Maurizio Tomasi

Comparison of wheat albumin inhibitors of α-amylase and trypsin

Abstract Wheat albumins were extracted from whole wheat flour with 150 mM sodium chloride solution and precipitated between 0·4 and 1·8 M ammonium sulphate. The albumin precipitate was separated by gel filtration on Sephadex G100 into five peaks. Three peaks (II, III, and IV), whose MWs were 60 000, 24 000 and 12 500 daltons respectively, were active toward several insect α-amylases, whereas only peak III inhibited human saliva and pancreatic α-amylases. Peaks III and IV also inhibited trypsin. In each active peak, we found several α-amylase inhibitors slightly different in their electrophoretic mobilities in a Tris—glycine buffer system (pH 8·5), whereas only one major trypsin inhibitor was present in peaks III and IV. In contrast to α-amylase inhibitors that were all anodic, trypsin inhibitors migrated to the cathode under our experimental conditions. From a quantitative standpoint, wheat albumins that inhibit trypsin are negligible, whereas about 2/3 of the total albumin inhibits amylases from different origins. All inhibitor components of peak III were active toward both insect and mammalian α-amylases. Moreover, they reversibly dissociated in the presence of 6 M guanidine hydrochloride giving two similar subunits.

Further characterization studies of the α-amylase protein inhibitor of gel electrophoretic mobility 0.19 from the wheat kernel

A highly purified amylase protein inhibitor from the kernels of hexaplois wheat, designated 0.19 according to its gel electrophoretic mobility, has been characterized according to its circular dichroism spectra determined at different pH values and in the presence or absence of dissociating and reducing agents. The 0.19 albumin has also been characterized according to the specificity with which it inhibits 21 alpha-amylases from different origins and according to its sensitivity to a number of chemical and enzymatic treatments of its inhibitory action on human saliva and Tenebrio molitor L. larval midgut alpha-amylases. Inhibitory activity of 0.19 toward human saliva amylase significantly increased when the inhibitor was incubated with the enzyme before the addition of starch, but it was not affected by the preincubation of 0.19 with starch. Maltose reversed the inhibition of human saliva by 0.19 and showed some inhibitory activity toward the enzyme. However, maltose concentrations that only slightly affected amylase activity were very effective in restoring the amylase activity inhibited by 0.19. The inhibitory action of 0.19 on human saliva and T. molitor L. amylases were equally resistant to trypsin and thermal treatments, but 0.19 was readily inactivated by incubation with pepsin or by reduction of disulfide bonds. The inhibition of the mammalian amylase by 0.19 was adversely affected by a treatment with CNBr (1:100 ratio of methionine residues to CNBr) whereas the inhibition of the insect amylase was not. As shown by circular dichroism measurements in the far ultraviolet, 0.19 is a protein with about 50% of ordered structure. Significant and largely reversible changes have been observed in the aromatic CD spectrum of 0.19 at alkaline pH values or in the presence of sodium dodecyl sulfate. These changes, which were associated with a partial loss of inhibitory activity, indicate that ionizable tyrosine groups contribute significantly to the ellipticity bands of 0.19 in the near ultraviolet.

Lipid interaction of diphtheria toxin and mutants with altered fragment B

The interaction of diphtheria toxin and its cross-reacting mutants crm 45,228 and 1001 with small unilamellar vesicles has been followed by a turbidity assay, electron microscopy, fluorescence energy transfer and membrane permeability. All toxins at pH lower than 6 induce the aggregation and fusion of liposomes containing negatively charged phospholipids; crm 45 and crm 1001 are less potent than diphtheria toxin. Isolated diphtheria toxin fragment B is very effective while isolated fragment A is ineffective. Liposome fusion induced by the toxins at low pH occurs without release of the internal content implying that fusion does not involve vesicle breakage and resealing. The pH dependence of the membrane interaction of diphtheria toxin monitored by turbidity is in close agreement with that monitored by fluorescence energy transfer. It shows that diphtheria toxin can alter the lipid bilayer structure in the pH interval 5-6. This pH range occurs in endosomes and suggests that histidyl and carboxyl residues are likely to be involved in the conformational change of diphtheria toxin triggered by acidic pH.

Conjugation of cholera toxin or its B subunit to liposomes for targeted delivery of antigens

Several immunoadjuvant systems have been proposed to enhance mucosal immune responses of orally administered purified antigens. Cholera toxin (CT) or its B subunit (CTB) have been found to promote immune responses to antigens when they are co-administered via mucosal routes. Oral administration of antigens incorporated into liposomes has also been shown to result in enhanced mucosal immune responses. Here, we describe the covalent coupling of CT and CTB to small unilamellar liposomes for targeting these vesicles to Peyers patch M cells, following their oral administration. Conjugation was done by means of a thioether bond using succinimidyl(4-N-maleimidomethyl)cyclohexane-1-carboxylate to modify the dipalmitoylphosphatidyl-ethanolamine constituent of liposomes and N-succinimidyl-3-(2-pyridyldithio)propionate to thiolate CT or CTB. The biological activity of CT or CTB bound to liposomes was confirmed by a hemagglutination assay using GM1-enriched human erythrocytes. Furthermore, oral administration of CT-conjugated liposomes to rats resulted in the induction of serum IgG and salivary IgA anti-CT responses. CT-conjugated liposomes may prove to be a useful system for targeted delivery and immunoenhancement of weakly immunogenic antigens.

