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At least 19 recordsLinked to original sources
PubMed1982

Relationship of plasma leucine and alpha-ketoisocaproate during a L-[1-13C]leucine infusion in man: a method for measuring human intracellular leucine tracer enrichment.

The keto analog of leucine, alpha-ketoisocaproate (KIC), is formed intracellularly from leucine and is released, in part, into the systemic circulation. Therefore. KIC can be used to estimate intracellular leucine tracer enrichment in man during labeled-leucine tracer experiments without requiring tissue biopsy samples. This approach was studied in young, healthy, male adults maintained on different dietary protein intakes from generous (1.5 g kg-1d-1) to deficient (0.0 g kg-1d-1) for 5-7 day periods. At the end of each dietary period, the volunteers were given a primed, continuous infusion of L-[1-13C]leucine either after an overnight fast (postabsorptive state) or while being fed hourly aliquots of the same diet. The plasma concentrations of all 3 branched-chain amino and keto acid pairs were measured from early morning blood samples taken from 4 subjects at 4 different levels of protein intake. Leucine concentration showed a weak correlation, and valine concentration showed a strong correlation with protein intake; isoleucine and the 3 keto acids did not. However, each branched-chain amino acid concentration was strongly correlated with its corresponding keto acid concentration. In plasma samples obtained during the L-[1-13C]leucine infusions, the ratio of [1-13C]KIC to [1-13C]leucine enrichment ratio remained relatively constant (77 +/- 1% over the wide range of dietary protein intakes and for both the fed and postabsorptive states. For the tissues from which the plasma KIC originates, the rate of plasma leucine into cells will account for approximately 77% of the intracellular leucine flux with the remaining 23% coming primarily from leucine release via protein breakdown. The constant nature of the plasma KIC to leucine 13C enrichment ratio implies that relative changes in leucine kinetics will appear the same under many dietary circumstances regardless of whether plasma leucine or KIC enrichments are used for the calculations.

Adult↗
PubMed1981

Semisynthesis and biological properties of the [B24-leucine]-, [B25-leucine[- and [B24-leucine, B25-leucine]-analogues of human insulin.

Trypsin-catalyzed coupling of porcine desoctapeptide-insulin with synthetic octapeptides produced the [LeuB24]- (I), [LeuB25]- (II) and [LeuB24, LeuB25]- (III)analogues of human insulin. I, II and III displayed respectively 20--30%, 1--2% and 0.5% of the receptor binding activity of the normal hormone. Biological activities of these analogues seemed to be proportional to their binding potencies when assayed in vitro, while in an in vivo assay analogue I was fully active and II exhibited 10--20% of normal activity. III was less active than II in all assays tested.

Adipose Tissue↗
PubMed1988

Failure to detect beta-leucine in human blood or leucine 2,3-aminomutase in rat liver using capillary gas chromatography-mass spectrometry.

It has been reported (Poston, J. (1976) J. Biol. Chem. 251, 1859-1863; (1982) 255, 10067-10072; (1984) 259, 2059-2061) that mammalian tissues contain an adenosylcobalamin-dependent enzyme, leucine 2,3-aminomutase, which catalyzes the interconversion of beta-leucine and leucine. It was also reported that beta-leucine is detectable in normal human serum (mean = 4.8 mumol/liter, n = 37) and is elevated in serum from patients with cobalamin deficiency (mean = 24.7 mumol/liter, n = 17). Serum levels of leucine were claimed to be decreased in the cobalamin deficient patients (mean = 52 mumol/liter) as compared with the normal subjects (mean = 81 mumol/liter). It was also reported that rat liver supernatant catalyzed the formation of beta-leucine, leucine, or both amino acids from iso-fatty acids, and that the generation of leucine from iso-fatty acids was stimulated by adenosylcobalamin and inhibited by unsaturated cobalamin-binding protein. We have synthesized t-butyldimethylsilyl derivatives of beta-leucine and leucine and have used capillary gas chromatography-mass spectrometry for their analysis. Using forms of beta-leucine and leucine that contain several deuterium atoms in place of several hydrogen atoms as internal standards, techniques have been developed which make it possible to detect and quantitate as little as 0.1 mumol/liter of beta-leucine or leucine in human serum and in incubations containing rat liver supernatant. beta-Leucine was not detectable, i.e. less than 0.1 mumol/liter, in any sera from 50 normal human subjects or in any sera from 50 cobalamin-deficient patients. The mean level of leucine in the 50 cobalamin-deficient sera was 219 mumol/liter, which was not decreased with respect to that in the 50 control sera (167 mumol/liter). Experiments in which beta-leucine, leucine, isostearic acid, or isocaproic acid were incubated with rat liver supernatant in the presence or absence of adenosylcobalamin or cobalamin-binding protein failed to demonstrate the formation of leucine or beta-leucine or their interconversion under any of the conditions studied. We conclude that beta-leucine is not present in human blood and that the existence of leucine 2,3-aminomutase in mammalian tissues remains to be established.

