生物化学技术

Preparations of calcium-tolerant cardiac myocytes for studies on intracellular calcium (i) signaling and contraction have been difficult owing to the susceptibility of these cells to the enzymatic digestion process. This often leads to the cells acquiring a bricklike contracted ...

Posttranslation covalent modification of proteins by phosphorylation represents an important mechanism in the control of many cellular functions (1,2). Protein kinases catalyze the transfer of the γ-phosphoryl group of ATP to an acceptor protein substrate. The activity of the enz ...

Phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks; EC 2.7.1.68) synthesize phosphatidylinositol 4,5-bisphosphate (PI 4,5-P2) from phosphatidylinositol 4-phosphate (PI 4-P). The hydrolysis of PI 4,5-P2 by phosphoinositide-specific phospholipase C generates the s ...

Signal-activated phospholipase-catalyzed hydrolysis of phosphatidylcholin involves three distinct enzymes: phospholipase A2, phospholipase C (PLC), and phospholipase D (PLD) (1). PLC-catalyzed hydrolysis generates sn-1,2-diacylglycerol (DAG) and choline phosp ...

The importance of diacylglycerol (DAG) as a second messenger for calcium-mobilizing hormones, neurotransmitters, and cytokines was underscored by the discovery of an effector enzyme, protein kinase C, which was also a cellular receptor for phorbol ester tumor promoters (reviewed in ...

Phospholipase A2 (PLA2, EC 3.1.1.4, phosphatide sn-2 acyl hydrolase) is a ubiquitous enzyme that plays a role in the response of a wide variety of cells to specific stimuli. PLA2 is an important component of the cellular machinery that is activated by inflammatory stimuli and it maintains cell homeo ...

Traditionally, phospholipase activation is monitored in static incubations by the intracellular accumulation of lipidic byproducts of phospholipid hydrolysis. Thus, for phospholipase C (PLC), diacylglycerol accumulation can be assessed, as can intracellular accumul ...

Arachidonic acid has been implicated as a second messenger in numerous systems, and many agonists stimulate the intracellular accumulation of arachidonic acid (1,2). Arachidonic acid may be generated by phospholipase A2 hydrolysis of phospholipids, or by hydrolysis of diacylgly ...

We have summarized in Chapter 16 the recent advances in the mechanisms of regulation of leukotriene and prostaglandin biosynthesis and their pathways of synthesis. We have outlined a detailed summary for measuring prostaglandins, thromboxane, and leukotrienes via radioimmuno ...

Sphingomyelin (SM) is the latest addition to the family of membrane phospholipids that is a source of molecules behaving as intracellular messengers (1). Ceramide is one of the products of sphingomyelin hydrolysis by specific sphingomyelinases (SMases) and appears to be involved in the s ...

We have summarized in Chapter 16 the recent advances in the mechanisms of regulation of leukotriene and prostaglandin biosynthesis and their pathways of synthesis. This chapter focuses on the measurement of the leukotrienes and other LOX products by high-pressure liquid chromatogr ...

The minor inositol-containing membrane phospholipids, the phosphoinositides, play a central role in cell signal transduction. Activation of a hormone-sensitive phospholipase C (phosphoinositidase C) at the plasma membrane results in the rapid catabolism of the polyphosph ...

The phosphorylation of long chain sphingoid bases on the primary alcohol group occurs in cells by the action of sphingosine kinase (1–3). Sphingosine kinase is present in the cytosolic fraction of most cells (4–7) and in the membrane fraction of certain tissues and organisms (8,9). The reaction pr ...

As described in Chapter 19, a demonstration of changes in sphingomyelin (SM) and ceramide cellular contents (in response to regulatory molecules and/or other stimuli) is essential to stablish their involvement in the signaling proccess. The study of variations in SM and ceramide cellul ...

The generation of the lipid-signal molecules diacylglycerol (DAG) and phosphatidic acid (PA) from phospholipids like phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositols (PIs) has been implicated in the transduction events essential f ...

Separation of PLs or the lipid mediators DAG and PA into individual molecular species is a complex multistep procedure. For example, first, phospholipid classes must be quantitatively isolated in amounts large enough for further analysis. In Chapter 22, we described the multiple steps and ...

Ceramides (CER) have been proposed to be the intracellular mediators of responses to such agents as interferon-γ (IFN-γ), dexamethasone, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β, and vitamin D3. These agents induce hydrolysis of the plasma membrane sphingomyelin (SM) by a ...

In Chapter 1, procedures for cell labeling with inositol, stimulation with agonists, and extraction of the phosphoinositols and phosphoinositides are described. In this chapter, simple low-resolution chromatography techniques capable of separating phosphoinositols and ...

Individual phosphoinositols and phosphoinositides in tissues and cells are commonly analyzed by thin-layer chromatography (TLC), liquid chromatography, and high-performance liquid chromatography (HPLC) after labeling the phospholipid with a radionucleotide such as ...

In Chapters 1 and 2, the extraction procedures for recovery of the phosphoinositols and phosphoinositides from cells in suspension or culture are described, together with simple separation procedures to resolve them into their general classes (InsP1, InsP2, and so on, and PtdIns, PtdIns ...