Impact of endothelial lipase on cellular lipid composition

Using mass spectrometry (MS), we examined the impact of endothelial lipase (EL) overexpression on the cellular phospholipid (PL) and triglyceride (TG) content of human aortic endothelial cells (HAEC) and of mouse plasma and liver tissue. In HAEC incubated with the major EL substrate, HDL, adenovirus (Ad)-mediated EL overexpression resulted in the generation of various lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) species in cell culture supernatants. While the cellular phosphatidylethanolamine (PE) content remained unaltered, cellular phosphatidylcholine (PC)-, LPC- and TG-contents were significantly increased upon EL overexpression. Importantly, cellular lipid composition was not altered when EL was overexpressed in the absence of HDL. [¹⁴C]-LPC accumulated in EL overexpressing, but not LacZ-control cells, incubated with [¹⁴C]-PC labeled HDL, indicating EL-mediated LPC supply. Exogenously added [¹⁴C]-LPC accumulated in HAEC as well. Its conversion to [¹⁴C]-PC was sensitive to a lysophospholipid acyltransferase (LPLAT) inhibitor, thimerosal. Incorporation of [³H]-Choline into cellular PC was 56% lower in EL compared with LacZ cells, indicating decreased endogenous PC synthesis. In mice, adenovirus mediated EL overexpression decreased plasma PC, PE and LPC and increased liver LPC, LPE and TG content. Based on our results, we conclude that EL not only supplies cells with FFA as found previously, but also with HDL-derived LPC and LPE species resulting in increased cellular TG and PC content as well as decreased endogenous PC synthesis.     

Lysophosphatidylethanolamine is – in contrast to – choline – generated under in vivo conditions exclusively by phospholipase A2 but not by hypochlorous acid

Inflammatory liver diseases are associated with oxidative stress mediated particularly by neutrophilic granulocytes. At inflammatory loci, hypochlorous acid (HOCl) is generated by myeloperoxidase. HOCl reacts with a large variety of molecules and induces (among other reactions) the formation of lysophosphatidylcholine (LPC) from polyunsaturated phosphatidylcholines (PC).As liver tissue contains huge amounts of polyunsaturated PC species enhanced LPC concentrations are detectable under these conditions. However, human liver contains also major amounts of polyunsaturated phosphatidylethanolamine (PE). It is so far widely unknown, if PE oxidation by HOCl leads to the generation of LPE in a similar way as observed in the case of PC. Using MALDI-TOF mass spectrometry (MS) and ³¹P NMR spectroscopy, LPC generation from unsaturated PC could be verified in the presence of HOCl. In contrast, unsaturated PE led exclusively to chlorohydrins and other oxidation products but not to LPE.Although these data were obtained with a quite simple model system, it is obvious that LPC is a much more suitable biomarker of oxidative stress than LPE: LPC is more readily generated and also more sensitively detectable by means of mass spectrometry and other spectroscopic methods. Nevertheless, it will also be shown that the nitrile of LPE is also generated. However, this compound is exclusively detectable as negative ion.     

Effect of feeding water washed neem (Azadirachta indica) seed kernel cake on the quality,lipid profile and fatty acid composition of goat meat

Three groups of five indigenous male goats 5–6 months of age, were offered control concentrate mixture (Group I) and those of Groups II and III were fed experimental concentrates containing 15 and 25% of water washed neem (Azadirachta indica) seed kernel cake (NKC). After 180 days of feeding the goats were slaughtered. Longissimus dorsi (LD) muscle from lumbar region was analysed for certain physico-chemical characteristics, organoleptic quality, detailed lipid profile and fatty acid composition; phosopholipids were fractionated into phosphatidyl inositide (PI), phosphatidyl serine (PS), lysophosphatidyl choline (LPC), lysophosphatidyl ethanolamine (LPE), sphingomyelin (SPH), phosphatidyl choline (PC) phosphatidyl ethanolamine (PE) and phosphatidic acid (PA). Fatty acids; capric, lauric, myristic, myristoleic, palmitic, palmitoleic, heptadecanoic, stearic, oleic, linoleic acid were also identified. Feeding of NKC did not affect slaughter weight and dressing out yield. The dressing percentage ranged between 42.2 and 43.8%. The pH, colour, moisture, total crude protein, different protein fractions; water extractable, salt extractable and content of NPN did not differ among the groups. There was a significant decrease in the total lipid content of LD of experimental groups. PC and PE were the major fractions accounting for 60% of total phospholipids. There was a significant increase in PC and LPC fractions while LPE+SPH, PE and PA fractions decreased in goats fed NKC. A significant increase was noticed in unsaturated fatty acid content and decrease in total saturates. It is concluded that NKC feeding has the ability of reducing lipid content and increasing the unsaturated fatty acids, which are considered to be beneficial in reducing the cholesterol level. It may be used beneficially as an alternative for costly conventional oil cakes for economic lean chevon production without affecting the quality of goat meat.     

