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Summary: T cell stimulation is metabolically demanding. To exit quiescence, T cells rely on environmental nutrients, including glucose and the amino acids glutamine, leucine, serine, and arginine. The expression of transporters for these nutrients is tightly regulated and required for T cell activation. In contrast to these amino acids, which are essential or require multi-step biosynthesis, alanine can be made from pyruvate by a single transamination. Here, we show that extracellular alanine is nevertheless required for efficient exit from quiescence during naive T cell activation and memory T cell restimulation. Alanine deprivation leads to metabolic and functional impairments. Mechanistically, this vulnerability reflects the low expression of alanine aminotransferase, the enzyme required for interconverting pyruvate and alanine, whereas activated T cells instead induce alanine transporters. Stable isotope tracing reveals that alanine is not catabolized but instead supports protein synthesis. Thus, T cells depend on exogenous alanine for protein synthesis and normal activation. : In health, T lymphocytes are in a resting state. However, stimulation with their cognate antigen induces massive growth and proliferation. Ron-Harel et al. demonstrate that T cells rely on extracellular alanine for activation. Consumed alanine is used primarily for protein synthesis, and alanine deprivation inhibits T cell metabolism and effector functions. Keywords: T cells, T cell activation, protein synthesis, metabolism, alanine

Alkaloids are a large cluster of molecules found in Mother Nature all over the world. They are all secondary compounds and collection of miscellaneous elements and biomolecules, derived from amino acids or from transamination. This diverse chemical group is categorized, based on the amino acids that deliver their nitrogen atom and part of their skeleton. Alkaloids from a similar origin or having the same basic nucleus may have dissimilar biosynthetic pathways and different biological activity. They are derived from l -phenylalanine, l -tyrosine, anthranilic acid or acetate, l -histidine, l -ornithine, nicotinic acid, and l -lysine. Apart from other types of alkaloids, indole, tropane, and isoquinoline alkaloids are very important. People from all over the world are using them in their everyday life. Alkaloids can also have an animal origin, which may be endogenous or exogenous. This chapter highlights the phytochemistry of the main representatives among the diverse group of alkaloids. This chapter also focuses on their extraction, purification, fractionation, identification, and quantification procedures. A fundamental understanding of the biological activity of important alkaloids is also highlighted in this chapter. With the potential of revealing new compounds and diverse pharmacological properties, alkaloids still embrace a great potential for the future of drug discovery.

Enzyme catalysis is the primary activity in energy and information metabolism and enzyme cofactors are key to the catalytic ability of most enzymes. Pyridoxal 5'-phosphate (PLP) cofactor, derived from Vitamin B6, is widely distributed in nature and has significant latitude in catalytic diversity. X-ray crystallography has revealed the structures of diverse PLP dependent enzymes from multiple families. But these structures are incomplete, lacking the positions of protons essential for understanding enzymatic mechanisms. Here, we review the diversity of PLP and discuss the use of neutron crystallography and joint X-ray/neutron refinement of Fold Type I aspartate aminotransferase to visualize the positions of protons in both the internal and external aldimine forms. Strategies used to prepare extremely large crystals required for neutron diffraction and the approach to data refinement including the PLP cofactor are discussed. The observed positions of protons, including one located in a previously unknown low-barrier hydrogen bond, have been used to create more accurate models for computational analysis. The results revealed a new mechanism for the transaminase reaction where hyperconjugation is key to reducing the energy barrier which finally provides a clear explanation of the Dunathan alignment.

Vitamin B6 comprises six compounds based on a 2-methyl-3-hydroxypyridine structure with differing subunits at positions C4 and C5 that are interconvertible. These are pyridoxine, pyridoxamine, and pyridoxal and their phosphorylated derivatives pyridoxine-5-phosphate, pyridoxamine-5-phosphate, and pyridoxal-5-phosphate (PLP). PLP is a cofactor in > 150 different enzyme reactions including transamination and decarboxylation enzyme systems. The symptoms of vitamin B6 deficiency include peripheral neuropathy, pellagra-like syndrome with seborrheic dermatitis and glossitis. Prolonged deficiency can lead to depression, confusion, and in severe cases causes abnormalities in EEG signals and seizures. The assessment of vitamin B6 status can be undertaken through direct measurement of vitamin B6 compounds or using indirect markers that reflect the effect of deficiency on the various pathways involving PLP-dependent enzymes, e.g., kynurenines, transaminases, and various amino acids.

Metabolism of amino acids. Hyperammonemia is the condition discussed. Other topics in this chapter are as follows: urea cycle, transamination, transamidation, deamination, amino acid racemization, l -amino acid decarboxylation, metabolism of amino acids to active substances, formation of catecholamines, melanin, phenylalanine and tyrosine blockages, and diseases and catabolism of amino acids. The chapter ends with a summary, a list of papers and books as suggested reading, summary multiple-choice questions, and a case-based problem.

To investigate the effect of low light (LL, 50% natural light) during grain filling (GF) stage on rice transamination, amino acid (AA) accumulation, nutritional value, and cooking quality in three different rice genotypes, transaminase activities and AA levels in grains during GF stage and the traits that significantly affected rice quality (physical appearance, cooking quality, and nutritional value) were analyzed. LL did not disturb transamination in rice grains during GF stage, as minimal impact was found on alanine and aspartate transaminase activities. Nevertheless, most AAs in caryopses, including lysine and threonine, increased in response to LL, except for sulfur-containing AAs. These results suggest that AA metabolism and accumulation in rice grains were rarely suppressed by LL during GF stage. Rice nutritional ingredients at harvest, such as major protein components including glutelin and most important essential amino acids (EAAs) including lysine and threonine, increased significantly in response to LL, whereas most protein and EAA ratios were rarely affected. However, LL markedly affected physical appearance of rice grains by reducing brown rice rate, milled rice rate, and 1000-grain weight and increasing the chalkiness rate. In addition, cooking qualities decreased in response to LL, while breakdown values and amylose levels decreased and setback values increased. We concluded that LL during GF stage decreased the cooking quality of rice, but could potentially improve the nutritional value of rice.

