Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for `aminoacetone synthase''

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.     

The specific radioactivity of the tissue free amino acid pool as a basis for measuring the rate of protein synthesis in the rat in vivo

1. Rats were infused in vivo with [U-(14)C]glycine for periods of 2-6h, during which time the specific radioactivity of the free glycine in plasma and tissue approached a constant value. 2. Free serine also became labelled. The ratio of specific radioactivity of serine to that of glycine in the protein of liver, kidney, brain, jejunum, heart, diaphragm and gastrocnemius muscle was closer to the ratio in the free amino acid pool of the tissue than that of the plasma. 3. The kinetics of incorporation of [(14)C]glycine and [(14)C]serine into the protein of gastrocnemius muscle further suggested that the plasma free amino acids were not the immediate precursors of protein. 4. Infusion of rats with [U-(14)C]serine resulted in labelling of free glycine. The ratio of specific radioactivity of glycine to serine in the protein of liver, kidney, brain, jejunum and heart again suggested incorporation from a pool similar to the free amino acid pool of the tissue. 5. Rates of tissue protein synthesis calculated from the incorporation into protein of both radioactive glycine and serine, either infused or derived, were very similar when the precursor specific radioactivity was taken to be that in the total free amino acids of the tissue. Except for gastrocnemius muscle and diaphragm during the infusion of radioactive serine, the rates of tissue protein synthesis calculated from the specific radioactivity of the free glycine and serine in plasma differed markedly.     

The Salmonella typhimurium glycine cleavage enzyme system

Summary A glycine cleavage enzyme system, inducible by glycine, has been demonstrated in Salmonella typhimurium. The induced enzyme levels, however, are only about 20% of the induced levels found in Escherichia coli. Starting with a serine auxotroph, mutants were isolated that grow with a serine supplement, but not with a glycine supplement. Three independently isolated mutants have reduced or nondetectable glycine cleavage enzyme levels. The new mutations, designated gcv, were mapped between the serA and lys genes at 62.5 min on the S. typhimurium chromosome.Abbreviations C1 one-carbon - GCV glycine cleavage - GM glucose minimal - L agar Luria agar - LB Luria broth - Tc tetracycline     

Catabolism of arginine and ornithine in the perfused rat liver: effect of dietary protein and of glucagon

The rates of oxidation of arginine and ornithine that occurred through a reaction pathway involving the enzyme ornithine aminotransferase (EC 2.6.1.13) were determined using (14)C-labeled amino acids in the isolated nonrecirculating perfused rat liver. At physiological concentrations of these amino acids, their catabolism is subject to chronic regulation by the level of protein consumed in the diet. (14)CO(2) production from [U-(14)C]ornithine (0.1 mM) and from [U-(14)C]arginine (0.2 mM) was increased about fourfold in livers from rats fed 60% casein diets for 3-4 days. The catabolism of arginine in the perfused rat liver, but not that of ornithine, is subject to acute regulation by glucagon (10(-7) M), which stimulated arginine catabolism by approximately 40%. Dibutyryl cAMP (0.1 mM) activated arginine catabolism to a similar extent. In retrograde perfusions, glucagon caused a twofold increase in the rate of arginine catabolism, suggesting an effect of glucagon on arginase in the perivenous cells.     

Gluconeogenesis from glycine and serine in fasted normal and diabetic rats.

1. Non-anaesthetized normal and diabetic rats were fasted for 1 day, and [U-14C]glycine, or [U-14C]serine, or [U-14C]- plus [3-3H]-glucose was injected intra-arterially. The rates of synthesis de novo/irreversible disposal for glycine, serine and glucose, as well as the contribution of carbon atoms by the amino acids to plasma glucose, were calculated from the integrals of the specific-radioactivity-versus-time curves in plasma. 2. The concentrations of both glycine and serine in blood plasma were lower in diabetic than in fasted normal animals. 3. The rates of synthesis de novo/irreversible disposal of both amino acids tended to be lower in diabetic animals, but the decrease was statistically significant only for serine (14.3 compared with 10.5 mumol/min per kg). 4. Of the carbon atoms of plasma glucose, 2.9% arose from glycine in both fasted normal and diabetic rats, whereas 4.46% of glucose carbon originated from serine in fasted normal and 6.77% in diabetic rats. 5. As judged by their specific radioactivities, plasma serine and glycine exchange carbon atoms rapidly and extensively. 6. It was concluded that the turnover of glycine remains essentially unchanged, whereas that of serine is decreased in diabetic as compared with fasted normal rats. The plasma concentration of both amino acids was lower in diabetic rats. Both glycine and serine are glucogenic. In diabetic rats the contribution of carbon atoms from glycine to glucose increases in direct proportion to the increased glucose turnover, whereas the contribution by serine becomes also proportionally higher.     

