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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.28, #1)

Allosteric mechanisms in ACT domain containing enzymes involved in amino acid metabolism by J. S. Liberles; M. Thórólfsson; A. Martínez (pp. 1-12).

An important sequence motif identified by sequence analysis is shared by the ACT domain family, which has been found in a number of diverse proteins. Most of the proteins containing the ACT domain seem to be involved in amino acid and purine synthesis and are in many cases allosteric enzymes with complex regulation enforced by the binding of ligands. Here we explore the current understanding of the ACT domain function including its role as an allosteric module in a selected group of enzymes. We will further describe in more detail three of the proteins where some understanding is available on function and structure: i) the archetypical ACT domain protein E. coli 3PGDH, which catalyzes the first step in the biosynthesis of L-Ser, ii) the bifunctional chorismate mutase/prephenate dehydratase (P-protein) from E. coli, which catalyzes the first two steps in the biosynthesis of L-Phe, and iii) the mammalian aromatic amino acid hydroxylases, with special emphasis on phenylalanine hydroxylase, which catalyzes the first step in the catabolic degradation of L-phenylalanine (L-Phe). The ACT domain is commonly involved in the binding of a small regulatory molecule, such as the amino acids L-Ser and L-Phe in the case of 3PGDH and P-protein, respectively. On the other hand, for PAH, and probably for other enzymes, this domain appears to have been incorporated as a handy, flexible small module with the potential to provide allosteric regulation via transmission of finely tuned conformational changes, not necessarily initiated by regulatory ligand binding at the domain itself.

Keywords: Keywords: ACT domain – Allosterism – Amino acid biosynthesis – Regulatory domain

Morphinome – proteome of the nervous system after morphine treatment by A. Bodzon-Kulakowska; A. Bierczynska-Krzysik; A. Drabik; M. Noga; A. Kraj; P. Suder; J. Silberring (pp. 13-19).

Proteome is a natural consequence of the post-genome era when the HUGO project (Human Genome Organization) has almost been completed. Here, a specifically aimed proteome in drug dependence – morphinome, is described, including tasks, strategies and pitfalls of the methodology.

Keywords: Keywords: Morphine – Dependence – Cell culture – Brain – Nervous system – Proteome – Proteins – Analysis – Peptide map – Sequence – Mass spectrometry – Electrophoresis

Involvement of polyamines in evening primrose extract-induced apoptosis in Ehrlich ascites tumor cells by T. Arimura; A. Kojima-Yuasa; Y. Tatsumi; D. O. Kennedy; I. Matsui-Yuasa (pp. 21-27).

We previously demonstrated that evening primrose extract (EPE) induced apoptosis and inhibited the DNA synthesis in Ehrlich ascites tumor cells (EATC) and suggested that EPE-induced inhibition of the growth of EATC are via at least two pathway differentially modulated by reactive oxygen species, notably intracellular peroxides. These are (a) the EPE-induced apoptosis pathway which is dependent on increases in hydrogen peroxide and (b) the EPE-induced inhibition of cell proliferation which is hydrogen peroxide independent. In this study, EPE brought about a significant decrease in intracellular polyamine levels. Furthermore, the addition of polyamines reversed the EPE-induced decrease in cell viability and suppressed the EPE-induced increase in intracellular hydrogen peroxides. However, the addition of polyamines did not reverse EPE-induced decrease in DNA synthesis and phosphorylation of Rb protein, and EPE-induced translocation of AIF. These results suggest the involvement of polyamines in the EPE-induced apoptosis pathway which is dependent on increase in hydrogen peroxide.

Keywords: Keywords: Evening primrose extract – Apoptosis – Polyamines – Hydrogen peroxide – DNA synthesis – Ehrlich ascites tumor cells

Using cellular automata to generate image representation for biological sequences by X. Xiao; S. Shao; Y. Ding; Z. Huang; X. Chen; K.-C. Chou (pp. 29-35).

