Lecithin:cholesterol acyltransferase (LCAT) was isolated from hog plasma and basic physicochemical properties and functionally important regions were investigated. Approximately one milligram of the enzyme was purified to apparent homogeneity with approximately a 20,000-fold increase in specific activity. In the plasma, hog LCAT was found to associate with high-density lipoproteins (HDL) probably through hydrophobic interactions with apolipoprotein A-I. HDL was the preferred lipoprotein substrate of the enzyme as its macromolecular substrate. The enzyme was found to contain 4 free sulfhydryl groups; at least one of these appeared to be essential for catalytic activity. The enzyme had a tendency to aggregate at high concentrations. More than half of the tryptophan and none of the tyrosine residues of the enzyme were shown to be exposed to the aqueous environment based on fluorescence and absorbance studies, respectively.

No description available.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 7, 2008). Includes bibliographical references (p. 114-133).

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Embargoed. Vita. Bibliography: 217-242.

En plus de leurs propriétés structurales comme constituants majeurs des membranes biologiques des cellules, les lipides jouent de nombreux rôles dans la signalisation cellulaire, le stockage d’énergie et le transport de protéines. Leurs importances biologiques ont mené à une augmentation accrue des méthodes analytiques pour la caractérisation d’espèces moléculaires uniques. De récents progrès en spectrométrie de masse ont amené à la caractérisation et à la quantification des espèces moléculaires des lipides dans des extraits lipidiques bruts (Han and Gross, 2005; Murphy et al., 2001). Par exemple, les espèces moléculaires de phospholipides peuvent être identifiées spécifiquement par leur tête polaire, la nature de leurs chaînes d’acide gras et leur positionnement au niveau du squelette glycérol. / In addition to their structural properties as main constituents of biological membranes, lipids play a multitude of roles such as in cell signalling, energy storage, and protein transport. Their biological importance has led to an increasing focus on analytical methods for the characterisation of their individual molecular species. Improvements in mass spectrometric technology has provided a great advantage for the characterisation and quantification of molecular lipid species in total lipid extracts (Han and Gross, 2005; Murphy et al., 2001). For instance, phospholipid molecular species can be identified on the basis of a characteristic fragment of the lipid class, the nature of the acyl chains and their positions on the glycerol backbone.A method allowing the quantitative profiling of the yeast lipidome was developed in a recent study using automated shotgun infusion strategy (Ejsing et al., 2009). We applied this method to characterise several lysophospholipid acyltransferase yeast mutants produced using reverse-genetics. These enzymes are involved in essential biological processes like de novo synthesis or remodelling of the phospholipid membrane component (Testet et al., 2005; Le Guedard et al., 2009). The comparative analysis of phospholipid molecular species from the wild-type strain and the corresponding deletion mutants has allowed us to identify lipid compositional changes, and has given us significant indications about the in vivo function of the encoded lysophospholipid acyltransferases.

No description available.

