Structural and genetic relationships between the O-antigens of Escherichia coli O118 and O151.

O-antigen (O-polysaccharide) is a highly variable part of the lipopolysaccharide present in the outer membrane of Gram-negative bacteria, which is used as the basis for bacterial serotyping and is essential for the full function and virulence of bacteria. In this work, the structure and genetics of the O-antigens of Escherichia coli O118 and O151 were investigated. Both O-polysaccharides were found to contain ribitol phosphate and have similar structures, the only difference between their backbones being one linkage mode (ß1→3 in E. coli O118 vs. ß1→2 in E. coli O151), which, most probably, is the linkage between the oligosaccharide repeats (O-units). The O-antigen gene clusters of the two bacteria are organized in the same manner and share high-level identity (>99%). Analysis of the wzy genes from E. coli O118 and O151 strains, which are responsible for the linkage between O-units, revealed only one nucleotide substitution, resulting in one amino acid residue substitution. The possible genetic events that may lead to the structural difference between two O-antigen structures are discussed. Salmonella O47 has the same O-unit backbone and a similar O-antigen gene cluster (OGC) (the DNA identity ranges from 74% to 83%) as E. coli O118 and O151. It was suggested that the OGCs of the three bacteria studied originated from a common ancestor.

A comprehensive method for the quantification of the non-oxidative pentose phosphate pathway intermediates in Saccharomyces cerevisiae by GC-IDMS.

A gas chromatography isotope dilution mass spectrometry (GC-IDMS) method was developed for the quantification of the metabolites of the non-oxidative part of pentose phosphate pathway (PPP). A mid-polar GC column (Zebron ZB-AAA, 10m, film composition 50% phenyl 50% dimethyl polysiloxane) was used for the chromatographic separation of the intermediates. The optimized GC-MS procedure resulted in improved separation performances and higher sensitivities compared to previous methods. Furthermore, the use of (13)C-labeled cell extracts as internal standards improved the data quality and eliminated the need to perform a recovery check for each metabolite. The applicability of the new method was demonstrated by analyzing intracellular metabolite levels in samples derived from aerobic glucose-limited chemostat cultures of Saccharomyces cerevisiae at steady state as well as following a short-term glucose pulse. The major achievements of the proposed quantitative method are the independent quantification of the epimers ribulose-5-phosphate and xylulose-5-posphate and the measurement of compounds present at very low concentrations in biological samples such as erythrose-4-phosphate and glyceraldehyde-3-phosphate.

Comparative structural and molecular characterization of ribitol-5-phosphate-containing Streptococcus oralis coaggregation receptor polysaccharides.

The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfbeta1-6GalNAcbeta1-3Galalpha1-2ribitol-5-PO(4)(-)-6Galfbeta1-3Galbeta1](n), thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and ribitol-5-phosphate, which is alpha1-2 in strain SK144 versus alpha1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.

The metabolic significance of octulose phosphates in the photosynthetic carbon reduction cycle in spinach.

