Generation of 3HOH from D-[6-3H]glucose by erythrocytes: role of pyruvate alanine interconversion

Human and rat erythrocytes were found to generate 3HOH from D-[6(N)-3H]glucose. The rate of 3HOH production represented 7-10% of the glycolytic flux. The generation of 3HOH appeared attributable, in part at least, to the detritiation of [3-3H]pyruvate during the interconversion of the 2-keto acid and L-alanine in the reaction catalyzed by glutamate-pyruvate transaminase. Indeed, purified pig heart glutamate-pyruvate transaminase, as well as homogenates prepared from rat erythrocytes or pancreatic islets, catalyzed the generation of 3HOH from L-[3-3H]alanine. When the production of tritiated pyruvate from L-[3-3H]alanine was coupled to the conversion of the 2-keto acid to L-lactate, the production of 3HOH accounted for one-third of the reaction velocity, the latter failing to display isotopic discrimination. In these experiments, the production of 3HOH was abolished by amino-oxyacetate. Likewise, in intact rat erythrocytes, aminooxyacetate inhibited the generation of 3HOH and tritiated L-alanine from D-[6-3H]glucose (or D-[1-3H]glucose), as well as the generation of 3HOH from L-[3-3H]alanine. In pancreatic islets, however, aminooxyacetate failed to affect significantly the generation of 3HOH from D-[6-3H]glucose. These findings indicate that the generation of 3HOH from D-[6-3H]glucose is mainly attributable to an intermolecular tritium transfer in transaminase reaction, at least in cells devoid of mitochondria.     

Metabolism of tritiated D-glucose anomers in rat erythrocytes

It was recently proposed that alpha-D-glucose 6-phosphate may undergo enzyme-to-enzyme channelling between glucokinase and phosphoglucoisomerase in rat pancreatic islets. The present study aims at exploring whether a different situation prevails in cells deprived of glucokinase, namely in erythrocytes. At anomeric equilibrium, the ratio between D-[2-3H]glucose and D-[5-3H]glucose conversion to 3HOH was lower in rat erythrocytes incubated for 60 min at 4 degrees C in the presence of 2.8 mM, rather than 8.3 mM, D-glucose. This coincided with both a greater relative increase in beta-D-[5-3H]glucose, as compared to alpha-D-[5-3H]glucose, conversion to 3HOH and an increase in the beta/alpha ratio for 3HOH generation from D-[5-3H]glucose in response to an increase in the anomeric concentration from 2.8 to 8.3 mM, the suppression of the difference between the beta/alpha ratios for 3HOH generation from D-[2-3H]glucose and D-[5-3H]glucose in the erythrocytes incubated at 8.3 mM, as distinct from 2.8 mM, alpha- and beta-D-glucose, and a [2-3H]/[5-3H] ratio for 3HOH generation lower than unity in erythrocytes exposed to alpha-D-glucose but not significantly different from unity in the presence of beta-D-glucose. These findings emphasize the relevance of alpha-D-glucose 6-phosphate channelling between hexokinase and phosphoglucoisomerase as a determinant of the difference between D-[2-3H]glucose and D-[5-3H]glucose conversion to 3HOH, and reveal that the regulation of such a tunnelling process by the concentration of the D-glucose represents, in rat erythrocytes, a mirror image of that observed in rat pancreatic islets. The regulation of this process thus tightly depends on the identity of the hexokinase enzyme mainly responsible for the phosphorylation of D-glucose in distinct cell types.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates. A model for D-[2-3H]glucose metabolism in human erythrocytes

When D-[2-3H]glucose 6-phosphate mixed with the unlabeled ester is converted to D-[1-3H]fructose 6-phosphate and 3HOH in the phosphoglucoisomerase reaction and then to D-[1-3H]fructose 1,6-bisphosphate in the phosphofructokinase reaction, the specific radioactivity of the latter metabolite and the production of 3HOH relative to the total generation of tritiated end products are both inversely related to the concentration of phosphofructokinase. In human erythrocytes, the modeling of D-[2-3H]glucose metabolism, based on the activity of phosphoglucoisomerase in cell homogenates and on the steady-state content of D-glucose 6-phosphate and D-fructose 6-phosphate in intact cells, indicates that the back-and-forth interconversion of these esters is about five-times higher than the net glycolytic flux. Yet, the production of 3HOH from D-[2-3H]glucose is about 20% lower than the net glycolytic flux, as judged from the production of 3HOH from D-[5-3H]glucose. Thus, an incomplete detriation of D-[2-3H]glucose is not incompatible with an extensive interconversion of hexose 6-phosphates in the reaction catalyzed by phosphoglucoisomerase.     

