The metabolism of [Me-14C]choline in the brain of the rat in vivo

[Me-(14)C]Choline was injected intracerebrally into the adult rat, and its uptake into the lipids and their water-soluble precursors in brain was studied. The radioactivity could be detected only in the choline-containing lipids and was confined to the base choline. The results indicated that initial phosphorylation of the free choline followed by the formation of CDP-choline and the subsequent transfer of the phosphorylcholine to a diglyceride is one of the principal routes by which choline lipids in brain are formed. Further evidence for this was obtained in experiments in which either phosphoryl[Me-(14)C]choline or [(32)P]orthophosphate was injected and the radioactivity in the choline-containing water-soluble and lipidbound components studied.     

The role of the plasma membrane in fatty acid uptake by rat liver parenchymal cells

1. Suspensions of isolated rat liver parenchymal cells incorporate [(14)C]palmitic acid into glycerides at about 40% of the rate obtained with liver slices. 2. At short time-intervals most of the incorporation is into phosphatidylcholine and this is recovered mainly in the plasma-membrane fraction. 3. At later times (5min to 2h) the [(14)C]palmitic acid is mainly found in triglyceride, but this is not recovered in the plasma-membrane fraction. 4. Addition of lysophosphatidylcholine increases incorporation of palmitic acid into both phosphatidylcholine and triglyceride, with maximum effect at about 0.1mm. 5. In vivo, 1min after injection of [(14)C]palmitic acid, radioactive phosphatidylcholine is concentrated in the plasma-membrane fraction, but the proportion present in this fraction declines rapidly. 6. The phosphatidylcholine of the plasma-membrane fraction has, at 1min after injection, a specific radioactivity 30-fold greater than that of the whole tissue. 7. This phosphatidylcholine reaches its maximum specific radioactivity before the tissue phosphatidic acid or diglyceride. 8. The phosphatidylcholine of the plasma-membrane fraction has a very rapid turnover. 9. It is proposed that the rapid formation of phospholipids in the plasma membrane is by acylation of their lyso-derivatives and the role of this process in fatty acid uptake is discussed.     

Comparative lipid composition of heterotrophically and autotrophically grown Sulfolobus acidocaldarius.

Complex lipids from the thermoacidophilic facultative autotroph Sulfolobus acidocaldarius, as well as a strictly autotrophic isolate, were compared between cells grown on yeast extract and elemental sulfur. Lipids from both organisms grown autotrophically were nearly identical. Each contained about 15% neutral lipids, 35% glycolipids, and 50% acidic lipids. Glycolipids and acidic lipids contained C40H82-76-derived glycerol ether residues. Major glycolipids included the glycerol ether analogues of glucosyl galactosyl diglyceride (5%) and glucosyl polyol diglyceride (75%). Acidic lipids were comprised mainly of the glycerol ether analogues of phosphatidyl inositol (7%), inositolphosphoryl glucosyl polyol diglyceride (72%), and a partially characterized sulfate- and phosphate-containing derivative of glucosyl polyol diglyceride (13%). The lipids from cells grown heterotrophically were similar to those from autotrophically grown cells, except that the partially characterized acidic lipid was absent. In addition, the two glycolipids as well as the respective inositolphosphoryl derivatives were each present in nearly equal proportions.     

Source and role of diacylglycerol formed during phagocytosis of opsonized yeast particles and associated respiratory burst in human neutrophils

The results presented in this paper demonstrate that in human neutrophils phagocytosis of C3b/bi and IgG-opsonized yeast particles is associated with activation of phospholipase D and that this reaction is the main source of diglycerides. The demonstration is based upon the following findings: 1) the challenge of neutrophils with these opsonized particles was followed by a rapid formation of [3H]alkyl-phosphatidic acid [( 3H]alkyl-PA) and [3H]alkyl-diglyceride [( 3H]alkyl-DG) in cells labeled with [3H]alkyl-lyso-phosphatidylcholine; 2) in the presence of ethanol [3H]alkyl-phosphatidylethanol was formed, and accumulation of [3H]alkyl-PA and [3H]alkyl-DG was depressed; 3) propranolol, by inhibiting the dephosphorylation of [3H]alkyl-PA, completely inhibited the accumulation of [3H]alkyl-DG and depressed by about 75% the formation of diglyceride mass. Evidence is also presented that phagocytosis of C3b/bi and IgG-opsonized yeast particles and associated respiratory burst can take place independently of diglyceride formation and of the activity of this second messenger on protein kinase C. In fact: a) propranolol while completely inhibited the formation of diglyceride mass did not modify either the phagocytosis or respiratory burst; b) these two processes were insensitive to staurosporine.     

