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PubMed2003

Radiolytic modification of basic amino acid residues in peptides: probes for examining protein-protein interactions.

Protein footprinting utilizing hydroxyl radicals coupled with mass spectrometry has become a powerful technique for mapping the solvent accessible surface of proteins and examining protein-protein interactions in solution. Hydroxyl radicals generated by radiolysis or chemical methods efficiently react with many amino acid residue side chains, including the aromatic and sulfur-containing residues along with proline and leucine, generating stable oxidation products that are valuable probes for examining protein structure. In this study, we examine the radiolytic oxidation chemistry of histidine, lysine, and arginine for comparison with their metal-catalyzed oxidation products. Model peptides containing arginine, histidine, and lysine were irradiated using white light from a synchrotron X-ray source or a cesium-137 gamma-ray source. The rates of oxidation and the radiolysis products were primarily characterized by electrospray mass spectrometry including tandem mass spectrometry. Arginine is very sensitive to radiolytic oxidation, giving rise to a characteristic product with a 43 Da mass reduction as a result of the loss of guanidino group and conversion to gamma-glutamyl semialdehyde, consistent with previous metal-catalyzed oxidation studies. Histidine was oxidized to generate a mixture of products with characteristic mass changes primarily involving rupture of and addition to the imidazole ring. Lysine was converted to hydroxylysine or carbonylysine by radiolysis. The development of methods to probe these residues due to their high frequency of occurrence, their typical presence on the protein surface, and their frequent participation in protein-protein interactions considerably extends the utility of protein footprinting.

Amino Acids↗
PubMed1993

Voltage dependence of facilitated arginine flux mediated by the system y+ basic amino acid transporter.

Two-microelectrode voltage clamp was used to measure membrane currents resulting from flux of cationic amino acids in Xenopus oocytes expressing the cloned dual-function ecotropic murine leukemia virus receptor/system y+ transporter. At membrane potentials ranging from +20 mV to -120 mV, arginine influx obeyed Michaelis-Menten kinetics. At concentrations from 0.01 to 1 mM, influx increased exponentially with membrane hyperpolarization (e-fold increase/-59 mV). Efflux from oocytes preloaded with arginine increased exponentially with depolarization (e-fold increase/+52 mV). Kinetic analysis based on an iso uni uni facilitated transport model suggests that the effect of voltage on steady-state flux arises largely from charge movement across the membrane field during the conformational transition of the unliganded transporter which switches the substrate binding site from one membrane face to the other. This charge movement would facilitate rapid increases in intracellular arginine concentrations in response to hyperpolarization, a property which could play a role in modulating nitric oxide synthesis in some types of cells.

Animals↗
PubMed2002

Role of pyridine hydrogen-bonding sites in recognition of basic amino acid side chains.

A series of three, new artificial receptors for guanidinium and ammonium guests has been synthesized. All three receptors have highly preorganized clefts bearing two carboxylate groups. They differ in the number of nitrogen atoms contained in their clefts, as follows: four N atoms in receptor 3, three N atoms in 4, and two nitrogens in 5. Crystallographic studies have produced the solid-state structures of the following guanidinium complexes of each receptor: 3.2CH(3)CH(2)NHC(NH(2))(2)(+), 4.2CH(3)NHC(NH(2))(2)(+), and 5.2C(NH(2))(3)(+). The conformations of the receptor molecules in all three complexes are very similar. N-Alkylguanidinium guests are bound in the clefts of 3 and 4 in similar manners, despite the loss of one hydrogen-bond acceptor nitrogen in 4 and the possible hindrance of the cavity by a CH group. In the guanidinium complex of 5, neither guest enters the cavity containing two CH groups. Complexation studies were conducted in methanol by (1)H NMR titration for several guanidinium and ammonium guests, including derivatives of the amino acids arginine and lysine. Receptor 5 binds all such guests weakly (K(s) < 4000), while 3 binds most guests very strongly (K(s) > 100 000). Receptor 3 is selective for arginine versus lysine, while 4 binds lysine better than does 3. The results generally underscore the importance of receptor preorganization and hydrogen-bonding complementarity in the design of receptors that can serve as probes for biomolecules.

Amino Acids↗
PubMed1986

Methotrexate analogues. 26. Inhibition of dihydrofolate reductase and folylpolyglutamate synthetase activity and in vitro tumor cell growth by methotrexate and aminopterin analogues containing a basic amino acid side chain.

Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a "self-potentiation" process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.

Aminobutyrates↗
PubMed1997

Site-directed mutagenesis in basic amino acid residues of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase.

Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V(max)/K(m)ADP. These results indicate that Arg76 is not involved in CO2 binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413-418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207-210], does not perform an essential function for the enzyme activity.

Arginine↗
PubMed1994

A requirement of hydrophobic and basic amino acid residues for substrate recognition by Ca2+/calmodulin-dependent protein kinase Ia.

