Abstract:
:This chapter outlines methods that can be applied to determine the levels of lipids in cells and tissues. In particular, the methods focus upon the extraction and analysis of those lipids critical for monitoring signal transduction pathways. The methods address the analysis of the phosphoinositides, the lipid agonists lysophosphatidic acid and sphingosine 1-phosphate, and the neutral lipid messengers diacylglycerol and ceramide. Additionally, because of the increasing need to determine the dynamics of signaling, the analysis of phospholipids synthesis using stable isotope methods is described. The use of these methods as described or adaptation to permit both approaches should allow investigators to determine changes in signaling lipids and to better understand such processes in most cell types. The increasing appreciation of the central roles played by lipid signaling pathways has dispelled the misconception that lipids are inert structural components that are involved solely in keeping a cell intact. Advances in our understanding of cell-signaling pathways have identified particular lipids that act to regulate the functions of a number of proteins either by controlling enzyme activity directly, or by localizing proteins to particular intracellular compartments where they perform a specialized role. These lipid-binding domains (e.g., PH, PX, FYVE) have been found in many proteins, and considerable detail is recorded of the structural basis of lipid protein interaction. Additional lipid-binding domains exist, which remain less well characterized (e.g., those that bind phosphatidic acid [PA] or ceramide); however, the important regulatory roles that these lipids play and the pathways involving these messengers are increasingly appreciated. While the downstream targets are thus being defined, the actual changes in lipid concentration in a stimulated cell or membrane are less characterized. The primary reason for this lack has been a deficiency in methodology. Much of the reported studies of lipid messengers in stimulated cells have depended upon monitoring changes in radio-labeled cells. Many well-documented problems are associated with this type of methodology, including lack of isotopic equilibrium, distinct pools with different turnover rates, and inadequate separation of radio-labeled metabolites; however, much important information has been generated. The second approach has been to make use of the lipid-binding properties of the target protein domains and to generate a tagged fusion protein, generally GFP, which permits identification of a region rich in a signaling lipid (Guillou et al., 2007). This has proved useful in monitoring PI-3-kinase activation in stimulated cells; however, considerable caveats must be raised, not least the problems associated with lipid specificity and the fact that many of these domains have associated protein-binding regions that can compromise the findings. A further problem associated with these two methodologies is that they tend to group lipids together and take no account of the multiple acyl chain structures that occur in all lipids. These concerns point to the need to determine actual changes in lipid compositions. Until relatively recently, such an analysis was unachievable; however, advances in both chromatographic separation and mass spectrometry (MS) have permitted the development of lipidomic analysis. This chapter outlines a number of methods that allow determination of changes in signaling lipids. Adaptation of the methods here for the analysis of other molecules should be relatively straightforward in the future. Much of the lipidomic research in the United Kingdom is focused upon signaling lipidomics, with particular foci upon phosphoinositide-related signaling in Birmingham and Cambridge (Wakelam) and London (Larijani), upon eicosanoids in Cardiff (O'Donnell), and steroids in London (Griffiths). Meanwhile, the use of stable isotopes has been particularly developed in Southampton (Postle).
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Wakelam MJ,Pettitt TR,Postle ADdoi
10.1016/S0076-6879(07)32010-7subject
Has Abstractpub_date
2007-01-01 00:00:00pages
233-46eissn
0076-6879issn
1557-7988pii
S0076-6879(07)32010-7journal_volume
432pub_type
杂志文章abstract::Two-component signal transduction systems, composed of histidine kinases and response regulators, enable bacteria to sense, respond, and adapt to changes in their internal and external conditions. The importance of these signaling systems is reflected in their widespread distribution and prevalence in the bacterial ki...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)23026-5
更新日期:2007-01-01 00:00:00
abstract::Vitamin K-dependent carboxylation, an essential posttranslational modification catalyzed by gamma-glutamyl carboxylase, is required for the biological functions of proteins that control blood coagulation, vascular calcification, bone metabolism, and other important physiological processes. Concomitant with carboxylati...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.10.015
更新日期:2017-01-01 00:00:00
abstract::gamma-Glutamyltranspeptidase is a heterodimeric glycoprotein that catalyzes the transpeptidation and hydrolysis of the gamma-glutamyl group of glutathione and related compounds. It is known that the enzyme plays a role in the metabolism of glutathione and in salvaging constituents of glutathione. In the adult animal, ...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)01025-6
更新日期:2005-01-01 00:00:00
