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PubMed1997

Interruption of G protein-coupling in CXCR2 does not alter ligand binding, but eliminates ligand-activation of GTPgamma35S binding, calcium mobilization, and chemotaxis.

CXCR2 is a seven-transmembrane receptor that transduces intracellular signals in response to the chemokines IL-8, MGSA/GRO, and other ELR motif-containing CXC chemokines by coupling to heterotrimeric GTP-binding proteins. In this study, we have mutated two putative G protein-coupling regions of CXCR2 and characterized the effects of these mutations on ligand-activated signal transductions: aspartic acid 89 in the second transmembrane domain and the HRAMR sequence (BBXXB motif, found in the third intracellular loop where B indicates a basic amino acid and X represents any amino acid). The Asp89 was replaced by either asparagine (D89N) or glutamic acid (D89E). For the BBXXB motif, the first two basic amino acids were mutated to two neutral isoleucines (HR-II), or alternatively, two isoleucines were inserted between alanine and methionine (II-insert). When expressed in human embryonic kidney 293 cells, the D89E mutant was localized intracellularly with no detectable cell surface expression. In contrast, D89N, HR-II, and II-insert mutants displayed cell surface expression, with Kd values and expression levels similar to that of the wild-type transfectant. The ability of the mutants to transduce signal was assessed by ligand-stimulated GTPgamma35S binding, mobilization of intracellular free Ca2+, and chemotaxis assays. Both D89N and HR-II mutants signaled similarly to a wild-type receptor in all three assays. However, the II-insert mutant exhibited a loss of ligand-stimulated GTPgamma35S binding, calcium mobilization, and chemotaxis. Unexpectedly, this receptor underwent ligand-induced sequestration comparable to wild-type CXCR2. These data indicate that Asp89 and the basic amino acids in the third intracellular domain do not play essential roles in ligand-induced signal transduction through CXCR2. However, proper secondary structure and orientation of the third intracellular loop of CXCR2 are essential for ligand-mediated signal transduction but not for receptor sequestration.

Calcium↗
PubMed2004

A predicted amphipathic helix mediates plasma membrane localization of GRK5.

G protein-coupled receptor kinases (GRKs) specifically phosphorylate agonist-occupied G protein-coupled receptors at the inner surface of the plasma membrane (PM), leading to receptor desensitization. GRKs utilize a variety of mechanisms to bind tightly, and sometimes reversibly, to cellular membranes. Previous studies demonstrated the presence of a membrane binding domain in the C terminus of GRK5. Here we define a mechanism by which this short C-terminal stretch of amino acids of GRK5 mediates PM localization. Secondary structure predictions suggest that a region contained within amino acids 546-565 of GRK5 forms an amphipathic helix, with the key features of the predicted helix being a hydrophobic patch of amino acids on one face of the helix, hydrophilic amino acids on the opposite face, and a number of basic amino acids surrounding the hydrophobic patch. We show that amino acids 546-565 of GRK5 are sufficient to target the cytoplasmic green fluorescent protein (GFP) to the PM, and the hydrophobic amino acids are necessary for PM targeting of GFP-546-565. Moreover, full-length GRK5-GFP is localized to the PM, but mutation of the hydrophobic patch or the surrounding basic amino acids prevents PM localization of GRK5-GFP. Last, we show that mutation of the hydrophobic residues severely diminishes phospholipid-dependent autophosphorylation of GRK5 and phosphorylation of membrane-bound rhodopsin by GRK5. The findings in this report thus suggest the presence of a membrane binding motif in GRK5 and define the importance of a group of hydrophobic amino acids within this motif in mediating its PM localization.

Amino Acid Motifs↗
PubMed1990

A host range mutant of Newcastle disease virus with an altered cleavage site for proteolytic activation of the F protein.

The primary structure of the F protein of a host range mutant of the Ulster strain of Newcastle Disease virus (NDV) has been determined by nucleotide sequence analysis and compared to that of the wild type and other NDV strains. The cleavage site of the mutant had the sequence Gly-Lys-Gln-Arg-Arg as compared to two isolated basic amino acids [Gly-Lys(Arg)-Gln-Gly-Arg] with the apathogenic strains and two pairs of basic amino acids [Arg-Arg-Gln-Lys(Arg)-Arg] with the pathogenic strains. The data indicate that the cleavability of the F protein of NDV increases with the number of arginine and lysine residues at the cleavage site and that the susceptibility of the pathogenic strains to ubiquitous host proteases depends on both pairs of basic amino acids.

Amino Acid Sequence↗
PubMed1993

Comparative analysis of structurally defined heparin binding sequences reveals a distinct spatial distribution of basic residues.