14C-Labelled octulose phosphates were formed during photosynthetic 14CO2 fixation and were measured in spinach leaves and chloroplasts. Because mono- and bisphosphates of D: -glycero- D: -ido-octulose are the active 8-carbon ketosugar intermediates of the L-type pentose pathway, it was proposed that they may also be reactants in a modified Calvin-Benson-Bassham pathway reaction scheme. This investigation therefore initially focussed only on the ido-epimer of the octulose phosphates even though 14C-labelled D: -glycero- D: -altro-octulose mono- and bisphosphates were also identified in chloroplasts and leaves. 14CO2 predominantly labelled positions 5 and 6 of D: -glycero- D: -ido-octulose 1,8-P2 consistent with labelling predictions of the modified scheme. The kinetics of 14CO2 incorporation into ido-octulose was similar to its incorporation into some traditional intermediates of the path of carbon, while subsequent exposure to 12CO2 rapidly displaced the 14C isotope label from octulose with the same kinetics of label loss as some of the confirmed Calvin pathway intermediates. This is consistent with octulose phosphates having the role of cyclic intermediates rather than synthesized storage products. (Storage products don't rapidly exchange isotopically labelled carbons with unlabelled CO2.)A spinach chloroplast extract, designated stromal enzyme preparation (SEP), catalysed and was used to measure rates of CO(2) assimilation with Calvin cycle intermediates and octulose and arabinose phosphates. Only pentose (but not arabinose) phosphates and sedoheptulose 7-phosphate supported CO2 fixation at rates in excess of 120 micromol h(-1) mg(-1) Chl. Rates for octulose, sedoheptulose and fructose bisphosphates, octulose, hexose and triose monophosphates were all notably less than the above rate and arabinose 5-phosphate was inactive. Altro-octulose phosphates were more active than phosphate esters of the ido-epimer. The modified scheme proposed a specific phosphotransferase and SEP unequivocally catalysed reversible phosphate transfer between sedoheptulose bisphosphate and D: -glycero- D: -ido-octulose 8-phosphate. It was also initially hypothesized that arabinose 5-phosphate, an L-Type pentose pathway reactant, may have a role in a modified Calvin pathway. Arabinose 5-phosphate is present in spinach chloroplasts and leaves. Radiochromatography showed that 14C-arabinose 5-phosphate with SEP, but only in the presence of an excess of unlabelled ribose 5-phosphate, lightly labelled ribulose 5-phosphate and more heavily labelled hexose and sedoheptulose mono- and bisphosphates. However, failure to demonstrate any CO2 fixation by arabinose 5-phosphate as sole substrate suggested that the above labelling may have no metabolic significance. Despite this arabinose and ribose 5-phosphates are shown to exhibit active roles as enzyme co-factors in transaldolase and aldolase exchange reactions that catalyse the epimeric interconversions of the phosphate esters of ido- and altro-octulose. Arabinose 5-phosphate is presented as playing this role in a New Reaction Scheme for the path of carbon, where it is concluded that slow reacting ido-octulose 1,8 bisphosphate has no role. The more reactive altro-octulose phosphates, which are independent of the need for phosphotransferase processing, are presented as intermediates in the new scheme. Moreover, using the estimates of phosphotransferase activity with altro-octulose monophosphate as substrate allowed calculation of the contributions of the new scheme, that ranged from 11% based on the intact chloroplast carboxylation rate to 80% using the carboxylation rate required for the support of octulose phosphate synthesis and its role in the phosphotransferase reaction.

Difference in the xylitol sensitivity of acid production among Streptococcus mutans strains and the biochemical mechanism.

Xylitol inhibits the glycolysis and growth of Streptococcus mutans, but to different degrees among strains. Thus, we studied the biochemical mechanism through which the inhibition varies, using S. mutans strains ATCC 31989, NCTN 10449, and NCIB 11723, which are highly sensitive, moderately sensitive, and resistant to xylitol, respectively, under strictly anaerobic conditions such as those found in deep layers of dental plaque. Xylitol (30 mM) decreased the rate of acid production from glucose (10 mM) in ATCC 31989, NCTC 10449, and NCIB 11723 by 86, 26, and 0%, respectively. The activities of the xylitol : phosphoenolpyruvate phosphotransferase system (PEP-PTS) relative to those of glucose : PEP-PTS were 120, 16, and 3%, respectively. In ATCC 31989 and NCTC 10449, intracellular accumulation of xylitol 5-phosphate and decreases of fructose 1,6-bisphosphate and glucose 6-phosphate were observed. Furthermore, in the presence of xylitol (30 mM), glucose : PEP-PTS activities decreased by 34, 17, and 0%, respectively. These findings indicated that the higher the xylitol : PEP-PTS activity was and the more effectively xylitol decreased glucose : PEP-PTS activity, the more sensitive the strain was to xylitol. These results suggest that the following inhibitory mechanisms are active in the xylitol-sensitive mutans streptococci: direct inhibition of glycolytic enzymes by xylitol 5-phosphate derived from xylitol : PEP-PTS and, possibly, indirect inhibition through competition for the phosphoryl donor, HPr-P, between glucose and xylitol : PEP-PTSs.

Identification and quantitation of phosphorus metabolites in yeast neutral pH extracts by nuclear magnetic resonance spectroscopy.

(31)P NMR spectroscopy offers a possibility to obtain a survey of all low-molecular-weight phosphorylated compounds in yeast. The yeast cells have been extracted using chloroform into a neutral aqueous phase. The use of high fields and the neutral pH extracts, which are suitable for NMR analysis, results in well-resolved (31)P NMR spectra. Two-dimensional NMR experiments, such as proton-detected heteronuclear single quantum ((1)H-(31)P HSQC) and (31)P correlation spectroscopy ((31)P COSY), have been used to assign the resonances. In the phosphomonoester region many of the signals could be assigned to known metabolites in the glycolytic and pentose phosphate pathways, although some signals remain unidentified. Accumulation of ribulose 5-phosphate, xylulose 5-phosphate, and ribose 5-phosphate was observed in a strain lacking transketolase activity when grown in synthetic complete medium. No such accumulation occurred when the cells were grown in yeast-peptone-dextrose medium. Trimetaphosphate (intracellular concentration about 0.2 mM) was detected in both cold methanol-chloroform and perchloric acid extracts.