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.     

Glycine formation during growth of Pseudomonas AM1 on methanol and succinate

1. The mechanism of regeneration of glycine during the growth of Pseudomonas AM1 on C(1) compounds has been investigated by brief incubation of bacterial suspensions with [2,3-(14)C(2)]succinate and observing the incorporation of radioactivity into various metabolites. 2. With the wild-type organism growing on methanol, radioactivity appeared rapidly in glycine and tricarboxylic acid-cycle intermediates, but there was a relatively slow labelling of serine and phosphorylated compounds. Serine became labelled predominantly in the C-2 position. 3. The proportion of radioactivity incorporated into glycine at earliest times was greatly diminished when succinate-grown cells were used. 4. Radioactivity was also incorporated from [2,3-(14)C(2)]succinate into glycine and serine by methanol-grown mutant 20S, which lacks phosphoserine phosphohydrolase. Both the glycine and serine were labelled mainly in C-2. 5. The formation of predominantly [2-(14)C]serine from [2,3-(14)C(2)]succinate in wild-type Pseudomonas AM1, and of [2-(14)C]serine and [2-(14)C]glycine in the mutant lacking the phosphorylated pathway from succinate to serine, is taken as strong evidence for a mechanism of glycine regeneration involving cleavage of a C(4) skeleton between C-2 and C-3, rather than by a direct combination of two C(1) units derived from the growth substrate. 6. The cleavage mechanism is quantitatively more significant during growth on methanol than on succinate.     

The glycine cleavage system: Composition,reaction mechanism,and physiological significance

Summary The glycine cleavage system catalyzes the following reversible reaction: Glycine + THF + NAD⁺ 5,10-methylene-THF + + CO₂ + NH₃ + NADHReversibility of the overall reaction was established through the studies with the enzymes prepared from liver mitochondria of rat and cock and from extracts ofArthrobacter globiformis grown on glycine. The glycine cleavage system is composed from four protein components. The four proteins were revealed to exist originally as an enzyme complex in the liver mitochondria. Partial reactions of glycine cleavage and glycine synthesis were studied in detail with partially purified individual protein components. Particularly a protein-bound intermediate of glycine metabolism could be isolated and its nature and role were clarified. A tentative scheme was presented to explain the whole process of the reversible glycine cleavage.The glycine cleavage system was shown to represent the major pathway of catabolism of both glycine and serine in vertebrates, including mammals, birds, reptiles, amphibians, and fishes. Serine catabolism in these animals proceeds mainly by way of the cleavage of serine to form methylene-THF and glycine rather than deamination by serine dehydratase. In ureotelic and ammonotelic animals methylene-THF formed from the -carbon of glycine as well as the-carbon of serine could be further oxidized to CO₂ in either the mitochondria or the soluble tissue fractions, while in uricotelic animals methylene-THF could hardly be oxidized to CO₂ and instead, was utilized mostly for purine synthesis. Glycine synthesis by the glycine cleavage system did not appear to have appreciable physiological significance in animals.Abbreviation THF dl,l-tetrahydrofolic acid. an invited article     

Degradation of glyphosate by Pseudomonas sp. PG2982 via a sarcosine intermediate

The bacterium Pseudomonas PG2982 metabolizes glyphosate (N-(phosphonomethyl)glycine) by converting it to glycine, a one-carbon unit, and phosphate. Here we show that this conversion involves the intermediate formation of sarcosine. When cells are incubated with [14C]glyphosate, the 14C can be entrapped in glycine or sarcosine. With added sarcosine, 14C from all three carbons of glyphosate is recovered solely in sarcosine. In experiments with glycine, radioactivity from the carboxymethyl moiety of glyphosate is trapped in glycine as well as serine, whereas radioactivity from the phosphonomethyl carbon is only incorporated into serine. These results are consistent with a pathway involving the conversion of glyphosate to sarcosine by cleavage of its carbon-phosphorus (C-P) bond, followed by the oxidation of sarcosine to glycine and formaldehyde.     