Enzyme-to-enzyme channelling in the early steps of glycolysis in rat pancreatic islets

The metabolism of D-glucose displays anomeric specificity in rat pancreatic islets. The aim of the present report is to investigate whether such a situation implies enzyme-to-enzyme tunnelling of metabolites in the early steps of glycolysis. For such a purpose, the modelling of alpha- and beta-D-glucose catabolism, itself based on available information concerning both the utilisation of these two anomers and the intrinsic properties of phosphoglucoisomerase, was first examined. According to a theoretical model with enzyme-to-enzyme channelling, the generation of 3HOH from D-[2-3H]glucose should be higher in islets exposed to beta-D-glucose rather than alpha-D-glucose, whilst the opposite situation should prevail in the case of D-[5-3H]glucose conversion to 3HOH. Experimental data collected in rat islets incubated for 60 min at 4 degrees C in the presence of either alpha- or beta-D-glucose mixed with tracer amounts of either alpha- or beta-D-[2- 3H]glucose and alpha- or beta-D-[5-3H]glucose indicate that the beta/alpha ratio for D-[2-3H]glucose conversion to 3HOH is indeed higher than the beta/alpha ratio for D-[5-3H]glucose conversion to 3HOH. These findings are consistent with the postulated enzyme-to-enzyme tunnelling of glycolytic intermediates between hexokinase isoenzyme(s), phosphoglucoisomerase and, possibly, phosphofructokinase.     

Hexose metabolism in pancreatic islets: enzyme-to-enzyme tunnelling of hexose 6-phosphates.

The fate of unlabelled D-glucose and D-[2-3H]glucose in pancreatic islets was simulated taking into account experimental values for glycolytic flux, intracellular concentration of D-glucose 6-phosphate and phosphoglucoisomerase activity. The model, which also takes into account the isotopic discrimination in velocity and intramolecular transfer of tritium between D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate in the reaction catalyzed by phosphoglucoisomerase, revealed that the predicted generation of 3HOH from D-[2-3H]glucose was much higher than the true experimental value. Such a discrepancy is reinforced by the consideration that the generation of 3HOH from D-[2-3H]glucose in islet cells is not solely attributable to the phosphoglucoisomerase-catalyzed detritiation of hexose 6-phosphates metabolized in the glycolytic pathway. In order to reconcile experimental and theoretical values for 3HOH production, it was found necessary to postulate enzyme-to-enzyme tunnelling of hexose 6-phosphates in the hexokinase/phosphoglucoisomerase/phosphofructokinase sequence. It is proposed that such a tunnelling may favour the anomeric specificity of D-glucose metabolism in islet cells, by restricting the anomerization of hexose 6-phosphates.     

Induction of neoplastic transformation by low-dose-rate exposure to tritiated water

Exponentially growing mouse BALB/3T3 cells seeded at low density were incubated with various concentrations of tritiated water (3HOH). A dose-dependent increase in the yield of transformants occurred in cells incubated for 100 h with 25 to 500 micrograms/ml of 3HOH. The dose-response curve rose linearly at low doses, with no evidence of a threshold or quadratic component. The transformation frequencies were similar when the same total dose of radiation was given over periods of 5 to 168 h, although survival was slightly lower with 5 h exposure compared with longer intervals. Acute X-ray exposure appeared to be more efficient in inducing transformation than protracted exposure to 3HOH at doses of 200-400 rad, but slightly less effective in the 25-100 rad range. When normalized for survival, 3HOH was more effective than X rays even at the higher doses. This result is very similar to previous findings with [3H]thymidine. A comparison of [3H]thymidine and 3HOH results suggest that the transmutational effect of tritium decay for radioactivity incorporated into DNA may be a significant factor in cytotoxicity but not in transformation; the enhanced effectiveness of both types of tritium exposure for transformation and mutagenesis may result from a higher relative biological effectiveness for the tritium beta particle for these end points.     

