Stimulation of glycogen synthesis in hepatocytes by added amino acids is related to the total intracellular content of amino acids

Katz et al. [Katz, J., Golden, S. & Wals, P.A. (1976) Proc. Natl Acad. Sci. USA 73, 3433-3437] were the first to report that in hepatocytes isolated from fasted rats and incubated with either dihydroxyacetone, glucose or other sugars, glycogen synthesis was greatly accelerated by addition of amino acids. We have looked for possible mediators responsible for this effect and have tested the effect of alanine, proline, asparagine, glutamine or a combination of ammonia with either pyruvate or lactate in activating glycogen synthesis from dihydroxyacetone. The following observations were made. 1. Stimulation of glycogen synthesis by alanine, proline or asparagine does not require production of glutamine since the effect also occurs in periportal hepatocytes which lack glutamine synthetase. 2. Under various conditions, stimulation of glycogen synthesis by added amino acids directly correlated with increases in the intracellular content of amino acids, expressed in osmotic equivalents. 3. 3-Mercaptopicolinic acid, the inhibitor of phosphoenolpyruvate carboxykinase, further enhances stimulation of glycogen synthesis by amino acids because it increases the intracellular accumulation of aspartate and glutamate. 4. The previously reported enhancement by leucine of the stimulation of glycogen synthesis by glutamine [Chen. K. S. & Lardy, H. A. (1985) J. Biol. Chem. 260, 14683-14688] can be ascribed to inhibition of urea synthesis by leucine which results in accumulation of glutamate and of ammonia, the essential activator of glutaminase. It is concluded that activation of glycogen synthesis by added amino acids is due to an increase in intracellular osmolarity following their uptake and the accumulation of intracellular catabolites. This results in an increase in hepatic volume which stimulates glycogen synthesis [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959].     

The reaction of ornithine aminotransferase with ornithine.

Rat liver ornithine aminotransferase is found to exchange the pro-S hydrogen on the delta-carbon atom of ornithine exclusively, thus showing that the mammalian enzyme transfers the delta-amino group and not the alpha-amino group as has been demonstrated with the plant enzyme [Mestichelli, Gupta & Spenser (1979) J. Biol. Chem. 254, 640-647]. The enzyme also transfers the alpha-amino group of glutamate and the kinetics of the half reactions between the enzyme and both amino acids are compared. Rate and dissociation constants for both reactions are determined.     

Avian mitochondrial glutamine metabolism.

Intact avian liver mitochondria were shown to synthesize glutamine from glutamate in the absence of exogenous ATP and ammonia. With L-[U-14C]glutamate as the substrate, there was an approximate 1:1 stoichiometry between glutamate deaminated (as measured by the release of 14CO2 due to alpha-keto-[14C]glutarate oxidation) and glutamate amidated. With L-[15N]glutamate as the substrate, the isolated glutamine was shown by low and high resolution mass spectrometry of its phenylisothiocyanate derivative to contain 15N in both the alpha-amino and amide groups. Thus, for each mole of glutamate taken up, approximately 0.5 mol is deaminated and the other 0.5 mol serves as a substrate for glutamine synthetase previously localized in these mitochondria (Vorhaben, J. E., and Campbell, J. W. (1972) J. Biol. Chem. 247,2763). The permeability of L-glutamine to intact avian liver mitochondria was studied by a rapid centrifugation technique. Efflux as well as influx of L-glutamine were both rapid and appeared to occur by a passive, energy-independent process. These results indicate that the mitochondrial glutamine synthetase present in uricotelic species represents the primary ammonia detoxication reaction in that ammonia released intramitochondrially during amino acid catabolism is converted to glutamine for efflux to the cytosol where it may serve as a substrate for purine (uric acid) biosynthesis.     

Identical N-terminal peptide sequences of asymmetric forms and of low-salt-soluble and detergent-soluble amphiphilic dimers of Torpedo acetylcholinesterase. Comparison with bovine acetylcholinesterase

We have determined partial N-terminal sequences of acetylcholinesterase (AChE) catalytic subunits from Torpedo marmorata electric organs and from bovine caudate nucleus. We obtain identical sequences (23 amino acids) for the soluble ('low-salt-soluble' or LSS fraction) and particulate ('detergent-soluble', or DS fraction) amphiphilic dimers (G2 form) and for the asymmetric, collagen-tailed forms ('high-salt-soluble', or HSS fraction, A12 + A8 forms). There are two amino acid differences, at position 3 (Asp/His) and 20 (Ile/Val), with the sequences obtained for T. californica by MacPhee-Quigley et al. [(1985) J. Biol. Chem. 260, 12185-12189] for the soluble G2 form and the lytic G4 form which is derived from asymmetric AChE. The bovine sequence (12 amino acids) presents an identity of 4 amino acids (Glu-Leu-Leu-Val) with that of Torpedo, at positions 5-8 (Torpedo) and 7-10 (bovine). There is also a clear homology with the sequence of human butyrylcholinesterase [(1986) Lockridge et al. J. Biol. Chem., in press] indicating that these enzymes probably derive from a common ancestor.     