Adult↗
PubMed2002

Kinetics of sequential metabolism from D-leucine to L-leucine via alpha-ketoisocaproic acid in rat.

D-Leucine is considered to be converted into the L-enantiomer by two steps: oxidative deamination to form alpha-ketoisocaproic acid (KIC) and subsequent stereospecific reamination of KIC. We investigated the pharmacokinetics of leucine enantiomers and KIC in rats to evaluate how deamination of D-leucine, reamination of KIC, and decarboxylation of KIC were affected to the overall extent that converted D-leucine into the L-enantiomer. After intravenous administrations of D-[(2)H(7)]leucine, L-[(2)H(7)]leucine, or [(2)H(7)]KIC, their plasma concentrations together with endogenous L-leucine and KIC were determined by gas chromatography-mass spectrometry. The rapid appearances of [(2)H(7)]KIC and L-[(2)H(7)]leucine were observed after administration of D-[(2)H(7)]leucine, whereas no detectable amount of D-[(2)H(7)]leucine was found after administrations of [(2)H(7)]KIC or L-[(2)H(7)]leucine. The fraction of conversion from D-[(2)H(7)]leucine into [(2)H(7)]KIC (F(D-->KIC)) was estimated by using the area under the curve (AUC) of [(2)H(7)]KIC on the D-[(2)H(7)]leucine administration [AUC(KIC(D))] and that of [(2)H(7)]KIC on the [(2)H(7)]KIC administration (AUC(KIC)) to yield 70.1%. The fraction of conversion from [(2)H(7)]KIC to L-[(2)H(7)]leucine (F(KIC-->L)) was 40.2%. The fraction of conversion from D-leucine to the L-enantiomer (F(D-->L)) was considered to be the product of F(D-->KIC) and F(KIC-->L), indicating that 28.2% of D-[(2)H(7)]leucine was metabolized to L-[(2)H(7)]leucine via [(2)H(7)]KIC. These results suggested that the relatively low conversion of D-leucine into the L-enantiomer might depend on irreversible decarboxylation of KIC. Regardless of [(2)H(7)]KIC, F(D-->L) was also calculated directly using AUC(L(D)) and AUC(L) to yield 27.5%. There were no differences between the two F(D-->L) values, suggesting that almost all of the formation of L-[(2)H(7)]leucine from D-[(2)H(7)]leucine occurred via [(2)H(7)]KIC as an intermediate.

Animals↗
PubMed1998

Defective nonoxidative leucine degradation and endogenous leucine flux in cirrhosis during an amino acid infusion.

