Acid proteinase from the roe of the trout Salmo gairdneri R

Acidic proteinase from the trout spawn is 640 fold purified (yield 22%). Purification includes autolysis, acid treatment, ammonium sulphate fractionation, G-100 Sephadex gel-filtration, ion-exchange chromatography on DEAE-cellulose. Molecular mass of the enzyme under study is 70 kDa according to the data of gel-filtration. Acidic proteinase displays its greatest activity towards hemoglobin (pH 4.0, 37 degrees C) and is inhibited completely by EDTA, by 50%--by Pb2+ and soya inhibitor of trypsin and 2.8 times activated by Zn2+. Enzyme activity is not affected by dithiotreitol, iodine acetate, phenylmethylsulphonylfluoride parachloromercurybenzoate, Hg2+, Na+, Co2+, Ca2+.     

Novel inhibitors of procollagen C-terminal proteinase. Part 1: diamino Acid hydroxamates

The parallel synthesis of novel inhibitors of procollagen C-terminal proteinase is described. The synthetic strategy allowed for the facile synthesis of a large number of side-chain diversified diamino acid hydroxamates, of which the D-diaminopropionic acid derivatives were shown to be single digit nanomolar PCP inhibitors.     

Acid sensing ion channel 1 in lateral hypothalamus contributes to breathing control

Acid-sensing ion channels (ASICs) are present in neurons and may contribute to chemoreception. Among six subunits of ASICs, ASIC1 is mainly expressed in the central nervous system. Recently, multiple sites in the brain including the lateral hypothalamus (LH) have been found to be sensitive to extracellular acidification. Since LH contains orexin neurons and innervates the medulla respiratory center, we hypothesize that ASIC1 is expressed on the orexin neuron and contributes to acid-induced increase in respiratory drive. To test this hypothesis, we used double immunofluorescence to determine whether ASIC1 is expressed on orexin neurons in the LH, and assessed integrated phrenic nerve discharge (iPND) in intact rats in response to acidification of the LH. We found that ASIC1 was co-localized with orexinA in the LH. Microinjection of acidified artificial cerebrospinal fluid increased the amplitude of iPND by 70% (pH 7.4 v.s. pH 6.5∶1.05±0.12 v.s. 1.70±0.10, n = 6, P<0.001) and increased the respiratory drive (peak amplitude of iPND/inspiratory time, PA/Ti) by 40% (1.10±0.23 v.s. 1.50±0.38, P<0.05). This stimulatory effect was abolished by blocking ASIC1 with a nonselective inhibitor (amiloride 10 mM), a selective inhibitor (PcTX1, 10 nM) or by damaging orexin neurons in the LH. Current results support our hypothesis that the orexin neuron in the LH can exert an excitation on respiration via ASIC1 during local acidosis. Since central acidification is involved in breathing dysfunction in a variety of pulmonary diseases, understanding its underlying mechanism may improve patient management.     

The proteinase: mucus proteinase inhibitor binding stoichiometry.

In the nanomolar enzyme and inhibitor concentration range, 1 mol of mucus proteinase inhibitor (MPI) inhibits 1 mol of neutrophil elastase, cathepsin G, trypsin, and chymotrypsin. In the micromolar concentration range, the enzyme:inhibitor binding stoichiometry is still 1:1 for elastase but shifts to 2:1 for the three other proteinases. These data could be confirmed by three nonenzymatic methods: (i) fluorescence anisotropy measurements of mixtures of proteinases with 5-dimethylaminonaphthalene-1-sulfonylated or fluoresceinylated MPI, (ii) absorption spectrocospy of fluorescein-MPI-proteinase complexes isolated by gel filtration, (iii) analytical ultracentrifugation which showed that the molecular mass of the MPI-chymotrypsin complex is 56 kDa, whereas that of the MPI-elastase complex is 39 kDa. The binary MPI-elastase complex is unable to inhibit trypsin or cathepsin G. On the other hand, 1 mol of elastase displaces 2 mol of trypsin or cathepsin G from their ternary complexes with MPI.     

