Peptidases soluble proteins

Furthermore, peptidases are also structural probes of conformation of soluble proteins[331. Although X-ray crystallography1341 and two-dimensional NM R1351 are the methods of choice for the determination of the three-dimensional structure of globular proteins, some weaknesses of these techniques demand alternative methods even if these will provide structural information at a lower level of resolution. For example, limited proteolysis can be used to probe the structure and the dynamics of proteins in solution, which provide experimental data that are easy to obtain and complement well those results derived from the techniques mentioned above. The... 

Proteolytic enzymes such as proteases and peptidases are ubiquitous throughout the body. Sites capable of extensive peptide and protein metabolism are not only limited to the liver, kidneys, and gastrointestinal tissue, but also include the blood and vascular endothelium as well as other organs and tissues. As proteases and peptidases are also located within cells, intracellular uptake is per se more an elimination rather than a distribution process [13]. While peptidases and proteases in the gastrointestinal tract and in lysosomes are relatively unspecific, soluble peptidases in the interstitial space and exopeptidases on the cell surface have a higher selectivity and determine the specific metabolism pattern of an organ. The proteolytic activity of subcutaneous tissue, for example, results in a partial loss of activity of SC compared to IV administered interferon-y. 

Wickner (1980) proposed an alternative mechanism of protein secretion, called the membrane trigger hypothesis. This model proposes that the signal sequence influences the precursor protein or a domain of the precursor to fold into a conformation that can spontaneously partition into the hydrophobic part of the bilayer. In prokaryotes, the membrane potential causes the protein to traverse the bilayer. The protein then regains a water-soluble conformation, and is expelled into the medium. Signal peptidase removes the signal sequence during or after this process. Thus, secretory proteins or domains are transported across the membrane posttranslationally without the aid of a proteinaceous secretory apparatus. An energy source, such as the membrane potential, is required for secretion. 

The water-insoluble fraction was more readily digested than the water-soluble fraction. Heat denaturation naturally made meat proteins more readily digested by these enzymes, and the effect of denaturation was more significant in the water-soluble fraction than the other fraction. As expected, hydrolysis of the native connective tissue by these enzymes as indicated by increased hydroxyproline was extremely low. Connective tissue after heat denaturation was readily hydrolyzed. Papaya peptidase A showed about a fivefold increase while papain and chymopapain increased their hydrolyses seven- to tenfold. 

This material was identified as the protein YPg (i) by labeling with ( 3H)amino acids (for example, lys, arg, leu, or tyr) (ii) by its sensitivity to pronase, protease K, or amino peptidase (iii) by its electrophoretic behavior on paper or in polyacrylamide gels (Figure 2) and (iv) by the fact that it is insoluble in chlorofom/ methanol or acetone, and only partially soluble in 5-20% trichloroacetic acid (for details, see ref. 8, 11, 12, I3). Attempts to remove the protein from the ENA prior to ENase T2 digestion by various physical methods failed (l1). Unambiguous proof that YPg was covalently linked to virion ENA came from the isolation of a nucleotidyl-protein (VPg-pUp) and a nonanucleotidyl-protein (VPg-pU-U-A-A-A-A-C-A-Gp) after digestion of virion ENA with ENase T2 and ENase T1, respectively (II, I3, I4). 

Degradability will be affected by such factors as the surface area available for microbial attack and the protective action of other constituents as well as the physical and chemical nature of the protein. Claims have been made that the solubility of a protein is correlated with ease of breakdown, but these do not survive critical examination. Thus, casein, which is readily degraded in the rumen, is not readily soluble whereas albumin, which is resistant to breakdown, is readily soluble. It has been suggested that a major factor affecting degradability is the amino acid sequence within the protein molecule. If this is so, then the nature of the microbially produced rumen peptidases is of considerable importance and it seems doubtful whether any simple laboratory test for degradability is possible. 

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