Energetics of sodium-dependent α-aminoisobutyric acid transport in the moderate halophile Vibrio costicola

The energetics of α-aminoisobutyric acid transport were examined in Vibrio costicola grown in a medium containing the NaCl content (1 M) optimal for growth. Respiration rate, the membrane potential (Δψ) and α-aminoisobutyric acid transport had similar pH profiles, with optima at 8.5–9.0. Cells specifically required Na⁺ ions to transport α-aminoisobutyric acid and to maintain the highest Δψ (150–160 mV). Sodium was not required to sustain high rates of O₂-uptake. Δψ (and α-aminoisobutyric acid transport) recovered fully upon addition of Na⁺ to Na⁺-deficient cells, showing that Na⁺ is required in formation or maintenance of the transmembrane gradients of ions. Inhibitions by protonophores, monensin, nigericin and respiratory inhibitors revealed a close correlation between the magnitudes of Δψ and α-aminoisobutyric acid transport. Also, dissipation of Δψ with triphenylmethylphosphonium cation abolished α-aminoisobutyric acid transport without affecting respiration greatly. On the other hand, alcohols which stimulated respiration showed corresponding increases in α-aminoisobutyric acid transport, without affecting Δψ. Similarly, N,N′-dicyclohexylcarbodiimide (10 μM) stimulated respiration and α-aminoisobutyric acid transport and did not affect Δψ, but caused a dramatic decline in intracellular ATP content. From these, and results obtained with artificially established energy sources (Δψ and Na⁺ chemical potential), we conclude that Δψ is obligatory for α-aminoisobutyric acid transport, and that for maximum rates of transport an Na⁺ gradient is also required.     

Amino acid uptake in the developing chick embryo heart. The effect of insulin on α-aminoisobutyric acid accumulation

1. The uptake of (14)C-labelled alpha-aminoisobutyric acid by 5-day-old chick embryo hearts was investigated in vitro, together with the effect of insulin thereon. 2. At equilibrium the distribution ratio of this amino acid analogue between intracellular and extracellular water attained values greater than unity. Insulin enhanced the rate of alpha-aminoisobutyric acid accumulation and increased the value of its final concentration in the cell water. 3. The rate of alpha-aminoisobutyric acid accumulation and the effect of insulin on it were independent of the presence of glucose in the incubation medium. Bovine and chicken insulin were equally effective, and the action of the hormone was specifically prevented by an anti-insulin serum but not by puromycin. 4. A linear relationship was observed between the intracellular accumulation of the analogue and the logarithm of the insulin concentration in the range 50muunits-100m-units/ml. of incubation medium. 5. Evidence was obtained for the occurrence of two different transport processes for alpha-aminoisobutyric acid in the chick embryo heart: one subject to saturation and one that was not saturated by reasonable concentrations of the analogue. Insulin increased the effectiveness of the saturable component, increasing the maximal velocity of transport without altering the concentration for half-maximal velocity of transport, and decreased the contribution of the non-saturable component.     

Hydrophobic channels in crystals of an α-aminoisobutyric acid pentapeptide

The crystal structure of the pentapeptide p-toluene-sulfonyl-(α-aminoisobutyryl)₅-methyl ester (Tosyl-(Aib)₅-OMe) has been determined in the space group P$\text{I}$. Pentapeptide molecules are folded in the 3₁₀ helical conformation and packed together, so as to yield a hydrophobic channel with a minimim diameter of 5.2 Å. The channel contains crystallographically disordered material. This structure provides a model for channel formation by hydrophobic peptide aggregates and should prove useful in studies of alamethicin, suzukacillin and related Aib containing membrane channels. Triclinic (P$\text{I}$) crystals of the pentapeptide are obtained in the presence of LiClO₄ in aqueous methanol, whereas crystallization from methanol alone yields crystals in the space group Pbca. The conformations of the peptide in the two crystal forms are very similar and only the molecular packing is dramatically different.     

The requirement for the protonated α-amino group for the transport of peptides in Escherichia coli

Many glycine peptides support growth of a glycine auxotroph of Escherichia coli. If the alpha-amino group of these peptides is methylated, the products are still utilized for growth, and also retain comparable ability with the unsubstituted peptides to compete with natural peptides for transport into the cell. In contrast, glycine peptides devoid of an alpha-amino group, or that have the alpha-amino group substituted by one of a number of acyl groups are not utilized, although E. coli possesses intracellular enzymic activity able to release glycine from such compounds; further, these derivatives do not compete with natural peptides for transport into the cell.     