Reaction of human skin chymotrypsin-like proteinase chymase with plasma proteinase inhibitors

The ability of plasma proteinase inhibitors to inactivate human chymase, a chymotrypsin-like proteinase stored within mast cell secretory granules, was investigated. Incubation with plasma resulted in over 80% inhibition of chymase hydrolytic activity for small substrates, suggesting that inhibitors other than alpha 2-macroglobulin were primarily responsible for chymase inactivation. Depletion of specific inhibitors from plasma by immunoadsorption using antisera against individual inhibitors established that alpha 1-antichymotrypsin (alpha 1-AC) and alpha 1-proteinase inhibitor (alpha 1-PI) were responsible for the inactivation. Characterization of the reaction between chymase and each inhibitor demonstrated in both cases the presence of two concurrent reactions proceeding at fixed relative rates. One reaction, which led to inhibitor inactivation, was about 3.5 and 4.0-fold faster than the other, which led to chymase inactivation. This was demonstrated in linear titrations of proteinase activity which exhibited endpoint stoichiometries of 4.5 (alpha 1-AC) and 5.0 (alpha 1-PI) instead of unity, and SDS gels of reaction products which exhibited a banding pattern indicative of both an SDS-stable proteinase-inhibitor complex and two lower Mr inhibitor degradation products which appear to have formed by hydrolysis within the reactive loop of each inhibitor. At inhibitor concentrations approaching those in plasma where inhibitor to chymase concentration ratios were in far excess of 4.5 and 5.0, the rate of chymase inactivation by both serpin inhibitors appeared to follow pseudo-first order kinetics. The "apparent" second order rate constants of inactivation determined from these data were about 3000-fold lower than the rate constants reported for human neutrophil cathepsin G and elastase with alpha 1-AC and alpha 1-PI, respectively. This suggests that chymase would be inhibited about 650-fold more slowly than these proteinases when released into plasma. These studies demonstrate that although chymase is inactivated by serpin inhibitors of plasma, both inhibitors are better substrates for the proteinase than they are inhibitors. This finding along with the slow rates of inactivation indicates that regulation of human chymase activity may not be a primary function of plasma.     

Purification of a proteinase and proteinase inhibitor from Tetrahymen a pyriformis

• 1.1. A cysteine proteinase and cysteine proteinase inhibitor have been purified from Tetrahymena.
• 2.2. The proteinase was purified by ammonium sulphate fractionation, gel filtration, ion exchange chromatography and affinity chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 28,000). It hydrolysed BAPNA, degraded azocasein, and converted 80S ribosomes to subunits. Thiol reagents inhibited these activities.
• 3.3. The inhibitor was purified by heat treatment, ammonium sulphate fractionation and ion exchange chromatography, and appeared homogeneous by gel filtration and electrophoresis (mol. wt approx 12.500). The inhibitor was heat stable and it inhibited papain, as well as the Tetrahymena proteinase.

     

Acid proteinase activity in fish--I. Comparative study of extraction of cathepsins B and D from Mujil auratus

Acid catheptic activity was measured in crude extracts of muscle, liver, heart, spleen and gonads from the fishes Mujil auratus, Sparus aurata and Lightonatus mormyrus. The spleen was the organ which showed the highest activity. A comparative study of the seven most commonly used extraction methods was made. Some were modified to account for the characteristics of the fish organs and the activity extracted from them. The Siebert method resulted as the best extraction method only if 1 mM EDTA was present in the medium. The activity from Mujil auratus muscle was strongly inhibited by iodoacetate, N-ethylmaleimide, p-hydroxy mercuribenzoate, and diazo-acetyl-DL-norleucine methyl ester. The results indicated the presence of a carboxyl-proteinase and a thiol-proteinase. According to inhibition studies, the levels of proteinase and amidase activities shown by different organs of Mujil auratus were re-examined. The spleen extract showed the maximum activity for both cathepsins, but muscle extract accounted for more than 95% of total catheptic activity.     

