Characterization of amino acid pools in the vacuolar compartment of Saccharomyces cerevisiae

Intact vacuoles are released from spheroplasts of Saccharomyces cerevisiae by means of a gentle mechanical disintegration method. They are purified by centrifugation in isotonic density gradients (flotation and subsequent sedimentation), and analyzed for their soluble amino acid content. The results indicate that about 60% of the total amino acid pool of spheroplasts is contained in the vacuoles. This may be an underestimate, as it presupposes no loss of amino acids from the vacuoles during the purification procedure. The amino acid concentration in the vecuoles is calculated to be approximately 5 times that in the cytoplasm if the total volumes of the two compartments are used for the calculation. The vacuolar amino acid pool is rich in basic amino acids, and in citrulline and glutamine, but contains a remarkably small amount of glutamate. Radioactive labeling experiments with spheroplasts indicate that the vacuolar amino acids are separated from the metabolically active pools located in the cytoplasm. This is particularly evident for the basic amino acids and glutamine; in contrast, the neutral amino acids and glutamate appear to exchange more rapidly between the cytoplasmic and the vacuolar compartments of the cells.     

Dynamics of Nitrogenous Assimilate Partitioning between Cytoplasmic and Vacuolar Fractions in Carrot Cell Suspension Cultures

Bulk vacuole isolation, gas chromatography-mass spectrometry,, and high-performance liquid chromatography have been used to investigate the accumulation and partitioning of assimilated nitrogen supplied as ¹⁵NH₄Cl between vacuolar and extravacuolar (cytoplasmic) fractions of protoplasts from suspension cultures of carrot (Daucus carota L. cv Chantenay). Glutamine was the most abundant amino acid in the vacuole of protoplasts from late-exponential phase cells, whereas alanine, glutamate, and γ-aminobutyric acid were located primarily in the cytoplasmic fraction. In ¹⁵N-feeding studies, newly synthesized glutamine partitioned strongly to the vacuole, whereas glutamate partitioned strongly to the cytoplasm, γ-aminobutyric acid was totally excluded from the vacuole, and alanine was distributed in both compartments. Comparison of the ¹⁵N-enrichment patterns suggests that initial assimilation to glutamine occurs within a subcompartment of the cytoplasmic fraction. The protoplast-feeding technique may be extended to investigate cytoplasmic compartmentation further.     

ON THE SITE OF ORIGIN OF TRANSMITTER AMINO ACIDS RELEASED BY DEPOLARIZATION OF NERVE TERMINALS IN VITRO

Abstract— —The site of origin of transmitter amino acids released by depolarizing agents from nerve endings was studied. The model used was the incubated and depolarized synaptosome preparation from which the component soluble, synaptic vesicle, membrane and mitochondrial sub-fractions were obtained. Synaptosomal amino acids were radioactively labelled from D-[U-¹⁴C]glucose in vivo by intraventricular injection and in vitro during subsequent incubation. The specific radioactivities of amino acids released in response to K⁺ (56 mM) or veratrine (75 μM) were found to closely resemble those of the soluble cytoplasmic fraction, in most cases differing significantly from those of the other fractions. The specific radioactivity of the GABA and aspartate released by K⁺ stimulation and the GABA and glutamate released by veratrine were significantly different from that of the vesicles in each case. The specific radioactivities of glutamate released by both agents, and also GABA with K⁺ stimulation, were approximately double that of the amino acid released in control conditions. Depletion of the soluble cytoplasmic pools of glutamate, GABA and aspartate occurred following stimulation, corresponding to the induced-release of these compounds. Turnover of the amino acids in the other subfractions was too low to account for their participation in the release process in addition to the soluble cytoplasmic pool. A cytoplasmic origin of release of neurotransmitter amino acids from nerve endings is proposed.     