Metabolism of α-aminoisobutyric acid in mungbean hypocotyls in relation to metabolism of 1-aminocyclopropane-1-carboxylic acid

1-Aminocyclopropane-1-carboxylic acid (ACC) is known to be converted to ethylene and conjugated into N-malonyl-ACC in plant tissues. When -amino[1-¹⁴C]isobutyric acid (AIB), a structural analog of ACC, was administered to mungbean (Vigna radiata L.) hypocotyl segments, it was metabolized to ¹⁴CO₂ and conjugated to N-malonyl-AIB (MAIB). -Aminoisobutyric acid inhibited the conversion of ACC to ethylene and also inhibited, to a lesser extent, N-malonylation of ACC and d-amino acids. Although the malonylation of AIB was strongly inhibited by ACC as well as by d-amino acids, the metabolism of AIB to CO₂ was inhibited only by ACC but not by d-amino acids. Inhibitors of ACC conversion to ethylene such as anaerobiosis, 2,4-dinitrophenol and Co²⁺, similarly inhibited the conversion of AIB to CO₂. These results indicate that the malonyalation of AIB to MAIB is intimately related to the malonylation of ACC and d-amino acids, whereas oxidative decarboxylation of AIB is related to the oxidative degradation of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyric acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid - MAIB -(malonylamino)-isobutyric acid - Mes 2-(N-morpholino)ethanesulfonic acid     

The effect of α-aminoisobutyric acid on wood decay and wood spoilage fungi

The amino acid analogue α-aminoisobutyric acid (AIB) decreased linear extension growth in fifteen out of sixteen wood decay and wood spoilage fungi. In Serpula lacrimans inhibition of extension growth by AIB was accompanied by an increase in the frequency with which the hyphae of the fungus initiated branches. AIB was shown to have a preservative effect against Lentinus lepideus, Serpula lacrimans and Pleurotus ostreatus when wood blocks were impregnated with this chemical prior to challenge by cultures of these fungi. The effectiveness of this compound in limiting growth in a large number of different fungi suggests that competitive inhibitors of nitrogen uptake and metabolism could be used to control fungi which decay wood and similar materials, and may also have wider applications.     

Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K^–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

Relationship between transport and metabolism of α-naphthaleneacetic acid, β-naphthaleneacetic acid and α-decalylacetic acid in segments of Coleus

Summary Transportand metabolism of -naphthaleneacetic acid -naphthaleneacetic acid, and -decalylacetic acid, all labelled with ¹⁴C in the carboxyl, group, were studied. Only -naphthaleneacetic acid is transported in a polar way. Most of the radioactivity in the tissue is in a low molecular form, either free or as immobilization products. The immobilization of -naphthaleneacetic acid is similar to that of -naphthaleneacetic acid. Immobilization of -decalylacetic acid is typically different. Bioassays showed -naphthaleneacetic acid as the sole biologically active component. It is concluded that stereo requirements necessary for biological activity are also required for polar auxin transport. It is further concluded that the observed specificity of the transport system is not related to the formation of immobilization products.     

The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers

The subcellular site of -amylase (EC 1.6.2.1) synthesis and transport was studied in barley aleurone layers incubated in the presence or absence of gibberellic acid (GA₃). Using [³⁵S]methionine as a marker, the site of amino-acid incorporation into organelles isolated from aleurone layers incubated with and without GA₃ was determined following purification by isopycnic sucrose-density-gradient centrifugation. Incorporation of radioactivity into trichloroacetic-acid-insoluble proteins was greatest in those fractions exhibiting activity of an endoplasmic reticulum (ER) marker enzyme. Further fractionation of densitygradient fractions by sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis showed that a major portion of the radioactivity in the ER fractions was present in a protein co-migrating with marker -amylase. This protein was identified as authentic -amylase by immunoadsorbent chromatography and affinity chromatography. The newly synthesized -amylase associated with the ER was shown to be sequenstered within the lumen of the ER by experiments which showed that the enzyme was resistant to proteolytic degradation. The labelled -amylase sequestered in the ER can be chased from this organelle when tissue is incubated in unlabelled methionine following a 1-h pulse of labelled methionine. The isoenzymic forms of -amylase found in tissue homogenates and incubation media of aleurone layers incubated with and without GA₃ were characterized after chromatography on diethylaminoethyl cellulose. In homogenates of GA₃-treated aleurone layers, five peaks of -amylase activity were detected, while in homogenates of aleurone layers incubated with-out GA₃ only three peaks of activity were found. In incubation media, four isoenzymes were found after GA₃ treatment and two were found after incubation without GA₃. We conclude that at least five -amylase isoenzymes are synthesized by the ER of barley aleurone layers and that this membrane system is involved in the sequestration and transport of four of these isoenzymes.Abbreviations CHA cyclohepataamylose - DEAE-cellulose diethylaminoethyl-cellulose - ER endoplasmic reticulum - GA₃ gibberellic acid - SDS-PAGE sodium-dodecyl-sulfate polyacrylamide-gel electrophoresis     