Amino acids (AA) from the diet join those generated by the degradation of endogenous proteins to form a common metabolic pool in the body. Once at the end of their life, proteins are degraded in lysosomes and in the ubiquitin–proteasome system. Nitrogen balance is the equilibrium that exists in normal well-fed adults between the intake of nitrogen (represented mainly by the protein content of the diet) and the nitrogen excreted via the urine and feces. AA are used in the synthesis of body proteins and other nitrogenous compounds or, if energy is needed, they are catabolized. The amine group is separated by transamination. This reaction transfers the α-amino group of AA to an α-keto acid. The reaction is catalyzed by transaminases or aminotransferases. Except for lysine and threonine, all AA participate in transamination reactions with pyruvate, oxaloacetate, or α-ketoglutarate to form alanine, aspartate, or glutamate, respectively, and the α-keto acids corresponding to the original AA. In turn, alanine and aspartate undergo transamination with α-ketoglutarate, with the amine groups of all AA converging to glutamate. Deamination of glutamate produces α-ketoglutarate and ammonia, which is converted to urea. Another mechanism of removal of ammonia is the formation of glutamine. Urea is formed in liver by a metabolic cycle and eliminated by the kidneys. The C skeletons of AA are metabolized to produce pyruvate or intermediates of citric acid cycle (glucogenic) and others, acetyl-CoA or acetoacetate (ketogenic). Other general mechanisms for AA degradation include decarboxylation, which leads to formation of biogenic amines. Transfer of monocarbon groups from AA (methyl, OH-methyl, formyl, and CO2) are used in various processes of syntheses, catalyzed by specific methyltransferases. Each AA has different metabolic pathways that catabolize them producing energy or different compounds. Thus, phenylalanine is converted to tyrosine, and can produce catecholamines and melanin. Tryptophan produces serotonin, melatonin, and nicotinic acid (a vitamin of B complex). Arginine generates nitric oxide. Glycine participates in biotransformation reactions, cysteine gives taurine, methionine and cysteine give S, which is used in sulfoconjugation. Arginine, glycine, and methionine participate in the synthesis of creatine, glutamate and cysteine in the synthesis of glutathione.

Trans-cinnamaldehyde, the major compound of cinnamon essential oil, is a potentially interesting natural antimicrobial food preservative. Although a number of studies have addressed its mode of action, the factors that determine bacterial sensitivity or tolerance to trans-cinnamaldehyde are poorly understood. We report the detailed characterization of a Listeria monocytogenes Scott A trans-cinnamaldehyde hypersensitive mutant defective in IlvE, which catalyzes the reversible transamination of branched-chain amino acids to the corresponding short-chain α-ketoacids. This mutant showed an 8.4 fold extended lag phase during growth in sublethal concentrations (4 mM), and faster inactivation in lethal concentrations of trans-cinnamaldehyde (6 mM). Trans-cinnamaldehyde hypersensitivity could be corrected by genetic complementation with the ilvE gene and supplementation with branched-chain α-ketoacids. Whole-cell fatty acid analyses revealed an almost complete loss of anteiso branched-chain fatty acids (BCFAs), which was compensated by elevated levels of unbranched saturated fatty acids and iso-BCFAs. Sub-inhibitory concentrations of trans-cinnamaldehyde induced membrane fatty acid adaptations predicted to reduce membrane fluidity, possibly as a response to counteract the membrane fluidizing effect of trans-cinnamaldehyde. These results demonstrate the role of IlvE in BCFA production and the role of membrane composition as an important determinant of trans-cinnamaldehyde sensitivity in L. monocytogenes. ispartof: Research in Microbiology vol:168 issue:6 pages:536-546 ispartof: location:France status: published

‘Systems Biocatalysis’ is a term describing multi-enzyme processes in vitro for the synthesis of chemical products. Unlike in-vivo systems, such an artificial metabolism can be controlled in a highly efficient way in order to achieve a sufficiently favourable conversion for a given target product on the basis of kinetics. However, many of the most interesting non-natural chemical reactions which could potentially be catalysed by enzymes, are thermodynamically unfavourable and are thus limited by the equilibrium position of the reaction. A good example is the enzyme ω-transaminase, which catalyses the transamination of a pro-chiral ketone into a chiral amine (interesting in many pharmaceutical applications). Here, the products are often less energetically stable than the reactants, meaning that the reaction may be thermodynamically unfavourable. As in nature, such thermodynamically-challenged reactions can be altered by coupling with other reactions. For instance, in the case of ω-transaminase, such a coupling could be with alanine dehydrogenase. Herein, the aim of this work is to identify thermodynamic bottlenecks within a multi-enzyme process, using group contribution method to calculate the Gibbs free energy change, Δ G r o ′ , of the overall cascade. The findings show that unfavourable reactions in the cascade can be improved by coupling to a favourable reaction giving more energetically stable products.

Alkaloids are a group of molecules with a relatively large occurrence in nature around the globe. They are very diverse chemicals and biomolecules, but they are all secondary compounds and are derived from amino acids or from the transamination process. Alkaloids are classified according to the amino acids that provide their nitrogen atom and part of their skeleton. Similar alkaloids can have quite different biosynthetic pathways and different bioimpacts. Alkaloids are derived from l-lysine, l-ornithine, l-tyrosine, l-tryptophan, l-histidine, l-phenylalanine, nicotinic acid, anthranilic acid, acetate, or amination and transamination reactions. The terpenoid, steroid, and purine alkaloids are also important. Millions of people around the globe use purine alkaloids every day whether starting the day with a cup of coffee or drinking a cup of tea in the afternoon. Alkaloids also occur in the animal kingdom. Unlike plants, the source of these molecules in an animal’s body can be endogenous or exogenous. Alkaloids are molecules participating in both producer and consumer chains in nature. They are vital in feeding and enjoy activation, aggression, and defense of various species. Homo sapiens is one of them.

Amino acids released from dietary or intracellular proteins are catabolized in similar fashions. Their degradation begins with the removal of the a-amino group, thus leaving an a-keto acid hydrocarbon skeleton. Alpha-amino group removal from amino acids occurs through either transamination or deamination reactions. Transamination reactions are catalyzed by transaminases (also called aminotransferases). There are four coenzymes intimately involved in amino acid catabolism: pyridoxal phosphate (derived from vitamin B6), tetrahydrofolic acid (THFA), biotin (a CO2 shuttler), and vitamin B12 (cobalamin). The oxidative deamination of Glu, catalyzed by mitochondrial GLDH, results in the direct release of ammonium ion (NH4 +), which can then be used for mammalian urea synthesis in the liver. The side chain amide groups of glutamine (Gln) and asparagine (Asn) can be released by hydrolysis, also resulting in NH4 + formation. Glutaminase and asparaginase, respectively, catalyze these reactions. During starvation, Ala is important in transferring amino groups from skeletal muscle to liver, where they can be incorporated into urea or Gln, and the pyruvate hydrocarbons remaining can be incorporated into glucose. Aspartate Aminotransferase is present in both the cytosol and mitochondria of liver cells, and both fractions can be separated electrophoretically. Other transaminases also exist in mammalian organisms (e.g., cysteine transaminase, ornithine transaminase, and tyrosine a-ketoglutarate transaminase). Since there are no reported metabolic defects associated with reactions catalyzed by any of the transaminases, like reactions of the tricarboxylic acid cycle, all are considered essential for life.