Hexose metabolism in pancreatic islets. Ca(2+)-dependent activation of the glycerol phosphate shuttle by nutrient secretagogues.

A method is proposed for the measurement of the flux through the glycerol phosphate shuttle in pancreatic islets. Such a flux is taken as the ratio between the production of 3HOH and the specific radioactivity of L-[2-3H]glycerophosphate in islets exposed to [2-3H]glycerol. D-Glucose and non-glucidic nutrient secretagogues, such as 2-ketoisocaproate and 2-aminobicyclo[2,2,1]heptane-2-carboxylate, stimulate, in a Ca(2+)-dependent manner, circulation in the glycerol phosphate shuttle. The shuttle flux is commensurate with the fraction of pyruvate generation which is not coupled with L-lactate production. These findings support the view that a rise in D-glucose concentration leads to activation of the FAD-linked mitochondrial glycerophosphate dehydrogenase through an increase in cytosolic Ca2+ concentration.     

Evidence from 18O exchange measurements for steps involving a weak acid and a slow chemical transformation in the mechanism of phosphorylation of the gastric H+, K+-ATPase by inorganic phosphate

Phosphorylation of the gastric H,K-ATPase by Pi has been studied by measuring the P18Oj16O4-j distribution as a function of time at different H+, K+, and [18O]Pi concentrations. The advantage of isotope exchange measurements is that the P18Oj16O4-j distribution depends on the relative rates of HOH loss to form the phosphoenzyme intermediate and Pi dissociation from the enzyme. Therefore, 18O exchange is a sensitive probe of mechanism. K+ increases the exchange rate (v(ex] but does not affect the partition coefficient (Pc) that determines the P18Oj16O4-j distribution. Conversely, H+ inhibits exchange. A single Pc describes the data at every pH, but the value increases from 0.04 at pH 8 to 0.64 at pH 5.5. Vex depends hyperbolically on [Pi]0. Km for Pi does not depend on pH, and Pc does not depend on [Pi]0. Individual rate constants in the phosphorylation mechanism are estimated. Formation of the E.Pi complex that looses HOH is 1-2 orders of magnitude slower at pH 5.5 than at pH 8 and is not diffusion controlled. The observed change in Pc with pH is compatible with catalysis occurring by a different mechanism when a group with pKa = 7.2 is protonated. Slower than diffusion-controlled formation of the E.Pi complex that splits out HOH is evidence for a relatively slow, unimolecular chemical transformation involving an additional intermediate in the phosphorylation mechanism, such as a protein conformational change.     

Interconversion of D-fructose 1,6-bisphosphate and triose phosphates in human erythrocytes.

Aldolase and triose phosphate isomerase both display strict specificity towards the enantiomers of [1-3H]glycerone 3-phosphate. The enantiomer generated from D-[1-3H]glyceraldehyde 3-phosphate produces 3HOH in the aldolase reaction, whilst the other enantiomer generated from D-[3-3H]fructose 1,6-bisphosphate is solely detritiated in the reaction catalyzed by triose phosphate isomerase. Advantage was taken of such a specificity to assess, in human erythrocytes exposed to either D-[3-3H]glucose or D-[3,4-3H]glucose, the extent of D-glyceraldehyde 3-phosphate sequential conversion to glycerone 3-phosphate and D-fructose 1,6-bisphosphate, relative to net glycolytic flux. At 37 degrees C and in the presence of 5.6 mM D-glucose, only 55% of the metabolites of D-[4-3H]glucose underwent detritiation in the reactions catalyzed by triose phosphate isomerase and aldolase. Such a percentage was further decreased at low temperature (8 degrees C) or lower concentrations of D-glucose (0.2 and 1.0 mM). However, when the erythrocytes were exposed to menadione, the increase in 3HOH production from either D-[3-3H]glucose or D-[3,4-3H]glucose indicated that the majority of the 3H atoms initially located on the C4 of D-glucose were recovered as 3HOH upon circulation through the pentose phosphate pathway. These findings suggest that, under physiological conditions, a large fraction of D-glyceraldehyde 3-phosphate generated from exogenous D-glucose may undergo enzyme-to-enzyme channelling in the glycolytic pathway.     

