Alanine Inhibition Research Articles

SLC36A4 (hPAT4) Is a High Affinity Amino Acid Transporter When Expressed in Xenopus laevis Oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.

The pH dependence of the allosteric response of human liver pyruvate kinase to fructose-1,6-bisphosphate, ATP, and alanine

The allosteric regulation of human liver pyruvate kinase (hL-PYK) by fructose-1,6-bisphosphate (Fru-1,6-BP; activator), ATP (inhibitor) and alanine (Ala; inhibitor) was monitored over a pH range from 6.5 to 8.0 at 37 °C. As a function of increasing pH, hL-PYK’s affinity for the substrate phosphoenolpyruvate (PEP), and for Fru-1,6-BP decreases, while affinities for ATP and alanine slightly increases. At pH 6.5, Fru-1,6-BP and ATP elicit only small allosteric impacts on PEP affinity. As pH increases, Fru-1,6-BP and ATP elicit greater allosteric responses, but the response to alanine is relatively constant. Since the magnitudes of the allosteric coupling for ATP and for alanine inhibition are different and the pH dependences of these magnitudes are not similar, these inhibitors likely elicit their responses using different molecular mechanisms. In addition, our results fail to support a general correlation between pH dependent changes in effector affinity and pH dependent changes in the corresponding allosteric response.

A role for alanine in the ammonium regulation of cephalosporin biosynthesis in Streptomyces clavuligerus.

It is known that excess ammonium supply decreases cephalosporin production and represses cephalosporin synthases. We wondered whether an additional important effect could be inhibition of synthase action by alanine. We had previously shown that ammonium addition induced alanine dehydrogenase and increased intracellular alanine and that alanine could inhibit resting cell synthesis of cephalosporins. In the present work we confirm the alanine inhibition of antibiotic production by resting cells. We found L-alanine inhibited three of the four synthases tested: ACV synthetase, cyclase and expandase; the epimerase was not inhibited. These data suggest that interference in cephalosporin production by growth in ammonium salts involves synthase inhibition by intracellular alanine, in addition to the known role of ammonium in synthase repression.

Differences in tissue-specific control of phosphofructokinase and pyruvate kinase activity in the cockle, Cardium tuberculatum during exercise and anoxia

1. 1. Kinetic properties of phosphofructokinase (PFK) and pyruvate kinase (PK) were used for identification of tissue-specific isoenzymes and their anaerobic modifications in the jumping cockle ( Cardium tuberculatum). 2. 2. PFK in foot muscle and gills of normoxic animals has nearly identical kinetic properties ( S 0.5(F6P) = 1.1 mM, N H = 2.2; K A(AMP) = 0.2 mM and K A(F-2,6-P 2) = 4.6 μM). 3. 3. After anoxia the PFK in gills exhibited a reduced substrate affinity for F6P, and for the enzyme of both tissues the activating effect of AMP was diminished. 4. 4. PK from foot muscle and gills differed in the affinity for the substrate phosphoenolpyruvate ( S 0.5 = 0.13 vs 0.25 mM PEP, respectively), in the effect of l-alanine on the PEP affinity ( S 0.5 = 0.25 vs 1.1 mM PEP at 1 mM alanine), and the affinity for the activator FBP ( K A = 0.64 vs 10.9 μM FBP). 5. 5. After anoxia, the PEP affinity of the PK in gills was lowered and the inhibitory influence of alanine was increased, while anoxic modification of PK properties in muscle tissue was indicated by the reduced effectiveness of FBP in reversing alanine inhibition. 6. 6. The anoxic modification of modulator affinities of the enzymes in muscle tissue preserves the opportunity of high glycolytic flux during exercise, while the direct alteration of substrate affinities causes tight regulation.

