Proteolysis proteolytic enzymes

Protenoids Proteoglycans Proteolysis Proteolytic enzymes Proteus... 

North, M.J. 1993. Prevention of unwanted proteolysis. In Proteolytic Enzymes a Practical Approach. R.J. Beynon and J.S. Bond, editors. IRL Press, Oxford,... 

The existence of flexible regions in ribosomal proteins can be explored by studying the action of proteolytic enzymes under mild conditions. It has been found that many E. coli ribosomal proteins consist of two domains one compactly folded and resistant to proteolysis, the other flexible and vulnerable to proteases (Littlechild et al, 1983). Some proteins (S15, S16, S17, and L30) are very resistant whereas others (S2, S6, S9, L2, L27, L29, and L33) are completely degraded without the appearance of discrete fragments. The remaining proteins yield fragments of various size under these conditions. 

A protein known as the amyloid precursor protein (APP) spans the plasma membrane of the neurone. It possesses an extracellular domain but its function is unknown. The extracellular domain is partially hydrolised by proteolytic enzymes, known as secretases. One of the products is the amyloid peptide, of which there are two forms. The larger form, contains 42 amino acids and readily polymerises to form plaques in the extracellular space, damaging the neurones. Some sufferers possess a mutated form of the APP protein which more readily produces the larger peptide upon proteolysis, so that more toxic plaques are produced. It is the progressive accumulation of these plaques that is considered to be one cause of Alzheimer s disease. 

The granules contain two types of proteins that result in death. First, compounds that produce holes (pores) in the membrane of the cells these are the proteins, perforin and granulysin. Both insert into the membrane to produce the pores. These were once considered to result in death by lysis (i.e. exchange of ions with extracellular space and entry of water into the cell). However, it is now considered that the role of the pores is to enable enzymes in the granules, known as granzymes, to enter the cell. These granzymes contain proteolytic enzymes. They result in death of the cell by proteolysis but, more importantly, activation of specific proteolytic enzymes, known as caspases. These enzymes initiate reactions that result in programmed cell death , i.e. apoptosis (Chapter 20). 

Figure 21.13 Regubtion of the rate of proteolysis of p53 by phosphorylation. The thick arrow indicates a high rate of proteolysis by the intracellular proteolytic enzyme, the proteasome. Phosphorylation of p53 reduces the rate of proteolysis (thin arrow) phosphorylation is catalysed by a DNA damage-sensitive protein kinase.

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... 

Kuehler and Stine (43) studied the functional properties of whey protein with respect to emulsifying capacity as affected by treatment with three proteolytic enzymes. Two microbial proteases and pepsin were examined. The emulsion capacity decreased as proteolysis continued, suggesting that there is an optimum mean molecular size of the whey proteins contributing to emulsification. 

Several variations of this procedure may be found in the literature. Often, homogenization is facilitated by adding a proteolytic enzyme to the original suspension in step 2, but the proteolysis must be carefully... 

The proteolysis of cellulases has been previously investigated. Nakayama et al. (14) found that mild proteolysis of endoglucanase from T. reesei by a protease prepared from the same fungus resulted in cellulase enzymes which still possessed cellulolytic activity. Earlier, Eriksson and Petterson (24) investigated the effect of various proteolytic enzymes on the cellulase activities on Penicillium notatum. They found that different proteases affected enzyme activities to different degrees. 

Previously, both in our laboratory and elsewhere, cellulases subjected to purification procedures were obtained from commercial sources (5,6, 8,9,10,13,39,46). Three cellobiases and several endoglucanases and cellobiohydrolases from commercial preparations were purified in our laboratory. While use of protease inhibitors in the fractionation procedures minimized proteolysis during enzyme purification, the existence of enzymes proteolytically modified, presumedly during prolonged fermentation (required for obtaining high titres for commercial production), was a source of confusion, as previously explained. Therefore, we prepared T. reesei cellulase harvested from young culture broth. This was used to carry out the enzyme purification procedures described below. 

