Phenoloxidase and trypsin in germ-free larvae of Artemia fed with cooked unicellular diets: Examining the alimentary and protective effects of putative beneficial bacterium, yeast and microalgae against vibriosis

Three putative beneficial unicellular organisms, the marine bacterium Roseobacter sp, the yeast Saccharomyces cerevisiae mnn9 strain (SC-mnn9) and the microalga Tetraselmis suecica were cooked and offered separately as diets to developing germ-free (GF) Artemia larvae, in order to analyze their alimentary and protective effects. GF Artemia larvae were able to grow with cooked Tetraselmis and SC-mnn9 but failed with cooked Roseobacter. In spite of its high dietary quality, Tetraselmis failed to provide protection against Vibrio proteolyticus infection, while Roseobacter failed as food as well to provide protection. Cooked SC-mnn9 appeared to possess both values, dietary for growth and protective against Vibrio infection. GF Artemia larvae were apparently rapid adapted to dietary swaps; from yeast to algal and from algal to yeast. While the diets swap from algal or yeast, to bacterial diet appeared to be detrimental. Phenoloxidase-L (PO-L) and trypsin-L were used as biochemical indicators of defense and digestive functions, respectively. Developmental trypsin-L patterns were similar when fed on yeast and microalgae diets, suggesting a good digestive adaptation to plant or fungal substrates at early stages. On the contrary, diets swap or Vibrio infection affected PO-L and trypsin-L suggesting a sort of ‘alteration’ of digestive and defense functions.     

Exploring the protease mediated conformational stability in a trypsin inhibitor from Archidendron ellipticum seeds

A Kunitz proteinase inhibitor from Archidendron ellipticum seeds (AeTI) was purified and complexed with bovine trypsin and chymotrypsin. The stoichiometric stability of AeTI with its interacting proteinases was then investigated using spectrophotometric, size exclusion chromatography (HPLC system), Western blotting and circular dichroism (CD) studies. All the methods were remarkably similar in revealing the preference of trypsin over chymotrypsin by AeTI for complex formation. Both Western blotting as well as spectrophotometry based assays for competition experiments indicated that trypsin displaces chymotrypsin from a previously formed AeTI-chymotrypsin complex. Chemical modification of lysine and arginine by TNBS and CHD treatments, respectively, suggested a lysine as the active site residue and also indicated the presence of a single protease-binding site for AeTI. CD of native AeTI showed a sharp minimum at 200 nm and deconvolution of the CD spectra revealed it to be an unordered protein possessing high beta-sheet content. Complex formation of AeTI with trypsin induces a fractional switchover of its unordered structure towards the beta-sheet fraction but lacked any such conversion in the presence of chymotrypsin. Prolonged exposure of excess trypsin generates conformational modifications both in the secondary and the tertiary structures.     

1-Anilino-8-Naphtalene Sulfonate Probes a Gastric HK-ATPase Potassium Site Whose Access Requires Ionophores

1-anilino-8-naphtalenesulfonate (ANS) is a hydrophobic dipole previously used to demonstrate that the proton for potassium exchange by the gastric HK-ATPase is electroneutral. In this paper, we demonstrate that ANS binds to gastric membranes and probes conformational changes of the HK-ATPase independently of any active H for K exchange. Conformational changes require the presence of potassium-valinomycin and are not triggered by sodium. Potassium effect is enhanced by ATP, in the presence and in the absence of magnesium and, by ADP, in the presence of magnesium. Labeling of the pig HK-ATPase K518 by fluorescein-5-isothiocyanate inhibits the enzyme activity and knocks out the ATP effect on ANS fluorescence. Scherring 28080 and the monoclonal antibody 95-111, two competitive inhibitors of K-activated ATPase dephosphorylation, do not modify K-effect on ANS fluorescence but inhibit ATP effects. This supports that ANS does not probe K-site between the H1–H2 loop. Treatment of gastric membranes with trypsin does not inhibit the ANS response to potassium but does inhibit the response to ATP. This suggests that the ATP site inducing the ANS response is cytoplasmic and the potassium site is intramembranous. Titration reveals that one mole of ANS interacts with one mole of ATPase. We suggest that ANS probes a hydrophobic potassium site of gastric ATPase and that addition of ATP and ADP-Mg embed that site. Received: 16 July 1997/Revised: 10 June 1998     

Amino acid sequence of a Bowman-Birk proteinase inhibitor from pea seeds

Trypsin inhibitors from winter pea seeds (c.v. Frilene) have been purified by ammonium sulfate precipitation, gel filtration, and anion and cation exchange chromatography and shown to consist of six protease inhibitors (PSTI I, II, III, IVa, IVb, and V). Their molecular weights were determined by electrospray mass spectrometry as 6916, 6807, 7676, 7944, 7848, and 7844 D, respectively, and the sequences of the first 20 N-terminal amino acid residues of these six inhibitors were found to be identical. The complete amino acid sequence of PSTI IVa was determined. This protein comprises a total of 72 residues and has 14 cysteines, all involved in disulfide bridges. Comparison of the sequence of PSTI IVa with those of other leguminous Bowman-Birk type inhibitors revealed that PSTI could be classified as a group III inhibitor, closely related toVicia faba andVicia angustifolia inhibitors.     

