Low density lipoprotein (LDL) and lipoprotein (a) [Lp(a)] were covalently labeled with the fluorescent dyes BODIPY succinimidyl ester (green) or Rhodamine iodoacetamide (red). The interaction of the fluorescent lipoproteins with HepG2 cells was visualized by means of a confocal laser scanning fluorescence microscope operating in the dual wavelength mode. If LDL or Lp(a) were incubated with the cells both lipoproteins bound to the cell surface at 4 degree(s)C or were internalized by the cells at 37 degree(s)C. In all cases larger amounts of LDL interacted with the cells compared with Lp(a). When mixtures of LDL and Lp(a), each labeled with a different dye, were incubated with cells again both lipoproteins bound to the cell surface (4 degree(s)C) or were internalized by the cells (37 degree(s)C). In addition, the major part of the lipoproteins colocalized either on the cell surface or inside the cells. thus, we conclude that interactions of Lp(a) with cells is mediated by LDL, probably via the LDL receptor, to a large extent.
Regulation of the activity of the mitochondrial enzyme phosphatidylserine decarboxylase (PSD) was measured in vitro by using membrane preparations from wild-type and mutant strains of Saccharomyces cerevisiae. PSD specific activity was not affected by carbon source, and on all carbon sources, the highest specific activity was observed in cells entering the stationary phase of growth. However, PSD activity was found to be regulated in response to soluble precursors of phospholipid biosynthesis. PSD specific activity was reduced to about 63% of the level observed in unsupplemented wild-type cells when the cells were grown in the presence of 75 microM inositol. The presence of 1 mM choline alone had no repressing effect, but the presence of 1 mM choline and 75 microM inositol together led to further repression to a level of about 28% of the derepressed activity. Regulatory mutations known to affect regulation or expression of genes encoding phospholipid-synthesizing enzymes also affected PSD specific activity. opi1 mutants, which are constitutive for a number of phospholipid-biosynthetic enzymes, were found to have high, constitutive levels of PSD. Likewise, in ino2 or ino4 regulatory mutants, PSD activity was found to be at the fully repressed level regardless of growth condition. Regulation of PSD activity was also affected in several structural-gene mutants under conditions of impaired phosphatidylcholine biosynthesis. Together, these data strongly suggest that PSD expression is controlled by the mechanism of general control of phospholipid biosynthesis that regulates many enzymes of phospholipid biosynthesis.
Subcellular membranes of Saccharomyces cerevisiae, including mitochondria, microsomes, plasma membranes, secretory vesicles, vacuoles, nuclear membranes, peroxisomes, and lipid particles, were isolated by improved procedures and analyzed for their lipid composition and their capacity to synthesize phospholipids and to catalyze sterol delta 24-methylation. The microsomal fraction is heterogeneous in terms of density and classical microsomal marker proteins and also with respect to the distribution of phospholipid-synthesizing enzymes. The specific activity of phosphatidylserine synthase was highest in a microsomal subfraction which was distinct from heavier microsomes harboring phosphatidylinositol synthase and the phospholipid N-methyltransferases. The exclusive location of phosphatidylserine decarboxylase in mitochondria was confirmed. CDO-diacylglycerol synthase activity was found both in mitochondria and in microsomal membranes. Highest specific activities of glycerol-3-phosphate acyltransferase and sterol delta 24-methyltransferase were observed in the lipid particle fraction. Nuclear and plasma membranes, vacuoles, and peroxisomes contain only marginal activities of the lipid-synthesizing enzymes analyzed. The plasma membrane and secretory vesicles are enriched in ergosterol and in phosphatidylserine. Lipid particles are characterized by their high content of ergosteryl esters. The rigidity of the plasma membrane and of secretory vesicles, determined by measuring fluorescence anisotropy by using trimethylammonium diphenylhexatriene as a probe, can be attributed to the high content of ergosterol.
A membrane‐bound phospholipase D (PLD) from Saccharomyces cerevisiae was solubilized from mitochondrial and plasma membranes and partially purified. The enzyme has an apparent molecular weight of approximately 60 kDa, is strictly Ca 2+ ‐dependent and preferentially hydrolyses phosphatidylserine and phosphatidylethanolamine. Enzyme activity is significantly increased in membranes from cells grown on a non‐fermentable carbon source. The Ca 2+ ‐dependent PLD is distinct from PLD encoded by the SPO14/PLD1 gene. The 195 kDa SPO14/PLD1 gene product is specific for PtdCho, Ca 2+ ‐independent and is activated by PIP 2 . Furthermore, Pld1p has transphosphatidylation activity in the presence of ethanol and thus resembles the prototypic PLD activity found in mammalian cells and plants. In contrast, the Ca 2+ ‐dependent PLD described here is not affected by PIP 2 and does not catalyze transphosphatidylation. Thus, the Ca 2+ ‐dependent PLD characterized in this study appears to be a member of a novel family of phospholipases D.
ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTConformation of phosphatidylserine in bilayers as studied by Fourier transform infrared spectroscopyW. Huebner, H. H. Mantsch, F. Paltauf, and H. HauserCite this: Biochemistry 1994, 33, 1, 320–326Publication Date (Print):January 11, 1994Publication History Published online1 May 2002Published inissue 11 January 1994https://pubs.acs.org/doi/10.1021/bi00167a042https://doi.org/10.1021/bi00167a042research-articleACS PublicationsRequest reuse permissionsArticle Views166Altmetric-Citations41LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts
