Use of 3H and 14C double-labeled glucose to assess in vivo pathways of amino acid biosynthesis in Escherichia coli.

3H and 14C tracing data concerning amino acid biosynthetic pathways in Escherichia coli K12 are presented. Thirteen acidic and neutral amino acids were isolated from protein hydrolysates of wild type E. coli K12 grown aerobically or anaerobically in the presence of [U-14C]glucose together with [1-3H]glucose, [3-3H]glucose, [4-3H]glucose, or [6-3H]glucose. The observed 3H/14C counts of the amino acids were compared with the ratios expected on the basis of the input substrate specific activities and present understanding of biosynthetic pathways. For nine amino acids, serine, valine, leucine, threonine, isoleucine, glycine, glutamate, proline, and phenylalanine, the agreement between anticipated and observed specific activities was satisfactory. For the remaining four, methionine, alanine, aspartate, and (in cells labeled with [3-3H]glucose) tyrosine, the anticipated and observed specific activities differed markedly. For alanine, aspartate, and tyrosine, the differences are probably due to exchange of tritium in the course of biosynthesis; for methionine, it may be that there is a principle source of the methyl group other than carbon 3 of serine.     

Conversion of [U-14C]threonine into 14C-labelled amino acids in the brain of thiamin-deficient rats.

In confirmation of the findings of Gaitonde et al. (1974), a decrease in the brain concentration of threonine and serine, and an increase in glycine, were observed in rats maintained on a thiamin-deficient diet. Similar changes were found in the blood, and the concentration of several other amino acids in the blood decreased significantly. There was a correlation between the concentrations of threonine, serine, aspartate and asparagine in the brain and blood. In experiments in which [U-14C]threonine was injected into rats most of the radioactivity in the brain and blood of control rats was, as expected, in threonine in the acid soluble metabolites. In contrast, a considerable proportion of radioactivity was also found in other amino acids, namely glutamate, glutamine, aspartate, gamma-aminobutyrate and alanine, in the brain of thiamin-deficient rats. [U-14C]Threonine was also converted into 14C-labelled lactate and glucose, but the extent of this conversion was severalfold higher in thiamin-deficient than in control rats. This finding gave evidence of the stimulation in thiamin-deficient rats of the catabolism of [U-14C]threonine to [14C]lactate by the aminoacetone pathway catalysed by threonine dehydrogenase, and into succinate via propionate by the alpha-oxobutyrate pathway catalysed by threonine dehydratase (deaminase). The measurement of specific radioactivities of glutamate, aspartate and glutamine after injection of [U-14C]threonine, indicated a stimulation of the activities of threonine dehydrogenase and threonine dehydratase (deaminase) in the brain of thiamin-deficient rats. The specific radioactivities of glutamate, asparatate and glutamine int he brain were consistent with an alteration in the metabolism of threonine, mainly in the 'large' compartment of the brain of thiamin-deficient rats. The measurement of relative specific radioactivity of proteins after injection of [U-14C]threonine indicated a marked decrease in the synthesis of proteins, mainly in the liver of thiamin-deficient rats.     

Separate beta subunits are derivatized with 14C and 3H when the bovine heart mitochondrial F1-ATPase is doubly labeled with 7-chloro-4-nitro[14C]benzofurazan and 5'-p-fluorosulfonylbenzoyl[3H]inosine.