The substrate recognition determinants of Ca2+/calmodulin-dependent protein kinase Ia were investigated by using peptide analogues based on the amino acid sequence around Ser-9 of synapsin I. The Km and Vmax for the synthetic peptide Leu-Arg-Arg-Arg-Leu-Ser-Asp-Ala-Asn-Phe are 3.9 microM and 18.5 mumol/(min.mg), respectively. Deletion of Leu at the -5 position lowers the Vmax/Km by 470-fold. The requirement for a hydrophobic residue at -5 was confirmed by the 90- to 2400-fold reduction in Vmax/Km produced by Arg, Ala, or Asp substitutions, but only 2.6-fold decrease after Phe substitution at this position. A hydrophobic residue is similarly required at the +4 position. Deletion of Phe at this position produces a 67-fold reduction, and substitution of Ala for Phe a 43-fold reduction in Vmax/Km. In contrast, substitution with Leu increases Vmax/Km by 1.8-fold. Arg at -3 is also required for recognition as shown by an approximately 240-fold decrease in Vmax/Km after Ala substitution at this position. Positions -2, -4, and +1 appear to play secondary roles in substrate recognition. Arg at -2 and -4 are positive determinants, since Ala substitution at these positions decreases Vmax/Km by 4.7- and 11-fold, respectively. Asp at +1 is a negative influence, since Ala and Leu substitutions at this position increase Vmax/Km by 2.3- and 6.3-fold, respectively. Substitution of Ala for Leu at -1 or Thr for Ser at the 0 position has little effect on phosphorylation kinetics. Thus, Ca2+/calmodulin-dependent protein kinase Ia has the minimal substrate recognition motif of Hyd-Xaa-Arg-Xaa-Xaa-(Ser*/Thr*)-Xaa-Xaa-Xaa-Hyd, where Hyd represents a hydrophobic amino acid residue.

Amino Acid Sequence↗
PubMed2001

Mutational analysis of the fractalkine chemokine domain. Basic amino acid residues differentially contribute to CX3CR1 binding, signaling, and cell adhesion.

Fractalkine (FKN/CX3CL1) is a unique member of the chemokine gene family and contains a chemokine domain (CD), a mucin-like stalk, a single transmembrane region, and a short intracellular C terminus. This structural distinction affords FKN the property of mediating capture and firm adhesion of FKN receptor (CX3CR1)-expressing cells under physiological flow conditions. Shed forms of FKN also exist, and these promote chemotaxis of CX3CR1-expressing leukocytes. The goal of the present study was to identify specific residues within the FKN-CD critical for FKN-CX3CR1 interactions. Two residues were identified in the FKN-CD, namely Lys-7 and Arg-47, that are important determinants in mediating an FKN-CX3CR1 interaction. FKN-K7A and FKN-R47A mutants exhibited 30-60-fold decreases in affinity for CX3CR1 and failed to arrest efficiently CX3CR1-expressing cells under physiological flow conditions. However, these mutants had differential effects on chemotaxis of CX3CR1-expressing cells. The FKN-K7A mutant acted as an equipotent partial agonist, whereas the FKN-R47A mutant had marked decreased potency and efficacy in measures of chemotactic activity. These data identify specific structural features of the FKN-CD that are important in interactions with CX3CR1 including steady state binding, signaling, and firm adhesion of CX3CR1-expressing cells.

Amino Acid Sequence↗
PubMed2001

Adjacent basic amino acid residues recognized by the COP I complex and ubiquitination govern endoplasmic reticulum to cell surface trafficking of the nicotinic acetylcholine receptor alpha-Subunit.

The nicotinic acetylcholine receptor in muscle is a ligand-gated ion channel with an ordered subunit arrangement of alpha-gamma-alpha-delta-beta. The subunits are sequestered in the endoplasmic reticulum (ER) and assembled into the pentameric arrangement prior to their exit to the cell surface. Mutating the Arg(313)-Lys(314) sequence in the large cytoplasmic loop of the alpha-subunit to K314Q promotes the trafficking of the mutant unassembled alpha-subunit from the ER to the Golgi in transfected HEK cells, identifying an important determinant that modulates the ER to Golgi trafficking of the subunit. The association of the K314Q alpha-subunit with gamma-COP, a component of COP I coats implicated in Golgi to ER anterograde transport, is diminished to a level comparable to that observed for wild-type alpha-subunits when co-expressed with the beta-, delta-, and gamma-subunits. This suggests that the Arg(313)-Lys(314) sequence is masked when the subunits assemble, thereby enabling ER to Golgi trafficking of the alpha-subunit. Although unassembled K314Q alpha-subunits accumulate in the Golgi, they are not detected at the cell surface, suggesting that a second post-Golgi level of capture exists. Expressing the K314Q alpha-subunit in the absence of the other subunits in ubiquitinating deficient cells (ts20) results in detecting this subunit at the cell surface, indicating that ubiquitination functions as a post-Golgi modulator of trafficking. Taken together, our findings support the hypothesis that subunit assembly sterically occludes the trafficking signals and ubiquitination at specific sites. Following the masking of these signals, the assembled ion channel expresses at the cell surface.