A novel approach to visualize biological sequences is developed based on cellular automata (Wolfram, S. Nature 1984, 311, 419–424), a set of discrete dynamical systems in which space and time are discrete. By transforming the symbolic sequence codes into the digital codes, and using some optimal space-time evolvement rules of cellular automata, a biological sequence can be represented by a unique image, the so-called cellular automata image. Many important features, which are originally hidden in a long and complicated biological sequence, can be clearly revealed thru its cellular automata image. With biological sequences entering into databanks rapidly increasing in the post-genomic era, it is anticipated that the cellular automata image will become a very useful vehicle for investigation into their key features, identification of their function, as well as revelation of their “fingerprint”. It is anticipated that by using the concept of the pseudo amino acid composition (Chou, K.C. Proteins: Structure, Function, and Genetics, 2001, 43, 246–255), the cellular automata image approach can also be used to improve the quality of predicting protein attributes, such as structural class and subcellular location.

Keywords: Keywords: Cellular automata images – Data visualization – Pseudo amino acid composition – Bioinformatics

Defective remethylation of homocysteine is related to decreased synthesis of coenzymes B2 in thyroidectomized rats by A. Ayav; J. M. Alberto; F. Barbe; L. Brunaud; P. Gerard; M. Merten; J. L. Gueant (pp. 37-43).

We investigated the influence of hypothyroidism on homocysteine metabolism in rats, focusing on a hypothetical deficient synthesis of FAD by riboflavin kinases. Animals were allocated in control group (n = 7), thyroidectomized rats (n = 6), rats with diet deficient in vitamin B2, B9, B12, choline and methionine (n = 7), thyroidectomized rats with deficient diet (n = 9). Homocysteine was decreased in operated rats (2.6 ± 1.01 vs. 4.05 ± 1.0 μmol/L, P = 0.02) and increased in deficient diet rats (29.56 ± 4.52 vs. 4.05 ± 1.0 μmol/L, P = 0.001), when compared to control group. Erythrocyte-Glutathione-Reductase-Activation-Coefficient (index of FAD deficiency) was increased in thyroidectomized or deficient diet rats (P = 0.004 for both). Methylenetetrahydrofolate-reductase and methionine-synthase activities were decreased in thyroidectomized rats but not in those subjected to deficient diet. Cystathionine-β-synthase was increased only in operated rats. Taken together, these results showed a defective re-methylation in surgical hypothyroidism, which was due in part to a defective synthesis of vitamin B2 coenzymes. This defective pathway was overcompensated by the increased Cystathionine-β-synthase activity.

Keywords: Keywords: Thyroidectomy – Homocysteine – Vitamin B2 – MTHFR – MTR – CBS

Branched chain amino acids as a parameter for catabolism in treated phenylketonuria by S. Illsinger; T. Lücke; U. Meyer; B. Vaske; A. M. Das (pp. 45-50).

This study was performed to study an association between nutritional status on one hand and BCAA- and Phe-concentrations on the other hand in PKU patients free of infection. AA profiles from 70 PKU patients were measured. 9 patients (subgroup I) with elevated Phe- and BCAA-concentrations as well as 23 patients (subgroup II) with only elevated Phe-levels were included. Dietary records were obtained from both groups; low caloric intake in subgroup I was increased with Duocal^® or p-am ANAMIX^® without modifying total protein- and Phe-intake. AA profiles were controlled after 2 weeks.Additionally, we investigated AA profiles from 26 liver transplanted patients with increased carbohydrate and caloric intake as an example for anabolism.In subgroup I Phe- and Isoleu-concentrations decreased sign. after dietary intervention. Leu, Val and Tyr levels decreased not sign. Initial Phe-levels correlated negatively with protein and caloric intake.BCAA concentrations of liver transplanted patients receiving high amounts of carbohydrates were in the lower range of normal.Increased caloric intake lowered most of the elevated Phe- and BCAA- concentrations.

Keywords: Keywords: PKU – BCAA – Catabolism – Diet – Compliance

Infantile dilated cardiomyopathy in Portuguese water dogs: Correlation of the autosomal recessive trait with low plasma taurine at infancy by J. Alroy; J. E. Rush; S. Sarkar (pp. 51-56).

