Quantitative Significance of Plasma Albumin Metabolism in the Whole Body Protein Turnover in Rats

The amount of extra- and intravascular albumin was estimated in two groups of rats, i.e., those fed a 20% casein (20% protein) diet and a 3% casein (low protein or 3% protein) diet.

The fractional turnover rate of whole body plasma albumin was also measured in the two groups of rats, employing the constant infusion method of Waterlow et al. At the same time, the fractional turnover rate of the whole body protein was measured.

When the diet was changed from the 20% protein to the 3% protein diet, the amount of albumin mass in both extra- and intravascular spaces decreased significantly. During 7 days on the diet, the extra- and intravascular albumin mass per 100 g of body weight did not change significantly in the rats fed the 20% protein diet. On the other hand, rats fed the 3% protein diet lost almost 30% of the extra- and intravascular albumin per lOOg body weight.

The fractional turnover rates of whole body albumin were estimated to be 31.7 and 19.8%/day in the 20% protein and the 3% protein diet-fed rats, respectively. The fractional turnover rates of whole body protein were 16.1 and 10.6%/day in the 20% protein and the 3% protein diet-fed rats, respectively.

The leucine fluxes to albumin synthesis and whole body protein synthesis were calculated to be 5.9 and 83 μmol/hr, respectively, in the 20% protein diet-fed rats. The leucine fluxes in the 3% protein diet-fed rats were 2.5 and 54μmol/hr for the albumin synthesis and for the whole body protein synthesis, respectively.

These results demonstrate the quantitative significance of albumin metabolism in the whole body protein turnover in rats fed on two levels of protein intake.     

Enteral arginase II provides ornithine for citrulline synthesis

The synthesis of citrulline from arginine in the small intestine depends on the provision of ornithine. To test the hypothesis that arginase II plays a central role in the supply of ornithine for citrulline synthesis, the contribution of dietary arginine, glutamine, and proline was determined by utilizing multitracer stable isotope protocols in arginase II knockout (AII(-/-)) and wild-type (WT) mice. The lack of arginase II resulted in a lower citrulline rate of appearance (121 vs. 137 μmol·kg(-1)·h(-1)) due to a reduced availability of ornithine; ornithine supplementation was able to restore the rate of citrulline production in AII(-/-) to levels comparable with WT mice. There were significant differences in the utilization of dietary citrulline precursors. The contribution of dietary arginine to the synthesis of citrulline was reduced from 45 to 10 μmol·kg(-1)·h(-1) due to the lack of arginase II. No enteral utilization of arginine was observed in AII(-/-) mice (WT = 25 μmol·kg(-1)·h(-1)), and the contribution of dietary arginine through plasma ornithine was reduced in the transgenic mice (20 vs. 13 μmol·kg(-1)·h(-1)). Dietary glutamine and proline utilization were greater in AII(-/-) than in WT mice (20 vs. 13 and 1.4 vs. 3.7 μmol·kg(-1)·h(-1), respectively). Most of the contribution of glutamine and proline was enteral rather than through plasma ornithine. The arginase isoform present in the small intestinal mucosa has the role of providing ornithine for citrulline synthesis. The lack of arginase II results in a greater contribution of plasma ornithine and dietary glutamine and proline to the synthesis of citrulline.     

Arginine catabolism by Thermanaerovibrio acidaminovorans

The arginine catabolism of Thermanaerovibrio acidaminovorans was investigated. T. acidaminovorans was able to produce approximately 0.4--0.5 mol citrulline and 0.5--0.6 mol ornithine from 1 mol of arginine. However, in a methanogenic coculture with Methanobacterium thermoautotrophicum Z245 1 mol arginine was converted to approximately 1 mol of propionate, 0.5 mol acetate, 4 mol ammonia and 4 mol hydrogen; citrulline and ornithine were not formed. Enzyme measurements indicated the presence of the arginine deiminase pathway (ADI) in cells of T. acidaminovorans growing on arginine.     

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.     