The metabolic fate of leucine's first and second carbon may be different depending on the tissue in which leucine is metabolized, as well as the prevailing hormonal milieu of that tissue. However, previous studies of leucine kinetics in humans have used only leucine labeled (as tracer) at the first carbon position. Because cirrhosis is associated with factors (such as insulin resistance and altered fuel substrate utilization) that may influence how leucine is degraded, the kinetics of leucine's first and second carbon using a simultaneous infusion of [1-14C] leucine and [2-13C] leucine were studied in the postabsorptive state and during an amino acid infusion in 6 stable cirrhotic patients and 6 matched controls. The data were normalized for different body compartments that were quantified from the dilution of H2 [180] and bromide. The body cell mass, but not body weight or fat-free body mass, was decreased in cirrhosis (P < .001). In response to the amino acid infusion, total leucine appearance from proteolysis and leucine's incorporation into protein increased significantly in both groups, but were higher in cirrhotic patients. Endogenous protein breakdown decreased in normals but remained unchanged in cirrhosis. These alterations in leucine metabolism became more prominent when data were expressed based on the body cell mass rather than on body weight. The oxidation of leucine's first carbon (C1) was decreased in cirrhosis, but the oxidation of leucine's second carbon (C2) did not differ between groups during both the postabsorptive period and the amino acid infusion, while nonoxidative leucine degradation [the difference between the oxidation of leucine's (C1) and (C2)] was also decreased in cirrhosis. In addition, there was a positive correlation between nonoxidative leucine degradation (which represents leucine incorporation into fat), and the respiratory quotient obtained from indirect calorimetry (r = .87; P < .001). These data suggest that the extent of leucine carbon oxidation is dependent on whether fat or carbohydrate is the prevailing fuel substrate. In addition, cirrhotic patients have decreased nonoxidative leucine degradation and are unable to suppress endogenous protein breakdown normally in response to amino acid administration. These abnormalities may contribute to the diminished fat stores and body cell mass commonly observed in cirrhosis.

Adult↗
PubMed1978

L-Leucine-induced secretion of glucagon and insulin, and the "off-response" to L-leucine in vitro. I. Characterization of the dynamics of secretion.

The effects of L-leucine, D-leucine, and L-isoleucine upon the secretion of glucagon and insulin were investigated using the isolated, perfused rat pancreas. All experiments were conducted in the presence of 5.6 mM D-glucose. Ten-minute perfusions of 2, 5, and 10 mM L-leucine induced the release of glucagon and insulin in a dose-related manner. The removal of L-leucine was followed by renewed release of insulin ("off-response") but not of glucagon. The magnitude of the off-response was greater when L-leucine was perfused over longer periods. L-Isoleucine evoked the release of both glucagon and insulin. When L-leucine was administered during perfusion of L-isoleucine, L-leucine-induced release of glucagon was inhibited, that of insulin was augmented, and the insulin off-response prevailed. When the perfusion of L-leucine immediately preceded that of L-isoleucine, L-isoleucine-induced release of glucagon was abolished and that of insulin was augmented. D-Leucine evoked the release of glucagon but not of insulin, and no off-response occurred. When the perfusion of D-leucine followed that of L-leucine, D-leucine-induced glucagon release was inhibited; the insulin off-response to L-leucine was not altered. We reached the following conclusions. 1) Glucagon release induced by L-leucine, D-leucine, or L-isoleucine is likely to be related to the occupancy by these analogous amino acids of transport and/or receptor sites which they share. 2) The insulin off response to L-leucine seems to be evoked by events which take place during the period of administration of L-leucine; these events are not likely to be the release of insulin that occurs during perfusion of L-leucine or the transport of L-leucine into or out of the beta cell. 3) Structurally or chemically similar compounds which are secretagogues both for glucagon and insulin affect the release of these hormones in different ways; these differences are likely to be due to dissimilar mechanisms governing the secretion of the two hormones.

Animals↗
PubMed1970

Biosynthesis of the branched-chain amino acids in yeast: a leucine-binding component and regulation of leucine uptake.

Use of an ion-exchange resin assay has shown that leucine is bound to a component of a dialyzed extract of yeast. Leucine binding may be related to in vivo uptake of the amino acid. A yeast strain with a 30-fold lower affinity for leucine uptake in vivo has a parallel reduction in affinity for in vitro leucine binding; the rate of leucine uptake in wild-type yeast can be increased four- to fivefold by growth on leucine as a sole nitrogen source. Under these conditions, the specific activity of the leucine-binding component also increases over threefold. Regulation of leucine uptake was studied by using wild-type strain 60615 and a mutant 60615/fl(2) with a constitutively elevated leucine uptake system. Leucine pool formation in the mutant was accompanied by an overshoot, leading to a loss of leucine from the pool. The phenomenon could be observed in the wild type under certain conditions. The mechanism of this process was examined. The leucine uptake system was found to be stable in the absence of protein synthesis. The rate of leucine uptake increased on reduction of the pool of amino acids, and in strain 60615/fl(2) the ability to overshoot was rapidly recovered on depletion of the leucine pool. The results suggest a control of leucine uptake by feedback inhibition, in which leucine or other amino acids, e.g., isoleucine, inhibit leucine uptake. The results do not exclude control by a rapidly activated-inactivated system.

