Proteinases containing enzymes

A large group of proteinases contain serine in their active center. The serine proteases include, for example, the digestive enzymes trypsin, chymotrypsin, and elastase (see pp. 94 and 268), many coagulation factors (see p. 290), and the fibrinolytic enzyme plos-min and its activators (see p. 292). 

Proteinase-containing samples are incubated with a variety of class- or enzyme-specific proteinase inhibitors, separated on a polyacrylamide gel, and activity stained as described in Basic Protocol 3. A clear zone will be evident in lanes where the proteinase is active (i.e., in the absence of inhibitor or in the presence of a mismatched inhibitor). This clear zone will be absent in the lane containing the properly matched proteinase inhibitor, which provides information about the class or type of proteinase detected in the band. 

Mix a proteinase-containing protein sample with 0.1 vol class- or enzyme-specific inhibitor solution. Include samples without inhibitor but with an appropriate amount of solvent used to prepare inhibitors, as well as known proteinases with their inhibitors as controls. 

Albumins and globulins 12-20 Extractable by water of dilute salt solutions (e.g., 0.1-0.5 M NaCl) Contains enzymes (amylases, proteinases, lipoxygenases) Rich in Lys, Asp, Ala, Cys, Leu, and Arg low in Met content... 

Crystallographic studies of native cysteine proteinases and enzyme-inhibitor complexes have been used to interpret much or the kinetic data for cysteine protemsse-caUlyzed hydrolysis of amide bonds. Analysis of the crystal structures of papain [16]. caricain [38], actinidain [56], etc. shows that these structures are closely related. The active site of all these cysteine proteinases contains the Cys-25 sulfhydryl group in close proximity to the His-159 imidazole ring nitrogens, where the latter can abstract the sulfhydryl proton to facilitate attack on the substrate amide carbonyl group [17]. 

In the family of subtilisin-type serine proteases, primary sequences of about 40 enzymes are known.423 Among them, no subtilisin produced by Bacillus species has cysteine residues. Aqualysin I (four cysteine residues per molecule),163 proteinase K (five residues),173 and thermitase (one residue)223 are cysteine-containing enzymes (Fig. 12.2). 

Pancreatic enzyme supplements are used to treat people who lack pancreatic secretions. Pancreatm, the British Pharmacopoeia standard, is an extract of pancreas, and contains enzymes with proteinase, amylase, and lipase activity most commercial formulations are similar or identical (SEDA16, 358). 

Transfer hardened blocks (or 1 cm tubing cylinders, or blocks cut from agarose drops in dishes) to conical tubes containing 50 vols. of buffer containing 10 mM Tris-HCI (pH 8.0), 0.1 M EDTA, 1% Sarkosyl and 100-400 pg/ml of proteinase K (enzyme added to stock solution just before use). Incubate for 24 h at 37°C, replace with fresh digestion solution and incubate for another 16-24 h at 37°C. Replace with 50 vols. of TE buffer and incubate overnight at 4°C. 

Cysteine proteinases contain a catalytically active cysteine sulfhydiyl group (Cys-25) and a histidine group (His-159) within the active center of the enzyme [51]. Alkylation of the active-center sulfhydryl group renders the product inactive. The minimal reaction mechanism is represented in Figure 4 [52]. 

Many plants detoxify heavy metals by sequestering them, either as phytochelatin complexes or without specific ligands, in the vacuoles (for reviews see e.g., [73,74]). It makes sense for hyperaccumulating plants to store metal in the vacuoles as well because this organelle only contains enzymes like phosphatases, lipases, and proteinases [75] that were never identified as targets of heavy metal toxicity. Vacuole sequestration is driven to an extreme form in hyperaccumulators, where... 

In the first edition of this book this chapter was entitled "Antiparallel Beta Structures" but we have had to change this because an entirely unexpected structure, the p helix, was discovered in 1993. The p helix, which is not related to the numerous antiparallel p structures discussed so far, was first seen in the bacterial enzyme pectate lyase, the stmcture of which was determined by the group of Frances Jurnak at the University of California, Riverside. Subsequently several other protein structures have been found to contain p helices, including extracellular bacterial proteinases and the bacteriophage P22 tailspike protein. 

While the majority of attention has focused on peptides contained within the nervous system, two other important methods for delivering peptides to the vicinity of the mast cell have been established (1) peptides produced and secreted by other cells of inflammation that may affect mast-cell function and (2) the local generation of mast-cell-active peptides by secreted enzymes acting on circulating protein precursors. Examples of the former include several, as yet ill-defined, peptide factors and cationic proteins from other immunocompetent cells [66-69], defined lymphokines such as the interleukin-1 [70] and interleukin-3 [71], and tumour necrosis factor [70], Examples of the latter include bradykinin [72] and a recently identified peptide produced by the action of acid proteinases on albumin [73, 74]. 

The tissue or cell sample is firstly homogenized in a buffer containing a detergent such as Triton X-100 and sodium deodecyl sulphate (SDS), which disrupts the cell and dissociates DNA-protein complexes. Protein and RNA are then removed by sequential incubations with a proteolytic enzyme (usually proteinase K) and ribonuclease. Finally the DNA is extracted into ethanol. Ethanol only precipitates long chain nucleic acids and so leaves the single nucleotides from RNA digestion in the aqueous layer. 

Aspartic Proteinases. This group of proteinases is named for the aspartic acid residue in the active site. Previously, this group of enzymes was often referred to as the "acid proteases" (4). Members of this group are generally found only in eukaryotic organisms. However, clear evidence has been presented that certain viruses, most importantly the virus (HIV-1) considered to give rise to autoimmune deficiency disease (AIDS), and the polio virus, contain coding sequences for a dimeric aspartic proteinase which is involved in the... 

The functional proteins in the cell have to be protected in order to prevent premature degradation. Some of the intracellularly active proteolytic enzymes are therefore enclosed in lysosomes (see p. 234). The proteinases that act there are also known as cathepsins. Another carefully regulated system for protein degradation is located in the cytoplasm. This consists of large protein complexes (mass 2 10 Da), the proteasomes. Proteasomes contain a barrel-shaped core consisting of 28 subunits that has a sedimentation coef cient (see p. 200) of 20 S. Proteolytic activity (shown here by the scissors) is localized in the interior of the 20-S core and is therefore protected. The openings in the barrel are sealed by 19-S particles with a complex structure that control access to the core. 

At the center of the apoptotic process lies a group of specialized cysteine-containing aspartate proteinases (see p. 176), known as cas-pases. These mutually activate one another, creating an enzyme cascade resembling the cascade involved in blood coagulation (see... 

This enzyme [EC 3.4.24.27], also known as Bacillus ther-moproteolyticus neutral proteinase, is a thermostable extracellular metalloendopeptidase containing zinc and four calcium ions. A member of the peptidase family M4, this enzyme catalyzes the hydrolysis of peptide bonds with a preference for Xaa—Leu > Xaa—Phe. 

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

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