Sphingomyelin synthesis in Fasciola hepatica

Whole worms and/or homogenates of F. hepatica incorporate label from cytidine-5-diphospho[methyl-14C]choline,[1-14C]palmitoylCoA,[U- 14C]serine,[2-14C]methionine, [U-14C]glycine, [U-14C]threonine and [U-14C]aspartate into the various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). This suggests that sphingomyelin synthesis in F. hepatica occurs by a pathway similar to that found in mammals. However, there is some evidence that in F. hepatica 3-ketosphinganine may be N-acylated prior to reduction and dehydrogenation.     

Flux of the L-serine metabolism in rabbit, human, and dog livers. Substantial contributions of both mitochondrial and peroxisomal serine:pyruvate/alanine:glyoxylate aminotransferase.

L-Serine metabolism in rabbit, dog, and human livers was investigated, focusing on the relative contributions of the three pathways, one initiated by serine dehydratase, another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Under quasi-physiological in vitro conditions (1 mM L-serine and 0.25 mM pyruvate), flux through serine dehydratase accounted for only traces, and that through SPT/AGT substantially contributed no matter whether the enzyme was located in peroxisomes (rabbit and human) or largely in mitochondria (dog). As for flux through serine hydroxymethyltransferase and GCS, the conversion of serine to glycine occurred fairly rapidly, followed by GCS-mediated slow decarboxylation of the accumulated glycine. The flux through GCS was relatively high in the dog and low in the rabbit, and only in the dog was it comparable with that through SPT/AGT. An in vivo experiment with L-[3-3H,14C]serine as the substrate indicated that in rabbit liver, gluconeogenesis from L-serine proceeds mainly via hydroxypyruvate. Because an important role in the conversion of glyoxylate to glycine has been assigned to peroxisomal SPT/AGT from the studies on primary hyperoxaluria type 1, these results suggest that SPT/AGT in this organelle plays dual roles in the metabolism of glyoxylate and serine.     

Regulation of the glycine cleavage system in the isolated perfused rat liver

The catabolism of glycine in the isolated perfused rat liver was investigated by measuring the production of 14CO2 from [1-14C]- and [2-14C]glycine. Production of 14CO2 from [1-14C]glycine was maximal as the perfusate glycine concentration approached 10 mM and exhibited a maximal activity of 125 nmol of 14CO2 X g-1 X min-1 and an apparent Km of approximately 2 mM. Production of 14CO2 from [2-14C]glycine was much lower, approaching a maximal activity of approximately 40 nmol of 14CO2 X g-1 X min-1 at a perfusate glycine concentration of 10 mM, with an apparent Km of approximately 2.5 mM. Washout kinetic experiments with [1-14C]glycine exhibited a single half-time of 14CO2 disappearance, indicating one metabolic pool from which the observed 14CO2 production is derived. These results indicate that the glycine cleavage system is the predominant catabolic fate of glycine in the perfused rat liver and that production of 14CO2 from [1-14C]glycine is an effective monitor of metabolic flux through this system. Metabolic flux through the glycine cleavage system in the perfused rat liver was inhibited by processes which lead to reduction of the mitochondrial NAD(H) redox couple. Infusion of beta-hydroxybutyrate or octanoate inhibited 14CO2 production from [1-14C]glycine by 33 and 50%, respectively. Alternatively, infusion of acetoacetate stimulated glycine decarboxylation slightly and completely reversed the inhibition of 14CO2 production by octanoate. Metabolic conditions which are known to cause a large consumption of mitochondrial NADPH (e.g. ureogenesis from ammonia) stimulated glycine decarboxylation by the perfused rat liver. Infusion of pyruvate and ammonium chloride stimulated production of 14CO2 from [1-14C]glycine more than 2-fold. Lactate plus ammonium chloride was equally as effective in stimulating glycine decarboxylation by the perfused rat liver, while alanine plus ammonium chloride was ineffective in stimulating 14CO2 production.     