Glycolytic and Associated Enzymes of Rainbow Trout (Oncorhynchus Mykiss) Red Cells: In Vitro And In Vivo Studies

ABSTRACT Studies were undertaken in vitro and in vivo to assess the maximal activities of 26 glycolytic and associated enzymes of rainbow trout (Oncorhynchus mykiss) red cells. The red cells possess a complete sequence of glycolytic enzymes capable of anaerobic oxidation of glucose to lactate. Red cell pyruvate kinase (PK) was inhibited by ATP (I50 ≈ 5 mmol I 1−1), but was not sensitive to alanine inhibition or fructose-1,6-bisphosphate activation. The properties of red cell PK were similar to those of the muscle-type enzyme. Lactate dehydrogenase (LDH) from the trout erythrocyte resembled LDH from trout heart in terms of pyruvate inhibition in vitro. Enzymes associated with phosphagen and amino acid metabolism as well as the pentose phosphate shunt were also present. However, enzyme indicators of glycogenolytic and gluconeogenic potential were either absent or present at very low levels. The capacity for aerobic respiration via the tricarboxylic acid (TCA) cycle was suggested by the presence of citrate synthase activity. The association of glycolytic enzymes with the particulate fraction of the red cell was assessed for six enzymes. No binding was detected for hexokinase, PK and LDH; low levels of binding by phosphofructokinase (5%), aldolase (1%) and glyceraldehyde-3-phosphate dehydrogenase (3%) were not altered by strenuous exercise. Glycolytic enzyme binding, therefore, does not appear to be an important regulator of energy metabolism in these cells. Intracellular glucose levels, in contrast, appeared to be regulated following exercise stress. An increase in intra-erythrocytic lactate levels at this time may reflect the importance of exogenously produced lactate as a substrate for ATP production. The description of trout red cell metabolism presented here provides a basis for further study of the relationships between organismic gas exchange and molecular-level adaptation of nucleated red cell function.

Regulation of glycolytic enzymes in the marine invertebrateHalicryptus spinulosus (Priapulida) during environmental anoxia and exposure to hydrogen sulfide

A particularly strong reduction of metabolic activity is a precondition for long-term survival ofHalicryptus spinulosus von Siebold under anoxic habitat conditions because of its relatively low fuel reserves (mainly glycogen). The present study analyses the mechanism of this metabolic slow-down. For this purpose the effects of environmental anoxia and exposure to hydrogen sulfide on the activity and selected kinetic properties of glycolytic enzymes [glycogen phosphorylase (GP), pyruvate kinase (PK)] and the concentrations of fructose-2,6-bisphosphate in the body wall ofH. spinulosus were analysed. Anoxia and hydrogen sulfide exposure stimulated modifications of the properties of the enzymes, in both cases due to probable covalent modification of the enzyme proteins. Under both conditions phosphorylase activity was depressed by about 1/3, the result of changes in the percentage of enzyme in the activea-form as well as the total amount of enzyme activity expressed (a +b). Effects of anoxia on the properties of pyruvate kinase included reducedV max , decreasedS0.5 for phospho-enolpyruvate, changes inK a for fructose-1,6-bisphosphate (an initial decrease was followed by a later increase). TheI50 forL-alanine of PK was extremely reduced under anoxia and showed an even greater sensitivity to the presence of hydrogen sulfide. Anoxia stimulated a slight reduction in the content of fructose-2,6-bisphosphate, whereas exposure to hydrogen sulfide caused a dramatic decrease of this allosteric activator of phos-phofructokinase. The study gives evidence that mechanisms of glycolytic rate depression are conserved within a wide variety of vertebrate and invertebrate phyla. With two exceptions (fructose-2,6-bisphosphate levels and alanine inhibition of PK) the responses to hydrogen sulfide were the same as those to anoxia, suggesting that at a metabolic level, the consequences of each stress on energy metabolism are similar.

Aspartate activation of pyruvate kinase from the kidney of Concholepas concholepas (gastropoda: muricidae)

1. 1. Kinetics of Concholepas concholepas kidney pyruvate kinase (EC 2.7.1.40) was sigmoidal with regard to phosphoenolpyruvate ( S 0.5 = 0.28 mM; n H = 1.92 ) and hyperbolic with respect to ADP ( m = 0.29 mM ). 2. 2. The enzyme was activated by aspartate ( a = 0.89 mM ) and fructose 1k,6-diphosphate ( K a = 9.8 μM ). Saturation with activators was hyperbolic. 3. 3. Co-operativity was observed with regard to the negative effector alanine ( I 0.5 = 0.23 mM; n H = 1.25 ). In the presence of 0.1 mM fructose 1,6-diphosphate, I 0.5 = 3.89 mM and n H = 2.16 . In the presence of 3 mM aspartate, I 0.5 = 0.56 mM and n H = 1.21 . Alanine inhibition was only partially reversed by the activators. 4. 4. Pyruvate kinase activity decreased from 3.45 ± 0.15 units/g wet wt in the aerobic kidney to 1.1 ± 0.10 units/g wet wt in the anoxic tissue. Kinetic parameters for substrates and allosteric effectors of the enzyme were not altered by anoxic stress. 5. 5. The results are discussed in connection with the adaptation to anoxia of the kidney of C. concholepas.

