Elastase Subject

The literature relating to collagenases and elastases has recently been the subject of a comprehensive review by Ines Mandl (1961) and no purpose would be served by attempting a similar description here. It is proposed instead to summarize briefly some of the salient features arising from the work of the last decade and to draw attention to a number of recent researches that have led to advances in the study of elastolysis by enzymes. 

Fig. 5. Proteolytic cleavage sites in apolipoprotein E. Top Linear representation of the structure of human apoE demonstrating cleavage sites when apoE3 was subjected to limited proteolysis with six enzymes O, trypsin 9, elastase , chymotrypsin O, subtili-sin , Staphylococcus aureus V8 , thrombin. Bottom Linear representation of the two thrombolytic fragments of apoE, the 22- and 10-kDa fragments.

Such a procedure adapted from Gross and Morell (1966) and Blumenfeld and Gallop (1962) is as follows. Visser et al. (1971) treated 2.5 to 10 mg of a modified elastase with 1 N NHjOH-HCl, adjusted to pH 9 by addition of sodium hydroxide, for 2 hr at 25°C. The excess hydroxylamine was removed either by dialysis or by precipitation of the protein at pH 3.0. The protein was then dissolved, brought to pH 8.0 by addition of NaOH and treated with an equal volume of a 1 % solution of l-fluoro-2,4-dinitrobenzene in ethanol. The pH of the solution was maintained at 8 by the continuous addition of NaOH. The reaction is complete when no additional alkali must be added for 5 min. The mixture is then extracted three times with ether and the aqueous phase subjected to the conditions of the Lossen rearrangement (i.e. heating to 100°C under alkaline condition (0.1 N NaOH) for 10 min). Acid hydrolysis followed by amino acid analysis permits the identification of either diaminopropionic or diaminobutyric acid which would result from either aspartate or glutamate modification, respectively. Diaminopropionic and diaminobutyric acids may be estimated on the short column of the amino acid analyzer. Diaminopropionic acid emerges with histidine. (Color values do not seem to be available.)... 

Alpha 1 Antitrypsin in CF. Alpha 1 antitrypsin (a 1-AT) can inhibit neutrophil elastase in epithelial lining fluid (ELF), restore ELF antineutrophil elastase capacity, and also reverse the inhibitory effect of CF ELF on neutrophil Pseudomonas killing [116]. Furthermore, aerosolized secretory leukoprotease inhibitor can reduce neutrophil number and also neutrophil elastase in CF subjects [117]. For these reasons some have suggested a possible role for a 1-AT in CF. This concept requires further exploration before clinical introduction. The treatment is exceedingly expensive, so use without good documentation would be inappropriate. 

In addition to the development of rhAAT products based on transgenic technology, clinical studies to investigate the use of gene therapy based on a recombinant ade-no-associated virus with the hAAT gene had been proposed to treat AAT-deficient human subjects [367]. Yeast-derived rhAAT developed for aerosol administration to treat AAT-deficient individuals was shown to be safe and resulted in increased lung antineutrophil elastase defenses [368]. 

While changes in the expression of pro- and anti-inflammatory cytokines and chemokines are clearly important in the initiation and maintenance of the inflammatory response in the airways, neutrophils also play a key role in the amplification of the inflammatory response. Neutrophils contribute to airway inflammation in several active ways, including the production of oxygen free radicals, the secretion of granule-associated enzymes (including neutrophil elastase), and the production of pro-inflammatory cytokines and chemokines (reviewed in 117). BAL studies of patients with cystic fibrosis have revealed the presence of abundant levels of neutrophil elastase, both complexed with a 1-antiprotease and as free elastase, compared to control subjects (70,118,119). 

Enzyme assays of the solutions containing unbound human neutrophil elastase were conducted in pH 7.6 buffer composed of O.IM sodium phosphate, 0.5 M NaCl, and 3.3 % DMSO and subjected to spectrophotomeric measurement of the release of p-nitroaniline at 410 nm from the enzymatic hydrolysis of MeOSuc-Ala-Ala-Pro-Val-pNA (Sigma) (9). The spectrophotometric kinetic assays were performed in a Bio-Rad Microplate Reader (Hercules, CA) with a 96-well format. Two hundred microliter iquots of a elastase solution (0.2 units) were assayed per welt, and 20 microliters of a 475 micromolar substrate solution was added to initiate the enzyme reaction. 

The system is particular in that the turkey ovomucoid inhibitor, TOMl (PDB access code IPPF), is an elastase inhibitor consisting of 56 residues (814 atoms), that is, characterized by a size drastically larger than the diflu-oroketone inhibitor, DFKi (PDB access code 4EST), with 70 atoms (Online Resource 1). Due to that particularity, it has been the subject of several studies [26-29] regarding their alignment using molecular similarity-based techniques. 

---