Amino Acids↗
PubMed2000

Downstream change in leucine aminopeptidase activity and leucine assimilation by epilithic microbiota along the River Swale, northern England.

Parallel determinations of epilithic extracellular leucine aminopeptidase activity and leucine assimilation were made at five sites along 112 km of the River Swale and also in two tributaries, the River Wiske and Cod Beck. Epilithic leucine aminopeptidase activity along the Swale increased with distance downstream; this increase was gradual, rather than stepwise in response to specific sewage-works outfalls. Epilithic leucine assimilation, in contrast, did not consistently increase along the river. Epilithic leucine aminopeptidase activity and leucine assimilation were both potentially controlled by epilithic microbial variables (bacterial abundance and chlorophyll a) while leucine aminopeptidase activity was also strongly related to water-quality variables, especially temperature, pH and conductivity. Epilithic leucine aminopeptidase activity and leucine assimilation were coupled, but the magnitude of aminopeptidase activity was always substantially greater than that of leucine assimilation. Arguments are presented, however, which suggest that this did not necessarily indicate the constant availability of excess leucine, and by inference amino-acid nitrogen, to epilithic bacteria. Values of epilithic leucine aminopeptidase activity and leucine assimilation, expressed relative to rates in overlying water, suggested that most activity and assimilation was epilithic rather than planktonic, although the planktonic contribution was proportionately greater at the deeper, more downstream, sites. In the tributaries, River Wiske and Cod Beck, values of epilithic leucine aminopeptidase activity and epilithic microbial abundance, as well as those of many water-quality variables, resembled values in the middle and lower Swale. Thus, these tributaries were essentially lowland, enriched watercourses being very different from the headstreams of the main river.

Animals↗
PubMed1988

Essential role of adenosylcobalamin in leucine synthesis from beta-leucine in the domestic chicken.

This study was designed to investigate a postulated relationship between vitamin B-12 and leucine metabolism in mature domestic chickens. Plasma amino acid analysis revealed the presence of beta-leucine at a concentration of 60 to 80 mumol/l. After 425 d on a vitamin B-12-deficient diet, plasma beta-leucine was 133% higher (P less than 0.06) and plasma leucine and methionine lower (P less than 0.03) than values in plasma from hens fed a diet adequate in vitamin B-12. Branched-chain-amino-acid aminotransferase (EC 2.6.1.42) (BCAT) activity was not enhanced by vitamin B-12 deprivation (P greater than 0.05). In contrast to leucine, beta-leucine was not utilized as substrate by BCAT for the formation of alpha-ketoisocaproate. Kidney extracts possessed leucine 2,3-aminomutase (EC 5.4.3.7) (LAM) activity, as evidenced by enhanced conversion of beta-leucine to alpha-leucine in the presence of adenosylcobalamin. LAM activity could not be demonstrated in liver or muscle extract, while leucine formation by pancreas extract was negligible. These data represent the first evidence of the presence of the amino acid beta-leucine in chicken plasma. In addition, the data support vitamin B-12-dependent leucine synthesis from beta-leucine in the chicken and highlight the kidney's role in leucine synthesis.

Amino Acid Isomerases↗
PubMed2000

Differential transport properties of D-leucine and L-leucine in the archaeon, Halobacterium salinarum.

The transport of D-leucine was compared with that of L-leucine in Halobacterium salinarum. When a high-outside/low-inside Na+ gradient was imposed, D-leucine as well as L-leucine accumulated in envelope vesicles, supporting the hypothesis that D-leucine is transported via a symport system along with Na+. Kinetic analyses, including inhibition experiments, indicated that both enantiomers are transported via a common carrier. However, a Hill plot indicated a single binding site for Na+ during L-leucine transport, but dual binding sites for Na+ during D-leucine transport. Furthermore, D-leucine transport was dependent on electrical membrane potential, suggesting that a transporter bound with D-leucine is positively charged. L-leucine transport was slightly, if at all, dependent on membrane potential, suggesting that a transporter bound with L-leucine is electrically neutral. These results indicate that the leucine carrier in Halobacterium salinarum translocates two moles of Na+ per mole of D-leucine, and one mole of Na+ per mole of L-leucine.

