Proteolysis, after mechanical

Cell fractionation by mechanical rupture has already come under investigation. Two separate studies of mechanical rupture of yeast showed different rates of release for enzymes in different cell locations (13,14). Wall-linked and periplasmic enzymes were released relatively faster than total protein, soluble cytoplasmic enzymes at about the same rate, and the mitochondrial enzyme fumarase later than total protein (13). Proteolysis by the yeast s own enzymes was not found to be a problem. Activities of the released enzymes declined slowly or not at all when disruption was continued after the end of protein release, and the effect of shear was not separated from the effect of proteolysis. Shetty and Kinsella (15) also found a low rate of proteolysis after mechanical disruption, though thiol reagents added to weaken the cell walls before disruption caused an important increase in the extent of protein breakdown. 

Another important protein of the Clp family is ClpB which possesses ATPase activity. In a clpB mutation, 45% of the denatured and aggregated protein arising transiently after the transfer of an E. coli culture from 30 to 45 °C is stabilized [14]. ClpB seems to play an important role in the renaturation or proteolysis of the aggregated proteins, but the mechanism of action of ClpB is not yet known. One can suppose that it might participate in the resolubilization of aggregated proteins. 

Wolf K, Mazo I, Leung H et al (2003) Compensation mechanism in tumor cell migration mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J Cell Biol 160 267-277... 

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). 

Sperm penetrate the zona pellucida only after completion of the acrosome reaction. A similar process occurs in nonmammalian species, where sperm must penetrate the vitelline coat. In abalone this is accomplished by release of lysin, an acrosomal protein that disperses the vitelline coat by a noncatalytic mechanism (Lewis et al., 1982 Shaw et al., 1993). In contrast, the generally accepted model for mammalian sperm penetration of the zona pellucida is the acrosin hypothesis in which proteolysis of zona pellucida matrix glycoproteins by acrosin, the acrosomal serine esterase, plays a trailblazing role in the sperm penetration process (Yanag-... 

For historical reasons many pharmaceutical enzymes are assayed with physiological or biopolymeric substrates (proteins, polysaccharides, bacteria, oil emulsions), which causes a number of theoretical and practical problems. The interpretation of results is difficult when natural substrates are converted into products that are substrates themselves for the next enzymatic attack. Reaction rates often depend on the position of the scissile bonds in the molecule and the chemical nature of the moieties. Hydrolysis can proceed simultaneously on various bonds at various rates. In proteolysis it is assumed that some products are liberated only after denaturation and that during the reaction course new peptide bonds become accessible for hydrolysis. In these cases the enzymatic mechanisms become exceedingly complex, kinetic parameters are apparent values, and experimental results are strongly influenced by the reaction conditions. Reproducibility problems can occur upon assaying proteinases with a limited specificity for particular casein types. Bromelain and pancreatic proteinase, FEP pharmaceutical enzyme standards, are assayed with a casein substrate. The extent of soluble peptide release is a measure of proteolytic activity. However, due to limited specificity, some proteinases release peptides with a nonrandom aromatic amino acid composition. Contamination of casein preparations with protein and of test enzyme substances with other proteinases biases the assay results. Under these conditions, relative assay methods are indicated. 

Acyl enzyme, an intermediate in the catalytic mechanism of serine proteases, such as trypsin and chymotrypsin. After the serine protease has bound a peptide substrate to form the Michaelis complex, Ser (in the case of chymotrypsin) nucleophilically attacks the peptide bond in the rate-determining step, forming a transition-state complex, known as a tetrahedral intermediate. The latter decomposes to the acyl enzyme, an extremely unstable intermediate, that bears the acyl moiety at the hydroxy group of Ser . The acyl enzyme intermediate is deacylated by water during proteolysis, or the attacking nucleophile is an amino component in case of kineticaUy controlled enzymatic peptide synthesis. 

Isoaspartyl dipeptidase (lAD), an enzyme catalyzing the hydrolytic cleavage of /3-aspartyl peptides ( isoaspartyl peptide bond). I AD is a member of the ami-dohydrolase superfamily. It has been assumed that the physiological function of lAD in E. coli is to prevent the accumulation of /3-aspartyl dipeptides after proteolysis of these proteins. The catalysis mechanism has been elucidated [E. E. Haley, J. Biol. Chem. 1968, 243, 5748 J. D. Gary et al., J. Biol. Chem. 1995, 270, 4076 R. Marti-Arbona et al.. Biochemistry 2005, 44, 7115]. 

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