生物化学技术

Although the majority of proteins expressed in organisms with relatively low protein complexity, such as Escherichia coli, can be resolved and detected in a single gel (1,2), this is not true for more complex organisms, such as Drosophila melanogaster. A smaller fraction of the total compleme ...

Shortly after the introduction of electrospray as a viable ionization technique for large molecules (1), electrospray tandem mass spectrometry (ES MS/MS) techniques, such as HPLC-ES MS/MS, were used successfully for peptide sequencing at picomole and subpicomole levels (2–4). In LC-M ...

The small nematode Caenorhabditis elegans (C. elegans, 1 mm in length) originally was introduced into the laboratory in 1965 by Brenner (1) and has since become an important model organism. Caenorhabditis elegans for a variety of reasons offers excellent conditions for the study of basic fea ...

Two-dimensional polyacrylamide gel electrophoresis (2-DE) provides the ability to resolve and quantify thousands of proteins from an unfractionated eukaryotic cell lysate. The use of 2-DE in studying eukaryotic cells has run the gambit from ozone-stressed Norway spruce needles ( ...

The protocol for extracting proteins from mouse and human tissues (organs) described in this chapter follows a strategy that is based on the intention to include all the various protein species of a particular tissue in a set of samples that are suitable for two-dimensional electrophoresis (2 ...

Differential detergent fractionation (DDF) represents an alternative method for cell fractionation that employs sequential extraction of cells or tissues with detergent-containing buffers to partition cellular proteins into structurally and functionally intact and ...

Mass spectrometric methods of determining protein ubiquitination are described. Characteristic mass shifts and fragment ions indicating ubiquitinated lysine residues in tryptic and gluC digests are discussed. When a ubiquitinated protein is enzymatically digested, a po ...

The first compartment encountered by newly synthesized secreted proteins is the endoplasmic reticulum (ER). Before secreted proteins can traffic beyond the ER they must fold into their final conformations, and components of multiprotein complexes must assemble. Not surprising ...

Recent studies have revealed that rabbit reticulocyte lysate (RRL) efficiently reconstitutes endoplasmic reticulum-associated degradation (ERAD) of mutant and misfolded membrane proteins. When supplemented with canine pancreas microsomal membranes, the RRL system ...

Despite the identification of numerous deubiquitinating enzymes (DUBs) in recent years, the large majority of this class of enzymes has not been well characterized. This chapter describes biochemical methods that can be used to characterize the function and substrate specificity of ...

The fast-track approval of a proteasome inhibitor, PS-341, to treat multiple myeloma spurred a wave of interest in both the proteasome itself and small-molecule compounds blocking its activities. Besides being candidates for drugs against cancer, autoimmune diseases, inflammati ...

Ubiquitin-activating enzyme is the archetype for a family of enzymes catalyzing the ATP-coupled activation of ubiquitin and other class 1 ubiquitin-like proteins required for their subsequent conjugation to cellular targets. The general physical and mechanistic features of the ...

Many eukaryotic proteins are regulated by the covalent attachment of ubiquitin or polyubiquitin chains. These include proteins involved in cell cycle control, tumor suppression, and many signaling pathways. Ubiquitination of proteins occurs through an enzymatic cascade of thr ...

Several important signaling processes depend on the tagging of cellular proteins with “polyubiquitin chains”—ubiquitin polymers whose building blocks are connected by isopeptide bonds between G76 of one ubiquitin and a specific lysine residue of the next one. Here we describe proc ...

The proteasome is a highly complex, ATP-dependent protease, consisting of over 30 subunits, and dedicated mainly to the degradation of ubiquitin-protein conjugates. Proteasomes are evolutionarily conserved in the eukaryotic kingdom, and those of yeast are well suited to serve as a gen ...

The 26S proteasome is composed of the core 20S proteasome in association with the 19S regulatory complex, or PA700. PA700 has multiple activities, including ATPase activity, polyubiquitin-chain binding activity, deubiquitination activity, chaperone-like activity, and subs ...

Although receiving little attention in biochemical and physiological textbooks, the polyamines have a long history and have accumulated a considerable literature (for reviews see refs. 1–11). In 1678 Antoni van Leeuwenhoek (12) described crystals that formed in samples of human semen ...

L-Ornithine decarboxylase (EC 4.1.1.17) (ODC) catalyzes the conversion of L-ornithine to putrescine and CO2. ODC is dependent on pyridoxal 5′-phosphate (PLP) and thiol-reducing agents for activity (1). At least two key active site residues of ODC are known. Lysine 69 was recently identified as ...

Ornithine decarboxylase (EC 4.1.1.17) (ODC) catalyzes the decarboxylation of ornithine to form putrescine. This reaction is the rate-limiting step in the biosynthesis of the naturally occurring polyamines, which regulate proliferation and which are closely linked to neoplastic ...

S-adenosylmethionine decarboxylase (AdoMetDC; EC 4.1 1.50) catalyzes the conversion of S-adenosylmethionine to S-5′-deoxyadenosyl-(5′)-3-methylthiopropylamine (decarboxylated S-adenosylmethionine) and CO2. The enzyme is unusual among decarboxylases in that it d ...