The signal molecule lysophosphatidylcholine in Eschscholzia californica is rapidly metabolized by reacylation

In cultured cells of California poppy (Eschscholzia californica), lysophosphatidylcholine (LPC) triggers a signal path that finally induces alkaloid biosynthesis. LPC is transiently generated by elicitor-activated phospholipase A(2) of the plasma membrane. Externally added LPC is rapidly acylated by a membrane-bound enzyme that shows the highest specific activity in the purified plasma membrane. The fatty acid incorporated into the sn-2 position of LPC is preferentially linoleic (18:2), which is the most abundant acyl component in the PC species of Eschscholzia cells, but a minor component of the pool of free fatty acids. The fatty acid at the sn-1 position of LPC is less important for substrate specificity. The capacity of LPC acylation by intact cells or isolated plasma membranes by far exceeds the rate of LPC generation by activated phospholipase A(2) and is not limited by the availability of acyl donors. Metabolites other than phosphatidylcholine (PC) were not significantly produced from labeled LPC within 20 min, indicating that lysophospholipases are not significantly contributing to the short-time metabolism of LPC. It is concluded that reacylation to PC is the dominating process in the detoxication of LPC and ensures the transient character of its steady state concentrations, even at maximum phospholipase A(2) activities.     

A rapid response to a plant hormone: auxin stimulates phospholipase A2 in vivo and in vitro

Addition of the active auxins indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid or alpha-naphthylacetic acid to cultured soybean (Glycine max L.) cells prelabeled with ethanolamine or choline increased the radioactivity in the lysophosphatidylethanolamine (LPE) or lysophosphatidylcholine (LPC) pool within 5 min. The inactive auxin analogue, beta-naphthylacetic acid, was inactive in this response. In membranes prelabeled in vivo, either with ethanolamine or choline, and subsequently isolated from zucchini (Cucurbita pepo L.) hypocotyls, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid stimulated the conversion of phosphatidylethanolamine (PE) to LPE and of phosphatidylcholine (PC) to LPC in vitro whereas the inactive auxin analogue 2,3-dichlorophenoxyacetic acid did not.     

A model to study physiological activation of phospholipase A2 and vasorelaxation by lysophosphatidylcholine

Earlier we demonstrated that micellar solutions of LPC caused endothelium-dependent relaxation of rabbit thoracic aorta and bovine intrapulmonary artery and vein through a cyclic GMP-dependent mechanism. The availability of LPC for vasorelaxation depends on its production by deacylation of PC by PLA2. We assessed the possible activation of PLA2 by commonly used vasorelaxants such as acetylcholine, bradykinin, calcium ionophore A23187 and thrombin and vasoconstrictors like histamine and phenylephrine in the presence of indomethacin in a model system where 14C PC was incorporated into bovine intrapulmonary arterial segments. Taking the ratio of 14C PC:LPC formed by exogenous PLA2 as an index of deacylation, we found that while all the agents relaxed the strips in an endothelium-dependent manner, only thrombin caused relaxation followed by an increase in 14C LPC and a concomittant decrease in 14C PC indicating activation of PLA2. Our data show that PC/PLA2 system can be activated to generate LPC for vascular relaxation under specific physiological conditions. This model system can be used to monitor PLA2 activity and LPC production to compensate flow and pressure induced changes in arteries.     

