CE also accumulated in vesicular structures. HPLC analysis of cellular lipid extracts fully supported these imaging results. It is concluded that PyrnCEs can be used to observe degradation of CEs directly in living cells. This should be particularly useful when exploring the mechanisms responsible for the accumulation of lipoprotein-derived CEs in complex systems such as the arterial intima.
Project description:Lysosomal phospholipase A2 (LPLA2) is characterized by broad substrate recognition, peak activity at acidic pH, and the transacylation of lipophilic alcohols, especially N-acetyl-sphingosine. Prior structural analysis of LPLA2 revealed the presence of an atypical acidic residue, Asp13, in the otherwise hydrophobic active site cleft. We hypothesized that Asp13 contributed to the pH profile and/or substrate preference of LPLA2 for unsaturated acyl chains. To test this hypothesis, we substituted Asp13 for alanine, cysteine, or phenylalanine; then, we monitored the formation of 1-O-acyl-N-acetylsphingosine to measure the hydrolysis of sn-1 versus sn-2 acyl groups on a variety of glycerophospholipids. Substitutions with Asp13 yielded significant enzyme activity at neutral pH (7.4) and perturbed the selectivity for mono- and double-unsaturated acyl chains. However, this position played no apparent role in selecting for either the acyl acceptor or the head group of the glycerophospholipid. Our modeling indicates that Asp13 and its substitutions contribute to the pH activity profile of LPLA2 and to acyl chain selectivity by forming part of a hydrophobic track occupied by the scissile acyl chain.
Project description:A graphical method is described that allows the determination of specific radioactivities of radioactively labelled hormones. This method combines the self-displacement technique, plotting bound/free ratios versus mass of unlabelled hormone or total radioactivity of labelled preparation added to the receptor preparation, and the maximal binding capacity of the labelled hormone. The procedure presented herein provides a more realistic specific radioactivity for use in all binding experiments. Application of the method is demonstrated for 125I-labelled ovine prolactin, and data are presented for 125I-labelled human choriogonadotropin and [3H]testosterone.
Project description:Rat somatotropin (growth hormone) was labelled biosynthetically by incubating anterior pituitary lobes with radioactive amino acids for 24 h in a simple buffered salts medium containing glucose. The labelled hormone was isolated by preparative polyacrylamide-gel electrophoresis or by chromatography on Sephadex G-100 and then DEAE-cellulose. The labelled material was pure by several criteria and cross-reacted immunologically with unlabelled rat somatotropin. When a mixture of 14C-labelled amino acids was used for labelling the protein, label could be introduced into these same amino acids of somatotropin, though relative specific radioactivities varied considerably. Somatotropin labelled by the procedures described in the present paper was suitable for structural studies and could be used for a variety of other biochemical experiments.
Project description:Previous studies have reported an increased turnover of phospholipid in isolated islets of Langerhans in response to raised glucose concentrations. The present investigation was thus undertaken to determine the nature of any phospholipases that may be implicated in this phenomenon by employing various radiolabelled exogenous phospholipids. Hydrolysis of 1-acyl-2-[14C]arachidonoylglycerophosphoinositol by a sonicated preparation of islets optimally released radiolabelled lysophosphatidylinositol, arachidonic acid and 1,2-diacylglycerol at pH 5,7 and 9 respectively. This indicates the presence of a phospholipase A1 and a phospholipase C. However, the lack of any labelled lysophosphatidylinositol production when 2-acyl-1-[14C]stearoylglycerophosphoinositol was hydrolysed argues against a role for phospholipase A2 in the release of arachidonic acid. Phospholipase C activity as measured by phosphatidyl-myo-[3H]inositol hydrolysis was optimal around pH8, required Ca2+ for activity and was predominantly cytosolic in origin. The time course of phosphatidylinositol hydrolysis at pH 6 indicated a precursor-product relationship for 1,2-diacylglycerol and arachidonic acid respectively. The release of these two products when phosphatidylinositol was hydrolysed by either islet or acinar tissue was similar. However, phospholipase A1 activity was 20-fold higher in acinar tissue. Substrate specificity studies with islet tissue revealed that arachidonic acid release from phosphatidylethanolamine and phosphatidylcholine was only 8% and 2.5% respectively of that from phosphatidylinositol. Diacylglycerol lipase was also demonstrated in islet tissue being predominantly membrane bound and stimulated by Ca2+. The availability of non-esterified arachidonic acid in islet cells could be regulated by changes in the activity of a phosphatidylinositol-specific phospholipase C acting in concert with a diacylglycerol lipase.