Arginine synthesis from enteral glutamine in healthy adults in the fed state

Recent studies have documented transfer of labeled nitrogen from [2-(15)N]glutamine to citrulline and arginine in fasting human adults. Conversely, in neonates and piglets we have shown no synthesis of arginine from [2-(15)N]glutamate, and others have shown in mice that glutamine is a nitrogen, but not a carbon donor, for arginine synthesis. Therefore, we performed a multitracer study to determine whether glutamine is a nitrogen and/or carbon donor for arginine in healthy adult men. Two glutamine tracers, 2-(15)N and 1-(13)C, were given enterally to five healthy men fed a standardized milkshake diet. There was no difference in plasma enrichments between the two glutamine tracers. 1-(13)C isotopomers of citrulline and arginine were synthesized from [1-(13)C]glutamine. Three isotopomers each of citrulline and arginine were synthesized from the [2-(15)N]glutamine tracer: 2-(15)N, 5-(15)N, and 2,5-(15)N(2). Significantly greater enrichment was found of both [5-(15)N]arginine (0.75%) and citrulline (3.98%) compared with [2-(15)N]arginine (0.44%) and [2-(15)N]citrulline (2.62%), indicating the amino NH(2) from glutamine is mostly transferred to arginine and citrulline by transamination. Similarly, the enrichment of the 1-(13)C isotopomers was significantly less than the 2-(15)N isotopomers, suggesting rapid formation of α-ketoglutarate and recycling of the nitrogen label. Our results show that the carbon for 50% of newly synthesized arginine comes from dietary glutamine but that glutamine acts primarily as a nitrogen donor for arginine synthesis. Hence, studies using [2-(15)N]glutamine will overestimate arginine synthesis rates.     

Arginine synthesis does not occur during first-pass hepatic metabolism in the neonatal piglet

We have shown that first-pass intestinal metabolism is necessary for approximately 50% of whole body arginine synthesis from its major precursor proline in neonatal piglets. Furthermore, the intestine is not the site of increased arginine synthesis observed during dietary arginine deficiency. Primed constant intravenous (iv) and intraportal (ip) infusions of L-[U-14C]proline, and iv infusion of either L-[guanido-14C]arginine or L-[4,5-3H]arginine were used to measure first-pass hepatic arginine synthesis in piglets enterally fed either deficient (0.20 g.kg(-1).day(-1)) or generous (1.80 g.kg(-1).day(-1)) quantities of arginine for 5 days. Conversion of arginine to other urea cycle intermediates and arginine recycling were also calculated for both dietary treatments. Arginine synthesis (g.kg(-1).day(-1)) from proline was greater in piglets (P < 0.05) fed the deficient arginine diet in both the presence (generous: 0.07; deficient: 0.17; pooled SE = 0.01) and absence (generous: 0.06; deficient: 0.20; pooled SE = 0.01) of first-pass hepatic metabolism. There was no net arginine synthesis from proline during first-pass hepatic metabolism regardless of arginine intake. Arginine conversion to urea, citrulline, and ornithine was significantly greater (P < 0.05) in piglets fed the generous arginine diet. Calculated arginine fluxes were significantly lower (P = 0.01) for [4,5-3H]arginine than for [guanido-14C]arginine, and the discrepancy between the values was greater in piglets fed the deficient arginine diet (35% vs. 20%). Collectively, these findings show that first-pass hepatic metabolism is not a site of net arginine synthesis and that piglets conserve dietary arginine in times of deficiency by decreasing hydrolysis and increasing recycling.     

Catabolic Rate of Body Protein in Adult Rat over an Entire Period of Protein Depletion

In order to clarify the magnitude of different labile body proteins and the over-all catabolizable body protein, the catabolic rate of total body nitrogen in adult rat was measured by nitrogen balance method up to the time of death due to protein depletion.