Conversely, the connective tissues and cartilage are much more resistant to proteolysis and will survive for a longer period of time, although they too will eventually succumb to the effects of putrefaction. Reticulin, epidermis, and muscle protein will resist breakdown for some time, whereas collagen and keratin may survive for longer periods (Linch and Prahlow 2001). Keratin is an insoluble fibrous protein found in the skin, hair, and nails, and its resistance to attack by most proteolytic enzymes (Gupta and Ramnani 2006) is the reason it is often found intact amongst skeletal remains, particularly in burial environments (Macko et al. 1999). 

Other measures of protein flexibility have been found to correlate with thermal stability. One is resistance to proteolysis (Daniel et al., 1982 Fontana, 1988). Another is the quenching of buried tryptophan fluorescence by acrylamide, used in a study by Varley and Pain (1991). Both these processes are mediated by the same combination of local and global unfolding events that determine rates of hydrogen exchange. Their rates will depend on the ability of another molecule, acrylamide or a proteolytic enzyme, to penetrate into normally buried regions of the protein in order to either quench fluorescence or cleave peptide bonds. 

C-Methyl-/3-Casein. 14C-Labeled proteins prepared by reductive methylation have potential as substrates in the study of proteolytic enzymes. A serious limitation is that complete methylation of lysine residues results in inhibition of proteolysis by enzymes with trypsin-like specificity (13). It was interesting to determine whether this problem could be overcome by incomplete methylation which left unaltered most of the lysine residues in more or less random distribution throughout the protein. /3-Casein was selected as a suitable protein for this study since it is cleaved by trypsin-like enzymes to well characterized fragments, the y-caseins, in addition to less well characterized fragments, the proteose peptones. We anticipated that this type of study could provide a basis for a general investigation of milk protein transformation by the native milk proteinase which has a specificity similar to trypsin (14). 

Drags that structurally resemble nutrients such as polypeptides, nucleotides, or fatty acids may be especially susceptible to enzymatic degradation. For example, the proteolytic enzymes chymotrypsin and trypsin can degrade insulin and other peptide drags. In the case of insulin, proteolysis was shown to be reduced by the coadmmistration of carbopol polymers at 1% and 4% (w/v%), which presumably shifted the intestinal pH away from the optimal pH for proteolytic degradation. 

All of the three phases of enzymatic methods were dominated by proteolytic enzymes. However, nonproteolytic ones were also addressed for special purposes to indirectly assist proteolysis, for example, by providing access to proteins to be hydrolyzed through the enzymatic decomposition of the matrix constituents. At the same time, the use of nonproteolytic enzymes might be promoted in the future to study the possibly nonprotein-associated Se macromolecules which have already been observed in the case of numerous real samples, for example, edible mushroom [58, 80], but so far have never been investigated in detail. 

In the diet, vitamin B12 is bound to proteins. Although some release of protein-bound vitamin B12 begins in the mouth, most of the release occurs in the stomach on exposure of food to gastric acid (HC1) and the proteolytic enzyme pepsin. For this reason, either hypo-chlorhydria (abnormally low concentration of HC1 in gastric fluid) or achlorhydria (the absence of HC1 in gastric fluid) may decrease the availability of dietary vitamin B12 for absorption by preventing the activation of pepsinogen to pepsin, the principal enzyme responsible for proteolysis in the stomach. Achlorhydric patients with adequate production of IF may have low normal or subnormal serum B12 concentrations because of failure to liberate B12 bound to food. 

A common problem associated with rupture of yeast cells and protein extraction is proteolysis. Yeast cells contain a full complement of intracellular proteolytic enzymes which may be liberated after the cells are broken either by autolysis or by mechanical disruption. These liberated proteolytic enzymes, unless inactivated during the isolation and purification of yeast proteins, hydrolyze the proteins causing poor yields of intact protein (55, 69,70). 

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