Beta-hydroxy-beta-methylbutyrate (HMB) is a metabolite of the amino acid leucine. The first step in HMB metabolism is the reversible transamination of leucine to α-ketoisocaproate (KIC), which occurs mainly extrahepatically. Under normal conditions the majority of KIC is converted into isovaleryl-CoA, in which approximately 5% of leucine is metabolized into HMB. An individual weighing 70 kg produces about 0.2 to 0.4 g of HMB per day from dietary L-leucine. The vast majority of studies have employed 3 g/day of HMB, grounded in evidence that this dose produces better results than 1.5 g/day and is equivalent to 6 g/day. This metabolite has received attention due to its anti-catabolic properties, through inhibition of muscle proteolysis and enhancement of protein synthesis. The aims of HMB supplementation are to counteract catabolic conditions, as well to enhance skeletal muscle mass and strength in athletes, improving performance. Moreover, HMB has been shown to produce an important effect in reducing muscle damage induced by mechanical stimuli of skeletal muscle. HMB acts as precursor to the rate-limiting enzyme HMG-coenzyme A reductase in cholesterol synthesis, which can enhance sarcolemmal integrity. For these reasons, HMB supplementation has been adopted as an alternative by those practicing resistance exercise or weight training, by individuals under extreme muscular stress, by elderly individuals, and by patients with diseases associated with muscle-wasting syndromes, such as cachexia. This chapter discusses: aspects of HMB metabolism; supplementation protocols; effects on contractile function and energetic metabolism; and mechanisms of action, emphasizing molecular mechanisms described by recent findings.

Branched-chain amino acids (BCAAs) leucine, isoleucine, and valine in the muscle are converted into branched-chain α -ketoacids (BCKAs) through transamination. BCKAs circulated to the liver then undergo oxidative decarboxylation, mediated by the mitochondrial BCKA dehydrogenase complex (BCKDC), to produce branched-chain acyl-coenzymes. Genetic defects in the BCKDC result in maple syrup urine disease (MSUD), characterized by often-fatal ketoacidosis, neurological derangements, and mental retardation. The rate-limiting step in BCAA oxidation is at the BCKDC, which is acutely regulated by phosphorylation (inactivation)/dephosphorylation (reactivation), in response to hormonal and nutritional stimuli. Among BCAAs, leucine serves as key nutrition and endocrine signals to promote protein synthesis in muscle and trigger insulin release from pancreatic β -islet cells. A murine knockout model with block at the transamination step provokes a leucine-mediated energy-wasting futile cycle, resulting in significantly reduced body weight and body fat mass. Similar effects were obtained with dietary supplements of leucine or BCAAs. In the brain, a leucine–glutamate cycle is responsible for maintaining a steady supply of glutamate, a major excitatory neurotransmitter for interneuronal communication. BCKAs accumulated in MSUD compromise brain energy metabolism by blocking the respiratory chain and inhibiting creatine kinase activity. The above advances indicate that BCAAs and BCKAs are important amino acids that play a multifaceted role in metabolism, neurophysiology, and clinical applications.

Alkaloids are a class of secondary metabolites with a relatively frequent occurrence in nature. Around 392 alkaloids have been isolated in several African plants, with isoquinoline alkaloids (19%) being the most abundant. They are very diverse chemicals and biomolecules, derived from l -amino acids or from the transamination process. Alkaloids have quite different biosynthetic pathways and different bioimpacts, derived from l -tryptophan, l -tyrosine, l -phenylalanine, l -lysine, l -arginine, l -histidine, l -anthranilic acid, and l -ornithine precursors, either alone or in combination with a steroidal, secoiridoid (e.g., secologanin), or other terpenoid-type moiety. Several alkaloid compounds exhibit various potentially useful activities such as enzyme inhibitory effects and anticancer, antimicrobial, and anti-inflammatory activity. This chapter aims to summarize as well as possible the alkaloids identified in African medicinal plants as well as their pharmacological potentials.

Vitamin B6 represents a group of substances with vitamin B activity and these substances are derivatives of 3-hydroxy-2-methylpyridine: pyridoxine (alcohol), pyridoxal (aldehyde), and pyridoxamine (amine), and their 5′-phosphorylated forms. Pyridoxal-5′-phosphate serves mainly as a coenzyme for about 100 enzymes in amino acid metabolism including transamination, decarboxylation, and elimination. Pyridoxamine-5′-phosphate acts exclusively as a coenzyme for transaminases (transferase, oxidoreductase, hydrolase, lyase). Vitamin B6 has many functions in various systems of the body (immune system, nervous system, gluconeogenesis, lipid metabolism, erythrocyte function, hormone modulation, gene expression, niacin formation). Deficiency symptoms are hard to detect and are similar to those due to deficiencies of niacin and riboflavin. Together with high homocysteine levels dementia may occur. Some drugs reduce vitamin B6 concentration, especially when they are taken over an extended period of time (then vitamin B6 status should be monitored): hydrazines, chelators, antibiotics, oral contraceptives, l-DOPA (l-3,4-dihydroxyphenylalanine), alcohol. In developed countries, a mixed diet is sufficient to supply the recommended 1.2 mg day-1.

Alkaloids are a group of molecules with a relatively large occurrence in nature around the Globe. They are very diverse chemicals and biomolecules, but they are all secondary compounds and they are derived from amino acids or from the transamination process. Alkaloids are classified according to the amino acids that provide their nitrogen atom and part of their skeleton. Similar alkaloids can have quite different biosynthetic pathways and different bioimpacts. Alkaloids are derived from L-lysine, L-ornithine, L-tyrosine, L-tryptophan, L-histidine, L-phenylalanine, nicotinic acid, anthranilic acid or acetate. The terpenoid, steroid and purine alkaloids are also important. Millions of people around the Globe use purine alkaloids every day whether starting the day with a cup of coffee or drinking a cup of tea in the afternoon. Alkaloids also occur in the animal kingdom. Differently from plants, the source of these molecules in an animal's body can be endogenous or exogenous. Alkaloids are molecules participating in both producer and consumer chains in nature. They are vital in feeding, and enjoy servations, agressivity and defence of the species. Homo sapiens is one of them.

Amino acids enter the body from dietary protein and are generated by the breakdown of tissue proteins. Nonessential amino acids can be synthesized from other metabolic intermediates. Amino acid disposal is dominated by protein synthesis, but amino acids are also oxidized by a series of complex pathways, some of which generate other useful small molecules. The nitrogen from amino acids is converted to urea in the liver and excreted in the urine. Amino acids circulate freely in the plasma and are distributed in all tissues. A series of specific proteins are responsible for transporting amino acids across cell membranes.

