Enzymes, proteolytic inhibition

Pharmacology Indinavir is an inhibitor of the HIV protease. HIV protease is an enzyme required for the proteolytic cleavage of the viral polyprotein precursors into the individual functional proteins found in infectious HIV. Indinavir binds to the protease active site and inhibits the activity of the enzyme. This inhibition prevents cleavage of the viral polyproteins resulting in the formation of immature... 

Irreversible inhibitors often provide clues to the nature of the active site. Enzymes that are inhibited by iodo-acetamide, for example, frequently have a cysteine in the active site, and the cysteinyl sulfhydryl group often plays an essential role in the catalytic mechanism (fig. 7.18). An example is glyceraldehyde 3-phosphate dehydrogenase, in which the catalytic mechanism begins with a reaction of the cysteine with the aldehyde substrate (see fig. 12.21). As we discuss in chapter 8, trypsin and many related proteolytic enzymes are inhibited irreversibly by diisopropyl-fluorophosphate (fig. 7.18), which reacts with a critical serine residue in the active site. 

Sometimes called suicide substrates, several protein inhibitors of proteases require proteolytic activity of the enzymes they inhibit, which leads to either covalent modification of the enzyme or releases charged groups that inhibit the catalytic machinery. In either case, this sort of activity-dependent inhibition is powerful and fundamentally different than the competitive mechanisms outlined above the inhibitor acts as a substrate and then uses the enzymes catalytic machinery to trap and then inhibit the enzyme. 

The effects of increasing concentrations of 8-OHQ-5SA, OP, and aa D on the activity of carboxypeptidase at a constant substrate concentration of 0.02 M CGP are shown in Fig. 2. (Vallee and Neurath, 1955.) Activity of the inhibited reaction was expressed as per cent of the proteolytic coefficient observed at zero inhibitor concentration. The conditions of preincubation are indicated. Recent and unpublished data indicate the time course of the inhibitory effects of these agents OP in concentrations of 1 X 10" M causes 90 % inhibition of the reaction in 60 minutes. 80 % of the inhibition occurs in the first 15 minutes (Fig. 3), Addition of 1 X 10" M zinc ions to the enzyme thus inhibited restores enzymatic activity, demonstrating the reversibility of inhibition (unpublished results). Since inhibition did not occur when chelating agents were first incubated with zinc, cupric, or ferrous ions to form the respective metal chelate, it appeared that the sites of chelation of these compounds are responsible for the observed inhibition. Inhibition is therefore not caused by any structural similarity between the inhibitors and the substrate. 

Indinavir (800 mg/t.i.d.) is an inhibitor of the HIV protease, which is an enzyme required for the proteolytic cleavage of the viral polyprotein precursors into the individual functional proteins found in infectious HIV. Indinavir binds to the protease active site and inhibits the activity of the enzyme. This inhibition prevents cleavage of the viral polyproteins resulting in the formation of immature noninfectious viral particles. Cross-resistance between indinavir and HIV reverse-transcriptase inhibitors is unlikely because the enzyme targets involved are different. Cross-resistance was noted between indinavir and the protease inhibitor ritonavir. Varying degrees of cross-resistance have been noted between indinavir and other HIV protease inhibitors. Indinavir is metabolized in the liver, and seven metabolites have been identified, and 20% of indinavir is excreted unchanged in the urine. 

Heparins and heparinoids form complexes with proteins and bases, and as shown by the ability to produce metachromasia with submicro quantities are very effective complexing agents in trace amounts. Hence, these substances in trace amounts affect many biological agents such as enzymes, etc. When heparin or a heparinoid is injected in animal or man, an enzyme appears in the blood plasma, lipoprotein lipase. When an oil emulsion is incubated with varying amounts of blood plasma obtained after injection of heparin, the oil is cleared. The heparin has caused the release of this enzyme from tissues to the blood. Heparin and heparinoids have a pronounced action on many enzymes - proteolytic enzymes, carbohydrases, etc. and may inhibit, activate. 

There are several reagents whose uses may be more restricted than those of inhibitors mentioned above but that nevertheless deserve discussion. Flavin-linked monoamine oxidase has been a fruitful enzyme for the development of new inhibitors. Besides being inhibited by acetylenic amines and olefinic amines, the enzyme is also inactivated by hydra-zides and cyclopropyl amines, e.g., the antidepressant drug tranylcypromine. Both of the latter reagents are first turned over by the enzyme before inhibition ensues, but the mechanisms of inhibition remain obscure. Recently, iV-nitroso compounds have been introduced as irreversible inhibitors of proteolytic enzymes. ... 