abstract::Circadian clocks are present in most cells and are essential for maintenance of daily rhythms in physiology, mood, and cognition. Thus, not only neurons of the central circadian pacemaker but also many other peripheral tissues possess the same functional and self-sustained circadian clocks. Surprisingly, however, thei...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/bs.mie.2014.10.023
更新日期:2015-01-01 00:00:00
abstract::Human solid tumors are invariably less well-oxygenated than the normal tissues from which they arose. This so-called tumor hypoxia leads to resistance to radiotherapy and anticancer chemotherapy as well as predisposing for increased tumor metastases. In this chapter, we examine the resistance of tumors to radiotherapy...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)35015-5
更新日期:2007-01-01 00:00:00
abstract::Genome sequencing projects have provided the substrate for an unimaginable number of biological experiments. Further, genomic technologies such as microarrays and quantitative and exquisitely sensitive techniques such as real-time quantitative polymerase chain reaction have made it possible to reliably generate millio...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)05012-5
更新日期:2009-01-01 00:00:00
abstract::The findings in this article illustrate the complexity residing in the regulation of reversible S-glutathionylation of proteins, such as GAPDH. This is clearly reflected in the design of suitable experimental approaches designed to cope with the interaction of several redox-dependent factors. Clear interactions are de...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)48636-3
更新日期:2002-01-01 00:00:00
abstract::In vivo genome manipulation through site-directed mutagenesis and chromosome rearrangements has been hindered by the difficulty in achieving high frequencies of targeting and the intensive labor required to create altered genomes that do not contain any heterologous sequence. Here we describe our approach, referred to...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)09019-1
更新日期:2006-01-01 00:00:00
abstract::In the near future, computational tools and methods based on the mathematical modeling of biomedically relevant networks and pathways will be necessary for the design of therapeutic strategies that fight complex multifactorial diseases. Beyond the use of pharmacokinetic and pharmacodynamic approaches, we propose here ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381270-4.00011-1
更新日期:2011-01-01 00:00:00
abstract::Signal transduction by vascular endothelial growth factors (VEGFs) through their cognate VEGF receptor tyrosine kinases follows the consensus scheme for receptor tyrosine kinases. Thus, binding of ligand induces receptor dimerization and activation of the tyrosine kinase through transphosphorylation between receptor m...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)02013-2
更新日期:2008-01-01 00:00:00
abstract::A variety of recent advances in single-molecule methods are now making possible the routine measurement of the distinct catalytic trajectories of individual enzymes. Unlike their bulk counterparts, these measurements directly reveal the fluctuations inherent to enzymatic dynamics, and statistical measures of these flu...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)75010-2
更新日期:2010-01-01 00:00:00
abstract::Anomalous small angle X-ray scattering (ASAXS) exploits contrast variation methods to highlight the scattering from one elemental component in a multielement sample, such as one ion species in an ion-DNA system. The ASAXS method has been applied to measure ions condensed around short nucleic acid duplexes. This chapte...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)69019-4
更新日期:2009-01-01 00:00:00
abstract::Structure-based protein design is a powerful technique with great potential. Challenges in two areas limit performance: structure scoring and sequence-structure searching. Many of the functions used to describe the relationship between protein sequence and energy are computationally expensive to evaluate, and the spac...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-394292-0.00008-4
更新日期:2013-01-01 00:00:00
abstract::Chromatin plays a vital role in regulating cellular processes that occur on the DNA. Modulation of chromatin structure is conducted through interactions with binding factors that direct critical actions such as posttranslational modifications, nucleosome remodeling, and incorporation of histone variants. Specific fact...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-391940-3.00011-1
更新日期:2012-01-01 00:00:00
abstract::Membrane-initiated steroid signaling (MISS) involves rapid second messenger based intracellular signaling without coupling to transcription or translation. MISS activates important cellular signaling cascades such as mitogen-activated protein kinase (MAPK) or adenylate cyclase pathways. Despite its vital role in signa...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2020.02.018
更新日期:2020-01-01 00:00:00
abstract::Research on circadian biology over the past decade has paid increasing attention to the photoreceptor mechanisms that align the molecular clock to the 24-h light/dark cycle, and some of the results to emerge are surprising. For example, the rods and cones within the mammalian eye are not required for entrainment. A po...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/S0076-6879(05)93037-1
更新日期:2005-01-01 00:00:00
abstract::This chapter deals with the use of functionalized carbon nanotubes (fCNTs) as supports for the development of nanobiocatalytic systems through the immobilization of enzymes. The surface characteristics, properties and production of carbon nanotubes are described, while an analysis in their biological applications is a...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2019.10.015
