Reactions proteolysis

Proteolytic cleavage has proven to be an efficient tool for exploring the structure and function of the Na,K-ATPase. Exposure and protection of bonds on the surface of the cytoplasmic protrusion provides unequivocal evidence for structural changes in the a subunit accompanying E1-E2 transition in Na,K-ATPase [52]. Localization of the proteolytic splits provided a shortcut to identification of residues involved in E1-E2 transition [33,53,54] and to detection of structure-function correlations [33]. Further proteolysis identifies segments at the surface of the protein and as the cytoplasmic protrusion is shaved off all ATP-dependent reactions are abolished. 

NCD-4 is a nonfluorescent carbodiimide derivative that forms a fluorescent adduct with the Ca -ATPase, accompanied by inhibition of ATPase activity and phos-phoenzyme formation [376-378]. Ca protected the enzyme against the inhibition by NCD-4 and reduced the extent of labeling, suggesting that the reaction may involve the Ca " " binding site. The stoichiometry of the Ca -protected labeling was i 2mole/mol ATPase. The fluorescence emission of the modified Ca -ATPase is consistent with the formation of a protein bound A-acylurea adduct in a relatively hydrophobic environment. After tryptic proteolysis of the NCD-4 labeled ATPase the fluorescence was associated with the A2 band of 24 kDa [376,379]. 

Piper and Fenton [10] indicated that extreme acidity or basicity of the gastric juice denaturalize the enzymatic activity of the pepsin, which shows has a higher activity at a pH = 2. At pH = 5 the enzyme starts to deactivate and at pH= 7, the enzyme irreversibly lose its activity. Fig. 3 shows the pepsin UV-visible spectra before and after interaction with the zeolites while Fig 4 shows the enzymatic activity of the denatured hemoglobin proteolysis versus reaction time. 

Figure 4. Enzymatic activity of the denatured hemoglobin proteolysis vs reaction time with selected samples (samples 5 and 6).

In Fig. 3, the pepsin dissolved in HC1, without interaction with any solid, showed a maximum at 272 nm. After interaction with the disordered cancrinite and the intermediate phase, a small decrease in the absorbance maximum of the pepsin spectrum was observed. This small decrease is due to the pepsin adsorption on the solid surfaces. The pepsin activity was also determined by the proteolysis reaction of a denatured haemoglobin solution at different times. Fig. 4 shows the obtained results. One can see, that the enzymatic activities (determined as absorbance), presented by the tested solids were very similar among them. These results show that pepsin enzymatic activity is not lost after the contact the pepsin with the tested solids. Therefore, the absorbance decrease observed in Fig. 4, is produced by the pepsin adsorption on the tectosilicate surface, and not by chemical reactions between pepsin and the tectosilicates... 

Reaction with Cr(III) Modified Plastocyanin. From thermo-lysin proteolysis experiments Farver and Pecht (20) have concluded that reduction of PCu(II) with labelled Cr0 20)52+ (1 1 mole amounts) at pH 7 gives a product in which Cr(III) is attached to the peptide chain 40-49. Coordination of the Cr at one or two carboxylates in the 42-45 patch is favoured. It has now been demonstrated that rate constants (25oC) for the reaction of PCu(I).Cr(III) + Co(phen)33+ are decreased by 16%. 

There were also less concrete considerations. In the early 1950s glycogenolysis was still believed to be completely reversible. UTP dependency and the glycogen synthase reactions had not yet been discovered nor had phosphofructokinase been shown to act irreversibly. The mechanism of protein synthesis was still a mystery. Laboratories studying proteolysis had shown that the peptide bond could be resynthesized by peptidases, although under very restricted conditions. Reversibility seemed to be an accepted property of the major metabolic pathways. 

There are many different types of fermented meats, each with its own particular process. The microbial and biochemical reactions during fermentation cause the characteristic acidification, proteolysis and drying that make the product safe. The distinctive flavor of sausage is also produced in these processes. The conditions under which fermented meats are produced are very favorable for the production of biogenic amines (Bover-Cid et al., 2000). Many factors contribute to the quality and acceptability of the final product. 

Armstrong attributed the increased resistance of dentin matrix to proteolysis to the blockage of susceptible sites by covalently bound carbohydrate. Later it became clear that the Maillard reaction induces the formation of covalent bonds (cross-links) between protein molecules, accounting for such resistance as well. The presence of non-degradable matrix proteins inhibits mineral dissolution (Chapter 2). In addition, both brown pigments and cross-linked proteins inhibit the production of extracellular polysaccharides by cariogenic streptococci (Kobayashi et al., 1990). 

In the course of dentin caries, both demineralization and reactions with the organic matrix take place. Matrix reactions include proteolysis and covalent modifications. From the introduction (Chapter 2) and the review on discoloration in caries (Chapter 3), it becomes clear that there are still few reports on the effect of matrix modifications on dentin caries. In Chapters 2, 4, and 5, the investigations were aimed at filling the information gap concerning the effect of reactions of dentin matrix on caries. To this end, degradation and modification of dentin were studied in demineralized specimens in vitro. In addition, specimens placed in dentures in situ and caries lesions in extracted teeth were analysed for modifications. 

Chapter 4 describes the in vitro reaction of glucose wifh demineralized dentin. Preliminary tests revealed that use of disfilled insfead of deionized water accelerated browning, consistent with the effect of frace metals on the Maillard reaction. The yellow discolored slices were more resistant than controls to pepsin-mediated breakdown, but not to trypsin-mediated breakdown. It would be worthwhile to investigate proteolysis of denfin collagen covalently bound by the Maillard reaction to proteins, which penetrate into a caries lesion. 

One of the questions evolving from the results of Chapters 3 and 5 is If C(, sugars are not involved in the Maillard reaction, which compounds are In addition, is the glycosylated dentin as resistant to proteolysis by cariogenic bacteria as it is to pepsin in vitro ... 

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 20.31 The principle of interconversion cycles in regulation of protein activity or changes in protein concentration as exemplified by translation/proteolysis or protein kinase/protein phosphatase. They result in very marked relative changes in regulator concentration or enzyme activity. The significance of the relative changes (or sensitivity in regulation) is discussed in Chapter 3. The principle of regulation by covalent modihcation is also described in Chapter 3. The modifications in cyclin concentration are achieved via translation and proteolysis, which, in effect, is an interconversion cycle. For the enzyme, they are achieved via phosphorylation and dephosphorylation reactions. In both cases, the relative change in concentration/activity by the covalent modification is enormous. This ensures, for example, that a sufficient increase in cyclin can occur so that an inactive cell cycle kinase can be converted to an active cell cycle kinase, or that a cell cycle kinase can be completely inactivated. Appreciation of the common principles in biochemistry helps in the understanding of what otherwise can appear to be complex phenomena.

The classical example is blood clotting, where successive steps involving enzyme-catalyzed proteolysis converts an inactive (or weakly active) proenzyme into its highly active form. Although unknown at the time of Wald s classical report, kinase-type and nucleotidyltransferase-type reactions (See Enzyme Cascade Kinetics) are frequently the source of biological signal transduction and amplification. 

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