Publisher Summary Ammonia is mainly produced in fish through catabolism of amino acids. Ingested proteins are hydrolyzed producing amino acids, the catabolism of which leads to the release of ammonia. Ammonia production takes place mainly in the liver in fish but other tissues are also capable of doing so. Ammonia is produced by the transamination of amino acids followed by the deamination of glutamate and by the deamination of adenylates in fish muscle during severe exercise. Most aquatic animals keep body ammonia levels low by simply excreting excess ammonia produced through digestion and metabolism. The dietary protein requirement of fish increases at higher temperatures, possibly due to increased oxidation of amino acids reviewed the effects of temperature on nitrogen metabolism and concluded that as temperature increased an increasing percentage of aerobic metabolism was fueled by oxidation of protein. That is, ammonia production increases with increasing temperature due to both increased metabolic rate and a switch to greater protein utilization. However, this conclusion was based on studies of temperate fishes and, therefore, may not be applicable to tropical fishes. Tropical fishes show a marked increase in food intake and ammonia excretion with increasing temperature but the effect on protein metabolism is not known.

30 p.-6 fig., The amino acid conversion to volatile compounds by lactic acid bacteria is important for aroma formation in cheese. In this work, we analyzed the effect of the lytic bacteriocin Lacticin 3147 on transamination of isoleucine and further formation of the volatile compound 2-methylbutanal in cheese. The Lacticin 3147 producing strain Lactococcus lactis IFPL3593 was fluorescently tagged (IFPL3593-GFP) by conjugative transfer of the plasmid pMV158GFP from Streptococcus pneumoniae, and used as starter in cheese manufacture. Starter adjuncts were the bacteriocin-sensitive strains L. lactis T1 and L. lactis IFPL730, showing branched chain amino acid aminotransferase and α-keto acid decarboxylase activity, respectively. Adjunct strains were selected to complete the isoleucine conversion pathway and, hence, increase formation of 2-methylbutanal conferring aroma to the cheese. The non-bacteriocin-producing strain L. lactis IFPL359-GFP was included as starter in the control batch. Fluorescent tagging of the starter strains allowed their tracing in cheese during ripening by fluorescence microscopy and confocal scanning laser microscopy. The bacteriocin produced by L. lactis IFPL3593-GFP enhanced lysis of the adjuncts with a concomitant increase in isoleucine transamination and about a two-fold increase of the derived volatile compound 2-methylbutanal. This led to an enhancement of the cheese aroma detected by a sensory panel. The improvement of cheese flavour and aroma may be of significant importance for the dairy industry. © 2003 Elsevier B.V. All rights reserved., The research was supported by the NRI Competitive Grants Program of the United States Department of Agriculture (Grant no. 2002-35204-11662) and Schering-Plough Animal Health Corp

This chapter discusses the process of catabolism of amino acids in cheese during ripening. Catabolism of amino acids plays a major role in flavor development in cheese during ripening. In particular, the catabolism of sulphur-containing amino acids (principally methionine), aromatic amino acids, and branched-chain amino acids to flavor, or perhaps off-flavor, compounds has received considerable attention. There appear to be two major pathways by which amino acids are catabolized. The first series of reactions is initiated by the action of an aminotransferase, which transfers the amino group from amino acid A to an α-keto acid B (usually ot-ketoglutaric acid) and results in the production of an α-keto acid corresponding to amino acid A and a new amino acid corresponding to α-keto acid B, α-Keto acids produced by the transamination of aromatic amino acids, branched-chain amino acids and methionine may be degraded further to other compounds by enzyme-catalyzed reactions or by chemical reactions. The second major series of reactions by which amino acids are catabolized is initiated by the action of amino acid lyases, which cleave the side chains of amino acids. These pathways are particularly important for the catabolism of aromatic amino acids and methionine. Other pathways by which amino acids may be catabolized include the production of amines by decarboxylases and the production of NH3 by deaminases. There are also specific pathways for the metabolism of threonine, aspartic acid, glutamic acid and arginine. The chapter also discusses their importance to cheese flavor, the catabolism of methionine, branched-chain and aromatic amino acids.

Branched chain aminotransferases (BCATs) catalyze transamination of the branched chain amino acids (BCATs) leucine, isoleucine, and valine. Except for the Escherichia coli and Salmonella proteins, which are homohexamers arranged as a double trimer, the BCATs are homodimers. Structurally, the BCATs belong to the fold type IV class of pyridoxal phosphate (PLP) enzymes. Other members are d -alanine aminotransferase and 4-amino-4-deoxychorismate lyase. Catalysis is on the re face of the PLP cofactor, whereas in other classes, catalysis occurs from the si face of PLP. Crystal structures of the fold type IV proteins show that they are distinct from the fold type I aspartate aminotransferase family and represent a new protein fold. Because the fold type IV enzymes catalyze diverse reactions, it is not surprising that the greatest structural similarities involve residues that participate in PLP binding rather than residues involved in substrate binding. The BCATs are widely distributed in the bacterial kingdom, where they are involved in the synthesis/degradation of the BCAAs. Bacteria contain a single BCAT. In eukaryotes there are two isozymes, one is mitochondrial (BCATm) and the other is cytosolic (BCATc). In mammals, BCATm is in most tissues, and BCATm is thought to be important in body nitrogen metabolism. BCATc is largely restricted to the central nervous system (CNS). Recently, BCATc has been recognized as a target of the neuroactive drug gabapentin. BCATc is involved in excitatory neurotransmitter glutamate synthesis in the CNS. Ongoing structural studies of the BCATs may facilitate the design of therapeutic compounds to treat neurodegenerative disorders involving disturbances of the glutamatergic system.

Publisher Summary The tissues of animals do not normally contain D-amino acids, and before an animal can utilize an ingested D-amino acid, it must convert the amino acid in the L form. This conversion, known as inversion, consists of two steps: (1) oxidative deamination of the α-keto analog; and (2) transamination of an amino group from glutamate. Amino acids differ greatly in the extent to which they can be inverted by animals and consequently, in the degree to which the D forms are bioavailable. Some amino acids are readily inverted and may be completely bioavailable, whereas others do not undergo inversion and therefore cannot be utilized by animals. In general, with the exception of tryptophan, avian species invert D-amino acids more efficiently than mammalian species do. Bioavailabilities of amino acid isomers and analogs include arginine, cystine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine. Most research on 2-hydroxy-4-methylthiobutyric acid, often referred to as methionine hydroxy analog (OH-M), has involved poultry, for which this methionine analog is an important commercial product. It is sold as either free OH‑M, a liquid, or OH‑M (Ca), a solid consisting of 2 mol of OH‑M bound to calcium by the two carboxyl carbons. Considerable controversy has surrounded the biological availability of DL‑OH‑M relative to DL-methionine. Recent research by Chung and Baker has indicated that young pigs utilize DL‑OH‑M with the same free molar efficiency as L-methionine.