The more labile body protein having 83.3% of fractional catabolic rate per day and occupying 2.8% of the whole body protein mainly represented the so-called protein reserves at the beginning of protein depletion. The less labile one, the remainder, namely 97.2%, having 0.79% of fractional catabolic rate almost wholly represented the exponential decrease of body protein during the first 40 days of protein depletion. Urinary and fecal nitrogen in this period showed a similar exponential decrease. In the next 40 days of the depletion, body protein decreased almost linearly giving the constant excretions of urinary and fecal nitrogen. In the last 40 days, it decreased drastically accompanied by a remarkable increase in urinary nitrogen.

After 118 days, on the average, the animals died of protein depletion at 35.7% level of the initial body nitrogen. Contributions of various organs to the total nitrogen deficit up to the time of death, were considerably different in different organs, where muscle was the greatest in total amount but with less catabolizability than the viscera, such as liver, pancreas and spleen.     

Protein turnover and cathepsin B activity in several individual tissues of foetal and senescent rats

The fractional rate of protein synthesis has been measured in vivo, and compared in the whole body and 12 major individual tissues of foetal and senescent rats. This synthetic rate was found to decrease in most tissues with increasing animal age. The rate of protein degradation was also determined and compared with cathepsin B activity within each tissue; both protein turnover and the endopeptidase activity decreased with ageing. Age-related changes in each tissue's contributions to the protein mass and synthetic rates of the whole animal are also summarized and related to developmental variations in physiological function.     

Effect of level of dietary protein on arginine-stimulated citrulline synthesis. Correlation with mitochondrial N-acetylglutamate concentrations.

Increases in dietary protein have been reported to increase the rate of citrulline synthesis and the level of N-acetylglutamate in liver. We have confirmed this effect of diet on citrulline synthesis in rat liver mitochondria and show parallel increases in N-acetylglutamate concentration. The magnitude of the effect of arginine in the suspending medium on citrulline synthesis was also dependent on dietary protein content. Mitochondria from rats fed on a protein-free diet initially contained low levels of N-acetylglutamate, and addition of arginine increased the rate of its synthesis. Citrulline synthesis and acetylglutamate content in these mitochondria increased more than 5-fold when 1 mM-arginine was added. A diet high in protein results in mitochondria with increased N-acetylglutamate and a high rate of citrulline synthesis; 1 mM-arginine increased citrulline synthesis in such mitochondria by only 36%. The concentration of arginine in portal blood was 47 microM in rats fed on a diet lacking protein, and 182 microM in rats fed on a diet containing 60% protein, suggesting that arginine may be a regulatory signal to the liver concerning the dietary protein intake. The rates of citrulline synthesis were proportional to the mitochondrial content of acetylglutamate in mitochondria obtained from rats fed on diets containing 0, 24, or 60% protein, whether incubated in the absence or presence of arginine. Although the effector concentrations are higher than the Ka for the enzymes, these results support the view that concentrations of both arginine and acetylglutamate are important in the regulation of synthesis of citrulline and urea. Additionally, the effects of dietary protein level (and of arginine) are exerted in large part by way of modulation of the concentration of acetylglutamate.     

Growth and Arginine Metabolism of the Wine Lactic Acid Bacteria Lactobacillus buchneri and Oenococcus oeni at Different pH Values and Arginine Concentrations

During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth of Lactobacillus buchneri CUC-3 and Oenococcus oeni strains MCW and Lo111 in wine were investigated. In contrast to L. buchneri CUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth of L. buchneri CUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.     

Protein synthesis in the whole body,liver, skeletal muscle and kidney cortex of lambs infected by the nematode Trichostrongylus colubriformis

