Serine proteinase inhibitor proteins

Brazzein is another small sweet-tasting protein whose solution structure has been recently solved by NMR. Brazzein tastes 2000 times sweeter than sucrose on a weight basis and is exceptionally thermostable. As indicated by NMR, the structure of this 54 residue, single-chain polypeptide does not change between 32 and 82 °C and retains its sweetness after incubation at 98 °C for two hours.Brazzein contains one a-helix and three strands of antiparallel jd-sheet stabilized by four intramolecular disulphide bonds. It has been proposed that the disulphide bonds could be responsible for the thermostability of brazzein by forming a compact structure at the tertiary level.The structure of brazzein does not resemble that of the other two sweet proteins with known structures, monellin and thaumatin, whereas sequence alignment and structural prediction indicate that brazzein shares the fold of a newly identified family of serine proteinase inhibitors. 

A compound that reduces the activity of an enzyme is known as an inhibitor. Inhibitors are usually small molecules but some are peptides or proteins. For example, there are a number of proteolytic enzymes in the blood that have serine in their active site. If the activities of these enzymes are too high, they can cause problems. Consequently, inhibitor proteins, known as serine proteinase inhibitors (serpins), are present in blood indeed, about 10% of all the plasma proteins are serpins (Box 3.4). 

Nilges, M., Gronenborn, A. M., Brunger, A. T. and Clore, G M. (1988). Determination of three-dimensional structures of proteins by simulated annealing with interproton distance restraints Application to crambin, potato carboxypeptidase inhibitor and barley serine proteinase inhibitor 2. Protein Eng. 2, 27-38. 

Since aiAT represents the archetype for the serpin (serine-proteinase inhibitor) superfamily of proteins, it is possible that similar oxidative or proteolytic mechanisms may function in the inactivation of other serpins that are important in controlling the inflammatory cascade. Some serpins contain a readily oxidised reactive-centre methionine residue (e.g. plasminogen activator-inhibitor [108] and a2-antiplasmin [109]), whilst all serpins (including antithrombin III [110] and protease nexin I [111]) contain an exposed loop which is susceptible to cleavage by proteinases. 

M. Nilges, A. M. Gronenborn, A. T. Brunger, and G. M. Clore, Protein Engin., 2,27 (1988). Determination of Three-Dimensional Structures of Proteins by Simulated Annealing with Interproton Distance Restraints. Application to Crambin, Potato Carboxypeptidase Inhibitor, and Barley Serine Proteinase Inhibitor 2. 

CHAPTER 36, FIGURE 7 A group of structurally similar protein inhibitors of the serine proteinases known as SERPINS (SERine Proteinase INhibitors). The structure shown is human antithrombin. The reference SERPIN, a j-proteinase inhibitor or a. -antitrypsin contains -30% a helix (9 helices) and 40% sheet (5 3 sheets). Other members of the SERPIN family contain both additional helices and p sheets. The reactive center loop of antithrombin, residues 378-396, contains the reactive site residues Arg and Ser . Upon reaction with the target proteinase or after cleavage by the target proteinase (a reaction that inactivates the inhibitor without inactivating the proteinase), the reactive center loop folds between the S3 and S5 sheets. 

A recent study reports the release profile of the Serp-1 proteinase from PVA-C [65]. Serp-1 is a serine proteinase inhibitor (serpin) secreted by the myxoma vims and is a potential new therapeutic for cardiovascular diseases. It has exhibited antiinflammatory activity through the modulation of immune cell responses [66]. The release profile of this protein in a buffer medium is typical of that of a diffusion controlled process. However, it is interesting to know that the release rate of Serp-1 and its final release level attained differ in human blood and in buffer. The release rate is twice as fast and in half of the time in blood than in buffer. The final release level is complete in blood and appears to level off at around 50% in buffer. It was suggested that there may be a difference in behavior between the two release media, which is important to consider because human whole blood represents a more realistic setting of the physiological environment in arteries. It is also possible that interaction between PVA-C and blood components play a role in determining the ultimate release rate (Fig. 9a, b) [65]. 

Enzyme inhibitors are also present, e. g., potatoes contain proteins which have an inhibitory effect on serine proteinases, while proteins from beans and cucumbers inhibit pectolytic enzymes. Protein and enzyme patterns, as obtained by electrophoretic separation, are often characteristic of species or cultivars and can be used for analytical differentiation. Figure 17.1 shows typical protein and proteinase inhibitor patterns for several potato cultivars. 

McPhalen, C. A., James, M. N. G. Structural comparison of two serine proteinase-protein inhibitor complexes Eglin-C-Subtilisin Carlsberg and CI-2-subtilisin novo. Biochemistry 27 (1988) 6582-6598... 

LASKOWSKi M Jr (1986) Protein inhibitors of serine proteinases-mechanism and classification. Adv Exp Med Biol. 199 1-17. 

In this laboratory, we also include the metal ion chelators EDTA (ethylene diamine tetraacetic acid binds, e.g., Mg2 1 -ions) and EGTA (ethylene glycol-bis(2-aminoethyl)-Al,iV,iV/,iV/,-tetraacetic acid binds, e.g., Ca2+-ions) in our lysis buffers. These agents help prevent phosphatase action (by the metal ion-dependent phosphatase PP2C, which is not inhibited by microcystin-LR), metal (Ca2+) dependent proteinases, and protein kinases, which require divalent cations such as Mg2 1 (and, in some cases, also Ca2+). We also use a mix of proteinase inhibitors that inhibit a broad range of proteolytic enzymes, including serine and cysteine proteinases. 

Based on the mixed-phase method, ACE is introduced for studying the interaction of heparin with the serine protease inhibitors, antithrombin III (ATIII) and secretory leukocyte proteinase inhibitor (SLPI) (85). An etched capillary, to which heparin has been covalently immobilized, was used in this study. This modified capillary both afforded an improvement in the separation of heparin-binding proteins and required a lower quantity of loaded protein. 

Figure B3.1.4 A 15% SDS-pdyacrylamide gel assayed for proteinase inhibitors. (A) Gel stained with Coomassie brilliant blue for total protein. (B) Gel assayed for serine-proteinase inhibitory activity against trypsin. Food-grade proteinase inhibitors used in surimi manufacture were assayed. Lane 1, whey protein concentrate. Lane 2, bovine plasma proteins. Lane 3, egg white. Each lane contains 15 pg protein. Lane M, molecular weight standards. The dark bands in (B) indicate proteins with proteinase inhibitory activity. Numerous proteins in egg white (lane 3) are shown to have inhibitory activity against trypsin. Adapted from Weerasinghe (1995).

Antithrombin is a member of the SERPIN superfamily of proteins, which includes the inhibitors a2 an1 Pbsniin, ar antichymotrypsin, and a -proteinase inhibitor (79). Antithrombin is considered to be the primary inhibitor of coagulation (80) and targets most coagulation proteases as well as the enzymes trypsin, plasmin, and kallikrein (81). Inhibition takes place when a stoichiometric complex between the active site serine of the protease and the ARG393-SER394 bond of antithrombin forms (82,83), The tertiary structure of antithrombin resembles a,-antitrypsin in that it is folded into N-terminal domain helices and (3-sheets. This tertiary structure is maintained by the formation of three disulfide bonds (71). Four glycosylation sites exist on human... 

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