The complete rate equation,including the explicit dependence on Na+ ions,for the influx of α-aminoisobutyric acid into mouse brain slices

Summary The rate equation, including dependence on Na⁺-ion concentration for the influx of -aminoisobutyric acid into mouse brain slices incubated in isotonic glucose medium at 37°C, isv=0.402S/{1.02(1+788/[Na⁺]²)+S}+0.0477S, wherev=influx in mol/min, g final wet wt of slices; [Na⁺]=concentration of Na⁺ ions in medium, inmm; andS=concentration of -aminoisobutyric acid in medium, inmm. This equation shows two kinetically independent, parallel pathways of concentrative uptake: one, saturable and dependent on Na⁺; the other, unsaturable and independent of Na⁺. Influx is independent of ionic strength, Cl^– ionper se, and a moderate increase in tonicity. The binding of substrate to the saturable carrier depends on the Na⁺ concentration; the maximum capacity of this carrier does not. For transport, 2 Na⁺ ions must interact with each saturable transport site. This does not imply coupling between the flux of Na⁺ and the flux of -aminoisobutyric acid.     

“Exchange diffusion”: Rate equations for the influx of α-aminoisobutyric acid into mouse cerebrum slices containing this amino acid

Rate equations for the gross influx of -aminoisobutyric acid (AIB) into mouse cerebrum slices containing AIB have a first-order term for unsaturable concentrative influx, identical to the corresponding term for unloaded slices, and a modified Michaelis-Menten term,V_max/(1+K _t /S), for saturable concentrative influx. [V_max v _L (1+K _t /S), wherev _L =saturable component of influx,S=AIB concentration in medium, andK _t =Michaelis constant for unloaded slices.] Below a tissue AIB (T) of 19 µmol/g final wet weight,V_max increases linearly followingV_max=V ₁+m ₁ T; above that value,V _max is virtually constant. The transition is sharp. This equation is consistent with a carrier model for active transport. At the transition, intracellular AIB is about 1 molecule for every 70 amino acid residues of tissue protein, vastly more than could be accommodated by AIB-binding sites in cell membranes. The transition may come from a slow process that does not fill all sites when the tissue AIB is below the transition concentration, or from an AIB-induced phase transition in the membrane.Nomenclature AIB -aminoisobutyric acid - A radioactivity of reference; unspecified amino acid - C counts in tissue sample; carrier for transport - C _i carrier in form that reacts with intracellular substrate - C _o carrier in form that reacts with extracellular substrate - C _R counts in reference - CS complex of substrate with carrier - (CS) _i complex of substrate with carrier in formC _i - (CS) _o complex of substrate with carrier in formC _o - G counts per gram of tissue - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - k _u rate constant for first-order unsaturable uptake - K,K ,K ,K ,K _d adjustable parameters in Eqs. (9)–(13) for v, analogous to the Michaelis constant - K _d dissociation constant - K _t Michaelis constant for saturable uptake - K _t Michaelis constant for gross saturable uptake by tissue containing substrate - m ₁,m ₂ slope in Eq. (5) or (6) expressing dependence ofV_max onT orT _i ^w in Region 1 or 2 - M binding site for amino acid A - n number of data points - P number of parameters to be determined; parameter in Stein's (1981) equation, Eq. (17) in this paper - P ₁,P ₂,P ₁₂ property of tissue with unoccupied binding sites, property of tissue with occupied binding sites, property of tissue with both unoccupied and occupied binding sites, respectively - Q parameter in Stein's (1981) equation, Eq. (17) in this paper - r Pearson's correlation coefficient - Relative error RE =100{[(observed quantity – calculated quantity)/calculated quantity]²/(n –P)}^1/2 - S concentration of substrate in medium; transport substrate - S _i intracellular transport substrate - S _int AIB in medium corresponding to intracellular AIB at intersection - S _o extracellular transport substrate - T observed concentration of substrate in tissue including substrate in extracellular space and adherent fluids - T _i intracellular concentration of substrate - T _int tissue AIB corresponding to intracellular AIB at intersection - T _i ^w ,T _i ^/30 intracellular concentration of substrate withw% (30%) extracellular and adherent fluids - U observed uptake of labeled substrate by incubated tissue including substrate in extracellular and adherent fluids - U _R observed uptake of labeled substrate referred to concentration of substrate in medium - U _max adjustable parameter in Eqs. (9)–(15) for v, analogous to the Michaelis-Menten maximum rate,V _max - v influx of substrate - v _L gross influx of substrate into tissue containing substrate - v _L contribution of saturable component to gross influx into tissue containing substrate - v incremental influx, that is, gross influx into tissue that contains substrate minus influx under the same conditions into tissue that does not contain substrate - V ₁,V ₂ intercept in Eq. (5) or (6) expressing dependence ofV_max onT orT _i ^w in Region 1 or 2, respectively - V _max maximum rate in Michaelis-Menten equation - V_max apparent maximum rate defined byV_maxv_max(1+K _t /S) - V_{max 1},V_{max 2} apparent maximum rate in Region 1 or 2, respectively - V_int apparent maximum rate at intersection defining boundary between Regions 1 and 2 - w weight of incubated tissue - W _d dry weight of tissue expressed as fraction by weight - W _e extracellular and surface space of incubated tissue expressed as percent by weight - , , adjustable parameters in modified expressions for gross unsaturable influx into tissue containing substrate - , , , exponents ofS orT in Eqs. (9)–(13) for v - parameter in Stein's (1981) equation, Eq. (17), corresponding more or less tom ₁ For my wife, Lynn.     

