Frank N. Chang

A new broad-spectrum protease inhibitor from the entomopathogenic bacterium Photorhabdus luminescens

A new protease inhibitor was purified to apparent homogeneity from a culture medium of Photorhabdus luminescens by ammonium sulfate precipitation and preparative isoelectric focusing followed by affinity chromatography. Ph. luminescens, a bacterium symbiotically associated with the insect-parasitic nematode Heterorhabditis bacteriophora, exists in two morphologically distinguishable phases (primary and secondary). It appears that only the secondary-phase bacterium produces this protease inhibitor. The protease inhibitor has an M:(r) of approximately 12000 as determined by SDS-PAGE. Its activity is stable over a pH range of 3.5-11 and at temperatures below 50 degrees C. The N-terminal 16 amino acids of the protease inhibitor were determined as STGIVTFKND(X)GEDIV and have a very high sequence homology with the N-terminal region of an endogenous inhibitor (IA-1) from the fruiting bodies of an edible mushroom, Pleurotus ostreatus. The purified protease inhibitor inactivated the homologous protease with an almost 1:1 stoichiometry. It also inhibited proteases from a related insect-nematode-symbiotic bacterium, Xenorhabdus nematophila. Interestingly, when present at a molar ratio of 5 to 1, this new protease inhibitor completely inactivated the activity of both trypsin and elastase. The activity of proteinase A and cathepsin G was partially inhibited by this bacterial protease inhibitor, but it had no effect on chymotrypsin, subtilisin, thermolysin and cathepsins B and D. The newly isolated protease inhibitor from the secondary-phase bacteria and its specific inhibition of its own protease provides an explanation as to why previous investigators failed to detect the presence of protease activity in the secondary-phase bacteria. The functional implications of the protease inhibitor are also discussed in relation to the physiology of nematode-symbiotic bacteria.

Purification and cloning of a Delta class glutathione S-transferase displaying high peroxidase activity isolated from the German cockroach Blattella germanica.

A highly active glutathione S‐transferase was purified from adult German cockroaches, Blattella germanica. The purified enzyme appeared as a single band of 24 kDa by SDS/PAGE, and had a different electrophoretic mobility than, a previously identified Sigma class glutathione S‐transferase (Bla g 5). Kinetic study of 1‐chloro‐2,4‐dinitrobenzene conjugation revealed a high catalytic rate but common substrate‐binding and cosubstrate‐binding affinities, with Vmax, kcat, Km for 1‐chloro‐2,4‐dinitrobenzene and Km for glutathione estimated to be 664 µmol·mg−1·min−1, 545 s−1, 0.33 mm and 0.76 mm, respectively. Interestingly, this enzyme possessed the highest activity for cumene hydroperoxide among insect glutathione S‐transferases reported to date. Along with the ability to metabolize 1,1,1‐trichloro‐2,2‐bis(p‐chlorophenyl)ethane and 4‐hydroxynonenal, this glutathione S‐transferase may play a role in defense against insecticides as well as oxidative stress. On the basis of the amino acid sequences obtained from Edman degradation and MS analyses, a 987‐nucleotide cDNA clone encoding a glutathione S‐transferase (BggstD1) was isolated. The longest ORF encoded a 24 614 Da protein consisting of 216 amino acid residues. The sequence had close similarities (∼ 45–60%) to that of Delta class glutathione S‐transferases, but had only 14% identity to Bla g 5. The putative amino acid sequence contained matching peptide fragments of the purified glutathione S‐transferase. ELISA showed that BgGSTD1 bound to serum IgE obtained from patients with cockroach allergy, indicating that the protein may be a cockroach allergen.

A simple method for assigning multiple immunogens to their protein on a two-dimensional blot and its application to asthma-causing allergens.

A “one‐step” procedure, that not only removes the color and blocking proteins used in the colorimetric immunodetection step but also stains the proteins originally on the blot, is presented. Following immunostaining and recording of immunoreactive spots, the blot was allowed to air‐dry overnight (or longer) at room temperature and then counterstained with a colloidal gold solution. This “air‐drying” process apparently altered the affinity of the blocking proteins (and possibly other proteins added subsequently to the blotting step) towards the nitrocellulose membrane causing them to be removed by the acidic colloidal gold solution while the “blotted” proteins were being stained. The sensitivity of this counterstained blot was comparable to that of the blot without going through the immunodetection process. Since both immunodetection and protein staining were carried out on the same blot, this allowed easy identification of many immunoreactive spots to their corresponding proteins when the two profiles were superimposed. Using this procedure, we have detected 25 immunoreactive spots (or allergens) from the whole body extract of the German cockroach (Blattella germanica)that contribute to asthma and assigned them to their corresponding proteins on a two‐dimensional (2‐D) protein map. The apparent Mr and pI for each of the allergens were determined. We have also located one of the major cockroach allergens, Bla g 5 (glutathione S‐transferase). Two‐dimensional zymography revealed the presence of ten gelatinase‐type proteolytic enzymes. Only one of the ten proteases comigrated with the immunoreactive proteins indicating that unlike other allergen‐producing systems, most of the cockroach allergens do not possess protease activity.