Glycine and serine catabolism in non-photosynthetic higher plant cells: their role in C1 metabolism

Glycine and serine are two interconvertible amino acids that play an important role in C1 metabolism. Using 13C NMR and various 13C-labelled substrates, we studied the catabolism of each of these amino acids in non-photosynthetic sycamore cambial cells. On one hand, we observed a rapid glycine catabolism that involved glycine oxidation by the mitochondrial glycine decarboxylase (GDC) system. The methylenetetra- hydrofolate (CH2-THF) produced during this reaction did not equilibrate with the overall CH2-THF pool, but was almost totally recycled by the mitochondrial serine hydroxymethyltransferase (SHMT) for the synthesis of one serine from a second molecule of glycine. Glycine, in contrast to serine, was a poor source of C1 units for the synthesis of methionine. On the other hand, catabolism of serine was about three times lower than catabolism of glycine. Part of this catabolism presumably involved the glycolytic pathway. However, the largest part (about two-thirds) involved serine-to-glycine conversion by cytosolic SHMT, then glycine oxidation by GDC. The availability of cytosolic THF for the initial SHMT reaction is possibly the limiting factor of this catabolic pathway. These data support the view that serine catabolism in plants is essentially connected to C1 metabolism. The glycine formed during this process is rapidly oxidized by the mitochondrial GDC-SHMT enzymatic system, which is therefore required in all plant tissues.     

Operation of the glycolate pathway in isolated bundle sheath strands of maize and Panicum maximum

Operation of the glycolate pathway in isolated bundle sheath (BS) strands of two C₄ species was demonstrated from ¹⁴C incorporation into two intermediates, glycine and serine, under conditions favourable for photorespiratory activity. Isolated BS strands fixing ¹⁴CO₂ under light at physiological rates incorporate respectively 3% (Zea mays L., cv. INRA 258) and 7% (Panicum maximum Jacq.) of total ¹⁴C fixed into glycine + serine, at low bicarbonate levels (less than the Km for CO₂ fixation, 0.8 mM). Higher bicarbonate concentrations depressed the percentage of incorporation into the two amino acids. No labelling was observed in the absence of added glutamate. Oxygen was required for glycine + serine labelling, since ¹⁴C incorporation into glycine was largely depressed by argon flushing, and labelling of the two amino acids was nearly suppressed by the addition of the strong reductant, dithionite, especially in maize. Two inhibitors of the glycolate pathway were tested. With α-hydroxypyridine-methanesulfonic acid, an inhibitor of glycolate oxidase, labelling of glycine and serine remained minimal whereas glycolate was accumulated. Isoniazid, an inhibitor of the transformation of glycine to serine induced a 50% increased labelling of glycine in maize BS, and a large decrease in serine labelling. In Panicum, the increase in [¹⁴C]-glycine was 90%. These results suggest that the pathway glycolate → glycine → serine operates in these plants. However, leakage of metabolites occurs in BS cells, especially in maize and a large part of newly formed glycolate, glycine and serine is exported out of the cells. Operation of ribulose-1,5-bisphosphate oxygenase activity in competition with ribulose-1,5-bisphosphate carboxylase is demonstrated by the lowering of total ¹⁴CO₂ fixation when O₂ is increased at low bicarbonate concentration. An interesting feature observed in maize BS, at low bicarbonate concentration, was an increase in ribulose-1,5-bisphosphate labelling when the O₂ level was decreased. This was accompanied by an increase in CO₂ fixation. This could indicate an increased rate in synthesis of ribulose-1,5-bisphosphate (which accumulated) due to a stimulation of ATP synthesis by cyclic photophosphorylation under anaerobic conditions.     