Publisher Summary This chapter explores the selective sequencing of peptides with N-terminal ser or thr in mixtures. It highlights the potential of a new protein sequencer designed around a distinctive approach to sample loading and retention. In a study described in the chapter, the peptide mixture gave two sequences at roughly equal levels, allowing for low recoveries of PTH-Ser and PTH-Trp. Periodate oxidation resulted in substantial loss of the signal from SYSMEHFRWG, consistent with modification of its N-terminus. Oxidation and transamination led to SYSMEHFRWG being detected in its product form of GYSMEHFRWG with little effect on relative signal strength compared to the control. The full three-step process permitted selective sequencing of the peptide GYSMEHFRWG, with near-complete elimination of the signal from RPVKVYPNGAEDESDEAFPLEF. There was a significantly reduced signal compared to the control, which appeared because of an effect of the acetylation step. Peptide elution by the hydrophobic acetic anhydride was a possible reason for this. The oxidized and transaminated peptide was ultimately sequenced at about 40% of its initial level.

Our studies have dealt with the synthesis and reactions of these important γ-glutamyl compounds whose metabolism and functions are interconnected. Glutamine transaminases, which contain vitamin B 6 , have several functions in amino acid and ammonia metabolism. Glutamine transaminase K also catalyzes β-elimination of S-conjugates of cysteine, which are formed from the corresponding conjugates of glutathione (GSH). β-Elimination is enhanced by α-keto acids, mediated by transamination of Enzyme-PMP to Enzyme-PLP, a control pathway shown previously for aspartate β-decarboxylase and other vitamin B 6 enzymes. Glutamine synthesis is inhibited by one of the four diastereoisomers (the L,S-isomer) of methionine sulfoximine (MSO). Administration of this isomer to mice leads to convulsions and death. On the other hand, certain higher homologs of MSO such as buthionine sulfoximine (BSO) do not produce convulsions or affect glutamine synthetase significantly, but selectively inhibit the first step of GSH synthesis (catalyzed by γ-glutamylcysteine synthetase). Enzyme inactivation by MSO and BSO involves ATP-dependent conversion of these compounds to the corresponding enzyme-bound amino acid sulfoximine N-phosphates, which bind tightly to the enzymes thus inactivating them. Depletion of GSH by inhibition of its synthesis has been useful in studies of GSH metabolism and transport and also for sensitization of tumor cells to radiation and chemotherapeutic agents. Experimental GSH deficiency produced without application of stress - and the reversal of such deficiency by GSH monoesters - has revealed that mitochondrial damage is a major effect of GSH deficiency in many tissues. GSH is not synthesized in mitochondria, but is imported from the cytosol by a multi-component transport system that includes a high affinity transporter. These studies thus elucidate an important physiological function of GSH that is essential for normal mitochondrial function.

Publisher Summary Nucleic acid hybridization techniques have a wide application in the biological sciences for the identification and quantification of specific nucleotide sequences in complex mixtures of nucleic acids. Most current hybridization assay systems use polynucleotide probes labeled with radioisotopes. Unpaired cytosine residues on deoxyribonucleic acids can be modified by the addition of sodium bisulfite to the 5,6-double bond of the pyrimidine base. The exocyclic amino group of the cytosine–bisulfite adducts can undergo substitution with various amines in the presence of ethylenediamine, transamination forms an N-substituted cytosine derivative that has a side chain terminating in a primary amino group. The extent of formation of the N4-substituted cytosine residue varies from 12 to 56%, depending on the pH and bisulfite concentration of the reaction mixture. The efficiency of the reaction between bisulfite–ethylenediamine-modified cytosine residues and N-biotinyl-ɛ-aminocaproic acid N-hydroxysuccinimide ester ranges from 77% for the most extensively modified nucleic acids to 100% for the lightly modified nucleic acids.

Publisher Summary This chapter describes the use of two classes of reagents—namely, hydrazide derivatives of biotin and pdiazobenzoylbiocytin (DBB). Biotin hydrazide (BHZ) and biocytin hydrazide (BCHZ) label cytidine residues in a one-step transamination reaction and DBB labels guanidine residues at position. The DNA sample is added to the DBB solution, and the reaction is allowed to proceed for 30 minutes at room temperature. The DNA is precipitated by adding successively 0.1 volume of acetate buffer and two volumes of ice-cold ethanol. Nitrocellulose membrane filters are spotted with heat-denatured, biotin hydrazide-labeled DNA in sequential half-logarithmic dilutions. The DNA sample is brought to 0.5 ml with the biotin hydrazide solution, and sodium bisulfite is added to a final concentration of I M. The contents of the dialysis bag are collected, placed into an Eppendorf tube, and concentrated to dryness in a SpeedVac Concentrator. The DNA is resuspended in the original volume of Tris–EDTA buffer.

Publisher Summary This chapter describes the DL-2-Keto-4-hydroxyglutarate (KHG). 2-Keto-4-hydroxyglutarate may be prepared by procedures such as: (a) nonenzymic transamination of 4-hydroxyglutamate with pyridoxal; (b) enzymic transamination of 4-hydroxyglutamate, catalyzed by glutamate-aspartate transaminase; (c) chemical condensation of glyoxylate with oxaloacetate followed by acidic conditions for decarboxylating fl-keto acids; and (d) enzymic condensation of glyoxylate with pyruvate, catalyzed by KHG-aldolase. Methods (a) and (b), which start with 4-hydroxyglutamate, can be used to obtain DL-, L-, or D-KHG, depending on the specific isomer(s) of 4-hydroxyglutamate utilized. The nonenzymic transamination of 4-hydroxyglutamate with pyridoxal, as described in the chapter, represents a simple and routine way for synthesizing KHG. Threo-4-Hydroxy-L-glutamate is commercially available; a preparative scheme for isolating this compound from leaves of Phlox decussata, the source in which 4-hydroxyglutamate was first detected, has been outlined. KHG can be determined chemically or enzymically. The chapter also discusses the methods for determining of KHG. KHG is cleaved by the aldolase and the glyoxylate or pyruvate liberated can be measured.

Publisher Summary ω -Amino acid-pyruvate aminotransferase ( β- alanine-pyruvate aminotransferase) was originally designated taurine-pyruvate aminotransferase and may correspond to β-alanine-pyruvate aminotransferase. The enzyme catalyzes the reversible transamination of various sulfur-containing ω-amino acids and ω-amino carboxylic acids with pyruvate to yield the corresponding aldehydic acids and L-alanine. The assay method is based on the measurement of aldehydic acid or L-alanine. Alanine in the supernatant fluid is determined with a conventional amino acid analyzer. The standard assay system consists of 20 μmol of β- alanine or other ω-amino acid, 20 μmol sodium pyruvate, 0. l μmol pyridoxal 5'-phosphate, 100 μmol potassium phosphate at pH 8.0, and enzyme in a final volume of 1.0 ml. In a control tube, β-alanine is replaced by water. After incubation at 37° for 30 min, the reaction is terminated by addition of 0.1 ml of 25% trichloroacetic acid followed by centrifugation at 17,000 g for 10 min.