Rat liver NAD(P)H:quinone reductase: isolation of a quinone reductase structural gene and prediction of the NH2 terminal sequence of the protein by double-stranded sequencing of exons 1 and 2

Recently two reports [J. A. Robertson et al. (1986) J. Biol. Chem. 261, 15794-15799 and R. M. Bayney et al. (1987) J. Biol. Chem. 262, 572-575] have appeared concerning the nucleotide sequence of quinone reductase cDNA clones. Although the cDNA clones are virtually identical, they diverge in the 5' region that encodes the NH2 terminus of the protein. In order to clarify the sequence of this region, we have isolated quinone reductase clones from a rat genomic library using a cDNA clone, pDTD55, isolated and characterized by our laboratory. We have determined the sequence of exons 1 and 2 of the structural gene by double-stranded sequencing using oligonucleotide primers. The sequence of exons 1 and 2 of the quinone reductase structural gene along with our previous nucleotide sequence analysis of pDTD55 as well as conventional amino acid sequence analysis of the purified protein indicates that quinone reductase is composed of 274 amino acids with a molecular weight of 30,946. These data agree with the published sequence of lambda NMOR1 reported by Robertson et al.     

Calculation of free-Mg2+ concentration in adenosine 5'-triphosphate containing solutions in vitro and in vivo

We have attempted to resolve the differences between the levels of free Mg2+ in muscle calculated by Wu et al. [Wu, S. T., Pieper, G. M., Salhany, J. M., & Eliot, R. S. (1981) Biochemistry 20, 7399-7403] (2.5 mM in guinea pig heart) and by Gupta and Moore [Gupta, R. K., & Moore, R. D. (1980) J. Biol. Chem. 255, 3987-3993] (0.6 mM in frog skeletal muscle) on the basis of substantially identical measurements by 31P NMR of the phosphate peaks in the spectrum of MgATP2-. The differences depend on the methods of calculation, including which reactions in which multiple equilibria are being considered. Biochemists and physical chemists customarily use different working definitions of the stability constant for MgATP2- in particular. Wu et al. used in their calculations, without reconciliation, methods involving three different operational definitions of the chelation equilibria involved. An algorithm for calculating Mg2+ and total ATP, which can be carried out with a hand calculator, is described here. With it, we calculated Mg2+ levels that agree with those determined by Gupta et al. [Gupta, R. K., Benkovic, J. L., & Rose, Z. B. (1978) J. Biol. Chem. 253, 6165-6171] with their in vitro systems. We therefore agree with the finding of Gupta and Moore that the Mg2+ level in skeletal and cardiac muscle is 0.6 mM.     

Identification of the metal-binding sites of restriction endonucleases by Fe2+-mediated oxidative cleavage