Isozymic Differentiation of Cardiac and Muscle Pyruvate Kinase in the Ringed Seal Phoca hispida

To determine whether the unusual capacity of muscle of diving mammals to undergo extended periods of anaerobiosis is due to the expression of specialized forms of the rate-limiting enzymes of glycolysis, we examined the isozymic nature of the pyruvate kinase expressed in various tissues of the ringed seal Phoca hispida. Our electrophoretic results indicate that heart and skeletal muscle contain different forms of pyruvate kinase. Both of these forms are distinct from the cathodally migrating type K isozyme in the liver. Kinetic results show that both these forms are distinct from other mammalian type M pyruvate kinases in having an acid pH optimum and a sensitivity to alanine inhibition. The affinity of the seal muscle pyruvate kinase for phosphoenolpyruvate (PEP) is lower than that of the heart form or of other mammalian type M isozymes. While the seal heart isozyme shows a decrease in affinity for PEP with a decrease in temperature, the PEP affinity of the muscle form is independent of temperature. This permits a more constant enzyme activity in the more heterothermic muscle. Muscle pyruvate kinase, with its acid pH optimum, lowered affinity for PEP, and increased sensitivity to alanine inhibition, is well suited for a tissue which undergoes prolonged anaerobic periods and then switches to aerobic utilization of lipids. We conclude that the differentiation of this distinct muscle pyruvate kinase is a special adaptation which may partially account for the ability of seal muscle to withstand considerable periods of anaerobiosis.

Pyruvate kinase in human brain tumours. Its significance in the treatment of gliomas.

Pyruvate kinase isozyme distribution was studied in 101 intracranial tumours of various nature and origin, and in normal human brain (both foetal and adult). In foetal brain, five different forms could be detected by electrophoresis (K4, K3M, K2M2, KM3, and M4). In adult brain, the M4 type, K3M hybrid, and K4 are present; the M type is largely predominant. Alanine inhibition of pyruvate kinase can be used to discriminate between M and K-type pyruvate kinase. The results obtained in an alanine inhibition test are in agreement with the electrophoretic pattern. Pyruvate kinase from foetal brain and brain of a newborn is more inhibited compared with pyruvate kinase from adult brain. In adult brain a high residual activity of pyruvate kinase is found in the presence of alanine. Well differentiated neuroepithelial tumours,i.e., astrocytomas, oligodendrogliomas, and ependymomas showed also relatively high residual activities, though less than in normal adult brain. On the contrary, in poorly differentiated gliomas low residual activity was found. Alanine inhibition of pyruvate kinase correlates well with degree of differentiation of these tumours. There is also a strong correlationship between alanine inhibition of pyruvate kinase and one year survival after “total or subtotal” resection of gliomas in adults. When in gliomas the residual activity is determined not in the centre of the tumour but more towards the periphery, much higher residual activity is found. It is suggested that brain biopsies in which a residual activity higher than 70% is found probably contain no tumour in the paraffin slides. Poorly differentiated gliomas were characterized by the presence of type K, and the hybrids K3M. In well differentiated gliomas, besides K4 and K3M, M4 was also present. Alanine inhibition was in agreement with the electrophoretic pattern in all tumours. In children (age 1–11 years) gliomas showed no correlation between the distribution of pyruvate kinase isozymes and the histological classification and grading. Of the non-neuro-epithelial tumours studied relatively high residual activities were found for pyruvate kinase in haemangioblastomas, chromophobe adenomas, and craniopharyngiomas. This was also found in an arteriovenous malformation. Other non-neuroepithelial tumours showed much less residual activity. These included benign tumours, meningiomas, neurilemmomas, malignant metastatic tumours, and fibrosarcomas. It was also found in cavernomas. The determination of pyruvate kinase activity in the presence of alanine may be useful for the diagnosis and treatment of intracerebral tumours, in particular gliomas of adults. The alanine inhibition test is a reliable quantitative procedure. It can be performed in 10 minutes, and may well fit in the scope of a surgical procedure.

