The small-scale production of [U-C]acetylene from BaCO3: Application to labeling of ammonia monooxygenase in autotrophic nitrifying bacteria

A small-scale method has been adapted from an established procedure for the generation of [U-¹⁴C]acetylene from inexpensive and commonly available precursors. The method involves the fusing of Ba¹⁴CO₃ with excess barium metal to produce Ba¹⁴C₂. The BaC₂ is reacted with water to generate acetylene which is then selectively dissolved into dimethyl sulfoxide (DMSO). The results presented demonstrate the effect of Ba:BaCO₃ ratio on the concentrations of various gases released during the hydrolysis reaction and quantify the selectivity of the DMSO-trapping process for each gas. [U-¹⁴C]-Acetylene generated by this method has been used to inactivate ammonia monooxygenase in three species of autotrophic nitrifying bacteria: Nitrosomonas europaea, Nitrosococcus oceanus, and Nitrosolobus multiformis. Our results demonstrate that acetylene inactivation of this enzyme in all three species results in the covalent incorporation of radioactive label into a polypeptide of apparent M_r of 25,000–27,000, as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography.     

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Detection of fecal coliforms in water by using [14C]mannitol

Interest in rapid bacterial detection methods for sanitary indicator bacteria in water prompted a study of the use of [U-14C]mannitol to detect fecal coliforms (FC). A simple method which used m-FC broth, membrane filtration, and two-temperature incubation (35 degrees C for 2 h followed by 44.5 degrees C for 2.5 h) was developed. [U-14C]mannitol was added to the medium, and the temperature was raised to 44.5 degrees C after 2 h at 35 degrees C. 14CO2 was collected as Ba14CO3 and assayed by liquid scintillation spectroscopy. Correlations were examined between FC cell numbers at the start of incubation (standard 24-h FC test) and Ba14CO3 counts per minute after 4.5 h. Results indicated that FC numbers ranging from 1 x 10(1) to 2.1 x 10(5) cells could be detected in 4.5 h. Within-sample reproducibility at all cell concentrations was good, but sample-to-sample reproducibility was variable. Comparisons between m-FC broth and m-FC broth modified by substituting D-mannitol for lactose indicated that the standard m-FC broth was the better test medium. Results from experiments in which dimethyl sulfoxide was used to increase permeability of FC to [U-14C]mannitol indicated no increase in 14CO2 production due to dimethyl sulfoxide. Detection of FC by this method may be useful for rapid estimation of FC levels in freshwater recreational areas, for estimating the quality of potable source water, and potentially for emergency testing of potable water, suspected of contamination due to distribution line breaks or cross-connections.     

Detection of fecal coliforms in water by using [14C]mannitol.

Interest in rapid bacterial detection methods for sanitary indicator bacteria in water prompted a study of the use of [U-14C]mannitol to detect fecal coliforms (FC). A simple method which used m-FC broth, membrane filtration, and two-temperature incubation (35 degrees C for 2 h followed by 44.5 degrees C for 2.5 h) was developed. [U-14C]mannitol was added to the medium, and the temperature was raised to 44.5 degrees C after 2 h at 35 degrees C. 14CO2 was collected as Ba14CO3 and assayed by liquid scintillation spectroscopy. Correlations were examined between FC cell numbers at the start of incubation (standard 24-h FC test) and Ba14CO3 counts per minute after 4.5 h. Results indicated that FC numbers ranging from 1 x 10(1) to 2.1 x 10(5) cells could be detected in 4.5 h. Within-sample reproducibility at all cell concentrations was good, but sample-to-sample reproducibility was variable. Comparisons between m-FC broth and m-FC broth modified by substituting D-mannitol for lactose indicated that the standard m-FC broth was the better test medium. Results from experiments in which dimethyl sulfoxide was used to increase permeability of FC to [U-14C]mannitol indicated no increase in 14CO2 production due to dimethyl sulfoxide. Detection of FC by this method may be useful for rapid estimation of FC levels in freshwater recreational areas, for estimating the quality of potable source water, and potentially for emergency testing of potable water, suspected of contamination due to distribution line breaks or cross-connections.     

Radioenzymatic determination of adenosine

Adenosine has been measured at the nanomolar level by an enzymatic radioactive assay. The nucleoside is converted into [U-14C]ribose-labeled inosine via the following reactions: adenosine + H2O----adenine + ribose (adenosine nucleosidase); adenine + [U-14C]ribose 1-phosphate in equilibrium with T[U-14C]ribose-adenosine + Pi (adenosine phosphorylase); [U-14C]ribose-adenosine + H2O----[U-14C]ribose-inosine + NH3 (adenosine deaminase). The radioactivity of inosine, separated by thin-layer chromatography, is a measure of the adenosine initially present.     

