The formation of a 1-5 phosphodiester linkage in the spontaneous breakdown of 5-phosphoribosyl-alpha-1-pyrophosphate.

The decomposition of 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP) in the presence of Mg2+ at pH=7.8 yields a combination of products including ribose 5-phosphate, ribose 1-phosphate, 5-phosphoribosyl 1,2 cyclic phosphate, inorganic phosphate, and pyrophosphate. Hydrogen decoupled 31P NMR analysis of the product mixture also exhibits a sharp peak (+2.6 ppm from phosphocreatine) in a chemical shift region which includes phosphodiester bonds. Alkaline phosphatase treatment of the product mixture results in cleavage of monophosphate esters such as ribose 1-phosphate and ribose 5-phosphate, but does not affect the unidentified peak. Homonuclear (1H) correlation spectroscopy (COSY) of a partially purified sample was successful in identifying the hydrogen spectra of this compound. Combined with results from the splitting patterns of selectively decoupled 31P spectra, the COSY data indicate that several hydrogens are directly coupled to the unknown phosphate group with J value matches to the hydrogen on carbon one and to the two hydrogens on carbon five. Heteronuclear (1H-31P) chemical shift correlation studies confirm these couplings and further substantiate the formation of a ribose 1-5 phosphate linkage during the degradation of PRPP under these conditions. It is presently unknown whether this is an intramolecular or intermolecular phosphodiester linkage, although some spectroscopic evidence suggest the intramolecular bond formation, i.e. a ribose 1,5-cyclic phosphate (R-1,5cP). The formation of R-1,5cP helps explain the observation that the 5-phosphate group from PRPP becomes labile during the spontaneous degradation of PRPP.

Spectrophotometric ribonuclease assays using dinucleoside monophosphate substrates.

A pair of ribonuclease assays have been developed which offer improvements in specificity, simplicity, and/or sensitivity over current procedures. The assays measure the rate of adenosine release upon ribonuclease hydrolysis of 3'-adenosyl dinucleoside monophosphate substrates. Adenosine formation is spectrophotometrically determined by combining a coupled-enzyme system (adenosine deaminase or an adenosine deaminase/nucleoside phosphorylase/xanthine oxidase combination) to the ribonuclease cleavage. As demonstrated by a brief characterization of the ribonuclease activities in several mouse tissues, the methods demonstrate the advantage of being able to discriminate between ribonucleases of differing substrate specificities. An interesting guanosyl(3'-5')adenosine-specific ribonuclease in mouse brain has been identified using these assay methods.

Protein quantitation of as low as 10-ng/ml concentrations by competitive binding to polystyrene latexes.

A protein quantitation method which offers protein detection as low as 10 ng protein/ml and accurate quantitation as low as 30-100 ng protein/ml, depending on the protein, has been designed. The assay, which is relatively quick and simple to perform, utilizes the strong, nonspecific adsorption of proteins onto polystyrene latexes. A competition is created between a marker enzyme and the analyte protein for a limited amount of latex surface area. Due to inactivation of the enzyme upon binding to a hydrophobic latex surface, measurement of enzyme activity allows determination of the bound/free enzyme ratio and thus the competing protein concentration. Considerations of sensitivity and simplicity are suggested to make this assay superior to others presently available.