Mechanically based protein release

These mechanically based protein release methods have several undesirable properties. One problem is that extensive fragmentation of the cells makes the subsequent centrifugation difficult (2.3). Adding to the problem of cell fragment removal is the high viscosity imparted to the solution by the released nucleic acids (4). A nucleic acid removal step is necessary to decrease the solution viscosity and avoid potential interference with fractional precipitation and chromatography (5,). Another undesirable property is that the harsh action of mechanical disruption causes the release... 

The partitioning of the activated inhibitor between direct covalent inactivation of the enzyme and release into solution is an important issue for mechanism-based inactivators. The partition ratio is of value as a quantitative measure of inactivation efficiency, as described above. This value is also important in assessing the suitability of a compound as a drug for clinical use. If the partition ratio is high, this means that a significant proportion of the activated inhibitor molecules is not sequestered as a covalent adduct with the target enzyme but instead is released into solution. Once released, the compound can diffuse away to covalently modify other proteins within the cell, tissue, or systemic circulation. This could then lead to the same types of potential clinical liabilities that were discussed earlier in this chapter in the context of affinity labels, and would therefore erode the potential therapeutic index for such a compound. 

When reactive metabolites are formed by metabolic activation, some of them can escape from the active site and bind to external protein residues or be trapped by reduced glutathione (GSH) or other nucleophiles. The remaining molecules that are not released from the active site will cause the suicide inhibition [7]. The ratio of the number of reactive molecules remaining in the active site and those escaping is a measure of the reactivity of the intermediates formed. The addition of scavengers or GSH to the incubation mixture does not affect and cannot prevent the CYP mechanism-based inhibition. However, GSH can reduce the extent of the nonspecific covalent binding to proteins by those reactive molecules that escape from the active site. In contrast, addition of substrates or inhibitors that compete for the same catalytic center usually results in reduction of the extent of inhibition. 

The mechanical properties of single hydrated dextran microcapsules (< 10 pm in diameter) with an embedded model protein drug have also been measured by the micromanipulation technique, and the information obtained (such as the Young s modulus) was used to derive their average pore size based on a statistical rubber elasticity theory (Ward and Hadley, 1993) and furthermore to predict the protein release rate (Stenekes et al., 2000). 

Based on our studies on the hydroperoxide-induced release of Ca from rat liver mitochondria a close correlation between protein mono(ADP-ribosylation) in the inner mitochondrial membrane and Ca " release is apparent. Whether this correlation represents a regulatory mechanism of Ca " release is presently not proven. It seems, however, justified to propose a regulation of a physiologically relevant Ca " release pathway by mono(ADP-ribosylation). Our results and the proposed mechanism of the hydroperoxide-induced release of Ca from rat liver mitochondria are summarized in Fig.l. 

We used 1,3-diketone hapten 1-carrier protein conjugate for immunization in our experiments to generate aldolase antibodies (Scheme 6.1) [4]. Class I aldolases use the e-amino group of a lysine in their active site to form a Schiff base vith one of their substrates and this substrate becomes the aldol donor substrate. Schiff-base formation reduces the activation energy for proton abstraction from the Ca atom and for subsequent enamine formation. The enamine, a carbon nucleophile, then reacts vith an aldehyde substrate, the aldol acceptor, to form a ne v C-C bond. The Schiff base is then hydrolyzed and the product is released [2]. The 1,3-diketone hapten acts as a mechanism-based trap of the requisite lysine residue in the active site and this lysine is necessary for formation of the essential enamine intermediate. The molecular steps involved in trapping the requisite lysine residue are essentially the same chemical steps as are involved in activating... 

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