Jones W. O. and Symons L. E. A. 1982. Protein synthesis in the whole body, liver, skeletal muscle and kidney cortex of lambs infected by the nematode Trichostrongylus colubriformis. International Journal for Parasitology12: 295–301. Tyrosine flux and the synthesis of protein in the whole body, liver, skeletal muscle and kidney cortex and of albumin in lambs infected with Trichostrongylus colubriformis and uninfected lambs fed ad libitum or pair-fed with the infected group, were measured by constant infusion of ¹⁴C-l-tyrosine. Live weight gain was lower in the infected than in pairfed lambs, but rates of whole body protein synthesis were similar in both groups. On the other hand, compared with control lambs, there was a faster rate of protein synthesis per unit of protein consumed in infected but not in pair-fed lambs. Rates of protein synthesis per unit of body weight in infected were higher than in pair-fed lambs, but similar to the rate in control lambs. The fractional synthetic rates (FSR) of albumin and liver proteins and the amount of liver protein synthesized per day were increased by infection. The FSR and amount of protein synthesized per day were depressed in skeletal muscle and kidney cortex. Anorexia did not explain any of these changes. Infection caused a loss of protein from each of these tissues, but this loss was due to anorexia in only the liver. There was generally good correlation between concentration of RNA per g fresh weight or per mg nitrogen and the FSR of protein. However, although the $\text{RNA}\text{DNA}$ ratio correlated well with synthesis in skeletal muscle, it was poorly correlated for liver proteins. The relationship between the rate of growth and protein synthesis in infected lambs is discussed.     

Arginine Metabolism in Developing Soybean Cotyledons : II. Biosynthesis

Tracer kinetic experiments were performed using [ureido-¹⁴C] citrulline, [1-¹⁴C]ornithine, and isotope trapping techniques to determine if arginine is synthesized via the urea cycle in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with the ¹⁴C-solution and incubated in sealed vials containing a CO₂ trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. In the ¹⁴C-citrulline feeding experiment argininosuccinate was the most highly labeled compound after 5 minutes and it was the first compound to lose ¹⁴C later in the time course. Carbon-14 was also recovered in free arginine, protein arginine, and CO₂ up to 4 hours after introduction of label. All of the ¹⁴C in free and protein arginine could be accounted for in the C-6 position. Metabolism of ¹⁴C-ornithine resulted in ¹⁴C-incorporation into citrulline and free and protein arginine and the evolution of ¹⁴CO₂. Citrulline was the most highly labeled compound after 15 minutes and was the first compound to reach a steady state level of ¹⁴C. With the addition of 800 nanomoles unlabeled citrulline to the ¹⁴C-ornithine feeding solution citrulline was the only compound labeled after 5 minutes and the steady state level of ¹⁴C-citrulline increased 12-fold. The appearance of ¹⁴C in free arginine and protein arginine was also delayed. In both ¹⁴C-ornithine feedings all of the ¹⁴C in free and protein arginine could be accounted for in the C-1 position. Together, the data support the reaction sequence: ornithine → citrulline → argininosuccinate → arginine → protein arginine.     

精氨酸激酶蛋白及分子生物学的研究进展

在无脊椎动物体内,精氨酸激酶(arginine kinase)在能量代谢过程中发挥着重要的作用.随着研究的深入,越来越多的数据表明,精氨酸激酶的作用不仅限于提供和维持能量的平衡,而且对生命活动的调控起到重要作用.综述无脊椎动物体内精氨酸激酶的进化历程、蛋白特性、特异表达及生物学功能等方面的研究进展,为深入研究无脊椎动物的抗性调控机制提供必要的参考.此外,对无脊椎动物精氨酸激酶的重要性和研究中存在的问题进行讨论.     

Protein turnover and growth in the whole body, liver and kidney of the rat from the foetus to senility.