Homogenates of MetruUum senile, possibly the world s most common large sea anemone, yield extracts that are powerfully hemolytic for washed mammalian erythrocytes (22). The active substance, metridiolysin, is a protein of molecular weight approximately 80,000. In contrast to the sphingomyelin-inhibitable toxins, metridiolysin is an acidic protein having a pi of about 5. It is thermolabile and is inactivat by proteolytic enzymes. The optimal pH for hemolysis is between 5 and 6, and at pH 8 the lysin is inactive. It can be dissociated into two subunits of unequal size. Besides being cytolytic in vitro, metridiolysin is lethal when injected intravenously into mice. As shown in Table IV erythrocytes from the horse or dog are about a hundred times as sensitive to lysis as those from the mouse, and erythrocytes from other animals tested are intermediate in sensitivity. 

Plasminogen activator inhibitors have been shown to be present in a large variety of different cells and tissues. These inhibitors are thought to play an important role in regulating tissue fibrinolysis. One of these inhibitors has been purified from cultured bovine aortic epithelial cells. This inhibitor has been shown to be a serine protease inhibitor and inhibits the function of two proteolytic enzymes urokinase and tissue plasminogen activator, both of which cleave and activate plasminogen. The mechanism by which this inhibitor functions is very similar to that described above with a-l-PI. Thus, the inhibitor forms a binary complex with the proteolytic enzyme and thereby inhibits its activity. Again in a situation comparable to that with a-l-PI, it was found that when the purified bovine aortic epithelial inhibitor was exposed to Al-chlorosuccinimide,... 

Proteinases and antiproteinases are part of the normal protective and repair mechanisms in the lungs. The imbalance of proteinase-antiproteinase activity in COPD is a result of either increased production or activity of destructive proteinases or inactivation or reduced production of protective antiproteinases. AAT (an antiproteinase) inhibits trypsin, elastase, and several other proteolytic enzymes. Deficiency of AAT results in unopposed proteinase activity, which promotes destruction of alveolar walls and lung parenchyma, leading to emphysema. 

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. 

The antiinflammatory mechanisms whereby corticosteroids exert their beneficial effect in COPD include reduction in capillary permeability to decrease mucus, inhibition of release of proteolytic enzymes from leukocytes, and inhibition of prostaglandins. 

Both the 26S proteasome and the RC hydrolyze all four nucleotide triphosphates, with ATP and CTP preferred over GTP and UTP [58]. Although ATP hydrolysis is required for conjugate degradation, the two processes are not strictly coupled. Complete inhibition of the peptidase activity of the 26S proteasome by calpain inhibitor I has little effect on the ATPase activity of the enzyme. The nucleotidase activities of the RC and the 26S proteasome closely resemble those of E. coli Lon protease, which is composed of identical subunits that possess both proteolytic and nucleotidase activities in the same polypeptide chain. Like the regulatory complex and 26S proteasome, Lon hydrolyzes all four ribonucleotide triphosphates, but not ADP or AMP [18]. 

Oxidized LDL alter cellular functions role in cell death Oxidized LDL seem to be poorly degraded by lysosomal enzymes and accumulate in lysosomes altering in turn their functionality (Dean et al., 1997). It has been proposed that inhibition of oxidized LDL degradation and subsequent lipid accumulation may induce a destabilization of the acidic compartment, and lysosomal rupture with a relocation of lysosomal enzymes in the cytosol (li W et al, 1998). This process, also called endopepsis , occurs early and could precede mitochondrial dysfunction and cell death (Lossel et al., 1994). Moreover, oxidized LDL trigger a dysfunction of the intracellular proteolytic ubiquitin/proteasome pathway (early activation followed by inhibition)... 

Aprotinin is a polypeptide consisting of a chain of 58 amino acid residues, which inhibits stoichiometrically the activity of several proteolytic enzymes such as chymotrypsin, kallikrein, plasmin, and trypsin. Aprotinin is obtained from bovine tissues and purified by a suitable process. It is stored as a bulk solution or lyophilized powder. The amount of two related substances des-Ala-des-Gly-aprotinin and des-Ala-aprotinin is determined by CZE with a 100% analysis. The relative migration times are 0.98 for des-Ala-des-Gly-aprotinin and 0.99 for des-Ala-aprotinin, and the specified limits are 8.0 and 7.5%, respectively. 

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