β-Cyanoalanine Formation by Chromobacterium violaceum

Nonproliferating cells of Chromobacterium violaceum incubated with glycine, methionine, and succinate as substrates accumulated beta-cyanoalanine in the culture fluid. Tracer experiments showed that carbons-2, -3, and -4 of beta-cyanoalanine are derived from the 2-carbon of glycine. When methionine-methyl-(14)C, succinate-1,4-(14)C, or succinate-2,3-(14)C was used as substrate, beta-cyanoalanine did not become labeled. If K(14)CN and serine were used as substrates, the cyano group of beta-cyanoalanine was labeled. Radioactive beta-cyanoalanine, labeled in the 3-carbon, was formed when glycine and H(14)CHO were used as substrates. (14)C-formic acid did not replace formaldehyde. Asparagine also accumulated in the incubated mixture and was found to be labeled in the amide carbon. Incubation of cells with beta-cyanoalanine-4-(14)C produced labeled aspartic acid in cell hydrolysates.     

Glyphosate catabolism by Pseudomonas sp. strain PG2982.

The pathway for the degradation of glyphosate (N-phosphonomethylglycine) by Pseudomonas sp. PG2982 has been determined by using metabolic radiolabeling experiments. Radiorespirometry experiments utilizing [3-14C]glyphosate revealed that approximately 50 to 59% of the C-3 carbon was oxidized to CO2. Fractionation of stationary-phase cells labeled with [3-14C]glyphosate revealed that from 45 to 47% of the assimilated label is distributed to proteins and that the amino acids methionine and serine are highly labeled. Adenine and guanine received 90% of the C-3 label found in the nucleic acid fraction, and the only pyrimidine base labeled was thymine. These results indicated that C-3 of glyphosate was at some point metabolized to a C-1 compound whose ultimate fate could be both oxidation to CO2 and distribution to amino acids and nucleic acid bases that receive a C-1 group from the C-1-donating coenzyme tetrahydrofolate. Pulse-labeling of PG2982 cells with [3-14C]glyphosate resulted in the isolation of [3-14C]sarcosine as an intermediate in glyphosate degradation. Examination of crude extracts prepared from PG2982 cells revealed the presence of a sarcosine-oxidizing enzyme that oxidizes sarcosine to glycine and formaldehyde. These results indicate that the first step in glyphosate degradation by PG2982 is cleavage of the carbon-phosphorus bond, resulting in the release of sarcosine and a phosphate group. The phosphate group is utilized as a source of phosphorus, and the sarcosine is degraded to glycine and formaldehyde. This pathway is supported by the results of [1,2-14C]glyphosate metabolism studies, which show that radioactivity in the proteins of labeled cells is found only in the glycine and serine residues.     

Purine biosynthesis de novo in rat skeletal muscle.

Purine biosynthesis by the 'de novo' pathway was demonstrated in isolated rat extensor digitorum longus muscle with [1-14C]glycine, [3-14C]serine and sodium [14C]formate as nucleotide precursors. Evidence is presented which suggests that the source of glycine and serine for purine biosynthesis is extracellular rather than intracellular. The relative incorporation rates of the three precursors were formate greater than glycine greater than serine. Over 85% of the label from formate and glycine was recovered in the adenine nucleotides, principally ATP. Azaserine markedly inhibited purine biosynthesis from both formate and glycine. Cycloserine inhibited synthesis from serine, but not from formate. Adenine, hypoxanthine and adenosine markedly inhibited purine synthesis from sodium [14C]formate.     

Cotranslational and posttranslational proteolytic processing of preprosomatostatin-I in intact islet tissue