Subcellular Distribution of Free Amino Acids in Relation to Protein Synthesis in Cells of Chara corallina

The influence of protein-synthesis inhibitors on the subcellular distribution of free amino acids was studied in internodal cells of Chara corallina. Use of an intracellular perfusion technique allowed separate measurements of amino acids in the vacuole, in the flowing sol endoplasm and in the gel layer. The sol endoplasm predominantly represents the cytosol, while the gel layer is occupied, for the most part, by chloroplasts. When cells were treated with 0.5 mM chloramphenicol (CRP) in the dark, both the total concentration of amino acids and the subcellular distribution were almost the same as in cells without treatment. In the light, however, the subcellular distribution changed dramatically, although the total concentration of amino acids was unchanged. The vacuolar concentration of amino acids was 3 times greater in CRP-treated cells than in the control. The concentrations of amino acids in the sol endoplasm and in the gel layer were only half of those in the control. Amino acid permeability of the chloroplast envelope, measured using the perfused internodal cells, slightly increased after the CRP treatment in the light. Time-dependent changes in concentrations of amino acids in the CRP-treated cells were also measured in the light. The total concentration of amino acids in the cytoplasm gradually decreased, while that in the vacuole increased commensurately. The concentration and/or subcellular distribution of alanine, glutamine, glutamate and glycine changed dramatically. The concentration of alanine increased considerably both in the vacuole and in the cytoplasm. The cytoplasmic concentration of glutamine increased transiently within 1 ?2 h after treatment with CRP. The cytoplasmic concentrations of glutamate and glycine decreased. Although the concentrations of some amino acids changed so markedly both in the vacuole and cytoplasm, only small differences in the activities of glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase and glutamine synthetase were detected between the control and the CRP-treated cells.     

The content of GABA and other amino acids in bovine cerebral cortex synaptic vesicles

The content of γ-amino butyric acid (GABA) and of other water soluble amino acids in bovine brain synaptic vesicles was determined by a modified automated amino acid analysis method. Following subcellular fractionation, GABA, glutamate and aspartate were distributed largely in the supernatant fractions and in the upper layer of the sucrose gradient. Only 10–20% of the total content was associated with the vesicular fraction. On the other hand, the other water soluble amino acids, such as serine, glycine and alanine, were evenly distributed between cytoplasmic and particulate fractions in a similar pattern to that observed with cytoplasmic enzyme markers. The results may indicate specific association of GABA, glutamate and aspartate with low density particles or cytoplasmic components.     

Alteration in the Amino Acid Content of Yeast During Growth Under Various Nutritional Conditions

Yeast cells grown under optimal and suboptimal concentrations of biotin were analyzed for the amino acid content of their soluble pool and cellular protein. Optimally grown yeast cells exhibited a maximum amino acid content after 18 hr of growth. Biotin-deficient cells were depleted of all amino acids at 26 and 43 hr, with alanine, arginine, aspartate, cysteine, glutamate, isoleucine, leucine, lysine, methionine, serine, threonine, and valine being present in less than half the concentration observed in biotin-optimal cells. At early time intervals, the amino acid pool of biotin-deficient yeast contained lower concentrations of all amino acids except alanine. After more prolonged incubation, several amino acids accumulated in the pool of biotin-deficient yeast, but citrulline and ornithine accumulated to appreciable levels. The addition of aspartate to the growth medium resulted in a decrease in the amino acid content of biotin-optimal cells but caused a marked increase in the concentration of amino acids in biotin-deficient cells. The pools of biotin-deficient yeast grown in the presence of aspartate displayed a marked reduction in every amino acid with the exception of aspartate itself. These data provide evidence that the amino acid content of yeast cells and their free amino acid pools are markedly affected by biotin deficiency as well as by supplementation with aspartate, indicating that aspartate plays a major role in the nitrogen economy of yeast under both normal as well as abnormal nutritional conditions.     

Purification and properties of dolphin muscle aspartate and alanine transaminases and their possible roles in the energy metabolism of diving mammals