Xylitol 5-P formation by dental plaque after 12 weeks' exposure to a xylitol/sorbitol containing chewing gum.

Five subjects used xylitol-containing chewing gum for 12 wk. Dental plaque was collected before and after the exposure to xylitol. The plaque samples were examined for their capacity to form xylitol 5-P by incubation with 14C labeled xylitol, extraction and separation on HPLC. It was found that the capacity of the plaque to form xylitol 5-P was not reduced during the xylitol exposure in any of the subjects. No other xylitol-derived metabolites were observed. The inhibitory capacity of xylitol thus appears to be maintained after 12 wk exposure to xylitol.

Evidence for xylitol 5-P production in human dental plaque.

The Turku sugar studies indicated that xylitol may possess a caries-therapeutic effect. More recent data show that xylitol exhibits a bacteriostatic activity on a wide range of bacteria based on uptake and expulsion of xylitol. Intracellular xylitol 5-P appears to be a key substance associated with inhibition of bacterial metabolism by xylitol. This has been shown in studies with pure strains of bacteria, mainly Streptococcus mutans. The aim of the present study was to examine if production of xylitol 5-P occurs in freshly collected dental plaque which is exposed to labeled xylitol. Plaque extracts were analyzed by thin-layer chromatography combined with autoradiography and high performance liquid chromatography. Strong indications were obtained that xylitol 5-P is readily produced by dental plaque. No other significant xylitol metabolites were identified. The bacteriostatic properties of xylitol in plaque are a mechanism which may well account for the caries-therapeutic effect of xylitol.

Radiometric measurement of phosphoribosylpyrophosphate and ribose 5-phosphate by enzymatic procedures.

Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response.

A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts.

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.

Modified phosphoribosylpyrophosphate (PRPP) radioenzymatic assay: increased sensitivity, technical simplification and new applications.

Phosphoribosylpyrophosphate in amounts as low as 25 pmol could be reliably and economically measured with a CO2-releasing radioenzymatic assay when appropriate technical modifications were introduced. The concentration of commercially available phosphoribosylpyrophosphate used for reference standards was ascertained by a method based on the utilization of phosphoribosylpyrophosphate by hypoxanthine catalyzed by hypoxanthine phosphoribosyltransferase from red blood cell lysates. The addition of inorganic phosphate increased intracellular phosphoribosylpyrophosphate levels in HL-60 cell lysates and can be used to amplify low levels of phosphoribosylpyrophosphate. This phosphoribosylpyrophosphate assay amplified by inorganic phosphate has been developed to assay perturbations in the purine biosynthetic nucleotide pathway in response to various chemotherapeutic agents, such as anti-folates, or as a result of folate deficiency.

Effects of uridine diphosphoglucose (UDPG) infusion on 5-phosphoribosyl pyrophosphate (PRPP) levels of mouse tissues.

Uridine diphosphoglucose (UDPG) has been shown to have tissue-specific effects that have proved to be of clinical value in the treatment of some liver ailments. In an effort to determine something about the mechanism of action, we investigated the effect of UDPG on the levels of 5-phosphoribosyl pyrophosphate (PRPP) and PRPP synthetase in mouse liver, spleen and transplanted tumors. Three strains of mice were studied with and without tumors under various experimental conditions. Balb/c mice were infused with UDPG intraperitoneally at levels of 0.16 g/kg/day (0.28 mmole) to 1.6 g/kg/day (2.8 mmoles) for 5 days. At the low dose rate the PRPP level in the liver was found to increase 3-fold. A slight increase was noted in the activity of PRPP synthetase. However, when the UDPG was infused at a level of 2.8 mmoles/kg/day, the increases in both the synthetase and PRPP were inhibited. Both CRF1 and CD8 mice were less sensitive to the effects of UDPG per se. However, the high level of PRPP in the tumors they carried was greatly affected by the UDPG infusion. The tumor-specific inhibition of PRPP suggests that this action might prove to be useful combination therapy with inhibitors of purine and pyrimidine nucleotide synthesis in various rescue regimens. UDPG was found to enter cells intact before it was cleaved into glucose phosphate and UMP. The fact that UDPG was also found in the membrane fraction suggests that either there is a specific transport mechanism or UDPG exerts its action via interaction with the cell membrane.

