细胞技术

An important goal for a biochemist studying a membrane transporter is to characterize various aspects of its transport function. Initial studies are often carried out in intact cells, but further work is often directed toward studying the functional properties of the purified protein. S ...

The E. coli JS200 strain carries a temperature-sensitive allele of DNA polymerase I that renders this strain conditional lethal. Growth under restrictive conditions is restored by small amounts of DNA polymerase activity. Even mutants with greatly reduced (1–10% of wild-type) catalyt ...

Genetic selection provides a powerful tool for the study of cellular processes. It is particularly useful in analyzing protein sequence constraints when used in conjunction with directed molecular evolution. Our lab has used this approach to analyze the function of enzymes involved in D ...

DNA-directed DNA polymerases have been broadly classified into seven families based on their sequence homology (1). It is surprising to learn that enzymes such as DNA polymerases, which carry out pivotal role during DNA replication, repair, and recombination, are poorly conserved amon ...

The evolution of proteins is more difficult than the evolution of nucleic acids both in principle and in practice. While nucleic acid sequence space has a dimensionality of 4n, where n is the size of the nucleic acid pool (i.e., G, C, A, and T), protein sequence space has a dimensionality of 20n. Similarly, while n ...

The physicochemical and mechanical properties of phospholipids dispersed in an aqueous medium provide the molecular framework for many of the dynamic properties of cell membranes (1,2). The ability of protein molecules embedded in the plasma membrane to move laterally from one locat ...

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 ...