1. Mitochondrial and supernatant aspartate transaminases (EC 2.6.1.1) and supernatant alanine transaminase (EC 2.6.1.2) were purified 89-, 204- and 240-fold respectively, from dolphin muscle. Starch-gel electrophoresis of crude and purified preparations revealed that all three enzymes exist as single forms. 2. K(m) values of alpha-oxoglutarate, alanine, pyruvate and glutamate for the alanine transaminase were 0.45, 8.2, 0.87 and 15mm respectively. For the aspartate transaminases, the K(m) values of alpha-oxoglutarate, aspartate, oxalacetate and glutamate were 0.76, 0.50, 0.10 and 9.4mm respectively, for the mitochondrial form and 0.13, 2.4, 0.06 and 3.2mm respectively, for the supernatant form. 3. In all cases, as the assay pH value was decreased from pH7.3, the K(m) values of the alpha-oxo acids decreased whereas those of the amino acids increased. 4. The apparent equilibrium constants for the aspartate transaminases were independent of pH. These values were 9.2 and 6.8 for the mitochondrial and supernatant forms respectively, where [Formula: see text] 5. Studies of the inhibition of the aspartate transaminases by dicarboxylic acids indicated that these enzymes may be controlled by pools of metabolic intermediates. 6. Three key roles are suggested for the transaminases in the energy metabolism of the diving animal. First, it is believed that a combined action of the transaminases could enhance energy production during hypoxia by providing (a) fumarate from aspartate for the ATP-producing reversal of succinate dehydrogenase, and (b) alpha-oxoglutarate from glutamate for the GTP-producing succinyl thiokinase reaction. Secondly, diving mammals probably accumulate more NADH than other mammals during hypoxia. The aspartate transaminases seem particularly well suited for restoring and maintaining redox balance via the malate-aspartate cycle after aerobic metabolism is resumed. Finally, since the preferred fuel for aerobic work is fat, the combined reactions of the transaminases could be instrumental in providing increased supplies of oxaloacetate for sparking the tricarboxylic acid cycle.     

Turnover rates of amino acid neurotransmitters in regions of rat cerebellum

The turnover rates of aspartate, gamma-aminobutyric acid (GABA), glutamate, glutamine, alanine, serine, and glycine were measured in five regions of rat cerebellum. Turnover rates of the putative neurotransmitters (aspartate, glutamate, and GABA) were 2-20-fold higher than those of alanine and serine, and generally consistent with the proposed neurotransmitter functions for these amino acids. However, glutamate turnover was high and similar in magnitude in the deep nuclei and granule layer, suggesting possible release, not only from parallel fibers, but from mossy fibers as well. The differential distribution of turnover rates for GABA supports its neuronal release by Purkinje, stellate, basket, and Golgi cells, whereas aspartate may be released by both climbing and mossy fibers. The distribution of glycine turnover rates is consistent with release from Golgi cells, whereas alanine may be released from granule cell parallel fibers. Turnover rates measured in two other motor areas, the striatum and motor cortex, indicated that utilization of these amino acid neurotransmitters is differentially distributed in brain motor regions. The data indicate that turnover rate measurements may be useful in identifying neurotransmitter function where content measurements alone are insufficient.     

Light-dependent synthesis of glutamate inRhodospirillum rubrum

Summary The relative sizes of the macro amino acid pools inRhodospirillum rubrum, as measured by the incorporation of [¹⁴C]-carbon dioxide, were approximately the same in the four different cultures examined; mid-exponential phase and initial stationary phase batch culture organisms and two steady state turbidostat continuous cultures. Glutamate was the dominant amino acid labelled with lower levels of labelling in glutamine, aspartate, alanine and threonine to complete the major amino acid pools. Glutamine synthesis was a light-dependent process in the four cultures examined whereas the synthesis of glutamate was light-dependent only in the stationary phase batch culture organisms and in low cell density turbidostat organisms. These results are presented as physiological evidence for the activity of the ATP-requiring GS-GOGAT system for ammonia assimilation under certain growth conditions inRhodospirillum rubrum.     

The tricarboxylic acid cycle in Dictyostelium discoideum. A model of the cycle at preculmination and aggregation.

A preliminary model of tricarboxylic acid-cycle activity in Dictyostelium discoideum is presented. Specific-radioactivity labelling patterns of intra- and extra-mitochondrial pools are simulated by this model and compared with the experimental data. The model arrived at by this method shows the following features. (1) The cycle flux rate is approx. 0.4 mM/min. (2) Both fumarate and malate are compartmentalized at approx. 1:5 between cycle pools and non-cycle pools. These may represent mitochondrial and cytoplasmic pools. Citrate is compartmentalized at 1:10. Succinate appears to exist in three compartments, two of which become labelled by [14C]glutamate and only one by [14C]aspartate (3) Two pools of aspartate with two associated pools of oxaloacetate are necessary for simulation. (4) Exchange between the cycle and non-cycle pools of both citrate and fumarate occurs at very low rates of about 0.003 mM/min, whereas exchange between the malate pools is about 0.004 mM/min. The exchange reaction glutamate in equilibrium 2-oxoglutarate runs at approx. 15 times the cycle flux. (5) A reaction catalysed by "malic" enzyme is included in the model, as this reaction is necessary for complete oxidation of amino acid substrates. (6) Calculation of the ATP yield from the model is consistent with earlier estimates of ATP turnover if the activity of adenylate kinase is considered.     

