Inhibition of starfish oocyte maturation by some inhibitors of proteolytic enzymes

Starfish oocyte maturation is triggered by a natural hormone, 1-methyladenine (1-MeAde), produced in the follicle cells, or artificially by dithiothreitol (DTT). These substances act on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which induces germinal vesicle breakdown (GVBD) and subsequent processes of meiotic maturation. Further, MPF is amplified in immature oocytes that have received the injection of MPF. In this paper the effect of leupeptin and antipain, protease inhibitors of microbial origin, on starfish oocyte maturation was investigated. The protease inhibitors were found to inhibit 1-MeAde-induced maturation when they were applied externally or injected into oocytes. DTT-induced maturation was also inhibited by injection of leupeptin. However, leupeptin did not inhibit the maturation-inducing action of MPF or MPF amplification. These results show that the protease inhibitors suppress the production of MPF by 1-MeAde or DTT, suggesting that some endogenous protease(s) acts in the production of MPF.     

Inactivation of proteolytic enzymes by cestodes

Inhibitor activity of cestodes from intestines of different hosts (sea birds, salt-water fish, and freshwater fish) was investigated. Alcataenia larina, Arctotaenia tetrabothrioides, Tetrabothrius erostris, T. minor, Wardium cirrosa, Bothriocephalus scorpii, Eubothrium rugosum, and Triaenophorus nodulosus were able to inhibit the activity of the commercial trypsin and activity of proteinase homogenates of the intestinal mucosa of the hosts. It was suggested that the inhibitor produced by the cestodes is specific for trypsin and protects them from the digestive enzymes of the host.     

Determination of proteolytic enzymes by flow-injection analysis

Quantitation of proteolytic enzymes using N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide has been adapted to flow-injection analysis. This procedure has been developed using two different proteases: subtilisin and chymotrypsin. For both enzymes the influence of substrate concentration on spectrophotometric response has been studied. The assay is based on the merging zones technique combined with a washing step. Results are obtained in less than 15 s and samples may be run at a rate of 90/h with good reproducibility. A linear relation between peak heights and enzyme concentrations was observed for 0-0.15 Anson unit/liter of subtilisin and for 0-30 mg/liter of a commercial preparation of chymotrypsin. The method requires only small sample volumes, and the consumption of the chromogenic substrate is reduced to a minimum by using intermittent pumping.     

Secretion of proteolytic enzymes by three phytopathogenic microorganisms

Serine proteinases from three phytopathogenic microorganisms that belong to different fungal families and cause diseases in potatoes were studied and characterized. The oomycete Phytophthora infestans (Mont.) de Bary and the fungi Rhizoctonia solani and Fusarium culmorum were shown to secrete serine proteinases. An analysis of the substrate specificity of these enzymes and their sensitivity to synthetic and protein inhibitors allowed us to refer them to trypsin- and subtilisin-like proteinases. The correlation between the trypsin- and subtilisin-like proteinases depended on the composition of the culture medium, particularly on the form of the nitrogen source. A phylogenetic analysis was carried out. In contrast to basidiomycetes R. solani, ascomycetes F. culmorum and oomycetes P. infestans produced a similar set of exoproteinases, although they had more distant phylogenetic positions. This indicated that the secretion of serine proteinases by various phytopathogenic microorganisms also depended on their phylogenetic position. These results allowed us to suggest that exoproteinases from phytopathogenic fungi play a different role in pathogenesis. They may promote the adaptation of fungi if the range of hosts is enlarged. On the other hand, they may play an important role in the survival of microorganisms in hostile environements outside their hosts.     

Conformational homogeneity in molecular recognition by proteolytic enzymes

Crystal structures for several hundred protease-inhibitor complexes have been analysed and their superimpositions have been used to demonstrate a universal relationship between inhibitor/substrate conformation and molecular recognition by all aspartic, serine, cysteine and metallo proteases. Proteases universally recognize an extended beta strand conformation in all their peptidic (and non-peptidic) inhibitors and substrate analogues without significant exceptions. This conformational homogeneity is illustrated here for a subset of 180 protease-inhibitor structures which are displayed as (a) structural overlays of multiple inhibitors for each of eight aspartic, eight serine, six metallo and five cysteine proteases; (b) single inhibitors each bound to different proteases; and (c) Ramachandran plots of peptide or pseudo-peptide dihedral angle pairs which demonstrate beta strands (Phi -54 degrees to -173 degrees, Psi 24 degrees to 174 degrees ) like those normally found paired in proteins as beta sheets. However, unlike beta sheets, alpha and 3(10) helices, beta and gamma turns, where the folded main chain amide components are intramolecularly hydrogen bonded and thus unavailable for interaction with proteins, an inhibitor/substrate in an isolated beta strand conformation provides maximum exposure of its hydrogen bonding donors/acceptors and side chain components to a putative protease receptor. This analysis highlights the advantages of a strand conformation over other elements of secondary structure for protease recognition and may lead to generic strategies for inhibitor design.     

Inhibition of amyloid fibrillization of hen egg-white lysozymes by rifampicin and p-benzoquinone

It has been reported that more than 20 different human proteins can fold abnormally, resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The current research, utilizing hen egg-white lysozymes as a model system, is aimed at exploring inhibitory activities of two potential molecules against lysozyme fibril formation. We first demonstrated that the formation of lysozyme amyloid fibrils at pH 2.0 was markedly enhanced by the presence of agitation in comparison with its quiescent counterpart. Next, via numerous spectroscopic techniques and transmission electron microscopy, our results revealed that the inhibition of lysozyme amyloid formation by either rifampicin or its analogue p-benzoquinone followed a concentration-dependent fashion. Furthermore, while both inhibitors were shown to acquire an anti-aggregating and a disaggregating activity, rifampicin, in comparison with p-benzoquinone, served as a more effective inhibitor against in vitro amyloid fibrillogenesis of lysozyme. It is our belief that the data reported in this work will not only reinforce the findings validated by others that rifampicin and p-benzoquinone serve as two promising preventive molecules against amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.