Infantile dilated cardiomyopathy (IDCM) in Portuguese water dogs (PWD) involves an autosomal recessive trait. Based on our previous studies we have tested the hypothesis that this disorder may be correlated with taurine deficiency. The plasma taurine levels of 249 puppies from 36 litters obtained from breeders at six and nine weeks of age, an early stage when usually the clinical symptoms are not manifested, were analyzed. Additional samples were collected from sixteen puppies that we raised from four litters. These litters were born to a dam that had low plasma taurine as a puppy and two known carrier sires. From the random samples obtained from the breeders, forty-eight pups from fourteen litters and twenty-nine pups from seven litters had low plasma taurine at least at one and at two time points, respectively. Also several puppies showing low plasma taurine died due to IDCM. Furthermore, from the sixteen pups we raised, fifteen had at least low taurine level at one and seven had at two time points. Considered together, these results strongly support the view that IDCM in PWD is associated with abnormal taurine metabolism that leads to low plasma taurine at early stages before the clinical symptoms appear.

Keywords: Keywords: Taurine deficiency – Canine infantile dilated cardiomyopathy – Low plasma taurine – Autosomal recessive trait

Using complexity measure factor to predict protein subcellular location by X. Xiao; S. Shao; Y. Ding; Z. Huang; Y. Huang; K.-C. Chou (pp. 57-61).

Recent advances in large-scale genome sequencing have led to the rapid accumulation of amino acid sequences of proteins whose functions are unknown. Because the functions of these proteins are closely correlated with their subcellular localizations, it is vitally important to develop an automated method as a high-throughput tool to timely identify their subcellular location. Based on the concept of the pseudo amino acid composition by which a considerable amount of sequence-order effects can be incorporated into a set of discrete numbers (Chou, K. C., Proteins: Structure, Function, and Genetics, 2001, 43: 246–255), the complexity measure approach is introduced. The advantage by incorporating the complexity measure factor as one of the pseudo amino acid components for a protein is that it can more effectively reflect its overall sequence-order feature than the conventional correlation factors. With such a formulation frame to represent the samples of protein sequences, the covariant-discriminant predictor (Chou, K. C. and Elrod, D. W., Protein Engineering, 1999, 12: 107–118) was adopted to conduct prediction. High success rates were obtained by both the jackknife cross-validation test and independent dataset test, suggesting that introduction of the concept of the complexity measure into prediction of protein subcellular location is quite promising, and might also hold a great potential as a useful vehicle for the other areas of molecular biology.

Keywords: Keywords: Pseudo amino acid composition – Complexity measure factor – Covariant-discriminant algorithm – Chou’s invariance theorem

Identification and characterisation of soluble epoxide hydrolase in mouse brain by a robust protein biochemical method by J.-H. Shin; E. Engidawork; J.-M. Delabar; G. Lubec (pp. 63-69).

The central nervous system is an important potential target for certain environmental prototoxins, but relatively little is known regarding brain-specific expression of biotransformation enzyme systems. On the other hand, developments in the field of molecular biology and advances in high-throughput screening methods continue to increase the number and amounts of available proteins. We used thus a robust and reliable technique, two-dimensional gel electrophoresis coupled to matrix assisted laser desorption/ionisation mass spectroscopy followed by tandem mass spectrometry and identified for the first time soluble epoxide hydrolase and added other biotransformation enzymes in the hippocampal region of mouse brain. Soluble epoxide hydrolase has an Mr of 61.5 kDa, pI of 5.9, twenty-six matching peptides and sequence coverage of 56% and was unambiguously identified by MS/MS. Since localised biotransformation events in regions of the central nervous system may account for pathologies and/or toxicities initiated by exposure to certain endogenous and/or environmental chemicals, identification of these enzymes would present an opportunity for developing novel therapeutic targets or would have critical toxicologic significance.

Keywords: Keywords: Soluble epoxide hydrolase – Mouse brain – Two-dimensional electrophoresis – Mass spectrometry – Biotransformation enzymes

The addition of fenugreek extract (Trigonella foenum-graecum) to glucose feeding increases muscle glycogen resynthesis after exercise by B. C. Ruby; S. E. Gaskill; D. Slivka; S. G. Harger (pp. 71-76).