This chapter discusses the catabolism of simple units. The pathways of catabolism can be divided into three stages: (1) stage 1, (2) stage 2, and (3) stage 3. Starting with a large number of compounds, each stage finishes with fewer. Thus, progression through the three stages means that more and more material is channeled through fewer pathways until the last stage consists of only one pathway with two inputs. Stage 1 takes a very large number of simple units and partially oxidizes them to give three major compounds, namely, acetyl coenzyme A, a-oxoglutarate, and oxaloacetate, and three minor ones, namely, pyruvate, fumarate, and succinyl coenzyme A. Stage 2 is the complete oxidation of the six compounds from stage 1 to give carbon dioxide. This is the major route in the cell for energy trapping and the remaining two-thirds of the energy is released. It is conserved as NADH, fpH 2, and a little as ATP directly. Stage 3 involves the reoxidation of the reduced coenzymes so that their hydrogen is released as water. The energy is finally transferred to ATP by the phosphorylation of ADP. For most amino acids, the first step in catabolism is transamination, that is, removal of the amino group. Each amino acid has its own pathway for the catabolism of its carbon skeleton. These compounds are partially oxidized until eventually one of the intermediates of the citric acid cycle or glycolysis is reached.

Publisher Summary This chapter focuses on immobilized aminotransferases for amino acid production. Aminotransferases catalyze the synthesis and breakdown of amino acids in microorganisms, plants, and animals by the transfer of an amino group from an α-amino acid to a 2-ketoacid. One feature of aminotransferases is the requirement for the small molecule, pyridoxal 5’-phosphate, for catalytic activity; this cofactor being bound through a Schiff base linkage to the є-amino group of an active-site lysine. Although the binding of pyridoxal 5'- phosphate to the enzyme is reversible, most aminotransferases show maximal catalytic activity at the cofactor concentrations of 100 μM or less. Such low saturating concentrations of pyridoxal phosphate are an important property of aminotransferases; at concentrations of 100 μM or less, the cost of the cofactor in biocatalytic transamination processes is a relatively minor component of the total cost. Although aminotransferases have been known for decades, these enzymes have seen little use as biocatalysts until now. However, as the normal function of aminotransferases is the biosynthesis and metabolism of amino acids, it is natural to look to these enzymes as potentially useful catalysts for the production of amino acids. In principle, almost any desired amino acid can be produced from the appropriate 2-ketoacid using an inexpensive amino acid as the amino donor. There are a number of advantages to the use of this kind of technology.

Publisher Summary This chapter describes the preparation and properties of hemoglobin modified with derivatives of pyridoxal. Hemoglobin can be modified readily at selected sites with a number of pyridoxal derivatives. The location of the substitution depends not only on the structure of the pyridoxal derivative used but also on the conformation of the hemoglobin during the modification reaction. In this way, hemoglobin derivatives with a very wide range of oxygen affinities can be prepared. The reactions involve first the formation of a Schiff's base between amino groups of the hemoglobin and the aldehyde(s) of the pyridoxal moiety, followed by the reduction of the imine with sodium borohydride to give a covalently linked pyridoxyl derivative in the form of a secondary amine. Pyridoxylation is confined to the N-terminal amino groups of hemoglobin if the pyridoxal compounds are used in the form of their Schiff's bases with Tris so that the pyridoxal residue is transferred to the protein by a transamination. The conformation of the hemoglobin during the reaction is also decisive in determining the site of substitution and the properties of the pyridoxylated derivative. The chapter also explains the preparation and properties of the reagents.

Publisher Summary This chapter provides an overview of glutamate-γ-aminobutyrate transaminase. γ-aminobutyrate (GABA) is a major inhibitory transmitter of the central nervous system of vertebrates. GABA arises via decarboxylation of L-glutamate and is subsequently metabolized via transamination to succinic semialdehyde. Succinic semialdehyde is then oxidized to succinate. The enzymes catalyzing reactions are called the GABA shunt (or bypath). The shunt allows four of the five carbons lost from the tricarboxylic acid cycle at the level of α-ketoglutarate to be reincorporated into the cycle at the level of succinate. In tissue homogenates where succinic semiaidehyde dehydrogenase is in excess, GABA transaminase may be estimated by using endogenous succinic semialdehyde dehydrogenase to convert formed succinic semialdehyde to succinate. The increase in absorbance at 340 nm because of NADH production can be used to estimate GABA transaminase activities.

Publisher Summary This chapter reviews the results of investigations into the biosynthesis of alkaloids as well as those of, the quite often related, microbial metabolites containing nitrogen. It covers the biosynthesis of pyrrolidine alkaloids, which include tropane and related alkaloids, nicotine, pyrrolizidine alkaloids, phenanthroindolizidine and related alkaloids. Piperidine alkaloids are a large group of alkaloids, whose structures contain a piperidine ring. They consist of alkaloids derived exclusively from acetate and alkaloids derived from lysine, such as simple piperidine bases, tetrahydroanabasine alkaloids, lythraceae alkaloids, lycopodium alkaloids, quinolizidine alkaloids, pipecolic acid and slaframine. Anthranilic acid is an intermediate on the biosynthetic pathway to tryptophan and it serves as a starting point for the elaboration of a number of metabolites of plant and microbial origin. Bases derived from the tryptophan pathway include gramine, dimethyltryptamine derivatives, indolmycin, pyrrolnitrin, sporidesmin, β-Carboline alkaloids, calycanthine and folicanthine, isoprenoid indole bases, such as echinulin and brevianamide, cyclopiazonic acid, ergot alkaloids and terpenoid indole alkaloids, phenoxazinones, damascenine, quinoline alkaloids, such as echinorine, pseudans, furoquinoline and related alkaloids, acridone alkaloids, graveoline, quinazoline alkaloids, rutaecarpine and evodiamine, benzodiazepine bases, benzoxazinones, phenazines, and shihunine. Many of the complex alkaloids, which have their origins in the aromatic amino acids phenylalanine and tyrosine arise by a combination of two units derived from these precursors.

