细胞功能测定

Cyclic adenosine 3′,5′-monophosphate (cAMP) and cyclic guanosine 3′,5′-monophosphate (cGMP) have between them been implicated in the mediation of many physiological phenomena. Over the last 30 years, a number of techniques have been employed to assess changes in the amounts of these two mo ...

Part A. Protein Kinase-Mediated Phosphorylation Events Protein kinases (ATP:protein phosphotransferases) regulate a wide range of cellular events, including the transduction of signals leading to cell growth. Although the protein kinase superfamily encompasses a large and ...

Part A. Identification of G-Proteins The past decade has seen the emergence of a rapidly expanding superfamily of regulatory proteins, the G-proteins, that transduce as diverse a range of biological functions as protein synthesis, transmembrane signaling, intracellular traffic ...

Part A. Sample and Standards Preparation In Chapter 18, the extraction procedures for recovery of the phosphoinositols and phosphoinositides from cells are described, together with simple separation procedures to resolve them into their general classes (InsP1; InsP2, and so on, and Pt ...

Part A. Biosynthesis and Extraction of Phosphoinositides and Phosphoinositols The minor inositol-containing membrane phospholipids, the phosphoinositides, play a central role in cell signal transduction. Activation of a hormone-sensitive phospholipase C (phosphoi ...

In order to determine the structure of a protein by X-ray crystallography, well ordered three-dimensional crystals are required. However, despite the wealth of experience accumulated in the course of the crystallization and structural analyses of several hundred soluble globular ...

Despite a 10,000-fold gradient of Ca2+ across the cell membrane, the concentration of cytosolic free calcium, i, is regulated with remarkable constancy. A combination of mechanisms precisely regulate i at nanomolar levels. These include influx of Ca2+ via plasma membrane calcium channe ...

Many cell functions are controlled by molecular signals (hormones, neurotransmitters, and so forth) that interact with cell-surface receptors and trigger specific intracellular responses. Intracellular signaling can be difficult to study in isolated, purified systems, be ...

Receptor-mediated endocytosis is the process used by all eukaryotic cells to internalize a variety of biologically important macromolecules, e.g., transport proteins that deliver nutrients to cells (1,2), plasma proteins (3), hormones and growth factors (4,5), and lysosomal enzym ...

Movement of ions across cell membranes is associated with processes such as cell signaling, regulation of cell volume, maintenance of cell composition and pH, and energy transduction. Even in the erythrocyte, a very specialized cell with limited biological function, 11 different trans ...

The study of proteins and glycoproteins as biologically active ligands is an expanding area of research, stimulated in part by the identification of growth factors and cytokines and, in particular, those involved in the regulation of hemopoiesis and the inmiune response. The exquisite se ...

The ability to compartmentalize function has enabled multicellular organisms to evolve sophisticated physiological systems. At the cellular level, compartmentalization of function is established by the maintenance of asymmetric structure and function between the apic ...

This method selectively labels glycoproteins on the cell surface. Tritiated borohydride reduction may follow chemical oxidation of vicinal hydroxyl groups by sodium periodate or enzymatic oxidation by galactose oxidase (GAO).** The periodate oxidation is more effi cient. It gene ...

Integral membrane proteins of physiological importance, such as ion channels, transporters, receptors, and enzymes, are usually minor components of the membrane. This low abundance, coupled with their hydrophobicity and frequent instability in detergent solution, renders th ...

Part A. Production of Hybridoma Cell Lines A strategy for raising monoclonal antibodies involves immunization of mice; fusion of the mouse spleen cells with a myeloma cell line; selection, cloning, and freezing of hybridoma cell lines; and screening for monoclonal antibody production. P ...

Purification of membrane proteins of necessity requires the use of detergents to solubilize the proteins prior to their isolation. Although reconstitution of membrane proteins has been achieved without the use of detergents (e.g., by sonication of membrane proteins with lipids), det ...

Protein-protein interactions in biological membranes can be studied using conventional chemical crosslinking methods, crosslinked products being detected either by altered molecular weights on SDS-polyacrylamide gels or using specific antibodies. Unfortunately, ...

Interactions between lipids and proteins have been studied using a variety of spectroscopic methods. Nuclear magnetic resonance (usually using deuterated phospholipid) can give information about conformational changes of lipid molecules on binding to membrane proteins, b ...

The basic principle for probing the transverse distribution of membrane lipids is simple. Lipids that are available for modification by enzymes or for covalent labeling in impermeable membrane vesicles are considered to be in the outer leaflet of the membrane bilayer, and those that are also ...

The transforming growth factor-β (TGF-β) pathway is important in the growth control of many different cell types, particularly epithelial cells, and is often disrupted in the development of cancer (1,2). The most common mutation in the TGF-β pathway detected thus far is mutation of the type II rec ...