A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate.

An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured.

The measurement of xylulose 5-phosphate, ribulose 5-phosphate, and combined sedoheptulose 7-phosphate and ribose 5-phosphate in liver tissue.

A modification of the method of Kauffman et al. (F. C. Kauffman, J. G. Brown, J. V. Passonneau, and O. H. Lowry (1969) J. Biol. Chem. 244, 3647-3653) for the spectrophotometric determination of xylulose 5-phosphate, ribulose 5-phosphate, and combined ribose 5-phosphate and sedoheptulose 7-phosphate in tissue extract is presented. Using commercially available enzymes all three assays come to a clear endpoint with the assays described. Values for these metabolites in liver in three dietary states are reported; 48 h starved, ad libitum feeding of standard NIH rat ration, and meal feeding of a fat-free diet. Xylulose 5-phosphate values were 3.8 +/- 0.3, 8.6 +/- 0.3, and 66.3 +/- 8.3 nmol/g. Ribulose 5-phosphate values were 3.4 +/- 0.3, 5.8 +/- 0.2, and 37.1 +/- 5.3 nmol/g. Combined ribose 5-phosphate and sedoheptulose 7-phosphate were 29.3 +/- 0.3, 38.2 +/- 1.2, and 108.2 +/- 14.5 nmol/g. The ratio of measured tissue content of [xylulose 5-phosphate]/[ribulose 5-phosphate] was found to be 1.12 +/- 0.07 in starved animals, 1.48 +/- 0.04 in ad libitum fed animals and 1.78 +/- 0.03 in low-fat meal fed animals. These data are in good agreement with the range of equilibrium constants reported for this reaction, suggesting that the ribulose 5-phosphate 3-epimerase reaction (EC 5.1.3.1) is a near equilibrium reaction despite a more than 10-fold change in the tissue content of these metabolites.

Rapid diagnosis of severe Haemophilus influenzae serotype b infections by monoclonal antibody enzyme immunoassay for outer membrane proteins.

A highly sensitive and specific enzyme immunoassay (EIA) for the detection of Haemophilus influenzae serotype b antigens in body fluids and broth cultures was developed, with a polyclonal antibody directed against polyribose phosphate as the solid-phase reagent and a biotinylated monoclonal antibody directed against H. influenzae type b outer membrane protein as the liquid-phase reagent. H. influenzae type b antigens could be detected in broth cultures containing as little as 50 organisms per ml. The sensitivity and specificity of this system were compared with those of two commercial kits and counterimmunoelectrophoresis. The overall detection of H. influenzae type b antigens in clinical specimens collected from children infected with H. influenzae type b was as follows: with Phadebact, 86 and 86% in cerebrospinal fluid and urine specimens, respectively; with Bactigen, 86, 80, and 92%, with counterimmunoelectrophoresis, 78, 73, and 75%, and with biotin-avidin EIA, 100, 100, and 100% for cerebrospinal fluid, serum, and urine specimens, respectively. In the biotin-avidin EIA, no positive reactions were noted in specimens collected from patients infected with other bacteria or from patients without evidence of bacterial infection, whereas false-positive reactions were found by counterimmunoelectrophoresis and the commercial kits. These results suggest that this monoclonal antibody reacting with the outer membrane protein is more specific and sensitive than the conventional methods using polyclonal antisera for the detection of H. influenzae type b antigens during severe infections in children.

Structure of the heparan sulfate-protein linkage region. Demonstration of the sequence galactosyl-galactosyl-xylose-2-phosphate.

We have treated bovine lung heparan sulfate with alkaline [3H]borohydride to end label the chains with [3H]xylitol. After subsequent periodate oxidation-alkaline elimination products were separated by gel permeation and ion exchange chromatography. The linkage region fragment expected to have 2 galactoses and 1 [3H]xylitol residue appeared in the tetra-/trisaccharide region after gel filtration and was bound to the anion exchange resin. A similar negatively charged fragment, expected to have 2 galactoses, 1 xylose and 1 serine, was isolated after periodate oxidation-alkaline elimination of unlabeled heparan sulfate. The negative charge was due to the presence of alkaline phosphatase-labile phosphate ester. The molar ratio of galactose:phosphate:xylose was 2.17:1.19:1.00. The phosphate ester was associated with the xylose/[3H] xylitol moiety as indicated by the formation of phosphoxylose/-xylitol by beta-galactosidase digestion of the phosphorylated trisaccharide. Furthermore, orcinol reactivity disappeared after periodate oxidation of the dephosphorylated trisaccharide. The phosphate ester must be located to C-2 of xylose/xylitol as the 1-3H radioactivity could be released by periodate oxidation when it was preceded by alkaline phosphatase treatment. It is estimated that almost every chain of heparan sulfate carries 2-phosphoxylose. It would be of interest to know if glycosaminoglycan chains that are artificially initiated onto exogeneous beta-D-xylosides also acquire the 2-phosphoxylose moiety.