Pancreatic fate of 14C-labelled hexoses

In order to assess the respective contribution of the exocrine and endocrine moieties of the pancreas to the overall net uptake of selected monosaccharides by the pancreatic gland, the apparent distribution space of L-[1-14C]glucose, 3-O-[14C-methyl]-D-glucose, D-[U-14C]glucose, D-[U-14C]mannose and D-[U-14C]fructose was measured in pieces of pancreas obtained from either control rats or animals injected with streptozotocin. Although the time course for the uptake of 3-O-[14C-methyl]-D-glucose, D-[U-14C]glucose, D-[U-14C]mannose and D-[U-14C]fructose was much slower in the pieces of pancreas than that previously documented in isolated pancreatic islets, no significant difference could, as a rule, be detected between the results obtained in pancreatic pieces of control and streptozotocin rats. A comparable situation prevailed in the pancreas of animals examined 3 min after the intravenous injection of 3-O-[14C-methyl]-D-glucose. D-Glucose inhibited the uptake of 3-O-[14C-methyl]-D-glucose and that of D-[U-14C]fructose. Likewise, 3-O-methyl-D-glucose inhibited the uptake of D-[U-14C]glucose. Cytochalasin B (20 microm) also inhibited the uptake of 3-O-[14C-methyl]-D-glucose and D-[U-14C]glucose, but not that of D-[U-14C]fructose. D-Mannoheptulose hexaacetate, but not the unesterified heptose, inhibited the metabolism of tritiated and 14C-labelled D-glucose, as well as the net uptake of D-[U-14C]glucose and D-[U-14C]mannose and, to a lesser extent, that of D-[U-14C]fructose. These findings indicate that despite marked differences between endocrine and exocrine pancreatic cells in terms of both the time course for the uptake of several hexoses and the inhibition of their phosphorylation by D-mannoheptulose, little or no preferential labelling of the endocrine moiety of the pancreas by the 14C-labelled hexoses is observed, at least when judged from their distribution space in pancreatic pieces or the whole pancreatic gland. Nevertheless, the findings made with D-mannoheptulose and its hexaacetate ester raise the view that this heptose could conceivably be used to achieve a sizeable preferential labelling of the endocrine pancreas under the present experimental conditions.     

Decreased incorporation of L-[U-14C]serine into phosphatidylserine by polymorphonuclear leukocytes during phagocytosis

A decreased rate of L-[U-14C]serine incoroporation into phosphatidylserine of polymorphonuclear leukocytes exposed to starch granules was observed. L-[U-14C]serine uptake was also depressed under identical conditions. The degree of reduction in specific radioactivity of phosphatidylserine was parallel to that of L-[U-14C]serine uptake. Both uptake and efflux of 45Ca2+ were enhanced in cells with starch granules, but no significant change in cellular calcium levels was detected. These results suggest that the reduced L-[U-14C]serine incorporation into phospholipids may be attributable to decreased availability of this amino acid. The involvement of Ca2+ fluxes in phosphatidylserine synthesis in intact leukocytes cannot, however, be excluded.     

Determination of evolved 14CO2 in decarboxylase reactions with application to measurement of [14C]oxalic acid.

An assay is described for the determination of the radioactive purity of [14C]oxalic acid preparations and the quantity of [14C]oxalic acid in biological samples. In this method oxalate decarboxylase is used to convert oxalate to formate and CO2. The entire procedure is carried out in a scintillation vial. The 14CO2 released in the enzymic reaction is allowed to diffuse off in a fume hood following acidification. Scintillation fluid is added to reacted and unreacted vials and the radioactivity measured. The loss of radioactivity from the reacted versus the unreacted vials provides the quantity of evolved 14CO2. This value is equal to 50% of the [14C]-oxalate (dpm) present. The radioactive purity of four preparations of [U-14C]oxalic acid was 99.0% while a fifth batch had a purity of 88%. A single batch of [U-14C]oxalic acid had a radioactive purity of 99.0% following storage of an aqueous solution, at -20 degrees C for 7 years. Recovery of [14C]oxalic acid from rat fecal extracts was 101.3%. Eight replicate analyses of a [U-14C]oxalic acid preparation gave a coefficient of variation of 0.3%. Following subcutaneous infusion of [U-14C]oxalic acid to rats, 100.2 +/- 2.9%, mean +/- SD, of the 14C in fecal extracts was present as [14C]oxalic acid (n = 10). The procedure provides a rapid, sensitive, and specific method to determine [14C]oxalic acid. It avoids the time consuming and inconvenient procedure for trapping and counting the evolved 14CO2. The approach used to determine the evolved 14CO2 may find application in other radiochemical methods that require its measurement.     