The phospholipid base-exchange system as a possible modulator of γ-aminobutyric acid transport in brain cells

Mixed cell suspensions from rabbit brain have been used to study the effect of base exchange in membrane phospholipids, on amino acid accumulation in vitro. -Aminobutyric acid (GABA), glutamic acid, and aminoisobutyric acid have been used. The accumulation of [³H]GABA, at concentrations employing the high-affinity uptake system, was measured after base-exchange reactions with ethanolamine, choline, orL-serine. Serine incorporation induced an increase of GABA uptake at all the concentrations used, while choline incorporation essentially led to inhibition of GABA accumulation. Ethanolamine exchange produced both stimulation and inhibition. The observed effects were not specific for GABA. Neuronal and glial cell perikarya and synaptosomes were studied in the same system in an attempt to resolve the complex type of response obtained with the mixed suspension. Cell specificity was found with respect to stimulation or inhibition of GABA transport after base exchange but, in some cases, the isolated fractions retained the multiphasic response observed with the mixed suspension.     

Use ofα-aminoisobutyric acid and isovaline as marker amino acids for the detection of fungal polypeptide antibiotics. Screening ofHypocrea

Summary Filamentous fungi of the genusHypocrea were grown on malt extract/peptone agar, the mycelia were extracted with dichloromethane/methanol, and the extracts were totally hydrolyzed with 6 N HCl (110°C, 24 h). The amino acids (AA) released from peptides were converted into theirN(O)-pen-tafluoropropionyl 1-propyl esters and investigated by gas chromatography and gas chromatography/mass spectrometry for the presence of the nonprotein AA-aminoisobutyric acid (Aib) and its homologue isovaline (Iva). In particular Aib served as specific marker compound for a particular group of fungal peptides named peptaibiotics,i.e. peptides containing Aib and having antibiotic activities. Screening of 24 species ofHypocrea revealed that the majority was capable of producing peptaibiotics. The reliability of the screening procedure was shown with the isolation of peptaibiotics fromHypocrea muroiana andHypocrea nigricans. These findings extend the list of genera of fungi already known to produce Aib-containing peptides and also establish that Aib and Iva are fairly common in the biosphere.     