Fenton chemistry [Fenton (1894) J. Chem. Soc. 65, 899-910] techniques were employed to identify the residues involved in metal binding located at the active sites of restriction endonucleases. This process uses transition metals to catalytically oxidize the peptide linkage that is in close proximity to the amino acid residues involved in metal ligation. Fe2+ was used as the redox-active transition metal. It was expected that Fe2+ would bind to the endonucleases at the Mg2+-binding site [Liaw et al. (1993) Biochemistry 32, 7999-4003; Ermácora et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 6383-6387; Soundar and Colman (1993) J. Biol. Chem. 268, 5264-5271; Wei et al. (1994) Biochemistry 33, 7931-7936; Ettner et al. (1995) Biochemistry 34, 22-31; Hlavaty and Nowak (1997) Biochemistry 36, 15515-15525). Fe2+-mediated oxidation was successfully performed on TaqI endonulease, suggesting that this approach could be applied to a wide array of endonucleases [Cao and Barany (1998) J. Biol. Chem. 273, 33002-33010]. The restriction endonucleases BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, BsoBI, EcoRI, EcoRV, MspI, and HinP1I were subjected to oxidizing conditions in the presence of Fe2+ and ascorbate. All proteins were inactivated upon treatment with Fe2+ and ascorbate. BamHI, FokI, BglI, BglII, PvuII, SfiI, BssSI, and BsoBI were specifically cleaved upon treatment with Fe2+/ascorbate. The site of Fe2+/ascorbate-induced protein cleavage for each enzyme was determined. The Fe2+-mediated oxidative cleavage of BamHI occurs between residues Glu77 and Lys78. Glu77 has been shown by structural and mutational studies to be involved in both metal ligation and catalysis [Newman et al. (1995) Science 269, 656-663; Viadiu and Aggarwal (1998) Nat. Struct. Biol. 5, 910-916; Xu and Schildkraut (1991) J. Biol. Chem. 266, 4425-4429]. The sites of Fe2+/ascorbate-induced cleavage for PvuII, FokI, BglI, and BsoBI agree with the metal-binding sites identified in their corresponding three-dimensional structures or from mutational studies [Cheng et al. (1994) EMBO J. 13, 3297-3935; Wah et al. (1997) Nature 388, 97-100; Newman et al. (1998) EMBO J. 17, 5466-5476; Ruan et al. (1997) Gene 188, 35-39]. The metal-binding residues of BglII, SfiI, and BssSI are proposed based on amino acid sequencing of their Fe2+/ascorbate-generated cleavage fragments. These results suggest that Fenton chemistry may be a useful methodology in identifying amino acids involved in metal binding in endonucleases.     

Reappraisal of the 20th-century version of amino acid metabolism

In this article, we advocate the radical revision of the 20th-century version of amino acid metabolism as follows. (1) Classic studies on the incorporation of [15N]ammonia into glutamate, once considered to be an epoch-making event, are not distinctive proof of the ability of animals to utilize ammonia for the synthesis of alpha-amino nitrogen. (2) Mammalian glutamate dehydrogenase has been implicated to function as a glutamate-synthesizing enzyme albeit lack of convincing proof. This enzyme, in combination with aminotransferases, is now known to play an exclusive role in the metabolic removal of amino nitrogen and energy production from excess amino acids. (3) Dr. William C Rose's "nutritionally nonessential amino acids" are, of course, essential in cellular metabolism; the nutritional nonessentiality is related to their carbon skeletons, many of which are intermediates of glycolysis or the TCA cycle. Obviously, the prime importance of amino acid nutrition should be the means of obtaining amino nitrogen. (4) Because there is no evidence of the presence of any glutamate-synthesizing enzymes in mammalian tissues, animals must depend on plants and microorganisms for preformed alpha-amino nitrogen. This is analogous to the case of carbohydrates. (5) In contrast, individual essential amino acids, similar to vitamins and essential fatty acids, should be considered important nutrients that must be included regularly in sufficient amounts in the diet.     

The metabolic fate of 13N-labeled ammonia in rat brain.

13N-labeled ammonia was used to study the cerebral uptake and metabolism of ammonia in conscious rats. After infusion of physiological concentrations of [13N]ammonia for 10 min via one internal carotid artery, the relative specific activities of glutamate, glutamine (alpha-amino), and glutamine (amide) in brain were approximately 1:5:400, respectively. The data are consistent with the concept that ammonia, entering the brain from the blood, is metabolized in a small pool of glutamate that is both rapidly turning over and distinct from a larger tissue glutamate pool (Berl, S., Takagaki, G., Clarke, D.D., and Waelsch, H. (1962) J. Biol. Chem. 237, 2562-2569). Analysis of 13N-metabolites, after infusion of [13N]ammonia into one lateral cerebral ventricle, indicated that ammonia entering the brain from the cerebrospinal fluid is also metabolized in a small glutamate pool. Pretreatment of rats with methionine sulfoximine led to a decrease in the label present in brain glutamine (amide) following carotid artery infusion of [13N]ammonia. On the other hand, 13N activity in brain glutamate was greater than that in the alpha-amino group of glutamine, i.e. following methionine sulfoximine treatment the expected precursor-product relationship was observed, indicating that the two pools of glutamate in the brain were no longer metabolically distinct. The amount of label recovered in the right cerebral hemisphere, 5 s after a rapid bolus injection of [13N]ammonia via the right common carotid artery, was found to be independent of ammonia concentration within the bolus over a 1000-fold range. This finding indicates that ammonia enters the brain from the blood largely by diffusion. In normal rats that were killed by a freeze-blowing technique 5 s after injection of an [13N]ammonia bolus, approximately 60% of the label recovered in brain had already been incorporated into glutamine, indicating that the t1/2 for conversion of ammonia to glutamine in the small pool is in the range of 1 to 3 s or less. The data emphasize the importance of the small pool glutamine synthetase as a metabolic trap for the detoxification of blood-borne and endogenously produced brain ammonia. The possibility that the astrocytes represent the anatomical site of the small pool is considered.     