Characterization of a highly stable trypsin-like proteinase inhibitor from the seeds of Opuntia streptacantha (O. streptacantha Lemaire)

A trypsin inhibitor from Opuntia streptacantha Lemaire (Prickly pear) seeds was purified and characterized. Of several proteases tested, this inhibitor showed specificity to trypsin-like enzymes. The major inhibitor present in these seeds showed distinctive characteristics, most notably a low molecular weight of 4.19 kDa, as determined by MALDI TOF, and an unusually high thermal stability, retaining most of the activity after heating the sample 1 h to 120 °C with a pressure of 1 kg/cm². Its complete amino acid sequence was obtained through mass spectrometry, this establishing presence a blocked N-terminal region. When comparing its sequence in the MEROPS database for peptidases and peptidase inhibitors, it showed 34.48% identity with a serine-proteinase inhibitor from the I15 family.     

Purification, characterization, sequence determination, and mass spectrometric analysis of a trypsin inhibitor from seeds of the brazilian treeDipteryx alata (leguminosae)

Dipteryx alata trypsin inhibitor (DATI) has been purified and completely sequenced. It showed homology to members of the Bowman-Birk family of inhibitors. The last step of DATI purification by RP-HPLC (narrow-bore C18 column) suggested the existence of some isoforms of the inhibitor due to the presence of a cluster of very close peaks in the chromatogram. By using electrospray ionization mass spectrometry (ESIMS) and laser desorption mass spectrometry (LDIMS), the identification of DATI isoforms was made possible. From the ESIMS data, the following molecular masses were found: 6803.22±0.92 for isoforma; 6890.94±0.73 forb; 6977.58±0.39 for c; 7065.07±0.67 ford; 7151.42±0.86 for e; and 7291.70±0.43 forf. Similar masses were found when using LDIMS. Isoformb was the most abundant and its molecular mass matched the molecular mass of 6893 calculated from the sequence of DATI. The mass differences betweena andb, b andc, c andd, andd ande were equal to 87, which corresponds to Ser. Isoforma might not have the N-terminal Ser present in isoformb, while the other additional Ser residues might comprise a row localized in the C- or N-terminal. The appearance of all these isoforms could result from posttranslational N- and C-terminal processing.     

Amino acid sequence of the acidic Kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC]

The primary sequence of trypsin inhibitor-2 (WBTI-2) fromPsophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 fromErythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors fromAdenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that thePsophocarpus andErythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.     

Plasmodium gallinaceum: Erythrocyte factor essential for zygote infection of Aedes aegypti

Zygotes of Plasmodium gallinaceum, fertilized in vitro and fed to Aedes aegypti mosquitoes through a membrane, formed oocysts only when a substance in the cytoplasm of uninfected erythrocytes was present. The relation between erythrocyte volume and infectivity was linear (1:1.2) up to a 50% hematocrit. The intraerythrocytic substance was both nondialyzable and poorly soluble in plasma. By carboxymethyl cellulose chromatography, cytoplasmic constituents eluted at pH 8.6 supported the same infection as control blood did; but higher and lower pH eluates supported none. Dialyzable factors present in the plasma, but absent from M199, enhanced infection but were not essential. Zygotes developed normally to ookinetes in the gut of plasma-fed mosquitoes, or when cultured in plasma or M199. Ookinetes from culture formed normal oocysts when fed to mosquitoes in blood or when injected with M199 into the hemocoels of unfed females. Mosquitoes fed infected blood containing lima bean or soybean trypsin inhibitor were unable to digest the erythrocytes and, although normal ookinetes developed, no oocysts formed. It appears from this and histological evidence that an erythrocyte substance, released by mosquito digestion, is needed for ookinete invasion of the gut epithelium.     

Reaction of the main proteolytic fraction of Schistosoma mansoni cercarial enzymes with synthetic substrates and inhibitors of proteolytic enzymes

The purified proteolytic fraction of Schistosoma mansoni cercarial enzymes (PCF) was inhibited by Diisopropylphosphofluoridate (DFP). Its esterolytic activity was not affected by either of two specific active center reagents of proteolytic enzymes: (1) 1-chloro-3-tosylamido-7-ammo-2-heptanone (trypsin) and (2) l-1-tosylamido-2-phenylethyl chloromethyl ketone (chymotrypsin). Furthermore, PCF did not destroy the biological activity of bradykinin on the isolated guinea pig ileum, nor did it release bradykinin from purified dog plasma bradykininogen.