Biological Transport↗
PubMed1990

Quantitative role of splanchnic region in leucine metabolism: L-[1-13C,15N]leucine and substrate balance studies.

The role of the splanchnic region (Sp) in whole body leucine metabolism was assessed in six chronically catheterized fasting mongrel dogs and in eight dogs during constant enteral feeding of a complete amino acid solution (0.24 g.kg-1.h-1). We used primed continuous intravenous infusions of L-[1-13C,15N]leucine and L-[1-14C]leucine and measurements of arteriovenous isotope and leucine balance across the gut, liver, and Sp. In the fasted condition, 3.5% of arterial leucine supply was oxidized in the Sp, accounting for 13% of total body leucine oxidation, with 10% by liver. With amino acid feeding 1) leucine carbon and nitrogen fluxes and oxidation were increased (P less than 0.01) at the whole body level; 2) the percent of whole body leucine oxidation occurring in the Sp and liver increased (P less than 0.01) to 41 and 27%, respectively; 3) fractional metabolic utilization of leucine delivered to the Sp was reduced (P less than 0.01) from 47 to 35%; 4) the deamination rate of leucine in the gut was increased (P less than 0.05), along with an increased reamination rate of alpha-ketoisocaproic acid in the Sp (P less than 0.05). These findings reveal that the Sp accounts for a small fraction of whole body leucine oxidation during the fasting condition, but it plays a quantitatively important role in total body leucine oxidation during amino acid feeding; the gut and liver play cooperative roles in controlling leucine supply to peripheral tissues.

Animals↗
PubMed1997

Effects of leucine on whole body leucine, valine, and threonine metabolism in humans.

We tested whether expansion of the plasma leucine pool distorts leucine or valine tracer kinetics, causing errors in the derived values of whole body proteolysis. Seven normal adults received a 10-h primed-continuous tracer infusion of L-[5,5,5-2H3]leucine, L-[(1-13)C]valine, and L-[(1-13)C]threonine, during the final 7 h of which L-leucine was infused at a rate that more than tripled the plasma leucine concentration. Leucine, valine, and threonine rates of appearance were converted to a common value of whole body proteolysis on the basis of their concentrations in body proteins. The conversion of labeled leucine and valine to their corresponding branched-chain alpha-keto and alpha-hydroxy acids was also monitored. Before the unlabeled leucine infusion, postabsorptive whole body proteolysis was estimated similarly by the three tracers (approximately 180 mg protein.kg-1.h-1. The leucine infusion reduced proteolysis by an average of 21% (P < 0.006), as estimated by use of valine or threonine kinetics, and by 10% by use of leucine kinetics (P < 0.02). No delay in the conversion of valine to alpha-ketoisovalerate occurred during the leucine infusion. Thus all three tracers indicated similar postabsorptive rates of whole body proteolysis and a reduction of proteolysis during leucine administration, although the magnitude of the effect was underestimated with use of the leucine tracer.

Adult↗
PubMed2001

Recovery of (13)CO(2) from infused [1-(13)C]leucine and [1,2-(13)C(2)]leucine in healthy humans.