Lipid profiles of Aspergillus niger and its unsaturated fatty acid auxotroph, UFA2

A comparative study of the mycelial lipid composition of a wild strain (V35) and one unsaturated fatty acid auxotroph (UFA2) of Aspergillus niger has been performed. The lipid composition of both strains are qualitatively the same but quantitatively different. All the strains contain the following phospholipids: cardiolipin, phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylcholine, and phosphatidylserine; and triglycerides, diglycerides, monoglycerides, ergosterol, and sterol esters as the neutral lipids; mono- and di-galactosyl diglyceride as the major glycolipids along with small amounts of the corresponding mannose analogs. Phosphatidylethanolamine and phosphatidylcholine constitute the bulk of the phospholipids. The mutant (UFA2) contains a higher level of glycerides and lower levels of sterol (both free and esterified form), phospholipids, and glycolipids than the wild type. Aspergillus niger contains C16 to C18 saturated and unsaturated fatty acids. Small amounts of long-chain (C20 to C24) and short-chain (C10 to C14) saturated and unsaturated acids are also present. Linoleic, oleic, and palmitic are the major acids, stearic and linolenic acids being minor ones. UFA2 grows only in the presence of unsaturated fatty acid (C16 or C18) and accumulates a higher concentration of supplemented acid which influences its fatty acid profile.     

Regulation of diacylglycerol levels in carbachol-stimulated pancreatic acinar cells: relationship to the breakdown of phosphatidylcholine and metabolism to phosphatidic acid.

Rat pancreatic acinar cells prelabeled with [14C]palmitic acid and then exposed to carbachol (CCh) exhibited a time-dependent increase in 1,2-[14C]diacylglycerol ([14C]DAG) levels, which was first detected at 2 min and then continued to rise in a linear manner. There was a concomitant increase in [14C]phosphatidic acid, which plateaued after 2 min and then remained at steady-state levels. CCh also promoted the release of phosphocholine, but not choline, within 60 s and caused a decrease in [14C]phosphatidylcholine in cells prelabeled with [14C]glycerol after 15 min. The inability to detect a rise in [14C]phosphatidylethanol accumulation and a fall in [14C]phosphatidate levels in [14C]palmitate prelabeled cells after exposure to CCh plus ethanol documented the absence of a phospholipase D-mediated pathway. The rapid phosphorylation of diglyceride in homogenates from unstimulated and carbachol-treated cells increased with increasing concentrations of exogenous substrate, thereby affirming that carbachol stimulates the phosphorylation of DAG by promoting the accumulation of the diglyceride. These collective findings provide evidence for the existence of an integrative control mechanism for regulating endogenous DAG levels during pancreatic acinar cell activation involving phosphatidylcholine-specific phospholipase C and DAG kinase.     

The role of valine in the biosynthesis of penicillin N and cephalosporin C by a Cephalosporium sp

1. The production of penicillin N, but not that of cephalosporin C, was inhibited by the addition of d-valine to suspensions in water of washed mycelium of Cephalosporium sp. 8650. The production of cephalosporin C was selectively inhibited by gamma-hydroxyvaline. 2. l-[(14)C]Valine was taken up rapidly and virtually completely by suspensions of washed mycelium but d-[(14)C]valine and alpha-oxo[(14)C]-isovalerate were taken up relatively slowly. 3. Part of the l-valine was rapidly degraded in the mycelium and part was incorporated into protein. Turnover of the valine in the amino acid pool was estimated to occur in 10-17min. 4. No detectable amount of l-[(14)C]valine was converted into the d-isomer in the mycelium. alpha-Oxo[(14)C]isovalerate was rapidly converted into l-[(14)C]valine in mycelium and mycelial extracts. 5. d-[(14)C]Valine was partially converted into the l-isomer in the mycelium and (14)C from d-valine was incorporated into protein. 6. The labelling of penicillin N and cephalosporin C by (14)C from l-[(14)C]valine was consistent with the view that l-valine is a direct precursor of C(5) fragments of both antibiotics and that any intermediates involved are present in relatively small pools in rapid turnover. 7. Labelling of the antibiotics with (14)C from d-[1-(14)C]valine appeared to occur after the latter had been converted into the l-isomer. Unlabelled d-valine did not decrease the efficiency of incorporation of (14)C from l-[1-(14)C]valine. 8. Intracellular peptide material which contained, among others, residues of alpha-aminoadipic acid, cysteine and valine, was rapidly labelled by (14)C from l-[1-(14)C]valine in a manner consistent with it being an intermediate in the biosynthesis of one or both of the antibiotics. 9. Labelling of penicillin N from l-[1-(14)C]valine occurred more rapidly than that of cephalosporin C. However, the effects of d-valine and gamma-hydroxyvaline on antibiotic production and the course of labelling of the antibiotics from l-[(14)C]valine could not readily be explained on the assumption that penicillin N was a precursor of cephalosporin C.     