The purpose of this study was to determine the effects of ingesting an oral supplement containing 4-Hydroxyisoleucine (4-OH-Ile, isolated from fenugreek seeds [Trigonella foenum-graecum]) with a glucose beverage on rates of post-exercise muscle glycogen resynthesis in trained male cyclists. Following an overnight fast (12 hr), subjects completed a 90-minute glycogen depletion ride after which a muscle biopsy was obtained from the vastus lateralis. Immediately and 2 hours after the muscle biopsy, subjects ingested either an oral dose of dextrose (Glu) (1.8 g·kg BW⁻¹) or 4-OH-Ile supplement (Glu+4-OH-Ile, including 2.0 mg·kg⁻¹ 4-OH-Ile with the same oral dose of dextrose) with a second muscle biopsy 4 hours after exercise. Post exercise muscle glycogen concentration was similar for both trials. Overall, there was a significant increase in glucose and insulin concentrations from time 0 throughout the majority of the 4-hour recovery period, with no significant differences between the two trials at any time point. Although muscle glycogen concentration significantly increased from immediately post exercise to 4 hr of recovery for both trials, the net rate of muscle glycogen resynthesis was 63% greater during Glu+4-OH-Ile (10.6±3.3 vs. 6.5±2.6 g·kg wet wt.⁻¹·hr.⁻¹ for the Glu+4-OH-Ile and Glu trials, respectively). These data demonstrate that when the fenugreek extract supplement (4-OH-Ile) is added to a high oral dose of dextrose, rates of post-exercise glycogen resynthesis are enhanced above dextrose alone.

Keywords: Keywords: Glucose uptake – 4-OH-Isoleucine – Insulin – Muscle recovery – Post-exercise nutrition

Biochemical properties of new synthetic carnosine analogues containing the residue of 2,3-diaminopropionic acid: the effect of N-acetylation by I. Cacciatore; A. Cocco; M. Costa; M. Fontana; G. Lucente; L. Pecci; F. Pinnen (pp. 77-83).

Three novel carnosine analogues 7–9 containing the residue of L(+)2,3-diaminopropionic acid with different degree of N-acetylation instead of β-alanine have been synthesized and characterized. Comparative analysis of hydrolysis by carnosinase revealed that the mono- and bis-acetylated compounds 8 and 9 are resistant to enzymatic hydrolysis and act as competitive inhibitors of this enzyme. The hydroxyl radical scavenging potential of the three analogues was evaluated by their ability to inhibit iron/H₂O₂-induced degradation of deoxyribose. The second-order rate constants of the reaction of compounds 7–9 with hydroxyl radical were almost identical to that of carnosine. These compounds were also found to act as protective agents against peroxynitrite-dependent damage as assessed by their ability to prevent nitration of free tyrosine induced by this species.

Keywords: Keywords: Carnosine – Scavengers – Peroxynitrite – Free radicals – Antioxidants – Peptidomimetics

Benzodiazepine receptor-dependent modulation of neutrophil (PMN) free amino- and α-keto acid profiles or immune functions by J. Mühling; J. Gonter; K. A. Nickolaus; R. Matejec; I. D. Welters; M. Wolff; A. Sablotzki; J. Engel; M. Krüll; T. Menges; M. Fuchs; M. G. Dehne; G. Hempelmann (pp. 85-98).

We have examined the effects of midazolam, Ro 5-4864 (agonist for “peripheral” [p] benzodiazepine receptors [BR]), PK 11195 (antagonist for pBR), flumazenil (antagonist for “central” BR), naloxone (antagonist for opiate receptors) and the combination of midazolam and Ro 5-4864, PK 11195, flumazenil or naloxone on intracellular amino- and α-keto acids and the immune function markers superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂) and released myeloperoxidase (MPO) activity in neutrophils (PMN). Only midazolam and Ro 5-4864 led to significant changes in the dynamic PMN free amino- and α-keto acid pools. Concerning PMN immune function markers, midazolam and Ro 5-4864 significantly decreased O₂⁻ and H₂O₂ formation and released MPO. When midazolam and Ro 5-4864 were applied together they appeared to act additively. Pre-incubation with PK 11195 partially neutralized the midazolam effects whereas flumazenil or naloxone showed no effects. We therefore believe that pBR are involved in the signal transmission of anesthetic-induced cellular metabolic changes in PMN.