Triacylglycerol (TAG) is the main storage lipid in plants, found both in seed and non-seed tissues (e.g. root, leaves, mesocarp etc.). Plants use TAGs as a carbon and energy source during seed germination while humans use plant lipids for biofuel production, industrial feedstocks and nutrition. It is expected that by 2030 the demand for biodiesel will be doubled. To meet this demand it is important to understand the regulation of rate limiting reactions involved in TAG accumulation in nonseed tissues because of their higher biomass relative to the seed tissues. In this study, avocado (Persea americana) is used as a model organism because it is a basal angiosperm and can store up to 70% oil content in the form of TAG in its mesocarp, a nonseed tissue. Typically, the last acylation of diacylglycerol (DAG) to form TAG in seed tissues is catalyzed by diacylglycerol acyltransferases (DGAT) and/or phospholipid:diacylglycerol acyltransferases (PDAT). Based on the transcriptome of avocado, it is hypothesized that both DGAT and PDAT are responsible for catalyzing the terminal step of TAG biosynthesis in mesocarp of avocado. Fulllength coding sequences for DGAT1 and PDAT1 were identified based on the avocado transcriptome data and expressed in TAG-deficient yeast strain (SCY-1998) for complementation. Total lipid extracts from complemented yeast lines will be analyzed for presence of TAG. Furthermore, the enzyme activity and substrate specificity for PaDGAT1 and PaPDAT1 will be determined from microsomal preparations of avocado and eukaryotic expression systems containing the avocado transgenes. This study is expected to identify the enzymes responsible for the terminal acylation step in TAG synthesis in avocado, thereby contributing to the basic understanding of TAG accumulation in nonseed tissues.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Les lipases/acyltransférases homologues à CpLIP2 de Candida parapsilosis forment un groupe phylogénétique marqué (au moins 56% d’identité entre les séquences protéiques) . Elles partagent le phénotype d’une activité significative d’acyltransfert, et ce, même dans un milieu aqueux avec une forte activité thermodynamique de l’eau (aW > 0.95), mais diffèrent dans leurs spécificités de substrats. L’identification et la caractérisation de nouvelles lipases/acyltransférases, CalLAc8 et CalLAc5 de Candida albicans et CduLAc de Candida dublininensis, ont apporté de nouveaux éclaircissements sur les relations structure/fonction au sein de cette famille particulière. Dans un premier temps, une définition claire et une méthodologie simple pour évaluer la capacité des enzymes lipolytiques à catalyser l’acyltransfert ont été élaborées. Puis, une stratégie d’ingénierie des protéines, basée sur une analyse comparative des structures 3D et de la mutagénèse dirigée, a été appliquée dans le but d’identifier les déterminants structuraux impliqués dans l’activité d’acyltransfert et la spécificité de substrat des lipases/acyltransférases. Il a été démontré que le caractère hydrophobe d’une cavité située sous le site actif était déterminant pour l’activité de transfert en favorisant les nucléophiles moins polaires que l’eau dans l’étape de désacylation du mécanisme catalytique. Ainsi, des mutants améliorés de plusieurs enzymes sauvages ont pu être élaborés. En parallèle, des enzymes chimériques ont été construites sur la base d’échanges rationnels de sous-domaines (corps principal, chapeau et volet C-terminal). Leur caractérisation a confirmé le rôle du chapeau dans la spécificité de substrat et le rôle principal de « l’acyltransfer pocket » dans la capacité d’acyltransfert. Une potentielle protéine ancestrale de la famille PaleoLAc a également été conçue pour trouver de nouveaux résidus clés et donner un aperçu de l’histoire évolutive de la spécificité de substrats. / Lipases/acyltransferases homologous to CpLIP2 from Candida parapsilosis constitute a consistent phylogenetic subgroup with at least 56% identity. They share the phenotype of a significant acyltransfer activity, even in aqueous media with a high thermodynamic activity of water (aW > 0.95), but are divergent in their substrate specificities. The identification and the characterization of new lipases/acyltransferases, CalLAc8 and CalLAc5 from Candida albicans and CduLAc from Candida dublininensis, brought new enlightenments to the structure/function relationships in this peculiar family. After the elaboration of a clear definition and a simple methodology to assess the acyltransferase character of lipolytic enzymes, a rational design strategy, based on comparative 3D structure analysis and site-directed mutagenesis, was applied to find structural determinants of the acyltransfer ability and the substrate specificities of lipases/acyltransferases. It was evidenced that the hydrophobicity of a cavity located under the active site was determinant for the acyltransfer activity. This allowed the improvement of the acyltransfer activity of several natural enzymes. In parallel, chimeric enzymes with rational exchanges of protein subdomains (main core, cap and C-term flap) were designed, and their characterization confirmed the role of the cap in the substrates specificity and the main role of the acyltransfer pocket in the acyltransfer ability. A putative ancestral protein of the family PaleoLAc was also designed to find new key residues and to give insights on the evolutionary history of the substrate specificities.