Multiple amino acid substitutions suggest a structural basis for the separation of biological activity and receptor binding in a mutant interleukin-1 beta protein.

Receptor binding and biological activity properties of human interleukin-1 beta can be dissociated by mutating a single amino acid, arginine 127, to glycine (IL-1 beta R----G) [Gehrke et al. (1990) J. Biol. Chem. 265, 5922-5925]. The mechanism underlying the reduced biological activity has been examined by replacing arginine 127 with several other amino acids, followed by determination of biological activity using a T-helper cell proliferation assay. Mutant IL-1 beta proteins containing lysine, glutamic acid, tryptophan, or alanine in place of arginine 127 maintain biological activity. These data strongly suggest that IL-1 beta biological activity is not directly dependent upon the specific properties of charge, hydrophobicity/hydrophilicity, or side-chain group presented by the residue at position 127. Molecular modeling analyses indicate that the structural integrity of the antiparallel beta-strand 1/12 pair is disturbed in the glycine 127 mutant protein. Collapse of beta-strand 1 into a hydrated space between strands 1, 2, and 4 could structurally alter a cleft in IL-1 beta that contains a cluster of highly conserved amino acids, including a key aspartic acid residue [Ju et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2658-2662]. Mutagenesis data and the differential activities of the IL-1 beta R----G and IL-1 receptor antagonist proteins in stimulating early and late gene expression [Conca et al. (1991) J. Biol. Chem. 266, 16265-16268] suggest that multiple receptor-ligand contacts, exclusive of those required for receptor binding, are required for the stimulation of full IL-1 biological activity.     

A new synthesis of adenosine 5'-([gamma(R)-17O,18O]-gamma-thiotriphosphate) and its use to determine the stereochemical course of the activation of glutamate by glutamine synthetase

A new synthetic route to adenosine 5'-([gamma(R)-17O,18O]-gamma-thiotriphosphate) is described which combines chemical methods for introducing the heavy oxygen isotopes and enzymic methods for achieving the enantiospecificity. This material was used as a substrate for the activation of glutamate catalyzed by glutamine synthetase from Salmonella typhimurium. Analysis of the chirality of the [16O,17O,18O]thiophosphate produced showed that the reaction proceeds with inversion of configuration on phosphorus. This result, taken together with the positional isotope exchange studies of Midelfort and Rose [Midelfort, C. F., & Rose, I.A. (1976) J. Biol. Chem. 251, 5881-5887], demonstrates that the activation of glutamate to form gamma-glutamyl phosphate proceeds by a direct "in-line" transfer of the phosphoryl group.     

The phosphocholine motif in membranes of Mycoplasma fermentans strains

Mycoplasma fermentans strains differ in the profile of choline-containing phosphoglycolipids (PGL) present in their cell membrane. MfGL-II [Z?hringer et al. (1997) J. Biol. Chem. 272, 26262-26270] was found to be the major PGL in most strains tested. However, in the pulmonary isolates, M52 and M39 the major choline-containing PGLs were MfGL-I [Matsuda et al. (1994) J. Biol. Chem. 269, 33123-33129] and MfEL, a unique choline-containing ether lipid recently identified by us [Wagner et al. (2000) Eur. J. Biochem. 267, 6276-6286]. MfGL-I, MfGL-II and MfEL were metabolically labeled by growing the cells with radioactive choline but only MfGL-I and MfGL-II [corrected] reacted with antiphosphocholine antibodies. All tested strains fused with Molt-3 cells at almost the same rate and to about the same extent and in all the strains membrane proteins that reacted with anti-phosphocholine antibodies were detected, indicating that some membrane proteins are decorated with phosphocholine moieties.     

Two human liver cDNAs encode UDP-glucuronosyltransferases with 2 log differences in activity toward parallel substrates including hyodeoxycholic acid and certain estrogen derivatives.