Tyrosine residues 311 and 345 of the beta subunit of the bovine heart mitochondrial F1-ATPase (MF1) are present on the same peptide when the enzyme is fragmented with cyanogen bromide. Maximal inactivation of MF1 with 7-chloro-4-nitro[14C]benzofurazan [( 14C]Nbf-Cl) derivatizes tyrosine-311 in a single beta subunit. Cyanogen bromide digests of MF1 containing the [14C]Nbf-O-derivative of tyrosine-beta 311 were submitted to reversed-phase HPLC, with and without prior reduction of the nitro group on the incorporated reagent with dithionite. The retention time of the radioactive cyanogen bromide peptide was shifted substantially by reduction. When a cyanogen bromide digest of MF1 inactivated with 5'-p-fluorosulfonylbenzoyl[3H]inosine [( 3H]FSBI), which proceeds with derivatization of tyrosine-345 in a single beta subunit, was submitted to HPLC under the same conditions, the fragment labeled with 3H eluted with the same retention time as the [14C]Nbf-O-derivative before reduction. Doubly labeled enzyme was prepared by first derivatizing Tyr-beta 311 with [14C]Nbf-Cl and then derivatizing tyrosine-beta 345 with [3H]FSBI with and without reducing the [14C]Nbf-O-derivative of tyrosine-beta 311 with dithionite before modification with [3H]FSBI. The doubly labeled enzyme preparations were digested with cyanogen bromide and submitted to HPLC. The 14C and 3H in the cyanogen bromide digest prepared from doubly labeled enzyme not submitted to reduction eluted together. In contrast, the 14C and 3H in the digest prepared from doubly labeled enzyme which had been reduced eluted separately. From these results it is concluded that different beta subunits are derivatized when MF1 is doubly labeled with [14C]Nbf-Cl and [3H]FSBI.     

Thin-layer mapping of peptides labeled with 3H or 14C via reductive methylation.

Two dimensional tryptic peptide maps have been obtained from 2 μg (40 pmol) of protein digest following labeling with ³H or ¹⁴C via reductive methylation. A simple labeling procedure is complete within 1 h; autoradiographs of ¹⁴C-labeled maps and fluorographs of ³H-labeled maps are obtained in 72 and 24 h, respectively. Tryptic peptide maps of ${}^{14}\text{C}{}^3\text{H}$ methylated α- and β-tubulin, and rabbit muscle, chick muscle, and chick brain actins show approximately the expected number of peptides. Methylation does not appear to measurably alter the map positions of the peptides relative to unmethylated peptides in the solvent systems used for either electrophoresis or chromatography.     

Glucose turnover in the young chicken (Gallus domesticus) using variously labeled (3H, U-14C) glucose tracers.

1. Various parameters of glucose metabolism--glucose replacement rate, percent recycling, mean transit time, and glucose mass were examined using various double labeled glucose tracers--2T-, U-14C; 3T-, U-14C; 4T-, U-14C; 5T-, U14C; and 6T-, U-14C. 2. Estimates of replacement rate were greatest for 2T-glucose (21.4 mg/min/kg), with 3T-, 4T-, 5T-, and 6T-glucose all having similar values (15.8, 15.6, 17.0, 16.0 mg/min/kg, respectively). 3. Calculated glucose mass based on all tritiated tracers (734-1086 mg/kg body weight) agreed closely with a direct determination of body glucose (969 mg/kg). 4. Reincorporation of tritium from 3H2O into glucose did not occur to any significant degree. 5. The young chick was found to have a very rapid rate of glucose turnover and high percent recycling compared to mammals.     

Decreased labeling of amino acids by inhibition of the utilization of [3H,14C]glucose via the hexosemonophosphate shunt in rat brain in vivo

Treatment of rats with 6-aminonicotinamide showed a small but significant decrease in the labeling of amino acids in the brain after injection of [³H]acetate. The results of these experiments also gave evidence of the presence of [³H]glucose and [³H]lactate, and an increase in [³H]glucose content in the brain of 6-aminonicotinamide treated rats. To apportion the contribution of [³H]glucose formed by gluconeogenesis from [³H]acetate to the labeling of amino acids a method was formulated based on the measurement of radioactivity of amino acids, lactate and free sugars in brain after injection of [6-³H]glucose or [1-³H]glucose relative to that after co-injection of [U-¹⁴C]glucose or [2-¹⁴C]glucose. In contrast to the expected formation of [1, 6-³H]glucose by gluconeogenesis from [³H]acetate,³H-labeled glucose isolated from brain, blood and liver showed the presence of [6-³H]glucose only. The values corrected for the presence of [6-³H]glucose showed that treatment with 6-aminonicotinamide had no effect on the labeling of amino acids by oxidation of [³H]acetate. These findings indicated that a significant decrease in the labeling of amino acids from [U-¹⁴C]glucose reported previously and again confirmed using [1-³H], [6-³H], [2-¹⁴C] or [U-¹⁴C]glucose in the present investigation was not due to the inhibition of the activities of enzymes of the citric acid cycle. These results support the postulated role of the hexosemonophosphate shunt for the utilization of glucose in providing neurotransmitter amino acids glutamate and -aminobutyrate.Dedicated to Professor K. A. C. Elliott on his 80th birthday.     

