细胞功能测定

In this chapter, we describe the preparation and use of b-PG, a biotinylated complex that specifically binds hyaluronan (1,2). The b-PG is derived from cartilage and consists of a trypsin fragment of the proteoglycan core protein and one of the link proteins. Because of its ability to bind to hyaluron ...

Cadherins constitute a family of transmembrane glycoproteins (Table 1) that mediate cell-cell adhesion by their ability to self-associate (1–4). For example, E-cadherin binds only to E-cadherin and not to N-cadherin. The homotypic interaction of cadherins is important for the sorting ...

The introduction of microinjection of trace amounts of a fluorescently labeled protein (1,2) into cultured cells leads to its incorporation into the cell’s pool of endogenous protein. Provided the microinjected protein has retained the properties of the native protein, it will become i ...

The manipulation of mice genome is a powerful technique to analyze biological processes that cannot be studied in tissue culture (1). The understanding of many developmental processes is today improving because of such techniques. This is also changing the way human genetic diseases are s ...

Formation of the vertebrate skeleton is an intricately orchestrated, multistep process. Mechanisms underlying skeletogenesis need to account for the global specification of skeletal pattern, commitment of cells to skeletal and hematopoietic lineages, local control of skel ...

Pax3, a transcription factor expressed in the developing embryo, is a critical factor for the proper formation of the mammalian nervous, cardiovascular, and muscular systems. In the mouse, spontaneous mutations in Pax3 resulting in complete loss of function have provided important mod ...

Disorders affecting craniofacial development represent a large fraction of birth defects (1). The advent of transgenic and gene knockout technology has led to the identification of a variety of genes that have roles in craniofacial development. These technologies, together with the t ...

The low solubility of a protein is one of the most frequent impediments for its structural and functional analysis and, on a more practical aspect, for its application as an industrial enzyme. The reason for low solubility can lie in low conformational stability (1), in a high number of surface-expos ...

For many applications proteins must retain their function for a long time and under a wide range of conditions, sometimes at elevated temperatures. However, the stability of natural proteins is often very low, just sufficient to ensure proper functioning under cellular conditions (1). It is s ...

Protein-protein interactions are involved in most biological processes and are an important target for drug design. Over the past decade, there has been an increased interest in the design of small molecules that mimic functional epitopes in protein-protein interactions. However, the ...

It is often possible to screen libraries of variant enzymes directly on agar plates. Colony-based solid-phase screening is an attractive option because of its relative ease and high throughput compared to liquid-phase screening in multi-well plates. As with any high throughput screeni ...

Enzyme thermostability is a property of great importance in the era of designed biocatalysts. While enzymes are capable of catalyzing reactions with exquisite specificity and selectivity, they are often limited by insufficient stability. Improvements in enzyme activity throu ...

We describe a high throughput screening setup for measuring α-amylase activity at temperatures far above the boiling point of water. The system consists of a sealed aluminum 384-well assay plate incubated between two preheated aluminum blocks. Samples consisting of starch solution and ...

Cytochromes P450 are a superfamily of enzymes that catalyze the monoxygenation of a variety of substrates, including aliphatic and aromatic compounds (1). They contain a noncovalently bound protoporphyrin IX, giving these enzymes characteristic spectral properties. This heme ...

The oxidation of aromatic compounds is important in producing chemical intermediates for the chemical and pharmaceutical industries (1,2). Conventional aromatic oxidation reactions are prone to byproduct formation and often require heavy-metal catalysts, extremes of tem ...

The United States consumes approximately 16–17 million barrels of crude oil per day, and the majority is used for electricity, heating, and transportation fuel (1). The main constituents of crude oil are linear aliphatics. Clearly, the selective hydroxylation of alkanes to more valuable pr ...

Peroxidases catalyze the decomposition of hydrogen peroxide coupled to the oxidation of a variety of organic and inorganic substrates. Produced by a broad variety of natural sources, most peroxidases use heme or vanadium as a cofactor at the redox active site, while some bacterial peroxid ...

Screening conditions should simulate the final application as closely as possible. This is especially a challenge when chromophore-free (e.g., aliphatic) substrates are used and no simple and reliable high-throughput method for quantitative analysis of the respective reaction ...

Oxidases remove hydrogen from substrates, transferring it to molecular oxygen to generate hydrogen peroxide or water. Since oxidases catalyze fundamental synthetic transformations, including alcohol-aldehyde and aldehyde-carboxylic acid conversions, they have att ...

In this chapter, we describe colorimetric methods for screening laccases using natural and nonnatural substrates. Laccases (EC 1.10.3.1) are blue-copper enzymes that oxidize phenols, polyphenols, and anilines (1,2). The catalytic capabilities of laccase can be greatly enhanced by t ...