Nicotinate, quinolinate and nicotinamide as precursors in the biosynthesis of nicotinamide–adenine dinucleotide in barley

1. The relative efficiencies of nicotinate, quinolinate and nicotinamide as precursors of NAD(+) were measured in the first leaf of barley seedlings. 2. In small amounts, both [(14)C]nicotinate and [(14)C]quinolinate were quickly and efficiently incorporated into NAD(+) and some evidence is presented suggesting that NAD(+) is formed from each via nicotinic acid mononucleotide and deamido-NAD. 3. [(14)C]Nicotinamide served equally well as a precursor of NAD(+) and although significant amounts of [(14)C]NMN were detected, most of the [(14)C]NAD(+) was derived from nicotinate intermediates formed by deamination of [(14)C]nicotinamide. 4. Radioactive NMN was also a product of the metabolism of [(14)C]nicotinate and [(14)C]quinolinate but most probably it arose from the breakdown of [(14)C]NAD(+). 5. In barley leaves where the concentration of NAD(+) is markedly increased by infection with Erysiphe graminis, the pathways of NAD(+) biosynthesis did not appear to be altered after infection. A comparison of the rates of [(14)C]NAD(+) formation in infected and non-infected leaves indicated that the increase in NAD(+) content was not due to an increased rate of synthesis.     

The metabolism of [14C]nicotine in the cat

The metabolism of [2'-(14)C]nicotine given as an intravenous injection in small doses to anaesthetized and unanaesthetized cats has been studied. A method is described for the quantitative determination of [(14)C]nicotine and [(14)C]cotinine in tissues and body fluids. Nanogram amounts of these compounds have been detected. After a single dose of 40mug. of [(14)C]nicotine/kg., 55% of the injected radioactivity was excreted in the urine within 24hr., but only 1% of this radioactivity was unchanged nicotine. [(14)C]Nicotine is metabolized extremely rapidly, [(14)C]cotinine appearing in the blood within 2.5min. of intravenous injection. [(14)C]Nicotine accumulates rapidly in the brain and 15min. after injection 90% of the radioactivity still represents [(14)C]nicotine. Metabolites of [(14)C]nicotine have been identified in liver and urine extracts. [(14)C]Nicotine-1'-oxide has been detected in both liver and urine.     

Metabolism of l-Threonic Acid in Rumex x acutus L. and Pelargonium crispum (L.) L'Hér

l-Threonic acid is a natural constituent in leaves of Pelargonium crispum (L.) L'Hér (lemon geranium) and Rumex x acutus L. (sorrel). In both species, l-[(14)C]threonate is formed after feeding l-[U-(14)C]ascorbic acid to detached leaves. R. acutus leaves labeled with l-[4-(3)H]- or l-[6-(3)H]ascorbic acid produce l-[(3)H]threonate, in the first case internally labeled and in the second case confined to the hydroxymethyl group. These results are consistent with the formation of l-threonate from carbons three through six of l-ascorbic acid. Detached leaves of P. crispum oxidize l-[U-(14)C] threonate to l-[(14)C]tartrate whereas leaves of R. acutus produce negligible tartrate and the bulk of the (14)C appears in (14)CO(2), [(14)C]sucrose, and other products of carbohydrate metabolism. R. acutus leaves that are labeled with l-[U-(14)C]threonate release (14)CO(2) at linear rate until a limiting value of 25% of the total [U-(14)C]threonate is metabolized. A small quantity of [(14)C]glycerate is also produced which suggests a process involving decarboxylation of l-[U-(14)C]threonate.     