Electrochemical investigation of active malic acid transport at the tonoplast into the vacuoles of the CAM plantKalanchoë daigremontiana

Summary The membrane potential of cells in leaf slices of the CAM plantKalanchoë daigremontiana Hamet et Perrier in the light and in the dark is –200 mV on the average; it is reversibly depolarized by the metabolic inhibitors FCCP (5×10^–6 m) and CN^– (5×10^–3 m); it shows the light-dependent transient oscillations ubiquitously observed in green cells; it is independent of the amount of malic acid accumulated in the cells (in a tested range between 30 and 140mm); and it is considerably hyperpolarized by the fungal toxin fusicoccin (30×10^–6 m). Fusicoccin inhibits nocturnal malic acid accumulation in intact isolated phyllodia of the CAM plantKalanchoë tubiflora (Harv.) Hamet but does not affect remobilization of malic acid during the day.Electrochemical gradients for the various ions resulting from dissociation of malic acid, i.e., H⁺, Hmal^– and mal^2–, were calculated using the Nernst equation. With a very wide range of assumptions on cytoplasmic pH and malate concentration results of calculations suggest uphill transport of H⁺ and Hmal^– from the cytoplasm into the vacuole, while mal^2– might be passively distributed at the tonoplast. On the basis of the present data the most likely mechanism of active malic acid accumulation in the vacuoles of CAM plants appears to be an active H⁺ transport at the tonoplast coupled with passive movement of mal^2– possibly mediated by a translocator (catalyzed diffusion), with subsequent formation of Hmal^– (2 H⁺+mal^2–H⁺+Hmal^–) at vacuolar pH's.     

Involvement of binding lipoproteins in the absorption and transport of α-tocopherol in the rat

1. Specific lipoproteins binding alpha-tocopherol but not its known metabolites have been isolated and identified from cytosol of rat intestinal mucosa and from serum. 2. A timestudy of the appearance of the orally administered alpha-[(3)H]tocopherol with these lipoproteins indicates that very-low-density lipoprotein of serum acts as a carrier of the vitamin. 3. The involvement of the mucosal lipoprotein in the absorption of the vitamin from the intestine has been inferred from observations on the amounts of alpha-tocopherol in serum of orotic acid-fed rats where release of lipoproteins from the liver to serum is completely inhibited. A considerable decrease in the association of alpha-tocopherol with serum very-low-density lipoprotein under this condition is interpreted to mean that serum lipoproteins are limiting factors for the transport of the vitamin across the intestine and that this is possibly effected by exchange of alpha-tocopherol between serum very-low-density lipoprotein and mucosal lipoprotein.     

The properties of “active” Cl transport across the cornea of the toad Bufo marinus

• 1.1. Active Cl transport occurs from the endothelial to epithelial side of the cornea of Bufo marinus. Na transport is much less and is in the opposite direction.
• 2.2. The rate of Cl transport is not saturable and is linearly related to the Cl concentration on the endothelial side.
• 3.3. Active efflux (endo to epi) of Cl is reduced (50%) by CN and abolished by IAA. Ouabain and Na-free solutions on the endothelial side also reduce Cl efflux.
• 4.4. O₂ consumption or lactate production are also decreased by ouabain or Na-free solutions. However, metabolism was not inhibited by Cl-free solutions or a specific Cl blocking drug bumetanide.
• 5.5. Cl transport exhibits some rather unusual characters and a model is proposed to account for them which involves an exchange of Cl for metabolically produced organic anions.

     

The detection of the messenger ribonucleic acid for the α-lactalbumin of guinea-pig milk (Short Communication)

RNA was extracted from the polyribosomes isolated from the mammary glands of a lactating guinea pig and injected into Xenopus oocytes. On incubation the oocytes effected the biosynthesis of alpha-lactalbumin.     

The role of amino acid residues in the active site of L-methionine γ-lyase from Pseudomonas putida

Cys116, Lys240*, and Asp241* (asterisks indicate residues from the second subunit of the active dimer) at the active site of L-methionine γ-lyase of Pseudomonas putida (MGL_Pp) are highly conserved among heterologous MGLs. In a previous study, we found that substitution of Cys116 for His led to a drastic increase in activity toward L-cysteine and a decrease in that toward L-methionine. In this study, we examined some properties of the C116H mutant by kinetic analysis and 3D structural analysis. We assumed that substitution of Cys116 for His broke the original hydrogen-bond network and that this induced a significant effect of Tyr114 as a general acid catalyst, possibly due to the narrow space in the active site. The C116H mutant acquired a novel β-elimination activity and lead a drastic conformation change in the histidine residue at position 116 by binding the substrate, suggesting that this His residue affects the reaction specificity of C116H. Furthermore, we suggest that Lys240* is important for substrate recognition and structural stability and that Asp241* is also involved in substrate specificity in the elimination reaction. Based on this, we suggest that the hydrogen-bond network among Cys116, Lys240*, and Asp241* contributes to substrate specificity that is, to L-methionine recognition at the active site in MGL_Pp.