Impaired enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase in rat pancreatic islets incubated at a low concentration of D-glucose

It was recently proposed that in rat pancreatic islets exposed to 8.3 mM D-glucose, alpha-D-glucose-6-phosphate undergoes enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase. To explore the identity of the hexokinase isoenzyme(s) involved in such a tunnelling process, the generation of 3HOH from the alpha- and beta-anomers of either D-[2-3H]glucose or D-[5-3H]glucose was now measured over 60 min incubation at 4 degrees C in pancreatic islets exposed only to 2.8 mM D-glucose, in order to decrease the relative contribution of glucokinase to the phosphorylation of the hexose. Under these experimental conditions, the ratio for 3HOH production from D-[2-3H]glucose/D-[5-3H]glucose at anomeric equilibrium (39.7 +/- 11.6%) and the beta/alpha ratios for the generation of 3HOH from either the D-[2-3H]glucose anomers (70.9 +/- 12.6%) or the D-[5-3H]glucose anomers (59.6 +/- 12.4%) indicated that a much greater fraction of alpha-D-glucose-6-phosphate escapes from the process of enzyme-to-enzyme channelling in the islets exposed to 2.8 mM, rather than 8.3 mM D-glucose. These findings suggest, therefore, that the postulated process of enzyme-to-enzyme channelling involves mainly glucokinase.     

Mechanistic studies with general acyl-CoA dehydrogenase and butyryl-CoA dehydrogenase: evidence for the transfer of the beta-hydrogen to the flavin N(5)-position as a hydride

Butyryl-CoA dehydrogenase from Megasphera elsdenii catalyzes the exchange of the alpha- and beta-hydrogens of substrate with solvent [Gomes, B., Fendrich, G., & Abeles, R. H. (1981) Biochemistry 20, 1481-1490]. The stoichiometry of this exchange was determined by using 3H2O label as 1.94 +/- 0.1 per substrate molecule. The rate of 3H label incorporation into substrate under anaerobic conditions is monophasic, indicating that both the alpha- and beta-hydrogens exchange at the same rate. The exchange in 2H2O leads to incorporation of one 2H each into the alpha- and the beta-positions of butyryl-CoA, as determined by companion 1H NMR experiments and confirmed by mass spectroscopic analysis. In contrast, with general acyl-CoA dehydrogenase from pig kidney, only exchange of the alpha-hydrogen was found. The beta-hydrogen is the one that is transferred (reversibly) to the flavin 5-position during substrate dehydrogenation. This was demonstrated by reacting 5-3H- and 5-2H-reduced 5-deaza-FAD-general acyl-CoA dehydrogenase with crotonyl-CoA. Only one face of the reduced flavin analogue is capable of transferring hydrogen to substrate. The rate of this reaction is 11.1 s-1 for 5-deaza-FAD-enzyme and 2.2 s-1 for [5-2H]deaza-FAD-enzyme, yielding an isotope effect of 5. These values compare with a rate of 2.6 s-1 for the reaction of native reduced enzyme with crotonyl-CoA. The two reduced enzymes (normal vs. 5-deaza-FAD-enzyme) thus react at similar rates, indicating a similar mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of D-glucose anomers in rat pancreatic islets exposed to equilibrated D-glucose.

