Vincenzo Lanzara

Characterization of Cholylglycine Hydrolase from a Bile-Adapted Strain of Xanthomonas maltophilia and Its Application for Quantitative Hydrolysis of Conjugated Bile Salts

ABSTRACT Purified bile salt hydrolase from bile-adapted Xanthomonas maltophilia displays Michaelis-Menten kinetics on cholylglycine and cholyltaurine and hydrolyzes bile salts also in crude bovine bile. The protein is a dimer and is resistant to proteinases and to heating at 55 to 60°C for up to 60 min, in agreement with calorimetric data.

G-actin modified by plasma membrane interaction polymerizes only in the presence of filamentous myosin

In non-muscle cells actin can occur in different forms depending on the immediate and local needs of the cell. This suggests the presence of regulatory mechanisms capable to allow an easy interconversion between polymerization states of actin ranging from functional microfilaments to oligomeric or even monomeric actin. We describe here a potential regulatory mechanism mediated by the interaction of G-actin with plasma membranes. After the interaction G-actin is modified insuch a way that it does not polymerize in the presence of 2 mM MgCl, but only in the presence ‘of myosin with the formation of contractile units.

On an Mg2+-dependent interaction of actin with glyceraldehyde-phosphate dehydrogenase The fundamental role of KCl in the organization of F-actin

In the presence of Mg2+, the formation of actin filaments is hindered by glyceraldehyde‐3‐phosphate dehydrogenase. This effect, which increases with the square of Mg2+ concentration, is counteracted by 0.15 M KCl. Thus KCl, at concentrations found in the intracellular compartment, appears to be strictly required for the correct formation of actin filaments in all tissues in which the glyceraldehyde‐phosphate dehydrogenase concentration is high.

The experimental basis of some recent hypotheses on the mechanism of the polymerization of actin: a reappraisal.

We have studied the effect of sonication on the fluorescence of N-(1-pyrenyl)iodoacetamide-labeled F-actin as well as of native actin-pyrenyl-actin mixed oligomers in which the subunits were covalently attached to each other by phenylenebismaleimide. In both cases the fluorescence of the solution was largely decreased by sonication. We have found that this effect is due (a) to a 20-30% decrease of the specific fluorescence of the polymers. These results question the validity of the novel mechanism for the polymerization of actin recently proposed (D. Pantaloni et al. (1984) J. Biol. Chem. 259, 6274-6283). In these studies, in fact, the implicit assumption was made that the quenching of the fluorescence of the solution under sonication was due exclusively to the conversion of F-actin into G-actin.

Fructose-1,6-bisphosphate aldolase from rabbit muscle. Half of the sites reactivity at low temperature

Abstract At −11°C, −13°C only two of the four dihydroxyacetone phosphate binding sites of aldolase are catalytically active. The substrate is very tightly bound to the two sites since it does not exchange with the free substrate of the medium.

Unfolding studies of tissue transglutaminase

Activation of tissue transglutaminase by calcium involves a conformational change which allows exposition of the active site to the substrate via movements of domains 3 and 4 that lead to an increase of the inter-domain distance. The inhibitor GTP counteracts these changes. Here we investigate the possible existence of non-native conformational states still compatible with the enzyme activity produced by chemical and thermal perturbations. The results indicate that chemical denaturation is reversible at low guanidine concentrations but irreversible at high concentrations of guanidine. Indeed, at low guanidine concentrations tissue TG-ase exists in a non-native state which is still affected by the ligands as in the native form. In contrast, thermal unfolding is always irreversible, with aggregation and protein self-crosslinkage in the presence of calcium. DSC thermograms of the native protein in the absence of ligands consist of two partly overlapped transitions, which weaken in the presence of calcium and merge together and strengthen in the presence of GTP. Overall, the present work shows, for the first time, the reversible denaturation of a TG-ase isoenzyme and suggests the possibility that also in in vivo, the enzyme may acquire non-native conformations relevant to its patho-physiological functions.

An ADPase from sarcoplasmic reticulum of rabbit muscle cleaves ADP bound to F-actin

We have found that sarcoplasmic reticulum from rabbit muscle contains an ADPase which cleaves ADP bound to F-actin. The interaction is not of the simple Michaelis-Menten type, the order of the reaction being larger than the first. A possible explanation of this behaviour could be that ADPase binds to two adjacent actin monomers with a preferred orientation thus cleaving preferentially the nucleotide of one of the two monomers.

Actin modifying system of the sarcoplasmic reticulum from rabbit muscle

Abstract By interaction with sarcoplasmic reticulum from rabbit muscle G-actin loses the capability to polymerize. The modification of actin is due to the concomitant action of the Ca 2+ ATPase and of an ADPase embedded in the sarcoplasmic reticulum vesicles. Methyl isobutyl xanthine increases the rate of modification of actin by the sarcoplasmic reticulum vesicles by increasing the rate of the exchange of the actin-bound nucleotide.