Diphtheria toxin and its mutant crm 197 differ in their interaction with lipids.

The interaction of diphtheria toxin and its enzymatically deficient mutants crm 176 and crm 197 with liposomes has been studied by turbidity measurement and hydrophobic photolabelling with photoactivatable phosphatidylcholines. Diphtheria toxin and crm 176 at neutral pH bind to the surface of lipid bilayers while crm 197 also appears to interact with the fatty acid chains of phospholipids. All proteins undergo a change in conformation over the same range of acidic pH and become able to insert in the lipid bilayer. The tighter lipid interaction of crm 197 may account for its higher cell association constant. The possibility is discussed that the binding of diphtheria toxin to cells is mediated by both a protein receptor and an interaction with the head group of phospholipids.

Recurrence quantification analysis reveals interaction partners in paramyxoviridae envelope glycoproteins

The paramyxovirus envelope fuses with the host cell membrane by cooperative interaction of two transmembrane glycoproteins: the hemagglutinin neuraminidase (HN) and the fusion (F) glycoprotein. The interaction appears to be finely regulated, as both proteins must derive from the same viral species to obtain a functional interaction. Because HN and F do not form stable complexes, this interaction is poorly characterized. This article demonstrates that a modification of a classical bioinformatic method based on the co‐evolution of interacting partners can detect the specificity of the HN and F interaction. The proposed approach relies on a relatively new nonlinear signal analysis technique, recurrence quantification analysis (RQA), applied to the hydrophobicity sequences of viral proteins. This technique is able to shed light on the interaction between HN and F proteins in the virus–cell fusion and, more generally, permits the quantitative comparison of nonhomologue protein systems. On the contrary, the same co‐evolution approach, based on the classical sequence alignment procedure, was unable to discriminate interacting partners from the general strict correlation existing between the evolution of viral proteins as a whole. The cooperation between HN and F in the fusion process is thus demonstrated by a bioinformatic, purely sequence‐dependent, perspective. Proteins 2002;46:171–176.

Interaction of Tenebrio molitor L. α-amylase with a wheat flour protein inhibitor

It is well known [ I,21 that crude preparations of larval Tenebrio molitor L. (yellow mealworm) cY-amylase (1,4-a-D-glucan glucano-hydrolase, EC 3.2.1. I ) are effectively inhibited by a number of highly purified albumins from wheat flour. The best known of these protein inhibitors has a mol. wt. 24 000 and consists of two similar subunits which are dissociated by chemical agents such as dodecyl sulphate or guanidine [2]. This inhibitor, coded 0.19 according to its gel electrophoretic mobility relative to bromophenol blue [3] and referred to here as 0.19 albumin, inhibits, in addition to the amylase from T. molitor, a large number of amylases of different origin [4]. The influence of several enzymatic and physico-chemical treatments on the activity of 0.19 [4] and other wheat albumin inhibitors [l] of T. moliror a-amylase has been studied. Recently, the a-amylase from T. molitor larvae has been purified by one-step chromatography on a Sepharose-albumin inhibitor column and extensively characterized [.5,6]. The enzyme has a single polypeptide chain with mol. wt. 68 000 and exhibits several properties similar to those of mammalian a-amylases [61. This paper deals with the influence of a number of physical and chemical parameters on the enzymeinhibitor reaction and provides conclusive evidence

Cytotoxicity acquired by ribosome-inactivating proteins carried by reconstituted Sendai virus envelopes

Association of the ribosome‐inactivating proteins (RIPs): pokeweed antiviral protein (PAP), gelonin, Momordica charantia inhibitor (MCI), with reconstituted Sendai virus envelopes (RSVE) was obtained without detectable loss of activities either of RIPs or of viral envelope glycoproteins. RIPs are inactive towards intact cells, but, once encapsulated in RSVE, they become cytotoxic. The concentration of RSVE‐associated PAP, which causes 50% inhibition of protein synthesis by Friend erythroleukemic cells, is 0.5 ng/ml. Substances capable to inhibit the viral activities block the acquired cytotoxicity of RIPs associated to RSVE.

Peptides derived from the heptad repeat region near the C-terminal of Sendai virus F protein bind the hemagglutinin-neuraminidase ectodomain

Previously, we showed that Sendai virus fusion protein (F) acts as an inhibitor of neuraminidase activity of hemagglutinin‐neuraminidase (HN) protein. Here we report that synthetic peptides derived from the heptad repeat region proximal to the transmembrane domain (HR2) of Sendai virus F inhibit fusion and enhance the enzymatic activity of the HN. This occurs on the virus‐bound HN and on its soluble globular head. The enhancing effect on virus‐bound HN is reversible and depends on the presence of F. The data indicate that, by binding to the HN ectodomain, the HR2 peptides abolish the F inhibition of HN and disrupt the communication between the F and HN essential to promote virus–cell fusion.

Hydrophobic photolabelling of pertussis toxin subunits interacting with lipids

The hydrophobic surfaces presumably involved in the membrane interaction of pertussis toxin have been mapped by a new detergent‐binding assay. This is based on the interdispersion among detergent micelles of trace amounts of radioactive photoreactive phospholipid analogues, able to cross‐link to the protein thereby labelling its detergent‐binding domains. The assay has proven to be very sensitive. Subunits B1, B2 and B3 of pertussis toxin were found to interact with the lipid micelles suggesting that they may be involved in the membrane penetration step of the intoxication process.