Capsular variation in experimental strains of Haemophilus influenzae.

This study sought to demonstrate that clinically untypable strains of Haemophilus influenzae are derivable from previously capsulated ones. Penicillin-induced forms were employed to explore in vivo and in vitro a possible mechanism of the reversible cycle vegetative to L-phase revertant. Normal H. influenzae type b (Rab), capsule-deficient strain ATCC 9333, and experimental L-phase and its revertants were used in this investigation. Capsular antigens, polyribose phosphate (PRP) content of each strain was assayed by orcinol and rocket immunoelectrophoretic methods. Intra- and inter-strain PRP differences were statistically analysed. Strain differences between in vivo and in vitro passaged extracts of strain 9333 and 9333 were significant (t-test P less than 0.01). There were also significant differences in vivo and in vitro between penicillin-treated, L-phase infected mouse isolates and penicillin-free, L-phase infected mouse isolates; and also between penicillin-treated, L-phase infected mouse isolates and revertant Rab infected mouse isolates (Mann-Whitney U-test P less than 0.02, P less than 0.01, respectively). These findings suggest that untypable isolates of H. influenzae are derivable from otherwise capsulated strains, depending on decapsulating factors in the microenvironment. Clinical implications of the findings are discussed.

Spectrophotometric and radioenzymatic determination of ribose-5-phosphate.

The present work describes an assay which is highly specific for ribose-5-phosphate. The method is based on the following three-stage enzymatic conversion: (1) ribose 5-phosphate in equilibrium ribose 1-phosphate (phosphopentomutase); (2) ribose 1-phosphate + adenine in equilibrium adenosine + Pi (adenosine phosphorylase); (3) adenosine + H2O----inosine + NH3 (adenosine deaminase). Ribose 5-phosphate may be determined either directly following the change in absorbance at 265 nm associated with the conversion of adenine to inosine, or radioenzymatically by measuring the radioactivity of inosine formed from [8-14C]adenine, after chromatographic separation of the nucleoside on polyethyleneimine-cellulose. The spectrophotometric assay was used to follow ribose 5-phosphate formation and ribose 1-phosphate consumption catalyzed by phosphopentomutase. Further, the ability of alkaline phosphatase, 5'-nucleotidase and crude extract of Bacillus cereus cells to act on ribose 5-phosphate was tested. The radioenzymatic assay was proved useful in determining the levels of ribose 5-phosphate in rat tissues.

Deoxyribose 1-phosphate: radioenzymatic and spectrophotometric assays.

A method has been developed to measure deoxyribose 1-phosphate in the presence of ribose 1-phosphate and other sugar phosphates. The specificity of the method is based on the observation that only deoxyribose 1-phosphate is hydrolyzed by heating at pH 7.4, while both deoxyribose 1-phosphate and ribose 1-phosphate remain unchanged when heated at pH 10. A tissue extract is heated at pH 10. The amount of deoxyribose 1-phosphate plus ribose 1-phosphate is determined from that of deoxyinosine plus inosine formed in a coupled enzymatic reaction, based on the following two-stage transformation: deoxyribose 1-phosphate (ribose 1-phosphate) + adenine in equilibrium deoxyadenosine (adenosine) + inorganic phosphate, catalyzed by adenosine phosphorylase; deoxyadenosine (adenosine) + H2O----deoxyinosine (inosine), catalyzed by adenosine deaminase. By taking advantage of its unique heat lability, deoxyribose 1-phosphate is eliminated by heating the tissue extract at pH 7.4, and ribose 1-phosphate is determined as above. The amount of deoxyribose 1-phosphate stems from the difference between the amount of deoxyinosine plus inosine measured in the tissue extract heated at pH 10 and that of inosine measured in the tissue extract heated at pH 7.4. Free deoxyribose 1-phosphate has been found in rat tissues, as well as in Bacillus cereus during stationary phase of growth.