Biosynthesis of an unusual amino acyl moiety contained in bestatin

The biosynthetic pathway of an unusual amino acyl [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl (AHP)] moiety which is contained in bestatin has been studied by testing the incorporation of potential precursors. L-[U-14C]-Phenylalanine, L-[U-14C]leucine, and [U-14C]acetic acid were efficiently incorporated into bestatin, but the radioactivity of L-[1-14C]phenylalanine, [1-14C]glyoxylic acid, and [14C]oxalic acid were not incorporated. Incorporation of acetic acid into 1- and 2-carbon of the AHP moiety was confirmed by incorporation of [13C]acetic acid. Thus, the AHP moiety was shown to be biosynthesized from L-phenylalanine and two carbon atoms of acetic acid, accompanied by decarboxylation of the phenylalanine.     

Studies on flavonoid metabolism. Metabolism of (+)-[14C]catechin in the rat and guinea pig

1. The fate of (+)-[U-(14)C]catechin and (+)-[ring A-(14)C]catechin has been studied in the guinea pig and rat. 2. (+)-[U-(14)C]Catechin was shown to give rise to labelled phenolic acids, labelled phenyl-gamma-valerolactones and (14)CO(2). 3. (+)-[ring A-(14)C]-Catechin did not give rise to labelled phenolic acids, but labelled phenyl-gamma-valerolactones were detected together with a higher proportion of (14)CO(2). 4. Administered [(14)C]delta-(3-hydroxyphenyl)-gamma-valerolactone gave rise to labelled m-hydroxyphenylpropionic acid in the rat whereas administered [(14)C]m-hydroxyphenylpropionic acid gave rise to a compound yielding labelled m-hydroxybenzoic acid on hydrolysis. 5. The distribution of radioactivity in the urine and faeces of (+)-[(14)C]catechin-fed animals is described; a high proportion of residual radioactivity was found in urine that had been exhaustively extracted with diethyl ether.     

Phospholipid biosynthesis in mature human erythrocytes

Mature human erythrocytes were tested for their ability to synthetize membrane phospholipids from simple precursors: [32P]-orthophosphate (32Pi), [U-14C] glycerol, [U-14C] glucose, [U-14C] serine, and [U-14C] choline. The incorporation of these labels into phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), lysophosphatidylcholine (lyso-PC), phosphatidylinositol-4-phosphate (PIP), and phosphatidylinositol-4,5-bisphosphate (PIP2) was measured. All the phospholipids tested incorporated 32Pi, glycerol, and glucose in a time dependent manner. According to the rate of 32Pi incorporation, three groups of phospholipids could be distinguished: 1) PA, PIP2, PIP, lyso-PC; 2) PI and PS; 3) PC and PE, which incorporated 5 x 10(3), 40, and 6 nmol 32Pi/mmol phospholipid per 1 h, respectively. Moreover, [U-14C] serine and [U14C] choline were found to incorporate into phospholipids, and PS-decarboxylase activity could be measured. The possibility that the observed incorporation was due to contamination with bacteria or other blood cells could be ruled out. Our results bring evidence for de novo phospholipid synthesis of human red blood cells.     

Over-estimation of glucose-6-phosphatase activity in brain in vivo. Apparent difference in rates of [2-3H]glucose and [U-14C]glucose utilization is due to contamination of precursor pool with 14C-labeled products and incomplete recovery of 14C-labeled metabolites

Significant dephosphorylation of glucose 6-phosphate due to glucose-6-phosphatase activity in rat brain in vivo was recently reported (Huang, M., and Veech, R.L. (1982) J. Biol. Chem. 257, 11358-11363). The evidence was an apparent more rapid 3H than 14C loss from the glucose pool and faster [2-3H]glucose than [U-14C]glucose utilization following pulse labeling of the brain with [2-3H,U-14C]glucose. Radiochemical purity of the glucose and quantitative recovery of the labeled products of glucose metabolism isolated from the brain were obviously essential requirements of their study, but no evidence for purity and recovery was provided. When we repeated these experiments with the described isolation procedures, we replicated the results, but found that: 1) the precursor glucose pool contained detritiated, 14C-labeled contaminants arising from glucose metabolism, particularly 2-pyrrolidone-5-carboxylic acid derived from [14C]glutamine; 2) [14C]glucose metabolite were not quantitatively recovered; 3) the procedure used to isolate the glucose itself produced detritiated, 14C-labeled derivatives of [2-3H,U-14C]glucose. These deficiencies in the isolation procedures could fully account for the observations that were interpreted as evidence of significant glucose 6-phosphate dephosphorylation by glucose-6-phosphatase activity. When glucose was isolated by more rigorous procedures and its purity verified in the present studies, no evidence for such activity in rat brain was found.     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Synthesis, characterization and properties of uridine 5′-(α-d-apio-d-furanosyl pyrophosphate)