Lipid Composition and Metabolism of Volvox carteri

The membrane structural lipids of somatic cells and gonidia isolated from Volvox carteri f. nagariensis spheroids have been characterized. The principal polar lipid components of both cell types are sulfoquinovosyl diglyceride, mono- and digalactosyl diglyceride, phosphatidylglycerol, phosphatidylethanolamine, and 1(3), 2-diacylglyceryl-(3)-O-4′-(N,N,N,-trimethyl)homoserine. Light-synchronized cultures of spheroids were shown to incorporate [¹⁴C]bicarbonate, [³⁵S]sulfate, [¹⁴C]palmitic acid, and [¹⁴C]lauric acid into complex lipids. [¹⁴C]Palmitic acid was incorporated mainly into diacylglyceryltrimethylhomoserine and was not significantly modified by elongation or desaturation. In contrast, [¹⁴C]lauric acid was incorporated into a wider variety of complex lipids and was also converted into longer chain saturated and unsaturated fatty acids. Volvox is a promising system for studying the role of membranes in algal cellular differentiation.     

A study of the biosynthesis of collagenous and non-collagenous proteins and of the proline/hydroxyproline ratios of collagens isolated from embryonic tissues

1. After incubation of chick-embryo skin slices with [(14)C]proline for 2hr. the specific activities of [(14)C]proline and [(14)C]hydroxyproline in soluble and insoluble collagens and [(14)C]proline in non-collagenous proteins were determined as well as the total amounts of both imino acids in these proteins. On the basis of these results it was demonstrated that soluble collagens having a high proline/hydroxyproline ratio are contaminated with non-collagenous proteins. 2. It was found that, in the presence of a mixture of amino acids in the incubation medium, the rate of synthesis of soluble collagen is significantly decreased. 3. The metabolic activity of collagenous proteins is related to their solubility, but that of non-collagenous proteins is not.     

Mineralization of Polycyclic Aromatic Hydrocarbons by the White Rot Fungus Pleurotus ostreatus

The white rot fungus Pleurotus ostreatus was able to mineralize to (sup14)CO(inf2) 7.0% of [(sup14)C]catechol, 3.0% of [(sup14)C]phenanthrene, 0.4% of [(sup14)C]pyrene, and 0.19% of [(sup14)C]benzo[a]pyrene by day 11 of incubation. It also mineralized [(sup14)C]anthracene (0.6%) much more slowly (35 days) and [(sup14)C]fluorene (0.19%) within 15 days. P. ostreatus did not mineralize fluoranthene. The activities of the enzymes considered to be part of the ligninolytic system, laccase and manganese-inhibited peroxidase, were observed during fungal growth in the presence of the various polycyclic aromatic hydrocarbons. Although activity of both enzymes was observed, no distinct correlation to polycyclic aromatic hydrocarbon degradation was found.     

Homologous recombination is involved in the repair response of mammalian cells to low doses of tritium

Radioactive compounds incorporated in tissues can have biological effects resulting from energy deposition in subcellular compartments. We addressed the genetic consequences of [(3)H] or [(14)C]thymidine incorporation into mammalian DNA. Low doses of [(3)H]thymidine in CHO cells led to enhanced sensitivity compared with [(14)C]thymidine. Compared with wild-type cells, homologous recombination (HR)-deficient cells were more sensitive to lower doses of [(3)H]thymidine but not to any dose of [(14)C]thymidine. XRCC4-defective cells, however, were sensitive to both low and high doses of [(3)H] and [(14)C]thymidine, suggesting introduction of DNA double-strand breaks, which were confirmed by gamma-H2AX focus formation. While gamma rays induced measurable HR only at toxic doses, sublethal levels of [(3)H] or [(14)C]thymidine strongly induced HR. The level of stimulation was in an inverse relationship to the emitted energies. The RAD51 gene conversion pathway was involved, because [(3)H]thymidine induced RAD51 foci, and [(3)H]thymidine-induced HR was abrogated by expression of dominant negative RAD51. In conclusion, both HR and non-homologous end-joining pathways were involved after labeled nucleotide incorporation (low doses); genetic effects were negatively correlated with the energy emitted but were positively correlated with the energy deposited in the nucleus, suggesting that low-energy beta-particle emitters, at non-toxic doses, may induce genomic instability.     

