Proteinase isoinhibitors from bovine spleen: primary structure of an intermediate in the processing of the precursor

The complete amino acid sequence of the proteinase inhibitor III from bovine spleen is reported. It consists of 62 amino acid residues and is identical to that of spleen inhibitor II (an isoinhibitor of the bovine pancreatic trypsin inhibitor, which shares with the latter 89% of sequence identity), except for four extra residues at the C-terminal side. Inhibitor III appears to be an intermediate in the processing of the putative 100-residue primary expression product, which leads to the mature inhibitor II. These results and those previously obtained for another intermediate, isoinhibitor I, are indicative of the following order for the last steps of the precursor processing inhibitor I----inhibitor III----inhibitor II. The mature protein and the two intermediates isolated have a very similar antiproteolytic activity. However, their in vivo target enzyme(s) are not yet known, as also the target enzyme of the bovine pancreatic trypsin inhibitor is not known. Thus, the available data would indicate that either the three isoinhibitors have a distinct functional role, by inhibiting different target enzymes, or inhibitors I and III are obligatory intermediates for directing the final targeting of the mature, functionally relevant inhibitor II.     

Quantitative profiling of the pathological prion protein allotypes in bank voles by liquid chromatography-mass spectrometry

The conversion of the cellular prion protein (PrP(C)) into a misfolded isoform (PrP(TSE)) that accumulates in the brain of affected individuals is the key feature of transmissible spongiform encephalopaties (TSEs). Susceptibility to TSEs is influenced by polymorphisms of the prion gene suggesting that the presence of certain amino acid residues may facilitate the pathological conversion. In this work, we describe a quantitative, fast and reliable HPLC-MS method that allowed to demonstrate that in the brain of 109(Met/Ile) heterozygous bank voles infected with the mouse adapted scrapie strain 139A, there are comparable amounts of PrP(TSE) with methionine or isoleucine in position 109, suggesting that in this TSE model the two allotypes have similar rates of accumulation. This method can be easily adapted for the quantitative determination of PrP allotypes in the brain of other natural or experimental TSE models.     

Different susceptibility of the two dimers of ribonuclease A to subtilisin. Implications for their structure

RNase A and its minor and major dimers were digested with subtilisin under controlled conditions. The major dimer was found to be slightly more resistant, the minor dimer markedly less resistant to subtilisin than monomeric RNase A. Two S-proteins formed for each RNase A species, one starting with Ser-21, the other with Ser-22. Their relative proportions indicate that the structure of the minor dimer, whose identity with that of a RNase A dimer shown to be 3D domain-swapped is strongly suggested by recent work [S. Sorrentino et al. (2000) FEBS Lett. 466, 35-39], makes its peptide bond between Ser-21 and Ser-22 more accessible to subtilisin than it is in RNase A and its major dimer. Moreover, (i) both subunits constituting the minor dimer are more susceptible to subtilisin than monomeric RNase A, and (ii) the susceptible bonds in one of its two exchanging N-terminal arms are more accessible to the protease than in the other. The properties of the major dimer suggest that its structure could be different.     

Structure of Pyridoxal Kinase from Sheep Brain and Role of the Tryptophanyl Residues

The primary structure of sheep brain pyridoxal kinase has been determined by direct chemical and physical methods. The enzyme contains 312 amino acid residues with an acetylated methionine at the N-terminus, yielding a molecular mass of 34,861 Da. The functional role played by the two tryptophanyl residues in positions 52 and 244 of the polypeptide chain has been investigated by fluorescence spectroscopy. The tryptophanyl residues are not completely exposed to the rapidly relaxing solvent and they are poorly accessible to collisional quenchers. Chemical modification with NBS abolishes the catalytic activity of the kinase. The amino acid sequence of the sheep brain enzyme shows high similarity (86.2% identity) with the human pyridoxal kinase recently reported [Hanna, Turner, and Kirkness, (1997), J. Biol. Chem. 272, 10756–10760]. Comparison of the mammalian proteins with bacterial and yeast putative pyridoxal kinases retrieved from the Swiss-Prot data bank shows a low degree of overall similarity. In particular, the putative ATP-binding domain is conserved, whereas the region that appears to be crucial in the binding of the pyridoxal substrate is not. Thus, the assignment of the bacterial and yeast cDNA-deduced proteins as pyridoxal kinases should be taken with caution.     

Artoindonesianins Q-T,four isoprenylated flavones from Artocarpus champeden Spreng. (Moraceae)

Four isoprenylated flavones, artoindonesianins Q-T, were isolated from the heartwood of Artocarpus champeden Roxb. The structures of these compounds were elucidated on the basis of their spectroscopic data.     

Glycosylation enhances functional stability of the chemotactic cytokine CCL2

The human chemokine CCL2 gene was expressed in the yeast P.pastoris and gave rise to a mixture of differently glycosylated recombinant proteins. In comparison to non-glycosylated E.coli-derived CCL2, glycosylated yeast CCL2L was 4-20 times less active in a chemotactic assay in vitro. However, CCL2L could maintain full activity upon prolonged incubation at 37 degrees C, whereas the non-glycosylated chemokine readily lost activity. It could be hypothesized that glycosylation is a mechanism used by the organism to modulate CCL2 stability. The partial loss of specific activity due to glycosylation is balanced by the advantage of prolonging the effectiveness of chemokine. Thus, differential glycosylation allows one to obtain highly effective short-lived CCL2 or less-effective long-lived CCL2 and may thus represent a novel mechanism of adaptation to pathological versus physiological conditions.     

Conformational study of proteins by SAXS and EDXD: the case of trypsin and trypsinogen

The radius of gyration (Rg) of bovine trypsinogen and beta-trypsin was measured by an energy-dispersive X-ray technique (EDXD) and by small-angle X-ray scattering (SAXS), under different solvent conditions. Both techniques gave superimposable results. The experimental evidence demonstrated that: (1) no structural modifications and/or damage occurred during the data acquisition by EDXD; (2) at pH 4 the active enzyme has one class of chloride binding sites in common with the zymogen, whereas the latter protease shows an additional class able to reverse the effects on Rg induced by chloride at low concentration; and (3) the pH profile of the Rg of both proteases does not resemble at all the pH effect on beta-trypsin activity, a result in line with the finding that the electrical potentials induced by surface charge are small in absolute magnitude and produce no gradient across the active site.     

Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-alpha-D-mannosidase

A cDNA encoding 1,2-alpha-D-mannosidase mds 1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56,266 Da and shows high similarity to the amino acid sequences of 1,2-alpha-D-mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris. Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae alpha-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analyzed. The enzyme was characterized as a class-I mannosidase.     

Cytotoxic properties of oligostilbenoids from the tree barks of Hopea dryobalanoides

A new modified stilbene dimer, diptoindonesin D (1), was isolated from the acetone extract of the tree bark of Hopea dryobalanoides, together with seven known compounds, parviflorol (2), (-)-balanocarpol (3), heimiol A (4), hopeafuran (5), (+)-alpha-viniferin (6), vaticanol B (7) and (-)-hopeaphenol (8). Cytotoxic properties of compounds 1-8 were evaluated against murine leukemia P-388 cells. Compound 8 was found to be the most active with IC50 of 5.7 microM.