This study aims at establishing the contribution of alpha- and beta-D-glucose to the total generation of (3)HOH by rat pancreatic islets exposed to D-[2 - (3)H]glucose or D-[5 - (3)H] glucose at anomeric equilibrium. The islets were incubated for 60 min at 4 degrees C in the presence of equilibrated D-glucose (2.8 and 8.3 mM) mixed with tracer amounts of either alpha- or beta-D-glucose labelled with tritium on either the C (2) or C (5) of the hexose. Relative to their respective concentrations, (3)HOH generation from the anomers labelled with tritium on the C (2) or C (5) of the hexose provided beta/alpha ratios comparable to those previously found at both 2.8 and 8.3 mM, when the islets were exposed to each anomer separately. The relative contributions of each anomer to the total generation of (3)HOH was also close to the theoretical values derived from mathematical models for the catabolism of D-glucose at anomeric equilibrium in rat islets at both 2.8 and 8.3 mM and in the case of both D-[2 - (3)H]glucose and D-[5 - (3)H]glucose. Thus, even in islets exposed to D-glucose at anomeric equilibrium, the metabolic fate of alpha-D-glucose differs vastly from that of beta-D-glucose, the enzyme-to-enzyme channelling between hexokinase isoenzymes, especially glucokinase, and phosphoglucoisomerase being restricted to alpha-D-glucose 6-phosphate.     

Vitamin K-dependent carboxylase. Demonstration of a vitamin K- and O2-dependent exchange of 3H from 3H2O into glutamic acid residues

The rat liver vitamin K-dependent carboxylase has been studied with t-butoxycarbonyl-Glu-Glu-Leu-OMe as a substrate. The crude enzyme preparation catalyzes incorporation of 3H from media 3H2O into the glutamic acid residues of the substrate. This incorporation is dependent on vitamin KH2 and O2, is stimulated by NaCN, and is inhibited by increasing the HCO3- concentration. These data lend support to an enzymatic mechanism involving a vitamin K- and O2-dependent formation of a carbanion at the gamma-position of the glutamic acid residue followed by attack of CO2 to form gamma-carboxyglutamic acid.     

Studies of the penetration of the blood brain barrier by atrial natriuretic factor

The atrial natriuretic factors (ANF) have been detected in various areas of the brain. To determine whether circulating blood borne ANF could contribute to the ANF content in the central nervous systems we examined the ability of ANF-99-126 or ANF-102-126 to penetrate the blood brain barrier. Carotid artery injections of [3H] inulin with [125I] ANF in anesthetized rabbits resulted in a comparably minimal brain uptake index (BUI) for each labeled substance as measured in cerebral cortex extracts. Injection of [3H] HOH and [125I] ANF resulted in a mean BUI in cortex of 4.9 +/- .6 (SEM)% for ANF relative to triated water; this low uptake was not significantly saturable. The BUI ratio for ANF/HOH in olfactory bulb was somewhat higher though still low, at 7.0 +/- 9%, possibly reflecting the high density of ANF receptors in this structure. Infusion of [125I] ANF into the carotid artery of anesthetized rabbits resulted in little radioactivity being detected in the cerebrospinal fluid. Infusion of unlabeled ANF, which raised plasma levels as high as 26.3 ng/ml, resulted in little change in CSF levels. Our results demonstrate that the uptake of ANF into the brain is minimal and supports the idea that local synthesis of ANF predominantly accounts for the brain pool of this peptide.     

Enzymatic synthesis of [4R-2H]NAD (P)H and [4S-2H]NAD(P)H and determination of the stereospecificity of 7 alpha- and 12 alpha hydroxysteroid dehydrogenase

The stereospecifically labeled coenzymes [4R-2H]NADH, [4R-2H]NADPH and [4S-2H]NAD(P)H were synthesized enzymatically in high yield and high isotopic purity (greater than or equal to 95%) with 2HCOO2H/formate dehydrogenase, (CH3)2C2HOH/alchol dehydrogenase from Thermoanaerobium brockii and [1-2H]glucose/glucose dehydrogenase, respectively. This set of deuterated coenzymes was used to determine the stereospecificity of the previously unstudied 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli (NAD-dependent) and 12 alpha-hydroxysteroid dehydrogenase from Clostridium group P (NADP-dependent). H-NMR and EI-MS of the nicotinamide moiety after enzymatic oxidation of deuterated NAD(P)H with dehydrocholic acid as substrate showed that both dehydrogenases are B-sterospecific.     

