Proteases action

Figure 9.11 A case of selective compartmentation in reverse micelles, permitting the synthesis of a peptide by the reverse protease action. The product C, produced in the water pool, is expelled into the outside hydrocarbon environment due to its insolubility in water. (Adapted from Barbaric and Luisi, 1981.)...

We have chosen to discuss enzyme modification of proteins in terms of changes in various functional properties. Another approach might have been to consider specific substrates for protease action such as meat and milk, legumes and cereals, and the novel sources of food protein such as leaves and microorganisms ( ). Alternatively, the proteases themselves provide categories for discussion, among which are their source (animals, plants, microorganisms), their type (serine-, sulfhydryl-, and metalloenzymes), and their specificity (endo- and exopeptidases, aromatic, aliphatic, or basic residue bond specificity). See Yamamoto (2) for a review of proteolytic enzymes important to functionality. 

The conditions necessary Tor the plastein reaction have been reviewed by Fujimaki et al. ( ), and compared to those necessary for proteolysis by Arai et al. ( ). The substrate for the synthetic reaction must consist of low molecular weight peptides, preferably in the tetramer to hexamer range. These are usually produced from proteins by protease action. A number of proteolytic enzymes and protein substrates have been investigated for producing plastein reaction substrates. The most often used proteases are pepsin JJ), and papain (12,13), but others... 

The formation of the oxyanion intermediate during serine protease action is also supported by the existence of tetrahedral forms of enzymes inhibited by substratelike aldehydes. The -OH group of Ser 195 can add to the carbonyl group to form hemiacetals. For example, a 13C-enriched aldehyde whose carbonyl carbon had a chemical shift of 204 ppm gave a 94 ppm resonance as it formed the tetrahedral hemiacetal with one of the inhibitory aldehydes, N-ace tyl-i -Len-i-Leu-L-arginal... 

Polgar, L. (1989) Mechanisms of Protease Action, CRC Press, Boca Raton, Florida... 

Attaching a dipeptide (112) to a sulfanilamide (113) gives a prodrug that may break down in vivo by protease action. In principle, either iV4-acyl- (114) or A -acyl-... 

Despite being more critical for intracellular products, protease action may also occur in extracellular media. For example, in the expression of a baculovirus vector, which is a lytic system, proteases are often expressed and secreted during recombinant protein production, and the problem may assume a critical level. A similar problem occurs in serum-free culture systems. The absence of proteins such as albumin and macroglobulin reduces the protection against proteolysis. In these cases, addition of an antiproteolytic agent to the culture medium is recommended. 

Treatment with chelating agents such as EDTA which removes divalent ions, leads to dissociation of cell monolayers and release of the cells into suspension without protease action. Perfusion of rat liver with citrate solutions has also been used in attempts to produce primary liver cell cultures. 

Albumin was effectively hydrolyzed by Sal3-PAD-Ac, with optimum activity at pH 3 -just as in the action of pepsin, the most typical aspartic protease. Protease action of Sal3-PAD-Ac at pH 3 appears to involve collaboration of proximal carboxyl groups, in analogy with pepsin. 

Aptamers inhibit virus protease action in vitro and in vivo. 

Proteases [DNase free ] are added to the DNA solution to decrease the amount of protein that can remain associated. After the protease action, the entire procedure of DNA extraction is repeated, and the DNA dissolved again in TE buffer and dialyzed against TE buffer to eliminate any proteases and peptides. Professors and students can also refer to the following website for DNA extraction and purification http //gmotraining.jrc.it/docs/ Session04.pdf... 

Kinase substrates can become resistant to the actions of proteases due to their phosphorylations. Thus, the fluorescence quench assays (described in Chapter 2 covering protease assays) can be used to measure kinase activity. The assays can be viewed as coupled because they require a second enzyme to convert a product or substrate into a detectable signal. With kinase assays, the formation of phosphopeptide inhibits the protease action on the peptide and the signal remains quenched and therefore decreased (Rodems et al., 2002). Inhibiting the kinase results in increases in protease sensitivity and in signal. 

Mechanism of HIV-1 protease action and search for inhibitors (with heterocyclic fragment) 03OBC5. 

Physically safer as a mechanistic criterion is reversible inhibition by boronic acids, RB(0H)2, which add the active site serine to form tetrahedral species [RB(0H)20Ser] , which mimic the tetrahedral intermediate/transition state. They also mimic the tetrahedral intermediates in aspartic protease action, however, and are therefore not as definitive. 

When cells are lysed, proteases (enzymes that break peptide bonds in proteins) are often activated. Degradation of proteins through protease action greatly complicates the analysis by 2D electrophoresis, so action should be taken to avoid this problem. If possible, it is advisable to inhibit proteases by disrupting the sample directly into strong denaturants such as 8 M urea, 10% trichloroacetic acid (TCA), or 2% SDS [45 7]. Proteases are less active at lower temperatures, so sample preparation at low temperature is recommended. In addition, proteolysis can often be inhibited by preparing the sample in the presence of Tris base, sodium carbonate or basic carrier ampholyte mixtures [48, 49]. 

The botulinum toxins are large polypeptide molecules (approximately 150 kDa) which contain Zn ions. They are synthesised as single polypeptides which are cleaved by endogenous protease action to yield the active forms, each of which consists of two polypeptide chains linked by a disulphide bond. The larger of the two chains, denoted the heavy (H) chain, has a mass of approximately 95 kDa and the smaller, the light (L) chain, a mass of approximately 50 kDa. The structure is illustrated in Figure 1. The H chain is composed of two domains of similar mass the C-terminal domain (He) is responsible for the specificity of binding of the toxin to peripheral motoneurones and the N-terminal domain (Hn) for penetration of the toxin into the neuronal cell. In the neuronal cytoplasm, the L chain is released by proteolysis and it is this subunit which is responsible for the disruption of the exocytotic apparatus, which causes blockade of transmitter release. 

The resulting protein hydrolysates, though free of nonprotein impurities, are accompanied by another problem in most cases—a problem caused by a bitterness resulting from protease action on proteins. As discussed below, the resynthesis reaction leading to plastein formation... 

The concept of a proteolytic mechanism in the production of inflammatory manifestations is supported by the demonstration of anti-inflammatory activity in various protease inhibitors, e.g. the trypsin inhibitors of the pancreas, soya bean, ovomucoid and potato . It is interesting to note that the inhibitor from potatoes exerts its effect even after the inflammation is well established , suggesting that protease action is a continuing feature of inflammation and not simply an initiating process. Various esterase inhibitors (dyflos, quinine, quinidine and chloroquine) also reduce capillary permeability induced by heat and the permeability globulins . 

Thyroxine and triiodothyronine are released from thyroglobulin through the action of a series of proteases. Both the protease action and the release of the thyroid hormones into the bloodstream are stimulated by pituitary thyrotropin Like glucagon... 

Hunkapiller, M. W., Smallcombe, S. H., and Richards, J. H. (1975). Org. Mag. Res. 7, 262. Mechanism of Serine Protease Action. Ionization Behaviour of Tetrahedral Adduct Between a-Lytic Protease and Tripeptide Aldehyde Studied by Carbon-13 Magnetic Resonance. 

Angiotensin-converting enzyme (ACE) inhibitors are used for the treatment of high blood pressure, and were designated using the carboxypeptidase structure as a model for Zn " protease action. Captopril is a small, potent, orally available, dipeptidyl inhibitor of ACE. 

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