Two human liver UDP-glucuronosyltransferase cDNA clones, HLUG25 [Jackson, M. R., et al. (1987) Biochem. J. 242, 581-588] and UDPGTh-2 [Ritter, J. K., et al. (1990) J. Biol. Chem. 266, 7900-7906] have previously been shown to encode isozymes active in the glucuronidation of hyodeoxycholic acid (HDCA) and certain estrogen derivatives (estriols and 3,4-catechol estrogens), respectively. Here we report that the UDPGTh-2-encoded isoform (udpgth-2) and the HLUG25-encoded isoform (udpgth-1) have parallel aglycon specificities. Following expression in COS-1 cells, each isoform metabolized three types of dihydroxy- or trihydroxy-substituted ring structures, including the 3,4-catechol estrogen (4-hydroxyestrone), estriol and 17-epiestriol, and HDCA, but the udpgth-2 isozyme is 100-fold more efficient than udpgth-1. udpgth-1 and udpgth-2 are 86% identical overall (76 differences out of 528 amino acids), including 55 differences in the first 300 amino acids of the amino terminus, a domain which confers isoform substrate specificity. The data indicate that a high level of conservation in the amino terminus is not required for the preservation of substrate selectivity. Analysis of glucuronidation activity encoded by UDPGTh-1/UDPGTh-2 chimeric cDNAs constructed at their common restriction sites, SacI (codon 297), NcoI (codon 385), and HhaI (codon 469), showed that nine amino acids between residues 385 and 469 are important for catalytic efficiency, suggesting that this region represents a domain which is critical for catalysis but distinct from that responsible for aglycon selection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple mRNA coding for phospholipid-transfer protein from Zea mays arise from alternative splicing

We have isolated a novel cDNA coding for maize phospholipid-transfer protein. The cDNA sequence is similar to the first one obtained by Tchang et al. [J. Biol. Chem. 263 (1988) 16849-16855] differing only by a mslal number of nucleotide substitutions and insertions. One of these insertions is 74 bp long and is flanked by consensus intron splicing sequences. The protein coded by the two cDNA has identical amino acids except in the C terminus. This difference derived from the presence of the 74-bp insert. The possible existence of an alternative splicing mechanism that could introduce heterogeneity in the sequence of these proteins is proposed.     

Metabolic cues from the microenvironment act as a major selective factor for cancer progression and metastases formation

Feature on: Serpa J, et al. J Biol Chem 2010. [Epub ahead of print]     

Superimposition of potent non-peptide AT1 receptor antagonists with angiotensin II

Summary The model of angiotensin II (ANG II) developed in our laboratory using a combination of NMR, fluorescence data and molecular graphics [Matsoukas, J.M. et al., J. Biol. Chem., 269 (1994) 5303] served as a template for a systematic superimposition of potent AT₁ receptor antagonists with ANG II. The key amino acids in this model, tyrosine, phenylalanine and histidine, form a charge-relay system. The studied ANG II AT₁ receptor antagonists were found to accommodate this relay system. The proposed model offers a motivation to synthetic chemists to develop ANG II antagonists that differ from the losartan prototype structure but possess an enhanced biological profile.     

Generation of mice deficient for macrophage galactose- and N-acetylgalactosamine-specific lectin: limited role in lymphoid and erythroid homeostasis and evidence for multiple lectins

Macrophage receptors function in pattern recognition for the induction of innate immunity, in cellular communication to mediate the regulation of adaptive immune responses, and in the clearance of some glycosylated cells or glycoproteins from the circulation. They also function in homeostasis by initiating the engulfment of apoptotic cells. Evidence has suggested that macrophage receptors function to recognize cells that are destined for programmed cell death but not yet overtly apoptotic. We have examined the function of a macrophage receptor specific for unsialylated glycoproteins, known as the mouse macrophage galactose- and N-acetylgalactosamine-specific lectin (mMGL) (Ii et al., J. Biol. Chem. 265:11295-11298, 1990; Sato et al., J. Biochem. [Tokyo] 111:331-336, 1992; Yamamoto et al., Biochemistry 33:8159-8166, 1994). With targeted disruption, we tested whether mMGL is necessary for macrophage function, controlled thymic development, the loss of activated CD8 T cells, and the turnover of red blood cells. Evidence indicates that mMGL may play a nonessential role in several of these macrophage functions. Experiments are presented that indicate the existence of another galactose- and N-acetylgalactosamine-recognizing lectin distinct from mMGL. This may explain the absence of a strong phenotype in mMGL-deficient mice.     