Heparin, among the best studied glycosaminoglycans, is well known for its involvement in a variety of physiological processes. Many proteins, whose activities are modulated via heparin binding, were identified, and the consequences of their interaction with heparin were characterized. However, in the absence of solid structural information regarding heparin-protein complexes, the mechanism by which heparin operates at the molecular level is still obscure. The structure of such a complex is hereby explored via the identification of a common motif in heparin binding sequences. To avoid ambiguity we included in our data base only sequences that have been shown experimentally to be directly involved in heparin binding. Then, a comparison of the spatial distribution of basic residues was conducted among those peptides for which three-dimensional structures were defined. Using computer graphics techniques we were able to identify a unique distribution shared by all of these segments. Two basic amino acids (most frequently arginine) are located at about 20 A apart, facing opposite directions of an alpha-helix. Other basic amino acids are dispersed between these two residues, facing one side, while nonpolar residues face the opposite side, forming an amphipathic structure. The distribution of basic amino acids in other heparin binding sequences that preserves the same spatial arrangement seems to be compatible with a beta-strand structure. The 20-A interval accommodates a glycosaminoglycan pentasaccharide, and the spatial distribution of the basic residues suggests an intertwinement of the heparin-protein complex. The dynamics of such an interaction may provide a clue regarding the ensuing change in protein activity.

Amino Acid Sequence↗
PubMed1985

Fluorescent pigments by covalent binding of lipid peroxidation by-products to protein and amino acids.

The fluorescent products formed on reaction of 12-oxo-cis-9-octadecenoic acid (12-keto-oleic acid) with about 20 different amino acids, polylysine and bovine serum albumin (BSA) were studied. Besides glycine, only the basic amino acids histidine, lysine and arginine gave products with strong fluorescence. N-Acetylation of amino acids greatly reduced the fluorescence of their reaction products. The formation of fluorescent products was inhibited strongly by SH-amino acids such as N-acetyl-cysteine and glutathione. Polyacrylamide gel electrophoresis showed that BSA treated with 12-keto-oleic acid was more acidic than untreated or ricinoleic acid-treated BSA, indicating that basic amino acid residues in BSA were modified by reaction with the keto fatty acid. None of the structural analogs of 12-keto-oleic acid tested--12-oxo-trans-10-octadecenoic acid, 12-oxo-octadecanoic acid, 12-hydroxy-cis-9-octadecenoic acid (ricinoleic acid), cis-9-octadecenoic acid (oleic acid) and linoleic acid--reacted with glycine to give a fluorescent product. The fluorescent products formed on reaction of 12-keto-oleic acid methyl ester with benzyl amine and glycine methyl ester were shown to be 8-(N-substituted-4,5-dihydro-4-oxo-5-hexyl-5-hydroxy-2-pyrrolyl) octanoic acid methyl esters. The fluorescence properties of these compounds were attributed to the chromophobic system NC = CC = O which contains 6 pi electrons. This investigation contributes to insight of the mechanism of formation of fluorescent pigments, probably by a similar reaction of other compounds of the beta, gamma-unsaturated carbonyl type.

Amino Acids↗
PubMed1979

Postnatal amino acid uptake by the rat small intestine. Energetics of membrane transport systems for amino acids in the developing jejunum.

The energetics of amino acid uptake by the developing small intestine was investigated in vitro. L-valine, L-leucine, L-phenylalanine, L-methionine, L-lysine and L-arginine were all actively transported by the newborn rat jejunum. Metabolic inhibitors (e.g. 2,4-dinitrophenol) significantly reduced uptake of all amino acids but uptake against a concentration gradient was not totally abolished. Uptake of all amino acids was reduced at low[Na+]. Inhibition of transport of neutral amino acids by reduced luminal [Na+] was greater than that of basic amino acids, and the tissue was barely able to concentrate the neutral amino acids. [Na+] affected the Michaelis constant (Km) of neutral transport systems for their substrates; for the basic amino acids Km values were unaffected by the presence or absence of Na+. Ouabain significantly inhibited neutral amino acid uptake but had no effect on L-lysine or L-arginine uptake. These results are discussed in terms of the Na+ gradient hypothesis for amino acid transport, and the site of energy input to active transport. The role of glycolysis in providing energy for intestinal transport in the neonatal rat and the efficiency of Na+ dependent and independent transport mechanisms are considered. It is concluded that the energetics of amino acid transport systems in neonatal and adult rats are essentially similar.

Amino Acids↗
PubMed1994

Electrogenic amino acid exchange via the rBAT transporter.