Hormone-induced changes in pyruvate kinase activity in isolated hepatocytes II. Relation to the hormonal regulation of gluconeogenesis gluconeogenesis

1. 1. Incubation of isolated hepatocytes with glucagon (10⁻⁶ M) or dibutyryl cyclic AMP (0.1 mM) causes a decrease in pyruvate kinase activity of 50%, measured at suboptimal substrate (phosphoenolpyruvate) concentrations and 1 mM Mg_free²⁺. The magnitude of the decrease in activity is not influenced by the applied extracellular concentrations of lactate (1 and 5 mM), glucose (5 and 30 mM) or fructose (10 and 25 mM). With all three substrates comparable inhibition percentages are induced by glucagon or dibutyryl cyclic AMP.

2. 2. The extent of inhibition of pyruvate kinase induced by incubation of hepatocytes with glucagon or dibutytyl cyclic AMP is not influenced by the extracellular Ca²⁺ concentration nor by the presence of 2 mM EGTA. The reactivation of pyruvate kinase seems to be inhibited by a high concentration of extracellular Ca²⁺ (2.6 mM) as compared to a low concentration of extracellular Ca²⁺ (0.26 mM).

3. 3. Incubation of hepatocytes in a Na⁺-free, high K⁺-concentration medium does not influence the magnitude of the pyruvate kinase inhibition induced by dibutyryl cyclic AMP. However, the reactivation reaction is stimulated under these incubation conditions.

4. 4. Incubation of hepatocytes with dibutyryl cyclic GMP (0.1 mM) leads to a 25% decrease in pyruvate kinase activity. The magnitude of the inhibition by dibutyryl cyclic (GMP) is not influenced by the presence of pyruvate (1 mM) or glucose (5 mM and 30 mM).

5. 5. The relative insensitivity of the pyruvate kinase inhibition induced by glucagon, dibutyryl cyclic AMP and dibutyryl cyclic GMP to the extracellular environment leads to the conclusion that the hormonal regulation of pyruvate kinase is not the only site of hormonal regulation of glycolysis and gluconeogenesis. It is concluded that hormonal regulation of pyruvate kinase activity is exerted by changes in the degree of (de)phosphorylation of the enzyme reflecting acute hormonal control as well as by changes in the concentration of the allosteric activator fructose 1,6-diphosphate. The latter depends at least in part on the hormonal control of the phosphofructokinase-fructose-1,6-phosphatase cycle.

Studies of glucose production in sheep using (6-3H)glucose and (U-14C)glucose.

Simultaneous primed-continuous intravenous infusions of [6-3H]glucose and [U-14C]glucose were performed on 13 fed, 4 fasted, and 4 dexamethasone-treated sheep. In 10 of the experiments on fed sheep, glucagon or insulin was infused intraportally for 2 h after control values were obtained. The 3H-labeled glucose gave glucose production values that were only 4.4 +/- 0.5, 5.4 +/- 1.0, and 5.8 +/- 0.8% higher than 14C-labeled glucose in the normal fed, fasted, and dexamethasone-treated sheep, respectively. Glucagon or insulin infusions did not significantly alter this recycling. It is condluced that a recycling of glucose carbon through metabolic intermediates is minimal in the sheep as compared with other species and also that it is not significantly altered by fasting or by hormones that affect glucose production.     

The effect of 6-aminonicotinamide on the levels of brain amino acids and glucose,and their labeling with14C after injection of [U-14C] glucose

The brains of rats paralysed at 4 hr after the administration of 6-aminonicotinamide were found to contain decreased levels of glutamate and -aminobutyrate. The glucose content of the brain of the treated rats was several fold higher than in controls. The incorporation of¹⁴C into brain amino acids at 30 min after the injection of [U-¹⁴C]glucose was decreased by 16%: this was attributed to mainly decreased labeling of glutamate and associated amino acids. The results are discussed in the light of previous findings that the administration of 6-aminonicotinamide resulted in the blockade of the direct oxidation of glucose by the pentose phosphate pathway.