Synthesis and characterization of a new bioactivated paramagnetic gadolinium(III) complex [Gd(DOTA-FPG)(H2O)] for tracing gene expression

A smart contrast agent for magnetic resonance imaging (MRI) can be used to exploit an enzymatic activity specific to the tissue or disease state signified by converting an MRI-inactivated agent to an activated MRI agent. In this study, a beta-galactopyranose-containing gadolinium(III) complex [Gd(DOTA-FPG)(H 2O)] was designed, synthesized, and characterized as being potentially suitable for a bioactivated MRI contrast agent. The (17)O NMR experiments were conducted to estimate the water exchange rate k e x 298 and rotational correlation time tau R 298 . The k ex 298 value of [Gd(DOTA-FPG)(H 2O)] is similar to that of [Gd(DO3A-bz-NO 2)(H 2O)]. The rotational correlation time value of [Gd(DOTA-FPG)(H 2O)] is dramatically longer than that of [Gd(DOTA)(H 2O)] (-) Relaxometric studies show that the percentage change in the T 1 value of [Gd(DOTA-FPG)(H 2O)] decreases dramatically in the presence of beta-galactosidase and human serum albumin. The T(1) change percentage of [Gd(DOTA-FPG)(H 2O)] (60%) is significantly higher than those of Egad and gadolinium(III)-1-(4-(2-(1-(4,7,10-triscarboxymethyl-(1,4,7,10-tetraazacyclododecyl)))-ethylcarbamoyloxymethyl)-2-nitrophenyl)-beta- d-glucopyronuronate. The signal intensity of the MR image for [Gd(DOTA-FPG)(H 2O)] in the presence of human serum albumin and beta-galactosidase (2670 +/- 210) is significantly higher than that of [Gd(DOTA-FPG)(H 2O)] in the sodium phosphate buffer solution (1490 +/- 160). In addition, the MR images show a higher-intensity enhancement in CT26/beta-gal tumor with beta-galactosidase gene expression but not for the CT26 tumor without beta-galactosidase gene expression. We conclude that [Gd(DOTA-FPG)(H 2O)] is a suitable candidate for a bioactivated MRI contrast agent in tracing gene expression.     

Enzymic generation of chiral acetates. A quantitative evaluation of their configurational assay.

1. R-Acetate was generated enzymically from R-acetate in the sequence acetate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. It was concluded that the formation of malate on malate synthase involves the operation of a normal isotopic effect combined with inversion of configuration. The malate synthase kH/k2H was determined as 3.7 +/- 0.5 by a method which yields results independently of the stereochemical purity of the chiral acetates used initially. 2. R-Acetate was also generated from R-acetate in the sequence acetate leads to citrate leads to malate leads to oxaloacetate leads to acetate, and S-acetate likewise from S-acetate. The conclusion is the same as given above, but refers to the formation of citrate on the re-synthase. 3. 2S,3R-[2-2H1,3-2H1,3H1]Malate and 2S,3S-[2-2H1,3-2H1]malate were prepared from 2S-[2,3-2H3]malate by treatment with fumarase in tritiated water and normal water, respectively. It was assumed that these malate specimens were pure with respect to chirality as generated by isotopic labelling. 4. These two malate specimens were partially converted (about 9%) to acetates in conditions where no racemization at the level of transiently formed oxaloacetate occurred. That no racemization took place was demonstrated experimentally. Oxidative enzymic hydrolysis of 2S,3R-[2-2H1,3-2H1,3H1]malate in normal water and of 2S,3S-[2-2H1,3-2H1]malate in tritiated water produced S-[2H1,3H1]acetate and R-[2H1,3H1]acetate, respectively. 5. The isolated R-[2H1,3H1]acetate and S-[2H1,3H1]acetate on configurational analysis yielded malates which in the presence of fumarase retained 79.7 +/- 0.7% and 20.3 +/- 0.9%, respectively, of their total tritium content. The symmetric deviation from the 50% value found with [3H1]acetate strengthens the conclusion that stereochemically pure chiral acetates were analyzed. The malate synthase kH/k2H was determined from the data of this study as 3.9 +/- 0.2. 6. The average of the values given under paragraphs 1 and 5 for the isotopic discrimination on malate synthase corresponds to kH/k2H=3.8 +/- 0.1. It was concluded that the configurational analysis of stereochemically pure R-[2H1,3H1]acetate and S-[2H1,3H1]acetate yields malates which in the presence of fumarase retain 79 +/- 2% and 21 +/- 2%, respectively, of their total tritium content. Hence, a deviation of 29 +/- 2% from the 50% value represents the actual amplitude of the configurational assay. 7. Outlines are given for an enzymic generation of chiral acetates in preparative scale.     