Phospholipid metabolism in human neutrophil subfractions

We describe a procedure for isolating human neutrophil subfractions by sucrose density centrifugation following nitrogen cavitation. Using this procedure we were able to isolate and characterize a cytosolic fraction, two separate plasma membrane-enriched fractions, and specific and azurophilic granule fractions. We used this procedure to examine the subcellular localization of the enzymes and substrates involved in the release of arachidonic acid from cellular phospholipids in response to whole cell stimulation. Whole cells were prelabeled for 2 h with [3H]arachidonic acid and [14C] stearic acid. When prelabeled cells were challenged with calcium ionophore A23187 (2 microM) for 5 min at 37 degrees C, each membrane-associated fraction, including both plasma membrane fractions and specific and azurophilic granule fractions, exhibited deacylation of phosphatidylinositol (PI) and phosphatidylcholine (PC). The specific granule fraction exhibited the greatest proportion of deacylation from PI while the more dense plasma membrane fraction was deacylated to a much lower extent than the other fractions. In terms of mass, the azurophilic granules deacylated the greatest amount of radiolabeled arachidonic acid. Although all membrane fractions may be sources of arachidonic acid to some extent, the azurophilic granule fraction may contain the largest pool of radiolabeled arachidonic acid that is released upon cell stimulation.     

Fatty acid remodelling of phosphatidylinositol under conditions of de novo synthesis in rat liver microsomes

Phosphatidylinositol (PI) is initially synthesized in mammalian cells with a fatty acid composition similar to that of its precursor, primarily monounsaturated forms of cytidine diphosphodiglyceride (CDP-DAG). However, at the steady state, over 80% of PI exists in the 1-stearoyl, 2-arachidonoyl form. The fatty acid remodelling of PI is due to a number of deacylation/reacylation mechanisms. In the preceding paper we demonstrated that de novo synthesized PI is rapidly deacylated and subsequently reacylated. In this report we present further evidence that cycles of deacylation and reacylation are involved in the remodelling of PI. Incubation of microsomes with CDP-DAG of different fatty acid composition results in quantitative and qualitative differences in lysoPI formation. Additionally, analyses of the resulting lysoPI and PI species reveal that multiple species of fatty acids are incorporated into the 1-position of both PI and lysoPI. Addition of acylation cofactors (fatty acyl CoAs or ATP plus CoA) potentiate reacylation in this system. The addition of stearoyl or myristoyl CoA during de novo synthesis of PI results in the incorporation of these added fatty acids into the I-positive of PI. In addition, some evidence is presented that multiple mechanisms for remodelling of the 1-position of PI may be active in the microsomes, including ATP- and CoA-dependent acylation, ATP-independent, CoA-dependent acylation and CoA-independent mechanisms. Finally, the disappearance of only a subset of lysoPI species upon the addition of acylation cofactors suggests that the reacylation step exhibits some substrate specificity.     

Effect of estradiol and progesterone on the rate of incorporation of radiolabeled arachidonate into phospholipids and triglycerides of the rat uterus

To obtain information on the effect of estradiol (E) and progesterone (P) on the overall rate of the acylation and deacylation reactions with [3H]arachidonate ([3H]AA) in the rat uterus, we investigated the effect of chronic treatment of ovariectomized rats with 2-200 micrograms/day E-dipropionate (EPP) and with 2 mg P alone or with the combination of these steroids on the extent and the time course of the in vitro incorporation of [3H]AA into various phospholipids (PLs) and the triacylglycerol fraction (TG). The results demonstrate that physiologic doses of E leads to a rapid equilibrium of the deacylation-acylation cycle only in the case of phosphatidylinositol (PI) whereas at high (200 micrograms/day) dose level it exerts the same effect on phosphatidylcholine (PC), phosphatidylethanolamine (PE) as well as on TG. P alone has no remarkable effects in the ovariectomized rat, but it decreases markedly the incorporation of [3H]AA into PI and TG in intact animals. Furthermore, P decreases the incorporation of [3H]AA into PI in the ovariectomized rat treated with 2 micrograms/day EPP as well as attenuating the enhanced labeling of PC, PE and PI caused by higher doses. Time-course studies provide evidence that all of these effects of progesterone can be accounted for by its ability to decrease the rate of deacylation and, therefore, to prolong the time needed to attain equilibrium in the rates of acylation and deacylation reactions of the various lipids with arachidonate. These data offer a novel outlook on the regulatory role of progesterone and estrogens in uterine function.     