A survey is presented of key steps in the investigation of reactions and enzymes of amino group transfer. These include: discovery of biological transamination reactions; early studies of their occurrence, substrate range and presumable enzymic mechanism; studies revealing the principal biological functions of transaminations as essential steps in nitrogen assimilation, in transformations and catabolism of amino-acids, and in the chemical integration of intermediary nitrogen and energy metabolism; further, the detection and investigation of the cofactor role of pyridoxal phosphate in enzymic transamination and other transformations of amino-acids; essentials of the theory of biological vitamin B6-dependent reactions; molecular-physical and bioorganic characterization of cytosolic aspartate transaminase (see Note a ); elucidation of its peptide sequence, identification of some functionally important groups (and their presumable roles); hypothetic dynamic schemes of the molecular mechanism of enzymic transamination, and an outline of the first results obtained in X-ray structural investigations of cytosolic aspartate transaminase from chicken heart.

Asparagine transaminase has been purified about 200-fold from rat liver. The enzyme has a broad specificity toward both amino acids and alpha-keto acids. Thus, amino acids substituted in the beta position such as asparagine, S-methylcysteine, phenylalanine, cysteine, serine, and aspartate are substrates. The enzyme is also active with alanine, methionine, homoserine, alpha-aminobutyrate, glutamine, and leucine. The enzyme has a high affinity for glyoxylate but the affinity falls off markedly through the series glyoxylate, pyruvate, alpha-ketoburyrate, alpha-Keto acids substituted in the beta or gamma position, such as alpha-ketosuccinamate, phenylpyruvate, p-hydroxyphenylpyruvate, alpha-keto-gamma-methiolburyrate, and alpha-keto-gamma-hydroxybutyrate, are substrates for the enzyme. Amino acids or alpha-keto acids possessing a branch point at the beta carbon are inactive. Kinetic analysis of the asparagine glyoxylate transamination reaction is consistent with a ping-pong mechanism.

Publisher Summary This chapter discusses the isolation and assay of hypoglycin and metabolically related inhibitors. Hypoglycin (L-2-amino-3-methylenecyclopropylpropionic acid) (I) is the toxic hypoglycemic principle of the unripe arillus of the ackee fruit, Blighia sapida, which occurs in several Caribbean islands, West Africa, and Florida. In animals, hypoglycin is converted to methylenecyclopropylpyruvlc acid (II) by transamination in the cytosol, and this is irreversibly decarboxylated to methylenecyclopropylacetyl-CoA (MCPA-CoA) (III). Four kilograms of ground ackee seeds are extracted three times with a total of 12 liters of 80% (v/v) ethanol. The combined extracts are left overnight, filtered, and then evaporated to dryness under reduced pressure at 50°. The purity of hypoglycin preparations can be determined by reaction with Br 2 and this reacts with the double bond of hypoglycin, but does not react with leucine. Any standard amino acid analyzer can be used to separate leucine from the bromination products of hypoglycin. The enzyme inhibition by hypoglycin and its metabolites is also discussed.

Publisher Summary This chapter provides an overview of aminotransferase activity in brain. Transamination is the transfer of an amino group from one molecule to another without the intermediate formation of ammonia. The enzymes that catalyze such a reaction are called “transaminases” or “aminotransferases.” The aminotransferases occupy a significant place in amino acid metabolism. They are the major degradative mechanism for the catabolism of many amino acids. Conversely, synthesis of nonessential amino acids occurs via the transamination of glutamic acid. These enzymes also provide a mechanism for the conversion of alanine, glutamate, and aspartate to pyruvate, α -ketoglutarate, and oxaloacetate, joining amino acid metabolism with carbohydrate metabolism. The aminotransferases are also crucial enzymes in other metabolic processes, such as urea synthesis in the liver, ammonia metabolism, or the degradation of neurotransmitter agents, such as γ-aminobutyric acid (GABA). The aminotransferases are discussed and their properties are described in this chapter.

Publisher Summary This chapter focuses on amino-acid catabolism in goldfish tissues. Active transamination is observed with aspartate in dorsal white muscle, with aspartate and branched-chain amino acids in lateral red muscle, and with alanine, aspartate, branched chain amino acids, histidine, and tyrosine in liver. In muscle tissues, the activities of glutamate dehydrogenase and the purine nucleotide cycle are of the same order, but in liver, glutamate dehydrogenase activity exceeds that of the purine nucleotide cycle with two orders of magnitude. The mechanism of ammonia production from amino acids has been studied in intact goldfish liver cells using the purine nucleotide cycle inhibitor hadacidine and the transaminase inhibitor amino-oxy-acetate. The results of these experiments suggest that in goldfish liver, ammonia is mainly produced by transdeamination and the hydrolysis of imino groups but not by purine nucleotide cycling.

Publisher Summary This chapter discusses aminotransferases in higher plants. The aminotransferases or transaminases are enzymes catalyzing the transfer of an amino group, plus a proton and an electron pair, from an amino donor compound to the carbonyl position of an amino acceptor compound. This process is known as transamination. Usually, an amino acid acts as the amino donor and a 2-keto acid as the amino acceptor. The result is the formation of a new amino acid plus the keto acid analogue of the amino acid, which originally serves as the amino donor. In some cases, aldehydes can serve as amino acceptor substrates and amines can act as donors. Most transaminations are freely reversible processes. The best characterized plant aminotransferase is glutamate-oxaloacetate transaminase, which catalyzes the reversible interconversions between glutamate and aspartate, and their 2-keto analogues. The enzymatic transfer of amino groups plays an important part in many metabolic processes, where the interconversion of nitrogen-containing molecules is involved. Nitrogen, following its initial assimilation into glutamine and glutamate, can be distributed to many other compounds by the action of aminotransferases.

Publisher Summary This chapter focuses on glutamate dehydrogenase (G1DH), which is a widely distributed enzyme. Because of its easy availability in liver, G1DH is one of the best studied enzymes. Measurement of G1DH activity can be used to check the purity of nonmitochondrial fractions during cell fractionation. G1DH is measured in the spectrophotometric assay of Warburg. All the necessary reagents are commercially available in A. R. or biochemical reagent grade and require no further purification. NADH/ADP, LDH, oxoglutarate solution should be stored and buffered at 4°C. For measurements in serum with G1DH values from 1 to 20 U/l, a coefficient of variation of 5.2% was obtained. In aqueous tissue extracts the following G1DH activities have been obtained with comparable methods. No effect of therapeutic measures on the actual assay is known. Like the deficiency of NADH, too high a concentration of NADH results in low values because of the extremely narrow concentration optimum. The main reaction is specific for G1DH activity because the amino acid concentrations in serum or tissue extracts are not high enough under the assay conditions to result in appreciable transamination, and the slight nonspecific reactions are corrected for by measurement of the preliminary reaction.

