Functional changes in the structure of the SRP GTPase on binding GDP and Mg2+GDP.

Ffh is a component of a bacterial ribonucleoprotein complex homologous to the signal recognition particle (SRP) of eukaryotes. It comprises three domains that mediate both binding to the hydrophobic signal sequence of the nascent polypeptide and the GTP-dependent interaction of Ffh with a structurally homologous GTPase of the SRP receptor. The X-ray structures of the two-domain 'NG' GTPase of Ffh in complex with Mg2+GDP and GDP have been determined at 2.0 A resolution. The structures explain the low nucleotide affinity of Ffh and locate two regions of structural mobility at opposite sides of the nucleotide-binding site. One of these regions includes highly conserved sequence motifs that presumably contribute to the structural trigger signaling the GTP-bound state. The other includes the highly conserved interface between the N and G domains, and supports the hypothesis that the N domain regulates or signals the nucleotide occupancy of the G domain.     

Determination of oxalyl thiolesters, N-oxalylcysteine and N-oxalylcysteamine in biological materials

A convenient method is described for the quantitative analysis of oxalyl thiolesters (OTEs), a newly discovered class of mammalian metabolites, in biological samples. By this particular technique the total concentration of all OTEs in the sample is determined. The method involves first reacting the biological material with cysteamine (2-aminoethanethiol) or cysteine under conditions that convert OTEs quantitatively to N-oxalylcysteamine (or N-oxalylcysteine), followed by reaction with monobromobimane to give a highly fluorescent derivative that is analyzed by reversed-phase ion-pair chromatography, with tetrabutylammonium ion as the counterion and N-(2-mercaptopropionyl)glycine as an internal standard. The method is capable of detecting as little as 0.6 pmol of the bimane derivative of the N-oxalyl compound in a single HPLC injection. The application of this method has led to the discovery that not only OTEs but also N-oxalylcysteine and N-oxalylcysteamine are normal mammalian metabolites. In various rat tissues the OTE concentration ranges up to 65 nmol/g (wet wt), the N-oxalylcysteine concentration is approximately 10 nmol/g, and the N-oxalylcysteamine concentration is 0-3 nmol/g.