Changes in the growth and protein turnover (measured in vivo) of the rat liver, kidney and whole body were studied between 16 days of life in utero and 105 weeks post partum. Tissue and whole-body growth were related to changes in both cellular hyperplasia (i.e. changes in DNA) and hypertrophy (protein/DNA values) and to the protein composition within the enlarging tissue mass. The suitability of using a single large dose of phenylalanine for measuring the rates of protein synthesis during both pre- and post-natal life was established. The declining growth rates in the whole animal and the two visceral tissues were then explained by developmental changes in the fractional rates of protein synthesis and breakdown, turnover rates being age-for-age higher in the liver than in the kidney, which in turn were higher than those measured in the whole animal. The declining fractional rates of synthesis in both tissues and the whole body with increasing age were related to changes in the tissues' ribosomal capacity and activity. The fall in the hepatic rate between 18 and 20 days of foetal life (from 134 to 98% per day) corresponded to a decrease in both the ribosomal capacity and the rate of synthesis per ribosome. No significant changes in any of these parameters were, however, found in the liver between weaning (3 weeks) and senility (105 weeks). In contrast, the fractional synthetic (and degradative) rates progressively declined in the kidney (from 95 to 24% per day) and whole body (from 70 to 11% per day) throughout both pre- and post-natal life, mainly as a consequence of a progressive decline in the ribosomal capacity, but with some fall in the ribosomal activity also during foetal life. The age-related contributions of these visceral tissues to the total amount of protein synthesized per day by the whole animal were determined. The renal contribution remained fairly constant at 1.6-2.9%, whereas the hepatic contribution declined from 56 to 11%, with increasing age. Approximate-steady-state conditions were reached at, and between, 44 and 105 weeks post partum, the half-life values of mixed whole-body, kidney and liver proteins being 6.4, 3.0 and 1.5 days, respectively, at 105 weeks.     

Arginine synthesis is regulated by dietary arginine intake in the enterally fed neonatal piglet

Arginine is conditionally indispensable in the neonate, and its synthesis in the intestine is not sufficient to meet requirements. It is not known how neonatal endogenous arginine synthesis is regulated and the degree to which proline and glutamate are used as precursors. Primed, constant intraportal and intragastric infusions of L-[U-14C]proline and L-[3,4-3H]glutamate, and intragastric L-[guanido-14C]arginine were used to measure whole body and first-pass intestinal arginine synthesis in 10 neonatal piglets fed generous (1.80 g.kg(-1).day(-1)) or deficient (0.20 g.kg(-1).day(-1)) quantities of arginine for 5 days. Glutamate tracer was not detected in arginine, indicating a biologically insignificant conversion of <1% of arginine flux. Endogenous arginine synthesis from proline had obligatory (0.36 g.kg(-1).day(-1)) and maximal (0.68 g.kg(-1).day(-1)) levels (P < 0.05, pooled SE 0.05). Although first-pass gut metabolism is responsible for 42-63% of whole body arginine synthesis, the gut is incapable of upregulating proline to arginine conversion during arginine deficiency, compared with a more than threefold increase without first-pass gut metabolism. These data suggest that upregulation of proline-to-arginine conversion occurs via increased arterial extraction of proline by the gut or in nonintestinal tissues. This study demonstrates that dietary arginine is an important regulator of endogenous arginine synthesis in the neonatal piglet and that proline, but not glutamate, is an important precursor for arginine synthesis in the neonate.     

The effect of total starvation and very low energy diet in lean men on kinetics of whole body protein and five hepatic secretory proteins

It is unclear whether the rate of weight loss, independent of magnitude, affects whole body protein metabolism and the synthesis and plasma concentrations of specific hepatic secretory proteins. We examined 1) whether lean men losing weight rapidly (starvation) show greater changes in whole body protein kinetics, synthesis, and circulating concentrations of selected hepatic secretory proteins than those losing the same amount of weight more slowly [very low energy diet (VLED)]; and 2) whether plasma concentrations and synthetic rates of these proteins are related. Whole body protein kinetics were measured using [1-(13)C]leucine in 11 lean men (6 starvation, 5 VLED). Fractional and absolute synthetic rates of HDL-apolipoprotein A1 (apoA1), retinol binding protein, transthyretin, alpha(1)-antitrypsin (alpha(1)-AT), and transferrin were measured using a prime-constant intravenous infusion of [(13)C(2)]glycine. Compared with VLED group, the starvation group showed greater increases (at a 5% weight loss) in whole body protein oxidation (P < 0.05); fractional synthetic rates of HDL-apoA1 (25.3 vs. -1.52%; P = 0.003) and retinol binding protein (30.6 vs. 7.1%; P = 0.007); absolute synthetic rates of HDL-apoA1 (7.1 vs. -3.8 mg.kg(-1).day(-1); P = 0.003) and alpha(1)-AT (17.8 vs. 3.6 mg.kg(-1).day(-1); P = 0.02); and plasma concentration of alpha(1)-AT (P = 0.025). Relationships between synthetic rates and plasma concentrations varied between the secreted proteins. It is concluded that synthetic rates of hepatic secreted proteins in lean men are more closely related to the rate than the magnitude of weight loss. Changes in concentration of these secreted proteins can occur independently of changes in synthetic rates, and vice versa.     