Preprosomatostatin-I (PPSS-I) is processed in anglerfish islets to release a 14-residue somatostatin (SS-14). However, very little is known regarding other processing events that affect PPSS-I. This is the first study to identify and quantify the levels of nonsomatostatin products generated as a result of processing of this somatostatin precursor in living islet tissue. The products of PPSS-I processing in anglerfish islet tissue were identified in radiolabeling studies using a number of criteria. These criteria included immunoreactivity, specific radiolabeling by selected amino acids, radiolabel sequencing, and chromatographic comparison to isolated, structurally characterized fragments of anglerfish PPSS-I using reverse-phase high performance liquid chromatography. Intact prosomatostatin-I (aPSS-I) was isolated from tissue incubated with [3H]tryptophan and [14C]leucine. Significant 14C radioactivity was observed in the products of 11 of the first 44 sequencer cycles in positions consistent with the generation of a 96- residue prosomatostatin. These results indicate that signal cleavage occurs after the cysteine located 25 residues from the initiator Met of PPSS-I, resulting in a signal peptide 25 amino acids in length. Nonsomatostatin-containing fragments of the precursor were also found in tissue incubated with a mixture of 3H-amino acids. Only a small quantity of the dodecapeptide representing residues 69-80 in the prohormone was found (10 nmol/g tissue). Two other fragments of aPSS-I, also observed to be present in low abundance, were found to correspond to residues 1-27 (16 nmol/g tissue) and to residues 1-67 (7 nmol/g tissue) of aPSS-I. No evidence for the presence of the fragment corresponding to residues 29-67 was found. However, large quantities of SS-14 were observed (287 nmol/g tissue), indicating that the major site of aPSS-I cleavage is at the basic dipeptide immediately preceding SS- 14. Recovery of much lower levels of the nonsomatostatin fragments of aPSS-I suggests that prohormone processing at the secondary sites identified in this study occurs at a low rate relative to release of SS- 14 from aPSS-I.     

Biosynthesis of intestinal mucins. Effect of puromycin on mucoprotein biosynthesis by sheep colonic mucosal tissue

1. Surviving sheep colonic mucosal tissue incorporated l-[U-(14)C]threonine when incubated in Krebs medium III at 37 degrees in an atmosphere of oxygen, into a well-characterized mucoprotein fraction, isolated by papain digestion of the incubated scrapings. 2. Acidic hydrolysis and chromatography of the labelled mucoprotein showed that threonine was the only constituent to become labelled. In the presence of puromycin the incorporation of l-[U-(14)C]threonine was considerably diminished (6.7 and 18.5% of control in duplicate experiments). Furthermore, puromycin also decreased incorporation of radioactivity from d-[U-(14)C]-glucose (48.0 and 31.6% of control) and (35)SO(4) (2-) (21.2 and 23.6% of control) into the mucoprotein fraction. 3. In a puromycin-inhibited system, with d-[U-(14)C]-glucose, where the overall specific radioactivity of the mucoprotein was 48% of control, the labelling of the individual monosaccharide constituents (as% of control) was: N-acetylneuraminic acid, 44%; N-glycollylneuraminic acid, 61%; hexosamines, 46%; fucose, 68%; galactose, 34%.     

13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae.

Saccharomyces cerevisiae has both cytoplasmic and mitochondrial C1-tetrahydrofolate (THF) synthases. These trifunctional isozymes are central to single-carbon metabolism and are responsible for interconversion of the THF derivatives in the respective compartments. In the present work, we have used 13C NMR to study folate-mediated single-carbon metabolism in these two compartments, using glycine and serine synthesis as metabolic endpoints. The availability of yeast strains carrying deletions of cytoplasmic and/or mitochondrial C1-THF synthase allows a dissection of the role each compartment plays in this metabolism. When yeast are incubated with [13C]formate, 13C NMR spectra establish that production of [3-13C]serine is dependent on C1-THF synthase and occurs primarily in the cytosol. However, in a strain lacking cytoplasmic C1-THF synthase but possessing the mitochondrial isozyme, [13C]formate can be metabolized to [2-13C]glycine and [3-13C]serine. This provides in vivo evidence for the mitochondrial assimilation of formate, activation and conversion to [13C]CH2-THF via mitochondrial C1-THF synthase, and subsequent glycine synthesis via reversal of the glycine cleavage system. Additional supporting evidence of reversibility of GCV in vivo is the production of [2-13C]glycine and [2,3-13C]serine in yeast strains grown with [3-13C]serine. This metabolism is independent of C1-THF synthase since these products were observed in strains lacking both the cytoplasmic and mitochondrial isozymes. These results suggest that when formate is the one-carbon donor, assimilation is primarily cytoplasmic, whereas when serine serves as one-carbon donor, considerable metabolism occurs via mitochondrial pathways.