Publisher Summary 3-Mercaptopyruvate, 3-mercaptolactate, and mercaptoacetate are compounds formed in the transamination pathway of cysteine. In this pathway, cysteine is first converted to 3-mercaptopyruvate via transamination. 3-Mercaptopyruvate may then be reduced by lactate dehydrogenase to 3-mercaptolactate or converted to mercaptoacetate by oxidative decarboxylation. All of these compounds are found in the urine of higher animals as mixed disulfides with cysteine; the disulfides are presumably formed by a thiol-disulfide exchange with cystine in the extracellular space. The determination of the transaminative metabolites of cysteine is not only of interest for studies of their metabolism but is also of clinical interest as human beings may be affected by an inborn error of metabolism, 3-mercaptolactate cysteine disulfiduria. A method for the assay of 3-mercaptopyruvate cysteine disulfide formed by the action of L-amino-acid oxidase has been reported, where the disulfide is separated by ion-exchange chromatography, reduced with dithiothreitol, and the 3-mercaptopyruvate formed is determined with lactate dehydrogenase. 3-Mercaptolactate has also been determined after aminoethylation with bromoethylamine using an amino acid analyzer, and mercaptoacetate has been determined by high-performance liquid chromatography after derivatization with N-(1-pyrene)maleimide.

Publisher Summary This chapter discusses the assays for various glutamate (α-ketoglutarate)-utilizing transaminases for comparative purposes. Several α-ketoglutarate utilizing transaminases, their purification and assay procedures are discussed in the chapter. Some of them include 6- N -acetyl-β-lysine, α-aminoadipate, cysteine, cysteine sulfinate, histidine, histidinol phosphate/imidazole acetol phosphate, methionine, and taurine/hypotaurine. Mitochondrial cysteine–glutamate transaminase is identical to mitochondrial glutamate–aspartate transaminase. A cysteine–glutamate transaminase that is apparently not identical to glutamate–aspartate transaminase is purified from rat liver. Histidinol phosphate catalyzes an important reaction in the biosynthesis of L-histidine. A taurine transaminase is purified from extracts of Achromobacter superficialis and then crystallized. The product of the reaction with taurine (sulfatoacetaldehyde) is relatively stable, whereas the transamination product of hypotaurine (sulfinoacetaldehyde) is unstable and spontaneously decomposes to acetaldehyde and sulfite.

Publisher Summary This chapter discusses asparagine synthesis. Asparagine is one of the common amino acid constituents of proteins and it is also found in the free-state in many animal and plant cells; it accumulates in considerable quantities in certain plants. Asparagine metabolism appears to be restricted to (1) utilization for protein synthesis, (2) conversion to aspartate and ammonia by enzymic hydrolysis, and (3) transamination with a-ketoacids to yield the corresponding amino acids and a-ketosuccinamic acid. Other metabolic pathways cannot be excluded; for example, it is possible that asparagine participates in amide nitrogen transfer reactions of the type observed with glutamine. Transamination of asparagine is reversible unless α-ketosuccinamic acid undergoes dimerization or deamidation. Thus, asparagine can be formed by transamination; however, no metabolic route to α-ketosuccinamic acid except transamination or oxidative deamination of asparagine is as yet known. The biosynthesis of asparagine in a number of mammalian tissues is catalyzed by glutamine-dependent asparagine synthetase. Asparagine synthesis also occurs when glutamine is replaced by ammonia. Glutamine-dependent asparagine synthetase is a glutamine amido transferase and its properties resemble those of such enzymes as glutamine-dependent carbamylphosphate synthetase, DPN synthetase, and formylglycinamide ribonucleotide amido transferase.

Publisher Summary This chapter provides an overview of L-lysine transaminase from Flavobacteriurn lutescens. L-Lysine-α-ketoglutarate 6-aminotransferase catalyzes the transamination of terminal amino group of L-lysine to α-ketoglutarate to produce Δ 1 -piperideine-6-carboxylate and L-glutamate. The enzyme is purified in a crystalline form from Flavobacterium lutescens. An improved purification method of the enzyme by affinity chromatography, subunit structures, and the stereochemical analysis of the enzyme reaction are described in the chapter. The affinity adsorbent used for purification of the enzyme is L-lysylacetamidododecyl-Sepharose 6B. Sepharose 6B is activated in a 10% (w/v) solution of CNBr (pH 10.5-11.0) at 4 ° . L-Lysine-α-ketoglutarate 6-aminotransferase from F. lutescens is composed of one each of four nonidentical subunits (A, B 1 , B 2 , and C). The molecular weights of the subunits are determined by three methods: sodium lauryl sulfate disc gel electrophoresis, sephadex G-75 column chromatography in the presence of 6 M guanidine-HCl, and equilibrium centrifugation in the presence of 8 M urea.

Publisher Summary This chapter provides an overview of taurine–glutamate transaminase. Taurine:α-ketoglutrate aminotransferase catalyzes the transamination of taurine with α-ketoglutarate to yield sulfoacetaldehyde and L-glutamate, and the reverse reaction. The assay method is based on the measurement of glutamate or sulfoacetaldehyde. Glutamate is determined with ninhydrin after the separation by circular paper chromatography. Sulfoacetaldehyde is determined by the reaction with 2,4-dinitrophenylhydrazine, or o-aminobenzaldehyde in the presence of glycine. The purification procedure consists of several steps including preparation of crude extract, diethylaminoethyl (DEAE)-cellulose column chromatography, first sephadex G-150 column chromatography, hydroxylapatite column chromatography, and ammonium sulfate fractionation. The crystalline enzyme is homogeneous by the criteria of ultracentrifugation and disc gel electrophoresis. The sedimentation coefficient of the enzyme is determined to be 9.1 S. The taurine transaminase activity is found only in the extracts of Achromobacter superficialis ICR B-89 and Achromobacter polymorph ICR B-88. The enzyme activity is induced by β-alanine, but not by taurine.

Publisher Summary This chapter reviews the studies on the hepatic glucocorticoid receptor and the studies on the hormonal modulation of specific mRNA levels during enzyme induction. The glucocorticoid hormones derive their names from the fact that their major physiologic effect is to place the animal in a negative nitrogen balance with a concurrent generation of carbohydrates, that is, to increase gluconeogenesis. These effects are partially the consequence of the involutionary influence of these hormones on lymphoid and muscle tissues resulting in the breakdown of some of their cellular proteins into amino acids. These amino acids are transported through the blood to the liver where they undergo transamination and deamination. The amino groups of the amino acids are ultimately converted to urea and excreted. The carbon skeletons of the glucogenic amino acids enter carbohydrate metabolism and are converted by gluconeogenesis to hepatic glycogen or blood glucose. This phenomenon of hormonal enzyme induction has attracted the attention of several laboratories that have attempted to elucidate the biochemical events by which the steroid hormones regulate hepatic enzyme levels. It was demonstrated that the liver is a direct target organ of these steroid hormones by the observation that enzyme induction occurred when inducing steroids were added to the perfusate bathing intact liver in vitro.