Animals↗
PubMed1979

[The metabolism of colostomised laying hens with 15N-labelled wheat. 4. 15N-Incorporation in the corpuscular components of blood, in the blood plasma and the basic amino acids].

Three colostomised Laying hybrids received over four days a dosage of 672 mg 15N-excess (15N'), 20.3 mg lysine 15N', 23.0 mg histidine-15N' and 66.7 mg arginine-15N' with a ration customary in production. After feeding the same nonlabelled ration for another four days the hens were killed and the N-content of the blood as well as of its fractions (corpuscular components, plasma, free amino acids of the plasma) was determined.

Animals↗
PubMed1984

[Studies of 15N-labeled lysine in colostomized laying hens. 6. 15N incorporation into basic amino acids of the liver, kidney, meat, bones and remaining body as well as 15N balance].

Three colostomised laying hybrids received 91.40 mg L-lysine-15N-excess (15N') each over a period of four days in a metabolism experiment with 15N-labelled lysine. After another four days, during which the hens received the same rations supplemented by commercial L-lysine, the animals were butchered and divided into individual fractions. After the hydrochloric hydrolysis of the organs and tissues the heavy nitrogen of lysine, histidine and arginine were separated, quantitatively evaluated, processed and measured with an emission spectrometer. Atom-% 15N' on an average amounted to 0.20 in the liver, 0.16 in the kidneys, 0.06 in the meat and 0.05 in the bones. Of the rediscovered 15N' applied, faeces contained 8.1%, urine 18.3%, the eggs 24.3%, the blood 4.9%, the meat 20.5%, the bones 5.2%, the gastro-intestinal tract with its contents 4.5%, the liver 3.5%, the kidneys 0.9%, the reproductive organs 3.7% and the rest 6.1%. The quota of rediscovery of the 15N' applied was 95.7%. 62% of the total 15N' was rediscovered in eggs, body and faeces as lysine 15N'. There was significantly more 15N' in the arginine in all fractions than in the histidine. The quota of the lysine-15N' of the total 15N' differed considerably in the fractions: less than 40% bones and blood; 48...56% gastro-intestinal tract, faeces, oviduct, kidneys; 62...63% remaining ovary, rest; 69...71% eggs, meat, liver. It could be proved that the alpha-amino group of lysine is to a large extent incorporated into other amino acids. Further proof that the amino acid metabolism proceeds in two phases was submitted, i.e. hight amounts of amino acids previously deposited in the body are used for egg synthesis.

Amino Acids↗
PubMed1997

Homoarginine influences voluntary feed intake, tissue basic amino acid concentrations and arginase activity in chickens.

Two experiments were conducted to investigate the factors responsible for the adverse effects of guanidinated proteins on feed intake in chickens. In Experiment 1, male broiler chicks were fed one of five purified diets containing casein or guanidinated casein (G-casein) as the sole source of protein (230 g crude protein/kg diet) from d 6 to 13 post-hatching. A casein-based diet containing 17.2 g lysine/kg, served as the control. In the experimental diets, casein was substituted by G-casein and lysine was added at 0, 5.6, 11.4 and 17.0 g/kg diet, respectively. Feed intake and weight gains of chicks fed the G-casein diet without added lysine were markedly depressed (P < 0.05), but this depression was largely overcome by additional lysine. The intake and gains of chicks fed the G-casein diet plus 17.0 g lysine/kg were lower (P < 0.05) than those fed the G-casein diet plus 11.4 g lysine/kg and this was associated with a higher plasma lysine:arginine ratio. Tissue analysis showed that homoarginine is distributed throughout body tissues following absorption. Brain lysine concentrations were lower (P < 0.05) in chicks fed diets containing G-casein without added lysine, but increased (P < 0.05) with supplemental lysine. In Experiment 2, the effect of homoarginine per se on feed intake was investigated in two short-term intake studies using 5-wk-old broiler chickens. Significant (P < 0.05) depressions in feed intake were observed within the first hour after oral administration of 400 mg homoarginine-HCl. The results suggest that both lysine deficiency and homoarginine per se were responsible for the adverse effects of guanidinated proteins on feed intake in chickens.

Amino Acids, Essential↗
PubMed1987

Site-directed mutagenesis of basic amino acid residues in the extension peptide of P-450(SCC) precursor: effects on the import of the precursor into mitochondria.

The precursor of cytochrome P-450(SCC) (preP-450(SCC], an inner membrane protein of adrenal cortex mitochondria, has an extension peptide consisting of 39 amino acids which is thought to play an essential role in the import of the precursor into mitochondria. The amino terminal portion of the extension peptide contains three positively charged amino acid residues, Arg(4), Arg(9), and Lys(14). To investigate their role in the import of preP-450(SCC) into mitochondria, they were replaced by other amino acids, Ser or Thr, by site-directed mutagenesis. The import of mutated preP-450(SCC)s with single amino acid substitution was much less efficient than with the original precursor. The mutated preP-450(SCC)s with two or three substitutions were not imported. These results suggest that the positively charged amino acid residues in the amino terminal portion of the extension peptide are essential for the import of preP-450(SCC) into mitochondria.

Amino Acids↗