Solution structure of carnobacteriocin B2 and implications for structure-activity relationships among type IIa bacteriocins from lactic acid bacteria

Carnobacteriocin B2 (CbnB2), a type IIa bacteriocin, is a 48 residue antimicrobial peptide from the lactic acid bacterium Carnobacterium pisicola LV17B. Type IIa bacteriocins have a conserved YGNGVXC sequence near the N-terminus and usually contain a disulfide bridge. CbnB2 seemed to be unique in that its two cysteines (Cys9 and Cys14) could be isolated as free thiols [Quadri et al. (1994) J. Biol. Chem. 26, 12204-12211]. To establish the structural consequences of the presence or absence of a disulfide bridge and to investigate if the YGNGVXC sequence is a receptor-binding motif [Fleury et al. (1996) J. Biol. Chem. 271, 14421-14429], the three-dimensional solution structure of CbnB2 was determined by two-dimensional (1)H nuclear magnetic resonance (NMR) techniques. Mass spectroscopic and thiol modification experiments on CbnB2 and on model peptides, in conjunction with activity measurements, were used to verify the redox status of CbnB2. The results show that CbnB2 readily forms a disulfide bond and that this peptide has full antimicrobial activity. NMR results indicate that CbnB2 in trifluoroethanol (TFE) has a well-defined central helical structure (residues 18-39) but a disordered N terminus. Comparison of the CbnB2 structure with the refined solution structure of leucocin A (LeuA), another type IIa bacteriocin, indicates that the central helical structure is conserved between the two peptides despite differences in sequence but that the N-terminal structure (a proposed receptor binding site) is not. This is unexpected because LeuA and CbnB2 exhibit >66% sequence identity in the first 24 residues. This suggests that the N-terminus, which had been proposed [Fleury et al. (1996) J. Biol. Chem. 271, 14421-14429] to be a receptor binding site of type IIa bacteriocins, may not be directly involved and that recognition of the amphiphilic helical portion is the critical feature.     

Amino acid sequence of silkworm (Bombyx mori) hemolymph antitrypsin deduced from its cDNA nucleotide sequence: confirmation of its homology with serpins

A cDNA clone of silkworm (Bombyx mori) larval hemolymph antitrypsin (sw-AT) has been isolated from a fat body cDNA library. The cDNA has an open reading frame which codes a 392-amino acid residue polypeptide comprising a 16-residue signal peptide and a 376-residue mature sw-AT of Mr 41,805. The reactive site of sw-AT for inhibition of bovine trypsin [Sasaki, T. et al. (1987) J. Biochem. 102, 433-441] was identified as Lys343-Val344. Alignment of the sw-AT amino acid sequence with those of 11 members of the serpin superfamily of proteins clearly confirmed the homology of sw-AT with serpins. The amino acid sequence of sw-AT is 56% identical with that of the proteinase inhibitor from a lepidopteron, Manduca sexta [Kanost, M.R. et al. (1989) J. Biol. Chem. 264, 965-972], but the sequence around the reactive site shows no homology and the inhibitory specificity for proteinases is very different.     

Identification of histidyl peptide labeled by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate in an ADP regulatory site of glutamate dehydrogenase

Bovine liver glutamate dehydrogenase reacts covalently with 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of 1 mol reagent/mol enzyme subunit and loss of one of the two ADP sites of native enzyme [S. P. Batra and R. F. Colman, J. Biol. Chem. 261, 15565-15571 (1986)]. Incorporation of reagent is prevented specifically by ADP. The modified enzyme has now been digested with trypsin. The nucleotidyl peptide has been purified by chromatography on phenylboronate-agarose, followed by reverse-phase HPLC. On the basis of amino acid composition following acid hydrolysis, and gas-phase sequencing, the modified tryptic peptide was established as Ala-Gln-His-Ser-Gln-His-Arg, corresponding to amino acids 80-86 of the known glutamate dehydrogenase primary structure. The evidence presented indicates that the target amino acid attacked by 2-BDB-TAMP is histidine-82 and that this residue is located within the high-affinity ADP-activating site of glutamate dehydrogenase. In the course of this work, it was found that the positions of Gln84 and His85 had been reported as reversed in the revised sequence of bovine liver glutamate dehydrogenase [J. H. Julliard and E. L. Smith, J. Biol. Chem. 254, 3427-3438 (1979)]. Three additional corrections are here reported in the amino acid sequence of the native enzyme on the basis of gas-phase sequencing of other peptides purified by HPLC: Asp168 (not Asn); His221-Gly222 (not Gly-His); and Glu355 (not Gln).