1. A method was developed for synthesizing UDP-apiose [uridine 5'-(alpha-d-apio-d-furanosyl pyrophosphate)] from UDP-glucuronic acid [uridine 5'-(alpha-d-glucopyranosyluronic acid pyrophosphate)] in 62% yield with the enzyme UDP-glucuronic acid cyclase. 2. UDP-apiose had the same mobility as uridine 5'-(alpha-d-xylopyranosyl pyrophosphate) when chromatographed on paper and when subjected to paper electrophoresis at pH5.8. When [(3)H]UDP-[U-(14)C]glucuronic acid was used as the substrate for UDP-glucuronic acid cyclase, the (3)H/(14)C ratio in the reaction product was that expected if d-apiose remained attached to the uridine. In separate experiments doubly labelled reaction product was: (a) hydrolysed at pH2 and 100 degrees C for 15min; (b) degraded at pH8.0 and 100 degrees C for 3min; (c) used as a substrate in the enzymic synthesis of [(14)C]apiin. In each type of experiment the reaction products were isolated and identified and were found to be those expected if [(3)H]UDP-[U-(14)C]apiose was the starting compound. 3. Chemical characterization established that the product containing d-[U-(14)C]apiose and phosphate formed on alkaline degradation of UDP-[U-(14)C]apiose was alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate. 4. Chemical characterization also established that the product containing d-[U-(14)C]apiose and phosphate formed on acid hydrolysis of alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate was d-[U-(14)C]apiose 2-phosphate. 5. The half-life periods for the degradation of UDP-[U-(14)C]apiose to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP at pH8.0 and 80 degrees C, at pH8.0 and 25 degrees C and at pH8.0 and 4 degrees C were 31.6s, 97.2min and 16.5h respectively. The half-life period for the hydrolysis of UDP-[U-(14)C]-apiose to d-[U-(14)C]apiose and UDP at pH3.0 and 40 degrees C was 4.67min. After 20 days at pH6.2-6.6 and 4 degrees C, 17% of the starting UDP-[U-(14)C]apiose was degraded to alpha-d-[U-(14)C]apio-d-furanosyl 1:2-cyclic phosphate and UMP and 23% was hydrolysed to d-[U-(14)C]apiose and UDP. After 120 days at pH6.4 and -20 degrees C 2% of the starting UDP-[U-(14)C]apiose was degraded and 4% was hydrolysed.     

Effect of CO2 concentration on phosphatidylcholine and phosphatidylglycerol metabolism in surfactant and residual lung fractions.

An investigation of the effect of change of total CO(2) concentration from 7 to 43 mM at pH 7.35 in the medium perfusing isolated rat lungs on [U-(14)C]glucose incorporation into lung phospholipids has been carried out. The incorporation of [U-(14)C]glucose into phosphatidylcholine and phosphatidylglycerol of the surfactant fraction and of the remaining lung tissue (residual fraction) was observed. Increased CO(2) concentration increased [U-(14)C]glucose incorporation into phosphatidylcholine of the surfactant fraction and residual fraction by 43 and 50%, respectively, during a 2 hr perfusion. Likewise, incorporation of [U-(14)C]glucose into phosphatidylglycerol was increased 22 and 34% into the surfactant and residual fractions, respectively. The percentage of [U-(14)C]glucose incorporated into the fatty acid moieties of phosphatidylcholine of both fractions increased as a result of increased CO(2) concentration. The increase in the incorporation of [U-(14)C]glucose into the fatty acid moieties of phosphatidylcholine was confirmed by an average increase of 56 and 77% in the specific activity of palmitic acid isolated from phosphatidylcholine of the surfactant and residual fraction, respectively, as a result of increased CO(2) concentration. The results suggest that alteration in extracellular CO(2) concentration affects the de novo synthesis from glucose of phosphatidylcholine and phosphatidylglycerol of the surfactant-lipoprotein fraction of lung.     

The stereochemistry of beta-lactam formation in penicillin biosynthesis.