Cytoplasmic accumulation of flavonoids in flower petals and its relevance to yellow flower colouration

It is widely accepted that the mix of flavonoids in the cell vacuole is the source of flavonoid based petal colour, and that analysis of the petal extract reveals the nature and relative levels of vacuolar flavonoid pigments. However, it has recently been established with lisianthus flowers that some petal flavonoids can be excluded from the vacuolar mix through deposition in the cell wall or through complexation with proteins inside the vacuole, and that these flavonoids are not readily extractable. The present work demonstrates that flavonoids can also be compartmented within the cell cytoplasm. Using adaxial epidermal peels from the petals of lisianthus (Eustoma grandiflorum), Lathyrus chrysanthus and Dianthus caryophyllus, light and laser scanning confocal microscopy studies revealed a significant concentration of petal flavonoids in the cell cytoplasm of some tissues. With lisianthus, flavonoid analyses of isolated protoplasts and vacuoles were used to establish that ca 14% of petal flavonoids are located in the cytoplasm (cf. 30% in the cell wall and 56% in the vacuole). The cytoplasmic flavonoids are predominantly acylated glycosides (cf. non-acylated in the cell wall). Flavonoid aggregation on a cytoplasmic protein substrate provides a rational mechanism to account for how colourless flavonoid glycosides can produce yellow colouration in petals, and perhaps also in other plant parts. High vacuolar concentrations of such flavonoids are shown to be insufficient.     

α-Latrotoxin Releases Both Vesicular and Cytoplasmic Glutamate from Isolated Nerve Terminals

alpha-Latrotoxin causes a massive release of endogenous glutamate from guinea-pig cerebrocortical synaptosomes. There appear to be two components to the release. In the first 2 min following addition of 1.3 nM alpha-latrotoxin, glutamate release is largely energy dependent. Superimposed upon this release is a more slowly developing but ultimately much more extensive release of cytoplasmic glutamate together with gamma-aminobutyric acid and nonvesicular amino acids such as aspartate and alpha-aminoisobutyrate. In parallel with this cytoplasmic release there is an extensive depletion of ATP, a massive rise in cytoplasmic free Ca2+ concentration, and a severe restriction of synaptosomal respiratory capacity. The cytoplasmic release is only partially Na+ dependent, eliminating a simple reversal of the plasma membrane acidic amino acid carrier. It is concluded that alpha-latrotoxin releases both transmitter and cytoplasmic pools of amino acids in synaptosomes and causes a major disruption of terminal integrity.     

Amino Acid Transport across the Tonoplast of Vacuoles Isolated from Barley Mesophyll Protoplasts : Uptake of Alanine, Leucine, and Glutamine

Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using ¹⁴C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors.     

The Acute Effect of Ammonium Acetate on Levels of Amino Acids in the Intact and Decorticated Rat Neostriatum

Unilateral frontal cortex ablations were performed in rats so that the glutamate terminals in the ipsilateral rostral neostriatum were removed. At 1 or 7 days later, intraperitoneal injections of ammonium acetate induced different changes in amino acid concentrations in the intact and deafferentated neostriatum. After 1 day, the level of glutamate decreased only in the intact side, whereas that of glutamine increased and that of aspartate decreased to the same extent on both sides following ammonia injection. After 7 days, the glutamate level decreased more in the intact than the decorticated side in both nonconvulsing and convulsing rats. The concentration of alanine increased most in the intact neostriatum, whereas glutamine levels increased and aspartate levels decreased to the same extent on both sides in nonconvulsing and convulsing rats. The results indicate that ammonia has a more pronounced effect on neuronal than glial glutamate pools.     

Metabolism of amino acids in Ascaridia galli: transamination

Ascaridia galli, using 2-oxoglutarate as an acceptor, transaminates alanine and aspartate at significantly high rates. Among other amino acids valine, phenylalanine, leucine, isoleucine, arginine, tyrosine and methionine are metabolised at moderate rates while lysine, serine, threonine, cysteine, glycine, histidine, tryptophan, DOPA and GABA appear to be inert in this respect. Body parts mimic the whole worm in the pattern of transamination of various amino acids with the exception of methionine. Alanine, aspartate and glutamate may transfer their amino group also to pyruvate and oxaloacetate. Alanine and aspartate: 2-oxoglutarate transaminases are located mainly in the cytosol and mitochondrial fractions.     