Glucose and alanine inhibition of phosphate transport in renal microvillus membrane vesicles.

Transport interactions among phosphate, glucose, and alanine were evaluated in brush border membrane vesicles isolated from the rabbit renal cortex. Both glucose and alanine inhibited the Na+ gradient-stimulated uphill accumulation of phosphate. Neither glucose nor alanine inhibited the phosphate uptake measured in the absence of Na+ or in the presence of a collapsed Na+ gradient shunted with amphotericin B. Phosphate and alanine inhibited the Na+ gradient-stimulated uphill accumulation of glucose but not the glucose uptake measured in the presence of a collapsed Na+ gradient shunted with amphotericin B. Alanine, which was more potent than glucose or phosphate in inhibiting Na+ gradient-stimulated solute transport, was also more potent than glucose or phosphate in stimulating the uptake of Na+ into the vesicles. We conclude that the inhibitory interactions among phosphate, glucose, and alanine probably represent indirect effects resulting from solute-induced alterations in the transmembrane electrochemical Na+ gradient rather than direct effects resulting from competition for a polyfunctional carrier or from allosteric interactions. Such a mechanism could explain the inhibitory interactions among phosphate, glucose, and alanine that have been observed in the intact proximal tubule.

Correlation between alanine inhibition of pyruvate kinase and composition of K-M hybrids

A method is described for determination of the percentage of K-subunits in the K-M set of pyruvate kinase. The method is based on the alanine inhibition of the K-subunits. A linear correlation ( r = 0.99) is found between the alanine inhibition and the percentage of K-subunits as calculated from the electropherograms, assuming (a) a subunit distribution as indicated by the suffix in K 4, K 3M, K 2M 2, etc., and (b) an equal contribution of K- and M-subunits to the intensity of the stain.

Pyruvate kinase from the liver and kidney of Arapaima gigas

Pyruvate kinases from the kidney and liver of the osteoglossid Arapaima gigas were partially purified and characterized kinetically. The two enzymes have different elect rophoretic mobilities at pH 7.0, and while they share some qualitative similarities they show quantitative differences in their catalytic and regulatory properties. Both enzymes are activated by fructose 1.6-bisphosphate and inhibited by low levels of alanine and MgATP. The liver isozyme shows hyperbolic phosphoenolpyruvate binding, with a K1 for alanine inhibition of 0.7 mM and a K1 for MgATP inhibition of 1.0 mM. In contrast, the kidney isozyme shows cooperative phosphoenolpyruvate binding, which is accentuated at low levels of ADP. MgATP inhibition does not increase the cooperativity and shows an apparent K1 of 1.68 mM. The inhibition of alanine leads to considerable increases in the cooperativity and is effective at 1 mM and lower levels. Fructose 1.6-bisphosphate completely reverses the inhibition by alanine for both isozymes, while only leading to a partial reversal of the MgATP inhibition. These regulatory properties of both the kidney and the liver isozymes suit them for function in tissues which undergo both glycolysis and gluconeogenesis.

The effects of various modulators on the activities of human pyruvate kinases isolated from normal adult and foetal liver and hepatoma tissue

1. 1. Type L pyruvate kinase from normal adult human liver displayed allosteric kinetics towards phospho- enol-pyruvate (PEP), which were enhanced by ATP, citrate, and alanine, and eliminated by fructose-1,6-diphosphate (FDP). 2. 2. Type M pyruvate kinases from human hepatoma and human foetal liver did not show allosteric properties towards phospho- enol-pyruvate and were unaffected by fructose-1,6-diphosphate. 3. 3. ATP and citrate were more potent inhibitors of the L type than of the M type pyruvate kinase, whereas alanine and AMP inhibited the M type enzymes to a slightly greater extent. 4. 4. Alanine inhibition of type M pyruvate kinase was the only one to be relieved by fructose-1,6-diphosphate.

Reversal by aromatic amino acids of 2-thiazole-DL-alanine inhibition of Salmonella typhimurium.

Of 21 l-amino acids tested (at 1.2 x 10(-4)m), only histidine and the aromatic amino acids (phenylalanine, tryptophan, and tyrosine) protect Salmonella typhimurium strains from inhibition of growth and immediately reverse the growth inhibition by 5 x 10(-4)m 2-thiazole-dl-alanine.