The existence of a group translocation transport mechanism in animal cells: Uptake of the ribose moiety of inosine

After exposure to inosine, transport-competent plasma membrane vesicles isolated from SV -40-transformed Balb/c 3T3 cells accumulate intravesicular ribose 1-PO₄ at a concentration 200-fold greater than the extravesicular concentration. An analysis of the purine nucleoside phosphorylase activity distribution in various subcellular fractions, relative to other enzyme activities, indicated the presence of plasma membrane-associated purine nucleoside phosphorylase activity. The plasma membrane vesicles appear relatively impermeable to hypoxanthine. However, hypoxanthine, which is a competitive inhibitor of the transport reaction, is the only compound tested capable of mediating efflux of already accumulated ribose 1-PO₄. In addition, hypoxanthine does not result in the efflux of transported uridine which is accumulated in these membrane vesicles as uridine. Exogenous ribose 1-PO₄ neither results in counterflow nor does it inhibit the original uptake reaction. The following transport reaction is proposed: uptake occurs by group translocation, mediated by membrane-localized purine nuceloside phosphorylase. The data are consistent with sites for inosine and hypoxanthine being on the outer membrane surface whereas the ribose 1-PO₄ site is only on the inner surface.     

The effect of ribose 5-phosphate and 5-phosphoribosyl-1-pyrophosphate availability on de novo synthesis of purine nucleotides in rat liver slices.

The effect of increasing cellular ribose 5-phosphate (ribose-5-P) availability by methylene blue-induced acceleration of the oxidative pentose phosphate pathway on the rate of 5-phosphoribosyl-1-pyrophosphate (P-ribose-PP) generation, was studied in slices of rat liver at varying Pi concentration. It was found that at Pi concentration prevailing in the tissue of extracellular physiological Pi concentration, ribose-5-P availability is saturating for P-ribose-PP generation, as gauged by the rate of adenine incorporation into tissue nucleotides. The effect of altering P-ribose-PP availability on the rate of de novo purine production gauged by the rate of formate incorporation into purines, was also studied. It was found that the physiological P-ribose-PP concentration in rat liver tissue is limiting for purine synthesis de novo. Depletion of cellular P-ribose-PP, achieved by increase of P-ribose-PP consumption, decelerated purine synthesis, while increase of P-ribose-PP availability, achieved by activation of P-ribose-PP synthetase occurring at elevated Pi concentration, resulted in acceleration of purine synthesis.     

Role of the ribose residue of substrates in reactions catalyzed by ribonuclease

The rate constants and Km for the hydrolysis of the optically active nonglycosidic analogues of the CpA and C greater than p catalysed by RNase A and RNase BS-I were measured. The rate of hydrolysis of the model substrates in 10(5) and 10(3) slower that for the appropriate dinucleoside phosphate and nucleoside cyclophosphate. However, substitution of the relatively rigid ribofuranose ring with flexible alifatic chains is accompanied by little variation in binding constants. The analyses based on the single substrate system indicate that the observed difference in rate constants must be accounted for by a difference between the binding of the substrates in the transition state to the RNase active site. Consequently, the "rigidity" of the ribose rings in RNA leads to large decreases in the free energy of activation for the reactions catalysed by RNases.     

The multiple Maillard reactions of ribose and deoxyribose sugars and sugar phosphates

Ribose 5-phosphate (R5P) undergoes the Maillard reaction with amines at significantly higher rates than most other sugars and sugar phosphates. The presence of an intramolecular phosphate group, which catalyzes the early stages of the Maillard reaction, provides the opportunity for the R5P molecule to undergo novel reaction paths creating unique Maillard products. The initial set of reactions leading to an Amadori product (phosphorylated) and to an alpha-dicarbonyl phosphate compound follows a typical Maillard reaction sequence, but an observed phosphate hydrolysis accompanying the reaction adds to the complexity of the products formed. The reaction rate for the loss of R5P is partially dependent on the pK(a) of the amine but also is correlated to the protonation of an early intermediate of the reaction sequence. In the presence of oxygen, a carboxymethyl group conjugated to the amine is a major product of the reaction of R5P with N-acetyllysine while little of this product is generated in the absence of oxygen. Despite lacking a critical hydroxyl group necessary for the Maillard reaction, 2-deoxyribose 5-phosphate (dR5P) still generates an Amadori-like product (with a carbonyl on the C-3 carbon) and undergoes phosphate cleavage. Two highly UV-absorbing products of dR5P were amine derivatives of 5-methylene-2-pyrrolone and 2-formylpyrrole. The reaction of dR5P with certain amines generates a set of products that exhibit an interesting absorbance at 340nm and a high fluorescence.     

Reaction of the ribose moiety of adenosine and AMP with periodate and carboxylic acid hydrazides

The dialdehyde (II–V) generated by periodate oxidation of the ribose moiety in adenosine or AMP reacts readily with carboxylic acid hydrazides yielding morpholine derivatives (VI, VII, VIII) which are stable over a wide range of pH and temperature. No side reactions have been observed. This reaction will allow introduction of various substituents into the 3′ end of RNAs, and the resulting modifications would permit investigations on the structure and function of such RNAs.