The iron-sulfur-cluster-containing L-serine dehydratase from Peptostreptococcus asaccharolyticus. Stereochemistry of the deamination of L-threonine.

The stereochemistry of the deamination of L-threonine to 2-oxobutyrate, catalyzed by purified L-serine dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled L-threonine in 2H2O and in 3HOH, as well as with L-[3-3H]threonine in unlabelled water. Isotopically labelled 2-oxobutyrate thus formed was directly reduced in a coupled reaction with L- or D-lactate dehydrogenase and NADH. The (2R)- or (2S)-2-hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H-NMR spectroscopy and, after degradation of 2-hydroxybutyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of L-threonine catalyzed by L-serine dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by serine dehydratase, therefore confirming the distinction of the L-serine dehydratase of P. asaccharolyticus as an iron-sulfur protein from those dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.     

Characterization of Receptors for Substance P in Human Astrocytoma Cells: Radioligand Binding and Inositol Phosphate Formation

UC11 cells, derived from a human astrocytoma, have a high density of functional substance P receptors. Radioligand binding studies were conducted with the highly selective neurokinin-1 receptor ligand [3H][Sar9,Met(O2)11]-substance P. Kinetic binding experiments conducted at 4 degrees C yielded an association rate constant k1 of 1.86 x 10(7) M-1 min-1, a dissociation rate constant k-1 of 0.00478 min-1, and a calculated kinetic KD of 257 pM. Saturation binding experiments yielded average values of KD = 447 +/- 103 pM, Bmax = 862 +/- 93 fmol/mg of protein. This Bmax corresponds to more than 150,000 binding sites/cell. Competition binding experiments with unlabeled [Sar9,Met(O2)11]-substance P yielded average values of KD = 491 +/- 48 pM and Bmax = 912 +/- 67 fmol/mg of protein. In [3H]inositol-labeled cells, substance P induced a robust inositol phosphate formation. Inositol trisphosphate levels increased as much as 20-fold within approximately 15 s of addition of substance P. This inositol trisphosphate formation was transient and had returned to baseline within the first 60-120 s. Inositol monophosphate formation, however, was linear for at least 2 h. Structure activity data on binding and inositol monophosphate formation confirmed the presence of a neurokinin-1 receptor subtype in these cells. Thus, the UC11 cell should be a useful model cell for delineating the physiological role of substance P receptors in astrocytes.     

Biosynthetic studies on the tropane ring system of the tropane alkaloids from Datura stramonium

Isotopic labelling experiments have been carried out in Datura stramonium root cultures with the following isotopically labelled precursors; [2H3]- [2-13C, 2H3]-, [1-13C, 18O2]-acetates, 2H2O, [2H3-methyl]-methionine, [2-13C]-phenyllactate, [3-2H]-tropine and [2'-13C, 3-2H]-littorine. The study explored the incorporation of isotope into the tropane ring system of littorine 1 and hyoscyamine 2 and revealed that deuterium from acetate is incorporated only into C-6 and C-7, and not into C-2 and C-4 as previously reported. Oxygen-18 was not retained at a detectable level into the C(3)-O bond from [1-13C, 18O2]-acetate. The intramolecular nature of the rearrangement of littorine 1 to hyoscyamine 2 is revealed again by a labelling study using [2'-13C, 3-2H]-littorine, [2-13C]-phenyllactate and [3-2H]-tropine.