Complement receptor-mediated phagocytosis is associated with accumulation of phosphatidylcholine-derived diglyceride in human neutrophils. Involvement of phospholipase D and direct evidence for a positive feedback signal of protein kinase.

Complement receptor (CR)-mediated phagocytosis is associated with an increased accumulation of diglyceride (sn-1,2-diacylglycerol and/or 1-O-alkyl-2-acyl-glycerol) in human neutrophils. The C3bi-mediated increase in diglyceride (5-20 min) was only partially impaired when phosphoinositide-specific phospholipase C (PLC) activity was abolished by reduction of cytosolic free Ca2+. At an early time point (1 min), however, diglyceride production was barely detectable in control cells, whereas production was considerable in cells with a reduced cytosolic free Ca2+ concentration. C3bi stimulation of 32P-labeled neutrophils caused a rapid and significant breakdown of [32P]phosphatidylcholine (PC) which was not affected by inhibition of Ca(2+)-dependent phosphoinositide-specific PLC. Thus, PC hydrolysis could be involved in C3bi-induced diglyceride formation. Stimulation of cells labeled with [3H]1-O-alkyl-lyso-PC ([3H]alkyl-lyso-PC), resulted in an increased formation of [3H]1-O-alkyl-phosphatidic acid ([3H]alkyl-PA) and a later and slower formation of [3H]1-O-alkyl-diglyceride ([3H]alkyl-diglyceride); this suggests activation of phospholipase D (PLD). When these labeled cells were stimulated in the presence of 0.5% ethanol a marked accumulation of [3H]1-O-alkyl-phosphatidylethanol ([3H]alkyl-PEt) was observed in both controls and calcium-reduced cells, further strengthening the suggested involvement of PLD activity. In parallel with the sustained increase in diglyceride formation, CR-mediated phagocytosis was also associated with phosphorylation of a cellular protein kinase C substrate (MARCKS). Therefore it seems reasonable to suggest a causal relationship between C3bi-induced PLD activation, which results in diglyceride formation, and activation of protein kinase C. In electropermeabilized cells which were incapable of ingesting particles, C3bi particles were still able to activate PLD and induce formation of diglyceride. This signaling event must therefore be triggered by binding of particles to the cell and not by the engulfment process. Most importantly, introduction of the protein kinase C inhibitor peptides, PKC(19-36) and PKC(19-31), into these permeabilized cells resulted in a clear reduction of the C3bi-induced production of diglyceride, indicating that CR-mediated activation of protein kinase C directly triggers a positive feedback mechanism for additional diglyceride formation. Taken together, these data further clarify the mechanisms of CR-mediated diglyceride formation and give added support to the concept that protein kinase C plays an important role in the phagocytic process.     

Changes in the ribonucleic acid metabolism of aging mouse tissues with particular reference to the prostate gland

1. Mouse mast-cell tumours P815 Y and HC were shown to contain glycoprotein material composed of glucosamine, galactosamine, sialic acid, galactose and mannose. 2. The major amino acids released after acid hydrolysis of Pronase-treated digests of the glycoprotein are aspartic acid, glutamic acid, serine, threonine, proline, glycine and alanine. The Pronase-digested material is not degraded in alkaline solution. 3. On incubation of mast cells with [(35)S]sulphate, heparin is the major radioactive product. However, [1-(14)C]glucosamine and d-[(14)C]glucose are incorporated largely into the glycoprotein. 4. The fate of [(35)S]sulphate-labelled and [1-(14)C]glucosamine-labelled material was studied. In each case high-molecular-weight radioactive material is released from the cells into the culture medium. The t((1/2)) of [(35)S]sulphate-labelled material in cells is 70hr. and that of [1-(14)C]-glucosamine-labelled material in cells is 40hr. 5. About 60% of the [(35)S]sulphate-labelled material is present in the mitochondrial and granular fraction. [1-(14)C]-Glucosamine-labelled material is present in both microsomal and mitochondrial and granular fractions, [(14)C]sialic acid being concentrated in the microsomal fraction.     