A cDNA clone was isolated from rabbit renal cortex using DNA-mediated expression cloning, which caused alanine-dependent outward currents when expressed in Xenopus oocytes. The cDNA encodes rBAT, a Na-independent amino acid transporter previously cloned elsewhere. Exposure of cDNA-injected oocytes to neutral amino acids led to voltage-dependent outward currents, but inward currents were seen upon exposure to basic amino acids. Assuming one charge/alanine, the outward current represented 38% of the rate of uptake of radiolabelled alanine, and was significantly reduced by prolonged preincubation of oocytes in 5 mM alanine. The currents were shown to be due to countertransport of basic amino acids for external amino acids using the cut-open oocyte system. This transport represents a major mode of action of this protein, and may help in defining a physiological role for rBAT in the apical membrane of renal and intestinal cells.

Alanine↗
PubMed1998

Identification and characterization of heparin binding regions of the Fim2 subunit of Bordetella pertussis.

Bordetella pertussis fimbriae bind to sulfated sugars such as heparin through the major subunit Fim2. The Fim2 subunit contains two regions, designated H1 and H2, which show sequence similarity with heparin binding regions of fibronectin, and the role of these regions in heparin binding was investigated with maltose binding protein (MBP)-Fim2 fusion proteins. Deletion derivatives of MBP-Fim2 showed that both regions are important for binding to heparin. The role of H2 in heparin binding was confirmed by site-directed mutagenesis in which basic amino acids were replaced by alanine. These studies revealed that Lys-186 and Lys-187 are important for heparin binding of MBP-Fim2, whereas Arg-179 is not required. Peptides derived from H1 and H2 (pepH1 and pepH2) also showed heparin binding activity. Using a series of peptides, in each of which a different basic amino acid was substituted for alanine, we demonstrated that the structural requirements for heparin binding differ significantly among pepH1 and pepH2 peptides. A Pepscan analysis of Fim2 revealed regions outside H1 and H2 which bind heparin and showed that not only basic amino acids but also tyrosines may be important for binding to sulfated sugars. A comparison of the heparin binding regions of Fim2 with homologous regions of Fim3 and FimX, two closely related but antigenically distinct fimbrial subunits, showed that basic amino acids and tyrosines are generally conserved. The major heparin binding regions identified in Fim2 are part of epitopes recognized by human antibodies, suggesting that the heparin binding regions are exposed at the fimbrial surface and are immunodominant. Since B. pertussis fimbriae show weak serological cross-reactivity, the differences in primary structure in the heparin binding regions of Fim2, Fim3, and FimX may affect antibody binding but not heparin binding, allowing the bacteria to evade antibody-mediated immunity by switching the fimbrial gene expressed.

Amino Acid Sequence↗
PubMed1983

[Some structural properties of 80S acid protein from pea ribosomes].

The 80S acid protein from pea ribosomes similar to the L7/L12 protein from E. coli was studied. This protein was found to be rich in alanine (18 mol.%) and to contain an acid amino acids excess over basic ones, the ratio of basic amino acids to acid ones was 0.42. As in the case of other eukaryotic L7/L12 homologs studied, the N-terminal amino acid of the protein is methionine. Using the double immunodiffusion technique, no crossreaction of E. coli anti-L7/L12 with 80S acid protein from pea ribosomes was observed. It was assumed that the protein molecule contains conservative sites responsible for the specific functioning of eukaryotic L7/L12 homologs.

Amino Acids↗
PubMed1995

A novel and simple method to assay the activity of individual protein kinases in a crude tissue extract.

Protein kinases and phosphatases play an important role in a variety of cellular functions. Thus, it is of interest to develop an assay system that can be used to quantify the activity of individual enzymes specifically in a crude cellular extract, is simple to perform, and is amenable to automation. Here we report on the development of a protein kinase assay that addresses these points and circumvents the pitfalls of existing methodologies. The assay is based on the high affinity and strong binding of streptavidin toward biotin-linked peptide substrates. The biotinylated peptide substrate is phosphorylated by the cognate protein kinase using [gamma-32P]ATP under optimal enzyme condition, and the phosphorylated peptide product is then captured by a streptavidin-linked disk. After removal of free [gamma-32P]ATP, the 32P incorporated into the peptide substrate can be used as an expression of enzyme activity. In contrast to the commonly used phosphocellulose method, only the phospho-, biotinylated peptide (and not other phosphorylated proteins present in the extract) will bind to the disks, thus giving a true estimate of enzymatic activity. In addition to specificity, this assay does not require the peptide substrate to contain basic amino acids or to be modified by the addition of basic amino acid residues as required for the phosphocellulose method which may result in altered specificity of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMedDate not supplied

Spike glycoprotein cleavage recognition site analysis of infectious bronchitis virus.