更新日期:2020-01-01 00:00:00
abstract::The reverse dot-blot method is a simple and rapid diagnostic procedure that allows screening of sample for a variety of mutations/polymorphisms in a single hybridization reaction. Several methods of immobilizing the oligonucleotide probes are discussed. The reverse dot-blot method has several unique properties that ar...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(93)18029-c
更新日期:1993-01-01 00:00:00
abstract::Benzylisoquinoline alkaloids (BIAs) constitute a diverse class of plant secondary metabolites that includes the opiate analgesics morphine and codeine. Collectively, BIAs exhibit a myriad of pharmacological activities, including antimicrobial, antitussive, antispasmodic, and anticancer properties. Despite 2500 known B...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.02.011
更新日期:2016-01-01 00:00:00
abstract::Biochemical analysis of the components of the mRNA decay machinery is crucial to understand the mechanisms of mRNA decay. The Lsm1p-7p-Pat1p complex is a key activator of decapping in the 5' to 3'-mRNA decay pathway that is highly conserved in all eukaryotes. The first step in this pathway is poly(A) shortening that i...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)02603-7
更新日期:2008-01-01 00:00:00
abstract::Peptides corresponding to the calmodulin-binding domain of MLCK and other target enzymes promise to be useful tools in the study of calmodulin action. Not only will they help to elucidate the molecular interactions of calmodulin with specific target enzymes, but they should also prove to be useful calmodulin antagonis...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(87)39079-2
更新日期:1987-01-01 00:00:00
abstract::Single-molecule Förster resonance energy transfer (smFRET) is a versatile tool for studying biomolecules in a quantitative manner. Multiple conformations within and interactions between biomolecules can be detected and their kinetics can be determined. Thus, smFRET has become an essential tool in enzymology. Ordinary ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.08.024
更新日期:2016-01-01 00:00:00
abstract::Virtually all eukaryotic cell functions and signaling pathways are regulated by protein phosphorylation. The Rad53 kinase plays crucial roles in the DNA damage response in Saccharomyces cerevisiae and is widely used as a surrogate marker for DNA damage checkpoint activation by diverse genotoxic agents. Most currently ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.09.032
更新日期:2017-01-01 00:00:00
abstract::This protocol describes the expression and analysis of membrane proteins produced in yeast, as illustrated with Yarrowia lipolytica and Pichia pastoris. Step by step, we explain how to generate a yeast strain expressing the membrane protein of interest, how to prepare a membrane protein sample from yeast, and how to a...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2014.12.010
更新日期:2015-01-01 00:00:00
abstract::Monte Carlo simulations of neighbor exclusion models have been used to demonstrate the importance of collecting and fitting data over a wide range of saturation. Low saturation data are important for good estimates of the affinity K of a drug or protein for the lattice site. High saturation data are important for dist...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(94)40065-2
更新日期:1994-01-01 00:00:00
abstract::Ubiquitin (Ub)-mediated protein degradation is a key cellular defense mechanism that detects and eliminates defective proteins. A major intracellular site of protein quality control degradation is the endoplasmic reticulum (ER), hence the term ER-associated degradation, or endoplasmic reticulum-associated degradation ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.12.035
更新日期:2019-01-01 00:00:00
abstract::The JNKs (c-Jun N-terminal protein kinases) play important roles in a variety of physiological and pathological functions including induction of apoptosis. A major pathway by which JNKs are activated in response to apoptotic stimuli includes the GTP*Rac1-binding scaffold protein POSH (plenty of SH3s). POSH acts as a s...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)06036-8
更新日期:2006-01-01 00:00:00
abstract::Chloroplast genomes of land plants and algae contain generally between 100 and 150 genes. These genes are involved in plastid gene expression and photosynthesis and in various other tasks. The function of some chloroplast genes is still unknown and some of them appear to be essential for growth and survival. Repressib...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2014.10.035
更新日期:2015-01-01 00:00:00
abstract::As new organisms are being sequenced on a daily basis, new DNA glycosylases that recognize DNA damage can be easily identified in an effort to understand both their phylogenetics and substrate specificities. As a practical matter, existing bacterial and human homologs need to be readily available as laboratory reagent...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)08002-5
更新日期:2006-01-01 00:00:00
abstract::Primary mixed liver cells were isolated from rats that had been fed an amino acid (AA) diet in which natural protein was replaced with a defined mixture of pure AAs. Nitric oxide (NO) production from these cells in vitro was monitored using a nitric oxide (NO)-selective fluorescent probe, diaminofluorescein, followed ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)96045-X
更新日期:2005-01-01 00:00:00