Glucose and amino acid metabolism in neonatal rat skeletal muscle in tissue culture

The characteristics of glucose and amino acid metabolism over a 98-hour incubation period were studied in a primary culture of neonatal rat skeletal muscle cells. The cells formed large myotubes in culture, were spontaneously highly contractile, and had cell phosphocreatine levels exceeding ATP concentrations. Medium glucose fell from 7.2±0.2 to 1.5±0.1 mM between 0 and 98 hours; intracellular glucose was readily detectable, indicating glycolysis was limited by phosphorylation, not glucose transport. Alanine levels in the medium increased from 0.06±0.01 to 0.82±0.04 mM between 0 and 48 hours and decreased to 0.72±0.04 mM by 98 hours. The period of net alanine production correlated with the rise in the cell mass action ratio of the alanine aminotransferase reaction. Cell aspartate, glutamate, and calculated oxalacetate levels were inversely related to the cell NADH/NAD⁺ ratio, as represented by the intracellular lactate/pyruvate ratio (r=0.78–0.88). The branched chain amino acids (leucine, isoleucine, valine) were actively utilized, e.g., medium leucine fell from 0.70±0.01 to 0.30±0.06 mM between 0 and 98 hours. In addition, arginine and serine consumption was observed in conjunction with ornithine, proline, and glycine production. Conclusions: (1) A major driving force for the high rates of alanine production by skeletal muscle cells in tissue culture is the active utilization of branched chain amino acids. (2) Intracellular aspartate and glutamate pools are linked, probably via the malate-aspartate shuttle, to the cell NADH/NAD⁺ redox state. (3) Muscle cells in tissue culture metabolize significant amounts of arginine and serine in association with the production of ornithine and proline, and these pathways may possibly be related to creatine production.     

The sequences of the coenzyme-binding peptide in the cytoplasmic and the mitochondrial aspartate aminotransferases from sheep liver.

The sequences of the coenzyme-binding peptide of both cytoplasmic and mitochondrial aspartate aminotransferases from sheep liver were determined. The holoenzymes were treated with NaBH4 and digested with chymotrypsin; peptides containing bound pyridoxal phosphate were then isolated. One phosphopyridoxyl peptide was obtained from sheep liver cytoplasmic aspartate aminotransferase. Its sequence was Ser-Ne-(phosphopyridoxyl)-Lys-Asn-Phe. This sequence is identical with that reported for the homologous peptide from pig heart cytoplasmic aspartate aminotransferase. Two phosphopyridoxyl peptides with different RF values were isolated from the sheep liver mitochondrial isoenzyme. They had the same N-terminal amino acid and similar amino acid composition. The mitochondrial phosphopyridoxyl peptide of highest yield and purity had the sequence Ala-Ne-(phosphopyridoxyl)-Lys-Asx-Met-Gly-Leu-Tyr. The sequence of the first four amino acids is identical with that already reported for the phosphopyridoxyl tetrapeptide from the pig heart mitochondrial isoenzyme. The heptapeptide found for the sheep liver mitochondrial isoenzyme closely resembles the corresponding sequence taken from the primary structure of the pig heart cytoplasmic aspartate aminotransferase.     

Role of aspartate aminotransferase and mitochondrial dicarboxylate transport for release of endogenously and exogenously supplied neurotransmitter in glutamatergic neurons