1. (2R,3S)-[U-14C,3-3H1]- and (2R,3R)-[U-14C,2,3-3H2] Cysteine hydrochlorides have been separately synthesised. The latter compound has been shown to have uniform distributions of tritium between C-2 and C-3. 2. The abvoe cysteines and (2R)-[U-14C,3,3,3',3'-3H4]cystine have been converted to samples of penicillin G by Penicillium chrysogenum. 3. Incorporation results indicate that all but 14% of the tritium is lost from the (2R,3S)-[3-3H1]isomer; that 42% of tritium is retained by the non-stereospecifically C-3 tritiated cystine; and that 58% of tritium is retained by the (2R,3R)-[2,3-3H2]isomer on conversion to penicillin G. 4. Degradation of the penicillin G derived from (2R,3R)-[U-14C,2,3-3H2]cysteine hydrochloride has indicated that in fact about 87% of the original C-3 tritium of cysteine is retained at C-5 of penicillin G. 5. The results indicate stereospecificity in the cyclisation giving rise to the beta-lactam ring in penicillin G in nature with loss of the 3-pro-S-hydrogen and rentention of the 3-pro-R-hydrogen of cysteine. Thus there is net retention of stereochemistry in the cyclisation.     

Studies on the incorporation of [U-14C]glucose and [35S]sulphate into the acid glycosaminoglycans of neonatal rat skin

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.     

Use of 3H and 14C doubly labeled glucose and amino acids in the study of hormonal regulation of gluconeogenesis in rats.

Double isotope procedures (3H and 14C) were used in vivo to investigate a) slow long-term gluconeogenic actions of adrenal glucocorticoids, and b) rapid stimulation of gluconeogenesis by glucagon. [U-14C,6-3H]Glucose was administered to normal and adrenalectomized rats. No effect was observed on the [6-3H]glucose half-life suggesting the dicarboxylic acid shuttle is unaffected by adrenalectomy; the Cori cycle is also not influenced. Loads of [14C]aspartate, [14C]glutamate, or [14C]alanine were given to normal and adrenalectomized rats. Simultaneously, in vivo transaminase activity was studied by measuring the appearance of 3H2O in body water after administration of [2-3H]aspartate, [2-3H]glutamate, or [2-3H]alanine, Adrenalectomy has no influence on the incorporation of glutamate or aspartate into glucose or on their in vivo transaminases. Diminution of incorporation of [14C]alanine into glucose and alanine transaminase activities occurs only when rats are given unphysiological loads. These studies support the contention that glucocorticoid rate-limiting actions occur in extrahepatic tissues to produce an increased flow of glucose precursors to the liver. [U-14C,3-3H]Glucose was used to investigate the effect of glucagon on the hepatic fructose-6-phosphate (F-6-P) cycle. Glucagon administration resulted in a rapid drop in the 3H/14C ratio of circulating glucose, suggesting an increase in F-6-P recycling caused by activation of FDPase with little or no decrease in phosphofructokinase. Such a change would direct substrate flux toward gluconeogenesis.     

D- and L-glucose transport across the pulmonary epithelium

Previously we observed what appeared to be augmented D-glucose transport across the pulmonary epithelium. To investigate this phenomenon we placed fluid containing L-[3H]glucose and D-[U-14C]glucose in the alveoli of isolated Ringer-perfused lungs from 4-wk-old rabbits. The appearance of radioactivity in recirculating glucose-free perfusate was measured. 3H appearing in the perfusate was associated with L-glucose. 14C, however, was associated with three compounds, with approximate molecular weights of 180 (glucose), 300, and 560. The nonglucose species were not identified. This 14C movement was inhibited by phlorizin, but not phloretin, in the alveolar fluid. A similar pattern of 14C movement occurred when D-[U-14C]glucose was replaced with 2-deoxy-D-[U14C]-glucose, but not with methyl-alpha-D-[U-14C]glucopyranoside. The activation energy of the 14C metabolism-transport process was found to be 34 kcal/mol, and L-glucose transport showed an unusual temperature dependence, with maximum conductance at 15 degrees C. It appears that some D-glucose crosses the pulmonary epithelium as does L-glucose. However, most enters epithelial cells and is incorporated into larger molecules which enter the vascular but not the alveolar space.     

Synthesis of fluoroacetate from fluoride, glycerol, and beta-hydroxypyruvate by Streptomyces cattleya.

Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]beta-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U-14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C; about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]beta-hydroxypyruvates to show that C-2 and C-3 of beta-hydroxypyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through beta-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.