Romano Felicioli

Mechanism of polyamine inhibition of a leaf protease

The inhibition of a highly purified alfalfa (Medicago sativa) leaf protease by naturally occurring polyamines is reported. The tetraamine spermine shows the highest inhibitory effect, with the maximum inhibition at 0.1 mM. Kinetic data indicate an apparent hyperbolic competitive inhibition. CD measurements show that in the presence of 0.1 mM spermine the enzyme undergoes a conformational change with the loss of 16% alpha-helix secondary structure content. Both the inhibition and the conformational change are prevented by high ionic strength. These data suggest a novel control mechanism of proteolytic activity in the leaf.

Purification and characterization of two leaf polypeptide inhibitors of leaf protease from alfalfa (Medicago sativa)

Two polypeptides with antiproteolytic activities have been isolated from alfalfa leaves. Polypeptide I resembles the previously described plant protease inhibitors in both structural and functional features; it has a molecular weight of 15,000, a random coil secondary structure, and inhibits exogenous protease as well as alfalfa leaf protease. Polypeptide II is a novel type of plant inhibitor with a molecular weight of 6300 and a highly organized structure with a high (40-50%) alpha-helix content. It only inhibits endogenous protease with a molar stoichiometry polypeptide/enzyme protein of 1.

Characterisation of a specific Phycocyanin-hydrolysing protease purified from Spirulina platensis

A novel protease has been identified, purified and partially characterised from complete medium grown Spirulina platensis, which could be responsible for the selective proteolysis of phycobiliproteins. It is an 80 kDa homodimeric enzyme; its N-terminal sequence is not related to any known protease sequence. It hydrolyses native phycocyanins in both crude extracts and reconstructed systems with purified Allo- or C-phycocyanin. It is inactive on several native proteins, including ribulose-1,5-bisphosphate carboxylase. The two phycocyanins are degraded at different velocities since C-phycocyanin is the better substrate, in agreement with the earlier observations on the progress of the phycobilisome disassembly. Specificity for synthetic substrates and inhibitors strongly suggests its assignment to the serine-protease family. The enzyme, however, is insensitive to the commercially available protein inhibitors of trypsin-like proteases.

Serum albumin fragmentation in end-stage renal disease patients - a pilot study

Abstract Background: The goal of this study was to detect modification in the expression of plasma proteins and/or post-translational modifications of their structure in patients with end stage renal disease. Methods: Serum samples from 19 adult patients treated by maintenance hemodialysis (MHD) were analyzed in comparison to sera from six healthy controls using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Spots of interest were identified by mass spectrometry analysis. In addition, the 2DE maps were incubated with a human anti-albumin polyclonal antibody. Results: SDS-PAGE gels, 2DE maps and matrix-assisted laser desorption/ionization time of flight analysis indicated over-expression of low-molecular weight proteins (LMWP) in sera from patients. Unexpectedly, another 15 spots with estimated Mr of 12.5–29 kDa from the 2DE maps of six patients were identified as fragments of albumin. 2D immunoblotting of sera from 12 other patients detected numerous albumin fragments. Conclusions: These results indicate that in addition to increased expression of LMWP, a relevant amount of albumin fragments are detectable in the serum of patients undergoing MHD. Uremia appears to facilitate the fragmentation of albumin and/or the retention of albumin fragments in blood. Clin Chem Lab Med 2009;47:1373–9.

Solid-phase synthesis and dimerization of an azobenzene-containing peptide as photoisomerizable proteinase inhibitor

The synthesis and biological activity of a photochromic compound, in which two Lys2 dipeptides are linked through their N-terminal amino groups by an azobenzene moiety, are reported. The synthesis was achieved by a novel solid-phase dimerization strategy, based on the reaction of 4,4′-azobenzene dibenzoyl chloride with two N-terminal amino groups of distinct Lys2 dipeptides directly on the resin. Both the trans form and the photostationary state mixture (59% cis and 41% trans) inhibit a plant proteinase which is known to be sensitive to polycationic compounds.

A Possible Mechanism of Proteolysis Modulation in the Life Cycle of alfalfa (Medicago sativa) Leaf.

Summary The molecular properties (e.g. low molecular weight and thermostability) of some known modulators of plant proteinases allowed the complete fractionation and the separate assay of the bulk of proteinases and of the bulk of modulators in extracts of leaves of different ages. No variations were observed in the total proteinase content throughout the life cycle; on the contrary, a high variability of the content of low molecular weight modulators was observed. After the two bulks were incubated together in the reconstructed systems, the modulation of the total apparent proteolytic activity was observed. This modulation is a function of the leaf age and resembles that reported for crude extracts by many authors. The data suggest that the control of proteolytic activity in the leaf may be due to the modulation of the inhibitors content.

The inhibition of a leaf proteinase by l-lysine homopolymers

The role of interlinked positively charged amino acids in the mechanism of inhibition of a monomeric trypsin-like proteinase has been investigated using high molecular mass L-lysine homopolymers ranging from 3.8 to 109 kDa. The data show that the degree of polymerization enhances the inhibitory efficiency which is maximal for homopolymers with more than eighteen interlinked lysine residues. The inhibition is cooperative and, under the maximal inhibition conditions, nine lysine residues of the polymer are involved in the electrostatic binding to the enzyme. A limited conformational change of the protein molecule accompanies the transition from a fully active to a fully inactivated enzyme.

The Role of Interlinked Positive Charges in the Polycation Inhibition of a Leaf Proteinase

Summary The role of the degree of polymerization and of interlinked positively charged groups in the mechanism of inhibition of a proteinase purified from alfalfa ( Medicago sativa ) leaf by polycations has been investigated using L-lysine oligomers as model. The monomer, dimer and trimer show competitive inhibition, do not induce conformational change of the enzyme molecule and are not sensitive to ionic strength; the pentamer shows a mixed type of inhibition and induces a conformational change in the enzyme molecule. Both pentamer inhibition and conformational change are ionic strength dependent.

The Use of a Native Enzyme Protein as Substrate for Plant Proteinase Assay

Summary Aldolase is very sensitive to proteolysis. In the present study native rabbit muscle aldolase is used as the substrate for crude extract leaf proteinase assay in a large range of pH. The proteolytic activity is directly measured by the decrease of aldolase activity after digestion. Furthermore the FDP/F-1-P cleavage ratio is used to discriminate between endo- and carboxy-peptidases. This novel method is described in detail and it is proposed as a general method for plant proteinase activity determination.

Characterization of the specificity of intracellular phosphodiesterases in Bacillus subtilis.

Abstract Previously described methods for the spectrophotometric determination of phosphodiesterases (orthophosphoric diester phosphohydrolase, EC 3.1.4.1) [P.L. Ipata and R.A. Felicioli, Eur. J. Biochem., 8 (1969) 174] have been employed to characterize intracellular phosphodiesterase in Bacillus subtilis during sporulation. Both 3′- and 5′-nucleoside monophosphate producing phosphodiesterases were found in vegetative and sporulating forms. In free spores the 3′-nucleoside monophosphate producing phosphodiesterase activity could not be detected with the methods employed.