Evoked release of glutamate and aspartate from cultured cerebellar granule cells was studied after preincubation of the cells in tissue culture medium with glucose (6.5 mM), glutamine (1.0 mM),d[³H] aspartate and in some cases aminooxyacetate (5.0 mM) or phenylsuccinate (5.0 mM). The release of endogenous amino acids and ofd-[³H] aspartate was measured under physiological and depolarizing (56 mM KCl) conditions both in the presence and absence of calcium (1.0 mM), glutamine (1.0 mM), aminooxyacetate (5.0 mM) and phenylsuccinate (5.0 mM). The cellular content of glutamate and aspartate was also determined. Of the endogenous amino acids only glutamate was released in a transmitter fashion and newly synthesized glutamate was released preferentially to exogenously suppliedd-[³H] aspartate, a marker for exogenous glutamate. Evoked release of endogenous glutamate was reduced or completely abolished by respectively, aminooxyacetate and phenylsuccinate. In contrast, the release ofd-[³H] aspartate was increased reflecting an unaffected release of exogenous glutamate and an increased psuedospecific radioactivity of the glutamate transmitter pool. Since aminooxyacetate and phenylsuccinate inhibit respectively aspartate aminotransferase and mitochondrial keto-dicarboxylic acid transport it is concluded that replenishment of the glutamate transmitter pool from glutamine, formed in the mitochondrial compartment by the action of glutaminase requires the simultaneous operation of mitochondrial keto-dicarboxylic acid transport and aspartate aminotransferase which is localized both intra- and extra-mitochondrially. The purpose of the latter enzyme apparently is to catalyze both intra- and extra-mitochondrial transamination of -ketoglutarate which is formed intramitochondrially from the glutamate carbon skeleton and transferred across the mitochondrial membrane to the cytosol where transmitter glutamate is formed. This cytoplasmic origin of transmitter glutamate is in aggreement with the finding thatd-[³H] aspartate readily labels the transmitter pool even when synthesis of endogenous transmitter is impaired in the presence of AOAA or phenylsuccinate.Special issue dedicated to Dr Elling Kvamme     

Nitrogen-15 spin-lattice relaxation times of amino acids in Neurospora crassa as a probe of intracellular environment

The nitrogen-15 spin-lattice relaxation time, T1, and the nuclear Overhauser enhancement (NOE) have been measured for intracellular glutamine, alanine, and arginine in intact Neurospora crassa mycelia to probe their various intracellular environments. The relaxations of 15N gamma of glutamine, 15N alpha of alanine, and 15N omega, omega ' of arginine in N. crassa were found, on the basis of their NOE values, to be predominantly the result of 15N-H dipolar relaxation. These relaxations are therefore related to the microviscosities of the various environments and associations of the respective molecules with other cellular components that act to increase the effective molecular sizes. For 15N gamma of glutamine in the cytoplasm, the intracellular T1 (4.1 s) was only slightly shorter than that in the culture medium (4.9 s). This indicates that the microviscosity of the cytoplasm surrounding the glutamine molecules is not much greater than 1.3 cP. By contrast, for 15N omega, omega ' of arginine, which is sequestered in vacuoles containing polyphosphates, the intracellular T1 (1.1 s) was only one-fourth of that in the medium (4.6 s). In model systems, the T1 of 15N omega, omega ' in a 1 M aqueous solution of arginine containing 0.2 M pentaphosphate was 0.95 s, whereas in an isoviscous (2.8 cP) solution without pentaphosphate, the T1 was 1.8 s. These results suggest either that the vacuolar viscosity is substantially above 2.8 cP or that the omega, omega '-nitrogens of vacuolar arginine are associated with a polyanion, possibly polyphosphate. The implications of these results for the properties of the vacuolar interior are discussed in relation to the mechanism of amino acid compartmentation.     

Branched-chain amino acid metabolism and alanine formation in rat muscles in vitro. Mitochondrial-cytosolic interrelationships.

Muscle branched-chain amino acid metabolism is coupled to alanine formation via branched-chain amino acid aminotransferase and alanine aminotransferase, but the subcellular distributions of these and other associated enzymes are uncertain. Recovery of branched-chain aminotransferase in the cytosol fraction after differential centrifugation was shown to be accompanied by leakage of mitochondrial-matrix marker enzymes. By using a differential fractional extraction procedure, most of the branched-chain aminotransferase activity in rat muscle was located in the mitochondrial compartment, whereas alanine aminotransferase was predominantly in the cytosolic compartment. Phosphoenolpyruvate carboxykinase, like aspartate aminotransferase, was approximately equally distributed between these subcellular compartments. This arrangement necessitates a transfer of branched-chain amino nitrogen and carbon from the mitochondria to the cytosol for alanine synthesis de novo to occur. In incubations of hemidiaphragms from 48 h-starved rats with 3mM-valine or 3mM-glutamate, the stimulation of alanine release was inhibited by 69% by 1 mM-aminomethoxybut-3-enoate, a selective inhibitor of aspartate aminotransferase. Leucine-stimulated alanine release was unaffected. These data implicate aspartate aminotransferase in the transfer of amino acid carbon and nitrogen from the mitochondria to the cytosol, and suggest that oxaloacetate, via phosphoenolpyruvate carboxykinase, can serve as an intermediate on the route of pyruvate formation for muscle alanine synthesis.