Manduca sexta hemolymph proteinase 21 activates prophenoloxidase-activating proteinase 3 in an insect innate immune response proteinase cascade

Melanization, an insect immune response, requires a set of hemolymph proteins including pathogen recognition proteins that initiate the response, a cascade of mostly unknown serine proteinases, and phenoloxidase. Until now, only initial and final proteinases in the pathways have been conclusively identified. Four such proteinases have been purified from the larval hemolymph of Manduca sexta: hemolymph proteinase 14 (HP14), which autoactivates in the presence of microbial surface components, and three prophenoloxidase-activating proteinases (PAP1-3). In this study, we have used two complementary approaches to identify a serine proteinase that activates proPAP3. Partial purification from hemolymph of an activator of proPAP3 resulted in an active fraction with two abundant polypeptides of approximately 32 and approximately 37 kDa. Labeling of these polypeptides with a serine proteinase inhibitor, diisopropyl fluorophosphate, indicated that they were active serine proteinases. N-terminal sequencing revealed that both were cleaved forms of the previously identified hemolymph serine proteinase, HP21. Surprisingly, cleavage of proHP21 had occurred not at the predicted activation site but more N-terminal to it. In vitro reactions carried out with purified HP14 (which activates proHP21), proHP21, proPAP3, and site-directed mutant forms of the latter two proteinases confirmed that HP21 activates proPAP3 by limited proteolysis. Like the HP21 products purified from hemolymph, HP21 that was activated by HP14 in the in vitro reactions was not cleaved at its predicted activation site.     

Potyviral NIa proteinase, a proteinase with novel deoxyribonuclease activity

The NIa proteinase from pepper vein banding virus (PVBV) is a sequence-specific proteinase required for processing of viral polyprotein in the cytoplasm. It accumulates in the nucleus of the infected plant cell and forms inclusion bodies. The function of this protein in the nucleus is not clear. The purified recombinant NIa proteinase was active, and the mutation of the catalytic residues His-46, Asp-81, and Cys-151 resulted in complete loss of activity. Most interesting, the PVBV NIa proteinase exhibited previously unidentified activity, namely nonspecific double-stranded DNA degradation. This DNase activity of the NIa proteinase showed an absolute requirement for Mg(2+). Site-specific mutational analysis showed that of the three catalytic residues, Asp-81 was the crucial residue for DNase activity. Mutation of His-46 and Cys-151 had no effect on the DNase activity, whereas mutant D81N was partially active, and D81G was completely inactive. Based on kinetic analysis and molecular modeling, a metal ion-dependent catalysis similar to that observed in other nonspecific DNases is proposed. Similar results were obtained with glutathione S-transferase-fused PVBV NIa proteinase and tobacco etch virus NIa proteinase, confirming that the DNase function is an intrinsic property of potyviral NIa proteinase. The NIa protein present in the infected plant nuclear extract also showed the proteinase and the DNase activities, suggesting that the PVBV NIa protein that accumulates in the nucleus late in the infection cycle might serve to degrade the host DNA. Thus the dual function of the NIa proteinase could play an important role in the life cycle of the virus.     

Photochemical oxidation of snake gourd proteinase A2, a serine proteinase

Snake gourd proteinase A₂ was rapidly inactivated by methylene blue catalysed photooxidation at pH 7.8 and 25°. The rate of inactivation was pH-dependent and became slower at lower pH values, suggesting the involvement of some histidine residues in the inactivation. Changes in amino acid composition occurred only with histidine residues. One mole or more of histidine residues in the molecule are of essential importance in the catalytic function of snake gourd proteinase A₂.     

Separation of cathepsin D-like proteinase and acid thiol proteinase of squid mantle muscle

When the proteinases of the squid mantle muscle were extracted in the presence of dithiothreitol (DTT), the acid proteinase activity increased, indicating that the squid mantle muscle contains a considerable amount of the acid thiol proteinase. The crude extract hydrolyzed neither alpha-N-benzoyl-D,L-arginine-p-nitroanilide (BAPA) nor azocasein, thus refuting the presence of cathepsins B and L in the mantle muscle. The cathepsin D-like proteinase and the acid thiol proteinase were separated by Sephadex A-50 column chromatography. Each of the above partially purified proteinases was able to degrade carp actomyosin at pH 2.5 and 5.0, respectively.