Acyl-Protein Thioesterase 2 Catalizes the Deacylation of Peripheral Membrane-Associated GAP-43

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.     

Acyl-protein thioesterase 2 catalyzes the deacylation of peripheral membrane-associated GAP-43

An acylation/deacylation cycle is necessary to maintain the steady-state subcellular distribution and biological activity of S-acylated peripheral proteins. Despite the progress that has been made in identifying and characterizing palmitoyltransferases (PATs), much less is known about the thioesterases involved in protein deacylation. In this work, we investigated the deacylation of growth-associated protein-43 (GAP-43), a dually acylated protein at cysteine residues 3 and 4. Using fluorescent fusion constructs, we measured in vivo the rate of deacylation of GAP-43 and its single acylated mutants in Chinese hamster ovary (CHO)-K1 and human HeLa cells. Biochemical and live cell imaging experiments demonstrated that single acylated mutants were completely deacylated with similar kinetic in both cell types. By RT-PCR we observed that acyl-protein thioesterase 1 (APT-1), the only bona fide thioesterase shown to mediate deacylation in vivo, is expressed in HeLa cells, but not in CHO-K1 cells. However, APT-1 overexpression neither increased the deacylation rate of single acylated GAP-43 nor affected the steady-state subcellular distribution of dually acylated GAP-43 both in CHO-K1 and HeLa cells, indicating that GAP-43 deacylation is not mediated by APT-1. Accordingly, we performed a bioinformatic search to identify putative candidates with acyl-protein thioesterase activity. Among several candidates, we found that APT-2 is expressed both in CHO-K1 and HeLa cells and its overexpression increased the deacylation rate of single acylated GAP-43 and affected the steady-state localization of diacylated GAP-43 and H-Ras. Thus, the results demonstrate that APT-2 is the protein thioesterase involved in the acylation/deacylation cycle operating in GAP-43 subcellular distribution.     

Inhibition of cathepsin G by 2-amino-3,1-benzoxazin-4-ones: kinetic investigations and docking studies

A series of benzoxazinones was used to investigate the interaction of human cathepsin G with acyl-enzyme inhibitors. With respect to the primary specificity of cathepsin G, inhibitors with hydrophobic or basic residues at position 2 were included in the study. Parameters of the enzyme acylation and deacylation were determined by slow-binding kinetics in the presence of a chromogenic substrate. For selected inhibitors, the time course of the enzyme-catalyzed conversion of the inhibitors was followed. This approach was suitable to elucidate a rate-determining deacylation step. Docking simulations of the noncovalent enzyme-inhibitor complexes were performed and several clusters were analyzed for each inhibitor. The amino acids of the active site that participate in the binding of the inhibitors were determined. The arrangements in several clusters of an inhibitor were not uniform with respect to the orientation by which the inhibitor was bound in the S(1) pocket. Docking of the basic piperazino derivatives 6 and 10 indicated an interaction with Glu 226 at the bottom of the S(1) specificity pocket. The (N-methyl)benzylamino derivative 1 showed the strongest acylation rate (k(on)=1200 M(-1) s(-1)), which was attributed to a high extent of pseudo-productive orientations of the noncovalent preassociation complex.     

A mechanistic model for butyrylcholinesterase.

A plausible mechanism of action of horse serum butyrylcholinesterase is proposed. It includes substrate activation at the level of deacylation. The rate constant for the acylation of the enzyme appears to be much greater than the rate constant for the deacylation, at low substate concentrations. At higher substrate concentrations the rate constants become more similar. No interaction between the four subunits in binding of inhibitors or in the catalysis was observed. There is one esteratic and one anionic site per subunit apparent from labelling studies with [32P]diisopropylfluorophosphate and binding studies with N-methylacridine. Although the tetrametric form of the enzyme appears to be the native one, the monomeric and several other aggregated and dissociated states are catalytically active.     