Glucomannan Biosynthesis Catalyzed by Pisum sativum Enzymes

The results of molecular weight studies, structural analysis of the [(14)C]polysaccharides, and enzymic properties indicate that the Pisum sativum guanosine diphosphosphate glucose: glucosyltransferase is an enzymic component involved in the biosynthesis of glucomannan chains. The properties of the Pisum sativum particulate enzyme are essentially identical to the glucomannan synthetase obtained from Phaseolus aureus. Also present in the particulate preparation is an enzyme which catalyzes the formation of a [(14)C]mannolipid, using guanosine diphosphate-[(14)C]mannose as a substrate. The [(14)C]mannolipid is hydrolyzed by treatment with 0.012 m HCl, but is stable to treatment with 0.09 m NaOH. The formation of the [(14)C]mannolipid is apparently reversed by guanosine diphosphate, but not by guanosine monophosphate. The chromatographic mobility of the [(14)C]mannolipid is identical to that of a similar mannolipid synthesized by a Phaseolus aureus enzyme.     

Isolation and characterization of glycolipids from some photosynthetic bacteria

Glycosyl glycerides have been found in substantial amounts in Chloropseudomonas ethylicum but could not be detected in two strains of Rhodopseudomonas palustris. Rhodospirillum molischianum possibly contains small amounts of monoglycosyl diglyceride. The glycolipids of C. ethylicum have been separated into two components. One of these, glycolipid I, is a monogalactosyl diglyceride. Glycolipid II, upon acid hydrolysis, yields galactose, rhamnose, and a third, unidentified sugar. The glycolipids or total lipids of the photosynthetic bacteria examined contained saturated and monounsaturated, but none of the more highly unsaturated, fatty acids.     

Rate studies of polysaccharide biosynthesis from guanosine diphosphate α-d-glucose and guanosine diphosphate α-d-mannose

With enzyme preparations from Phaseolus aureus seedlings, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose is not increased by additions of GDP-alpha-d-mannose. However, final incorporation is increased by addition of GDP-alpha-d-mannose, since the total reaction-time is extended. In contrast, the initial rate of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is increased by all concentrations of GDP-alpha-d-glucose that are less than that of the GDP-alpha-d-[(14)C]mannose. Maximum stimulation of the initial rate occurs at a GDP-alpha-d-[(14)C]mannose/GDP-alpha-d-glucose concentration ratio of about 4:1. However, eventual incorporation from GDP-alpha-d-[(14)C]mannose is decreased by the addition of GDP-alpha-d-glucose, since the reaction rate falls off sharply after about 2min. Reciprocal plots of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose result in biphasic graphs. The two straight-line portions of the plot are joined by a curved line in the concentration range between 2-3 and 50mum. Extrapolated K(m) values for the two linear components are 0.4-1.0 and 700-1500mum. The effect of GDP-alpha-d-glucose on the kinetics of (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]mannose is complex, and depends on relative concentrations of the two sugar nucleotides. (14)C-labelled polysaccharide formation from GDP-alpha-d-[(14)C]glucose also results in biphasic reciprocal plots. One component appears to have K(m) about 2-3mum, the other about 200-400mum. In this reaction, GDP-alpha-d-mannose appears to be a competitive inhibitor with K(i) 20-30mum. With particulate preparations of P. aureus, GDP-alpha-d-[(14)C]glucose appears to be a precursor for the synthesis of one polysaccharide, a glucomannan, the mannose moieties of which are derived from an intermediate existing in the particulate preparation. From the rate results, GDP-alpha-d-[(14)C]mannose appears to be a precursor for at least two polysaccharides, one of which is a glucomannan.     

Arginine catabolism in the cyanobacterium Synechocystis sp. Strain PCC 6803 involves the urea cycle and arginase pathway

Cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 supplemented with micromolar concentrations of L-[(14)C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of L-[(14)C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [(14)C]ornithine preceded that of [(14)C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with L-[(14)C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Delta(1)pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an argF (ornithine carbamoyltransferase) insertional mutant was impaired in the production of [(14)C]citrulline from [(14)C]arginine; (ii) a proC (Delta(1)pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [(14)C]proline, [(14)C]glutamate, and [(14)C]glutamine from [(14)C]arginine or [(14)C]ornithine; and (iii) a putA (proline oxidase) insertional mutant did not produce [(14)C]glutamate from L-[(14)C]arginine, L-[(14)C]ornithine, or L-[(14)C]proline. Mutation of two open reading frames (sll0228 and sll1077) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of argD (N-acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Delta(1)pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [(14)C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi-L-arginyl-poly(L-aspartic acid)], a reserve material unique to cyanobacteria.