Nitric oxide and L-arginine metabolism in a devascularized porcine model of acute liver failure

In acute liver failure (ALF), the hyperdynamic circulation is believed to be the result of overproduction of nitric oxide (NO) in the splanchnic circulation. However, it has been suggested that arginine concentrations (the substrate for NO) are believed to be decreased, limiting substrate availability for NO production. To characterize the metabolic fate of arginine in early-phase ALF, we systematically assessed its interorgan transport and metabolism and measured the endogenous NO synthase inhibitor asymmetric dimethylarginine (ADMA) in a porcine model of ALF. Female adult pigs (23-30 kg) were randomized to sham (N = 8) or hepatic devascularization ALF (N = 8) procedure for 6 h. We measured plasma arginine, citrulline, ornithine levels; arginase activity, NO, and ADMA. Whole body metabolic rates and interorgan flux measurements were calculated using stable isotope-labeled amino acids. Plasma arginine decreased >85% of the basal level at t = 6 h (P < 0.001), whereas citrulline and ornithine progressively increased in ALF (P < 0.001 and P < 0.001, vs. sham respectively). No difference was found between the groups in the whole body rate of appearance of arginine or NO. However, ALF showed a significant increase in de novo arginine synthesis (P < 0.05). Interorgan data showed citrulline net intestinal production and renal consumption that was related to net renal production of arginine and ornithine. Both plasma arginase activity and plasma ADMA levels significantly increased in ALF (P < 0.001). In this model of early-phase ALF, arginine deficiency or higher ADMA levels do not limit whole body NO production. Arginine deficiency is caused by arginase-related arginine clearance in which arginine production is stimulated de novo.     

Arginine Metabolism in Developing Soybean Cotyledons: III. Utilization

Tracerkinetic experiments were performed using l-[guanidino-¹⁴C]arginine, l-[U-¹⁴C]arginine, l-[ureido-¹⁴C]citrulline, and l-[1-¹⁴C]ornithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free ¹⁴C compounds and incubated in sealed vials containing a CO₂ trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the ¹⁴C metabolized from [guanidino-¹⁴C]arginine and [U-¹⁴C]arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours ¹⁴CO₂ was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra ¹⁴CO₂ was evolved during the catabolism of ornithine-derived glutamate; ¹⁴C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.     

Arginine Biosynthesis by Streptococcus bovis

The pathway of arginine biosynthesis in Streptococcus bovis was studied by radioactive tracer techniques. Cells were grown anaerobically with (14)CO(2) in a synthetic medium containing NH(4) (+) as the sole nitrogen source except for the trace present in nitrogen-containing vitamins. The protein fraction isolated from the labeled cells was acid-hydrolyzed, and (14)C-arginine was isolated from the protein hydrolysate by ion-exchange chromatography. The carboxyl carbon of the isolated arginine was removed with arginine decarboxylase, and the guanidino carbon was removed by simultaneous arginase-urease degradation. By manometric measurement and liquid scintillation counting of the CO(2) released by enzymatic degradation, 50% of the label was found in the carboxyl carbon and 50% in the guanidino carbon. Specific radioactivity determinations indicated that growth on (14)CO(2) resulted in twice as much label in arginine as with aspartate, glutamate, or lysine. These results are consistent with a glutamate --> ornithine --> citrulline pathway of arginine biosynthesis in S. bovis and provide further evidence for the synthesis of glutamate via the tricarboxylic acid cycle reactions from citrate through alpha-ketoglutarate.