Stefania Gini

Phosphorylase-mediated mobilization of the amino group of adenine in Bacillus cereus

Mobilization of the ribose moiety of purine nucleosides as well as of the amino group of adenine may be realized in Bacillus cereus by the concerted action of three enzymes: adenosine phosphorylase, adenosine deaminase, and purine nucleoside phosphorylase. In this pathway, ribose-1-phosphate and inorganic phosphate act catalytically, being continuously regenerated by purine nucleoside phosphorylase and adenosine phosphorylase, respectively. As a result of such a metabolic pathway, adenine is quantitatively converted into hypoxanthine, thus overcoming the lack of adenase in B. cereus.

In situ inactivation of E. coli uridylylation cycle is independent of the state of covalent modification of the components of the glutamine synthetase cascade

Abstract Permeabilization of nitrogen-starved cells of Escherichia coli W with Lubrol WX leads to a selective inactivation of the uridylyl-removing uridylyltransferase (UR/ UTase) enzyme of the glutamine synthetase (GS) cascade system; whereas similar treatment does not affect activity of UR/UTase in cells grown under conditions of nitrogen excess (10 m m glutamine) (Mura, U., and Stadtman, E. R. (1981) J. Biol. Chem. 256 , 13014–13021). The possibility that susceptibility to Lubrol inactivation is related to differences in the state of adenylylation of GS and/or in the state of uridylylation of the P II protein was investigated. Permeabilized cells from nitrogen sufficient as well as from nitrogen-limited growth medium were exposed to Lubrol after prior incubation under conditions that lead to high or low states of GS adenylylation and high or low P IID /P IIA ratios. Integrity of UR/UTase was monitored by measuring the capacity of UTP to stimulate the deadenylylation of GS in situ . The results showed that the inactivation of UR/UTase by Lubrol is not affected by the states of GS adenylylation or P II uridylylation.

Radioenzymatic determination of adenosine.

Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U-14C]ribose-labeled inosine via the following reactions: adenosine + H2O----adenine + ribose (adenosine nucleosidase); adenine + [U-14C]ribose 1-phosphate in equilibrium with T[U-14C]ribose-adenosine + Pi (adenosine phosphorylase); [U-14C]ribose-adenosine + H2O----[U-14C]ribose-inosine + NH3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.

In Situ Regulation Studies of Escherichia coli Glutamine Synthetase

Publisher Summary The involvement of glutamine in the synthesis of purine and pyrimidine nucleotides, glucosamine 6-phosphate, and p -aminobenzoic acid, and in the formation of all the amino acids makes this metabolite a key intermediate in the synthesis of important end products such as proteins, nucleic acids, and complex polysaccharides. The enzyme of Escherichia coli and other gram-negative bacteria has been widely studied and appears to possess an extraordinary capability to integrate quite different metabolic signals and react appropriately. The E. coli glutamine synthetase (GS) is regulated at least by four different regulatory mechanisms, including repression of its synthesis, cumulative feedback inhibition by several end products of glutamine metabolism, modulation by the divalent metal ions, and covalent modification. With the aim of verifying in vivo the exceptional capability of GS to answer with an appropriate’ catalytic potency and regulatory susceptibility to different microenvironmental conditions, a method has been devised to study the GS cascade system in situ . E.coli can be made permeable to low molecular weight compounds while retaining GS and its cascade components by treating the cells with the nonionic detergent.

Studies on IMP degradation and ribose 1-phosphate utilization in human erythrocytes.

1. Intact human red cells do not attack exogenous IMP. The nucleotide is readily broken down by the soluble erythrocyte fraction to inosine, hypoxanthine and ribose 1-phosphate, with a pH optimum of approx. 6.2. 2. Ribose 1-phosphate can be actively reutilized, in the presence of ATP and hypoxanthine, to give IMP, at pH 7.4. The velocity of the IMP salvage synthesis dramatically increases at more alkaline pH values. 3. The two curves relating the velocities of IMP breakdown and of IMP synthesis as a function of hydrogen ion concentration intersect at pH 7.4. 4. The observations might be relevant in the process of purine transport by red cells.

In situ stability of uridylyl removing-uridylyltransferase of Escherichia coli.

The difference in sensitivity of UR/UT toward Lubrol WX permeabilization treatment of stationary phase E. coli cells is not uniquely related to nitrogen availability during cellular growth. The sensitivity of UR/UT to detergent treatment appears to be related to differences in the balance between fermentative and oxidative glucose metabolism. The possible occurrence of a third cycle in the glutamine synthetase regulatory cascade mechanism is considered.