Carbon (C) in the 1-position of leucine is released as CO(2) with the decarboxylation of alpha-ketoisocaproate (KIC). Carbon in the 2-position of leucine undergoes several additional metabolic steps before entering the tricarboxylic acid (TCA) cycle in the 1-position of acetyl-CoA, where it can be released as CO(2) or be incorporated into other compounds. This study examined the metabolic fate of C in the 2-position of leucine. We infused 11 healthy subjects with [1-(13)C]leucine and [1,2-(13)C(2)]leucine for 3.5--4 h to measure leucine kinetics and the oxidation of the tracers from enrichments of (13)C in blood and expired CO(2). The fraction of leucine infused that was oxidized (f(ox)) was used to define the degree of recovery of the (13)C label(s) for each tracer. As expected, leucine appearance (means +/- SE) did not differ between tracers ((13)C(1): 92.1 +/- 3.1 vs. (13)C(2): 89.2 +/- 3.2 micromol x kg(-1) x h(-1)) when calculated using plasma leucine enrichments as an index of intracellular enrichment. A small (3%) but significant (P = 0.048) difference between tracers was found when KIC was used to calculate leucine appearance ((13)C(1): 118.0 +/- 4.1 vs. (13)C(2): 114.4 +/- 4.5 micromol x kg(-1) x h(-1)). The value of f(ox) was 14 +/- 1% for [1,2-(13)C(2)]leucine and was lower than the f(ox) for [1-(13)C]leucine (19 +/- 1%). From the f(ox) data, we calculated that the recovery of the 2-(13)C label in breath CO(2) was 58 +/- 6% relative to the 1-(13)C label. These findings show that, although a majority of the 2-(13)C label of leucine is recovered in breath CO(2), a significant percentage (approximately 42%) is retained in the body, presumably by transfer to other compounds, via TCA exchange reactions.

Adult↗
PubMed1987

Extrapancreatic effects of L-leucine infusion in leucine-sensitive and control subjects.

We studied the extrapancreatic effects of L-leucine infusion (2.5 mumol/kg/min) in six controls (three females, three males) and three members of a family with leucine-sensitive hypoglycemia (LSH). Total glucose disposal and endogenous glucose production (EGP) rates were assessed after an overnight fast (12 to 14 hour) during somatostatin (SRIF) infusion (500 micrograms/h) with insulin replacement (0.2 mU/kg/min), combined with D-(3-3H)-glucose infusion. Additional studies of forearm arterial-venous (A-V) balances of amino acids, glucose, and other substrates, combined with L-(1-14C)-leucine kinetics, were done in these two groups. L-leucine infusion resulted in a 15% decrease in whole-body total glucose utilization in controls (P less than .05) during SRIF-insulin infusion, but did not affect glucose utilization in LSH subjects. EGP was not affected by L-leucine infusion in either group. Control subjects demonstrated a significant reduction in forearm glucose uptake and greater lactate release during L-leucine infusion, compared to the basal state. In contrast, subjects with LSH showed a slight increase in forearm glucose uptake and significantly less lactate release during L-leucine infusion. Subjects with LSH demonstrated significantly lower forearm leucine uptake, and lower rates of appearance and oxidation of L-(1-14C)-leucine during leucine infusion, compared to controls. Our data suggest that members of this LSH kindred have a defect in intracellular leucine metabolism in skeletal muscle, resulting in a lack of leucine-induced inhibition of glucose utilization. This may contribution to the development of hypoglycemia in these subjects.

Adolescent↗
PubMed1986

Leucine kinetics at graded leucine intakes in young men.

A study was carried out with 12 young men to examine the relationships between the intake of leucine and indices of leucine kinetics, using L-[1-13C]leucine as a tracer. Six subjects received L-amino acid diets during 7-day periods supplying leucine in the range of 79 to 20 mg.kg-1.day-1 (Group I) and another six subjects (Group II) received leucine intakes ranging from 20 to 4 mg.kg-1.day-1. Estimations were made of leucine kinetics, at the end of each diet period, when subjects were receiving small isonitrogenous, isocaloric meals during the isotope infusion period. Leucine flux declined with reduced leucine intake and leucine oxidation tended not to change at intakes below 20 mg.kg-1.day-1 (slope not statistically different than zero). Plasma valine increased markedly with further restriction in leucine intake below this level. The daily mass balance of leucine, estimated from the difference between intake and oxidation, became negative at an intake of about 20 mg.kg-1.day-1. These findings are discussed in relation to the published mean and upper range of requirement for leucine in healthy adults, currently taken to be 11 mg.kg-1.day-1 and 14 mg.kg-1.day-1, respectively.

Adolescent↗
PubMed1988

Leucine kinetics at graded intakes in young men: quantitative fate of dietary leucine.