Lysophosphatidylcholine (LPC) attenuates macrophage-mediated oxidation of LDL

We have previously shown that paraoxonase 1 action on macrophages produced lysophosphatidylcholine (LPC) and significantly decreased cell-mediated LDL oxidation. Thus, in the present study, we questioned whether LPC can directly inhibit macrophage-mediated oxidation of LDL. Addition of increasing LPC concentrations (0-5 microM) to J774A.1 macrophages, mouse peritoneal macrophages (MPM), or to human monocytes-derived macrophages (HMDM) resulted in up to 83%, 67%, and 75% inhibition in cell-mediated oxidation of LDL, respectively. The mechanism for this LPC effect involves up to 60% inhibition of superoxide anion release from MPM in response to phorbol ester (PMA), 26% inhibition of PMA-induced NADPH oxidase activation (p47phox translocation from the cytosol to the plasma membrane), and a 2-fold stimulation of the macrophage paraoxonase 2 (PON2) lactonase activity. We thus conclude that inhibition of macrophage-mediated oxidation of LDL by LPC can contribute to attenuation of macrophage foam cell formation and atherosclerotic lesion development.     

Role of methionyl-transfer ribonucleic acid in the regulation of methionyl-transfer ribonucleic acid synthetase of Escherichia coli K-12.

A decrease in the in vivo acylation level of methionine transfer ribonucleic acid (tRNAmet) induced by methioninyl adenylate led to a specific derepression of methionyl-transfer ribonucleic acid (tRNA) synthetase formation. This derepression required de novo protein synthesis and was reflected by overproduction of unaltered enzyme. Two different strains of Escherichia coli K-12 that have normal levels of methionyl-tRNA synthetase were examined and the derepression of methionyl-tRNA synthetase was observed in both. Moreover, for one of these strains, the relation between the level of methionyl-tRNA synthetase and deacylation level of tRNAmet was established; under the growth conditions used, when more than 25% of tRNAmet was deacylated, methionyl-tRNA synthetase formation was derepressed and the level of derepression became proportional to the amount of tRNAmet deacylated. Concomitantly, the enzyme was subject to specific inactivation as a consequence of which the true de novo rate of derepression of the formation of this enzyme was higher than that determined by measurements of enzyme activity. These studies were extended to strains AB311 and ed2, which had a constitutive enhanced level of methionyl-tRNA synthetase. In these strains no derepression of enzyme formation was observed on reducing the acylation level of tRNAmet by use of methioninyl adenylate.     

Lysophospholipid regulates release and activation of latent TGF-beta1 from chondrocyte extracellular matrix

Transforming growth factor beta-1 (TGF-beta1) is released from the extracellular matrix of rat growth plate chondrocytes and activated by stromelysin-1 (matrix metalloproteinase 3, MMP-3), an enzyme that is stored in matrix vesicles. MMP-3 is released from these extracellular organelles by the direct action of 1alpha,25(OH)2D3 via activation of phospholipase A2 (PLA2), resulting in local production of lysophospholipids and matrix vesicle membrane destabilization. This effect of 1alpha,25(OH)2D3 is greater in matrix vesicles from growth zone chondrocyte cultures and PLA2 activity is higher in the growth zone in vivo, suggesting that it may depend on chondrocyte maturation state in the endochondral lineage. Previous studies have shown that latent TGF-beta1 can be activated by mild detergents in vitro, suggesting that lysophospholipids may act in vivo in a similar manner. To test this hypothesis, we determined if rat costochondral growth plate cartilage cells produce lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) in a maturation state-dependent manner and if LPC or LPE could release and activate latent TGF-beta1 from the extracellular matrix produced by these cells. Rat growth plate chondrocytes produced both lysophospholipids, with growth zone cells producing higher levels of LPE via PLA1, and resting zone cells producing higher levels of LPC via PLA2. LPC and LPE directly increased activation of recombinant human latent TGF-beta1 in a biphasic manner with a peak at 2 microg/ml. Phosphatidylcholine, phosphatidylethanolamine, and LPE plasmalogen (LPEP), but not choline, also activated TGF-beta1. Latent TGF-beta1 incubated with LPC or LPE, but neither lysophospholipid alone, stimulated [3H]-thymidine incorporation of resting zone cells, indicating the TGF-beta1 released was biologically active. LPC and LPE also released TGF-beta1 in a dose- and time-dependent manner when incubated with cell-free extracellular matrices produced by the cells. These results indicate that LPC and LPE have important roles as regulators of rat growth plate chondrocytes by directly and indirectly activating TGF-beta1 stored in the extracellular matrix.     

