Biomedical subjects
Publications and source records attributed to M Signorini.
The report describes a patient with 45 per cent BSA burns who developed Legionnaire's disease 3 days after the acute injury. The diagnosis of this life-threatening complication was late because most of its signs and symptoms can be encountered in the burned patient. This delay could have been fatal to the patient and required the evacuation of the burn centre for disinfection.
We have investigated the interaction between calcium ions and erythrocyte transglutaminase and the enzyme activation. The binding involves both high and low affinity sites, but only the former ones are relevant for activation. The binding of calcium and the activation are modified by treatment with NBD-Cl and with PLP suggesting the presence of cysteine and lysine residues at the high affinity binding sites. The interaction of the enzyme with calcium is not calmodulin dependent and is easily detected as a shift in electrophoretic mobility in the presence of SDS.
Addition of low concentrations of acrylamide to the assay mixture of erythrocyte transglutaminase leads to a strong inhibition of the enzyme with a mechanism consistent with a non-competitive inhibition against both substrates. For the quenching of the intrinsic fluorescence of the protein, much higher concentrations of acrylamide are required so that both phenomena appear independent of each other. In this particular case, therefore, acrylamide can be employed to obtain information on ligand-triggered conformational changes.
Erythrocyte transglutaminase was purified by anion-exchange chromatography, size exclusion and affinity chromatography. Homogeneity was achieved by an additional step of HPLC size-exclusion chromatography. The molecular mass of the purified enzyme was calculated to be 65,000 Da by size-exclusion chromatography and sucrose-gradient centrifugation, and 92,000 Da by SDS-PAGE, thus suggesting a high degree of asymmetry. The amino-acid composition of erythrocyte transglutaminase differed substantially from that of the guinea-pig liver enzyme, notably with respect to the number of histidine, cysteine and acidic amino-acid residues. The enzyme has an absolute requirement for divalent cations for activity: calcium, manganese, and the lanthanides terbium and gadolinium activate the enzyme in decreasing order of efficacy, while no activity is displayed in the presence of magnesium. In the presence but not in the absence of calcium ions, the enzyme is rapidly inactivated by N-ethylmaleimide and by diethylpyrocarbonate suggesting that the cation influences the reactivity of amino acids essential for catalysis. When erythrocyte proteins are employed as amine acceptors in the presence of calcium, the erythrocyte transglutaminase appears to preferentially modify membrane-associated proteins, although, in the absence of calcium ions and exogenous amines, it displays a pH-dependent interaction with soluble proteins.
When human endometrial transglutaminase was measured a 10-fold higher activity was detected during the secretive phase. This change was not related to either differences in solubility of the enzyme or to selective contamination by plasma factor XIII and rather appears to depend on the expression of the tissue form of transglutaminase, suggesting that this enzyme is regulated in vivo by progesterone.
Purified transglutaminase from human erythrocytes was shown to undergo time dependent inactivation by acrylamide added at millimolar concentration: this effect was wholly dependent on calcium ions and some protection was produced by glutamine substrates and GTP. The enzyme activity was recovered by addition of thiol based reducing agents, while ascorbic acid and sodium borohydrate were ineffective. These data are suggestive of an active site directed action of acrylamide.
The guanine nucleotides GTP, GDP and GMP inhibit the activity of erythrocyte transglutaminase (protein-glutamine:amine gamma-glutamyltransferase, EC 2.3.2.13) in a decreasing order of effectiveness. The inhibition is more apparent at low than at saturating levels of calcium ions and is not due to the chelation of Ca2+, but to an interference with the process of activation by the cation. This inhibition is likely to contribute to the latency of erythrocyte transglutaminase in physiological conditions.
The first cause of death in burned patients is still sepsis. A great number of studies point out a serious immunodeficiency due to several circulating mediators. Nowadays early excision and plasma exchange appear to be the most efficient procedures in order to remove from the patient these circulating agents. Therefore in this study the authors have treated 8 patients (average age 25 years), who presented an average of total burned surface of 49%, with plasma exchange performed early after injury. The results of PMN and lymphocyte function tests showed a significant therapeutic correction of these parameters in patients treated with plasma exchange. The clinical observations agreed with the results: in 7 patients with positive outcome, no invasive infections were recorded. The positive results obtained warrant further investigation with this procedure.
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Glycogen synthetase from skeletal muscle is rapidly inactivated by DEPC. In the presence of the substrate UDPG only 50% of the enzyme activity is lost. The concomitant addition of both UDPG and the allosteric activator glucose-6-phosphate almost completely prevents the inactivation by DEPC. Since glucose-6-phosphate alone does not prevent the inactivation by DEPC, it is concluded that it is effective through a potentiation of the effects of UDPG, possibly through a conformational change of the enzyme.
Uridine diphosphoglucose pyrophosphorylase was purified 2500-fold from rabbit skeletal muscle with a total recovery of 35% of the initial activity. The present procedure was made possible by an extensive use of hydrophobic chromatography. Purified pyrophosphorylase had a specific activity of 500 mumol/min/mg of protein and was homogeneous by chromatographic and electrophoretic criteria. The enzyme appears to be composed of eight subunits of 53,000 molecular weight each.
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The kinetic properties of rabbit muscle uridine diphosphoglucose (UDP-Glc) pyrophosphorylase have been studied, in both directions, with respect to substrate saturation, product inhibition, and cation requirement for activity. The results demonstrate that UDP-Glc pyrophosphorylase is a non-Michaelian enzyme: the synthetic reaction is characterized by a marked inhibition by glucose-1-phosphate (at concentrations higher than 0.3 mM) and by an hyperbolic saturation for UTP. In the reverse reaction, instead, the saturation function for UDP-Glc is hyperbolic and that for inorganic pyrophosphate is sigmoid, with a high Hill coefficient of (nH) 2.5. The study of the metal requirement indicates a distinctive ability of cations to stimulate the reactions of synthesis and degradation of the sugar nucleotide and a different stoichiometry of the metal chelates involved. The reaction mechanism is of the ordered-sequential type and the data of product inhibition allowed the identification of glucose-1-phosphate as the first substrate bound and UDP-Glc as the last product released. The inhibition pattern by UDP-Glc gives evidence for cooperativity also in the binding of this molecule.
Incubation of skeletal muscle glycogen phosphorylase with sodium periodate's results into irreversible loss of enzyme activity. The rate of inactivation is influenced by the ionic strength of the medium and by the presence of caffeine, but not by nucleotides. During the reaction, cysteine residues slowly reactive towards DTNB are modified and the coenzyme is released. These results suggest the presence of cysteine residues at the protein site involved in the binding of the phosphate group of pyridoxal phosphate.