To explore leucine metabolism in relation to leucine intake, five young adult men received an L-amino acid diet that supplied 40, 30, 20, and 10 mg leucine.kg-1.d-1 for 6 d. A stable-isotope-tracer infusion study was then conducted for 5 h while subjects received an intragastric infusion of the test diet. Primed, constant infusions of L-[1-13C]leucine (intragastric) and L-[2H3]leucine (intravenous) were given simultaneously. A final infusion study was conducted in subjects in the postabsorptive state after an additional 2 d with the 10-mg diet. Estimates were made of leucine flux and oxidation, rates of uptake and release of absorbed leucine by the splanchnic region, and leucine balance. The rate of appearance of dietary leucine in the systemic circulation (Leud) decreased (p less than 0.01) between the 40- and 10-mg diets. At the latter intake, splanchnic uptake was approximately 37% of absorbed leucine. The correlation between Leud and plasma leucine concentration was highly positive. A leucine intake of approximately 40 mg. kg-1.d-1 was close to that required to maintain leucine balance under these conditions.

Adult↗
PubMed1992

Role of the conserved leucines in the leucine zipper dimerization motif of yeast GCN4.

Yeast GCN4 belongs to the class of eukaryotic transcription factors whose bZIP DNA-binding domains dimerize via a leucine zipper motif that structurally resembles a coiled coil. The leucine zipper contains 4-5 highly conserved leucine residues spaced exactly 7 residues apart that are located within the alpha-helical hydrophobic interface between protein monomers. Here, we investigate the role of the four canonical leucines in the GCN4 leucine zipper by analyzing a series of mutated derivatives for their ability to activate transcription in vivo and to bind DNA in vitro. The GCN4 leucine zipper is surprisingly tolerant of mutations, with a wide variety of single substitutions at any of the four leucines including basic and acidic amino acids behaving indistinguishably from wild-type GCN4. Moreover, some derivatives containing two leucine substitutions display detectable though reduced function. These results indicate that other residues within the coiled coil are crucial for efficient dimerization, and they suggest that some eukaryotic transcriptional regulatory proteins lacking the conserved leucine repeat will dimerize through a structurally homologous motif. Interestingly, our results differ in several respects from those obtained by analyzing mutations in the GCN4 leucine zipper in the context of a lambda repressor-GCN4 zipper hybrid protein. These apparent differences may reflect a functional interrelationship between the leucine zipper and basic region subdomains for DNA-binding by bZIP proteins.

Base Sequence↗
PubMed1992

Nitrogen absorption from isonitrogenous solutions of L-leucyl-L-leucine and L-leucine: a study in the isolated perfused rat small intestine.

1. We assessed the efficacy of nitrogen absorption from luminal L-leucine (1.2 and 12 mmol/l) and from isonitrogenous L-leucyl-L-leucine (0.6 and 6.0 mmol/l) in a preparation of vascularly and luminally perfused rat small intestine by measuring luminal leucyl-leucine disappearance and venous leucine appearance. 2. No intact dipeptide was found in the vascular perfusate. Leucine-nitrogen absorption, as judged by venous leucine appearance, was as efficient from free leucine (29 +/- 3 and 245 +/- 19 ng-atom min-1 g-1) as from leucyl-leucine (27 +/- 4 and 211 +/- 58 ng-atom min-1 g-1). It was not reduced in the presence of glycyl-L-proline or of the brush-border dipeptidase inhibitors alanine-beta-naphthylamide and cilastatin. 3. After one passage of the whole small intestine, only trace amounts of dipeptide, but large amounts of free leucine, were detected in the luminal effluent. Peptidase activity in the luminal effluent was demonstrated in 100,000 g supernatant and was inhibited by p-hydroxy-mercuribenzoate, but not by brush-border dipeptidase inhibitors. 4. We propose that nitrogen absorption from luminal leucyl-leucine may proceed predominantly via intraluminal peptide hydrolysis and subsequent transport of free leucine. Nevertheless, our findings and conclusions are at variance with previous observations and current opinion on intestinal handling of dipeptides, which may be due, in part, to the different methodological approach.

Animals↗