Deciphering the roles of Arabidopsis LPCAT and PAH in phosphatidylcholine homeostasis and pathway coordination for chloroplast lipid synthesis

Phosphatidylcholine (PC) is a key intermediate in the metabolic network of glycerolipid biosynthesis. Lysophosphatidylcholine acyltransferase (LPCAT) and phosphatidic acid phosphatase (PAH) are two key enzymes of PC homeostasis. We report that LPCAT activity is markedly induced in the Arabidopsis pah mutant. The quadruple pah lpcat mutant, with dual defects in PAH and LPCAT, had a level of lysophosphatidylcholine (LPC) that was much higher than that in the lpcat mutants and a PC content that was higher than that in the pah mutant. Comparative molecular profile analysis of monogalactosyldiacylglycerol and digalactosyldiacylglycerol revealed that both the pah and pah lpcat mutants had increased proportions of 34:6 from the prokaryotic pathway despite differing levels of LPCAT activity. We show that a decreased representation of the C_16:0C_18:2 diacylglycerol moiety in PC was a shared feature of pah and pah lpcat, and that this change in PC metabolic profile correlated with the increased prokaryotic contribution to chloroplast lipid synthesis. We detected increased PC deacylation in the pah lpcat mutant that was attributable at least in part to the induced phospholipases. Increased LPC generation was also evident in the pah mutant, but the phospholipases were not induced, raising the possibility that PC deacylation is mediated by the reverse reaction of LPCAT. We discuss possible roles of LPCAT and PAH in PC turnover that impacts lipid pathway coordination for chloroplast lipid synthesis.     

Studies on the endogenous phospholipids of chick embryo myocardium and their in vitro hydrolysis by endogenous phospholipases during embryogenesis

The phospholipid profiles of the myocardium (from 10- and 18-day old chick embryos and 13-day old chick) and their in vitro response to the endogenous lipolytic enzymes (mainly of the phospholipase group) at pH 7.4 and 38 degrees C for 60 min were analyzed by TLC technology and densitometry. Cardiolipin (CL) was shown to be one of the major phospholipids of the chick embryo myocardium and its concentration increased as the chick embryo advanced in development. Monolysocardiolipin (MLCL) was produced subsequent to in vitro incubation of whole tissue homogenates in all myocardia studied as well as a concurrent reduction in CL. This deacylation of CL increased in magnitude as the chick embryo advanced in development indicating its age relatedness. The level of phosphatidyl ethanolamine (PE) plasmalogen was also high in all myocardia studied. Lyso alkenyl PE (LPE) was produced subsequent to in vitro incubation and its level increased as the chick embryo advanced in development, indicating PLA(2) action on the sn-2 fatty acid of PE. Phosphatidyl choline (PC) plasmalogen was also present in the chick embryo myocardium and its level increased gradually as the chick embryo advanced in development. In contrast, yolk-sac membrane contains very minute amounts of CL and PE. No PC was detected and no LPE was formed following in vitro incubation. The yolk of the unfertilized chicken egg has no CL and has very minute amounts of PE, no PC and no lysophospholipids were detected following in vitro incubation in all samples analyzed.     

Catalytic activity of α-chymotrypsin in which histidine-57 has been methylated

The properties of a derivative of alpha-chymotrypsin in which histidine-57 has been methylated have been examined. Although the modified enzyme binds substrate with the same affinity as does native alpha-chymotrypsin, acylation and deacylation occur at much decreased rates. As for native alpha-chymotrypsin, a basic group of pK(a) approx. 7 is involved in both acylation and deacylation. The significance of these results is considered in relation to the normal function of histidine-57.