Keywords: Keywords: Benzodiazepine receptors – Neutrophil – Amino acids – α-Keto acids – Immune function

Effect of pH and salts on the binding of free amino acids to the corn protein zein studied by thin-layer chromatography by T. Cserháti; E. Forgács (pp. 99-103).

The interaction of free amino acids with the corn protein zein was studied by thin-layer chromatography carried out on cellulose layers covered with zein and the effect of pH and salts on the strength of interaction was elucidated. Only the binding of Arg, His, Lys, Orn and Trp to zein was verified, other amino acids were not retained. Retention of Arg, His, Lys and Orn decreased linearly with increasing concentration of salts the mobile phase indicating the hydrophilic character of amino acid–zein interaction. Both alkaline and acidic pH influenced the strength of binding. Principal component analysis indicated the different character of the influence of pH and salts on the interaction. The results suggest that these amino acid residues may account for the binding of other peptides and proteins to zein.

Keywords: Keywords: Amino acids – Corn protein zein – Salt effect

Application of metal-chelate affinity chromatography to the study of the phosphoproteome by N. Imam-Sghiouar; R. Joubert-Caron; M. Caron (pp. 105-109).

With the increasing importance of proteome analysis, studying the phosphoproteome is a priority for functional studies. Therefore, a rational approach to simplifying the proteome is needed. In this work, we examined the use of immobilized metal affinity chromatography (IMAC) using ferric ions-chelated column for enriching crude cell extracts in phosphoproteins. The adsorption of the proteins on Fe³⁺ was obtained at an acidic pH 5.6, and their elution at a more basic pH in Tris buffer. To evaluate the separation, western blots were performed with either anti-phosphotyrosine or anti-phosphoserine/threonine. The analysis of the eluates demonstrated the selectivity of the separation, particularly for proteins phosphorylated on serine or threonine. In conclusion, the advantages and the limits of this approach are discussed.

Keywords: Keywords: Affinity – Chromatography – Phosphorylation – Proteomics

The influence of NMDA, a potent agonist of glutamate receptor, on behavioral activity of rats with experimental hyperammonemia evoked by liver failure by M. Fedosiewicz-Wasiluk; Z. Z. Hoły; R. J. Wiśniewska; K. Wiśniewski (pp. 111-117).

The study was designed to investigate the effects of NMDA receptor agonist on the behavioral activity in rats with experimental hyperammonemia. The experiments were performed on adult male Wistar rats. Experimental hyperammonemia was induced by intraperitoneal injections of tioacetamide (TAA, 200 mg/kg) for three consecutive days. Rats treated with saline (0.9%) served as control. Stimulation of the NMDA glutamatergic receptor was evoked by ip. injection of agonist N-methyl-D-aspartate acid (NMDA) in a dose of 30 mg/kg thirty minutes before experiments. Memory motivated affectively was evaluated in the passive avoidance responses. The speculative influence of the treatment on anxiety and motor activity was tested in elevated plus-maze and in open field respectively.To show change of NMDA receptor function after various doses of agonist, the seizures evoked by N-methyl-D-aspartate acid was carried out. This experiment showed that with rise of dose of NMDA time to appear of convulsions was contracted in rats with hyperammonemia as well as in control rats. Dose of NMDA caused convulsions was three times as less in rats with hyperammonemia than dose in control. Time of duration of convulsions was proportional to applied dose of NMDA and it lengthened with rise of agonist’s dose in both groups of studied animals.Furthermore, we observed that NMDA increased motor activity of control rats in open field test, but not in rats with hyperammonemia (treated tioacetamide). Hyperammonemia did not have significant influence on motor activity and on a passive avoidance latency. The NMDA given in control and in hyperammonemia, increased acquisition, consolidation and recall of a passive avoidance responses. Moreover, NMDA had anxiogenic-like profile in elevated plus-maze.In rats with hyperammonemia NMDA had no influence on locomotor activity but it significantly increased memory in a passive avoidance responses. Furthermore, we observed that reactivity of NMDA glutamate receptor in rats with hyperammonemia was higher than in control rats.

Keywords: Keywords: NMDA – Hyperammonemia – Behavior – Rats

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.28, #1)