The spike glycoprotein of infectious bronchitis virus (IBV), a coronavirus, is translated as a precursor protein (So), then cleaved into two subunits (S1 and S2) by host cell serine proteases. In this study, we compared the cleavage recognition site of 55 IBV isolates to determine if the cleavage recognition site sequence, which consists of five basic amino acid residues, correlates with host cell range, serotype, geographic origin, and pathogenicity as it does in orthomyxoviruses and paramyxoviruses. The most common cleavage recognition site observed (33 of 55 viruses) was Arg-Arg-Ser-Arg-Arg, representing at least 11 different serotypes. Thus, cleavage recognition site does not appear to correlate with serotype. We also determined that cleavage recognition site sequence does not correlate with pathogenicity because attenuated and pathogenic isolates (different passages of the same virus) contain identical cleavage recognition site sequences. In addition, nephropathogenic strains had the same cleavage recognition site sequence as many nonnephropathogenic isolates. Cleavage recognition site sequence does correlate with viruses in different geographic regions, which may be an important characteristic to examine in epidemiologic studies. An IBV monoclonal antibody neutralization-resistant mutant (NR 18) had an unusual substitution of Ile for Arg at the fourth position, giving the sequence Arg-Arg-Ser-Ile-Arg, which likely prevents cleavage and, thus, destroys the conformationally dependent monoclonal antibody binding epitope. Six residues on the amino-terminal side of the cleavage recognition site are conserved in 31% of the isolates and consist of only one or two basic amino acids. Thus, the number of basic residues around the cleavage recognition site does not appear to correlate with increased cleavability, host cell range, and increased virulence as it does with envelope glycoproteins in orthomyxoviruses and paramyxoviruses.

Amino Acid Sequence↗
PubMed1986

Pancreatic carboxypeptidase hydrolysis of bile acid-amino conjugates: selective resistance of glycine and taurine amidates.

To find a possible explanation for the selective hepatic conjugation of bile acids with glycine or taurine, the N-acyl amidates of cholic acid and a number of amino acids and amino acid analogues were synthesized, and their susceptibility to hydrolysis by pancreatic juice, gastric juice, serum, or small intestinal mucosal enzymes was measured. Deconjugation by pure carboxypeptidase A and B was also examined, and hydrolysis by these tissue fluids and enzymes was compared with that mediated by a bacterial cholylglycine hydrolase. Human pancreatic juice efficiently hydrolyzed cholyl conjugates of all neutral-L-amino acids (cholyl-L-alanine, cholyl-L-valine, cholyl-L-leucine, and cholyl-L-tyrosine), except cholylglycine. The net hourly rate of hydrolysis (in micromoles per milligram protein per hour) increased when the terminal residue was aromatic or branched aliphatic, and appeared to be specific for L-alpha-amino acids as cholyl-beta-alanine and cholyl-D-valine were not cleaved. From cholyl glycylglycine, only the terminal glycine was efficiently removed. Cholyltaurine and cholyl conjugates with the methyl and propyl analogues of taurine were resistant to hydrolysis. Two basic amino acid conjugates (cholyl-L-lysine and cholyl-L-arginine) were cleaved, whereas conjugates of acidic amino acids (cholyl-aspartate and cholyl-cysteate) were not cleaved. Studies using pure enzymes showed that bovine carboxypeptidase A hydrolyzed the cholyl conjugates of the neutral L-alpha-amino acids with similar specificity as observed for the human pancreatic juice, whereas bovine carboxypeptidase B cleaved the basic amino acid conjugates. Cholyl-L-lysine and cholyl-L-arginine were also cleaved by serum and plasma, which are known to possess carboxypeptidase activity. Cholyl conjugates were not cleaved by gastric juice, by trypsin, or by homogenates of rat small intestinal mucosa. In contrast, all cholyl conjugates were cleaved by a bacterial cholylglycine hydrolase. These experiments indicate that glycine and taurine amidates of cholic acid differ from a number of other conjugates with neutral and basic amino acid in being resistant to hydrolysis by pancreatic and plasma carboxypeptidases.(ABSTRACT TRUNCATED AT 400 WORDS)

Amidohydrolases↗
PubMed2001

Equilibrium between basic nitrogen compounds in lupin seeds with differentiated alkaloid content.

The results of studies on the content of the nitrogen basic compounds, viz. quinolizidine alkaloids, biogenic polyamines and basic amino acids in lupin seeds are presented. The investigations concerned three lupin species (Lupinus angustifolius L., Lupinus albus L. and Lupinus luteus L.) and 10 bitter and sweet cultivated varieties. Content of quinolizidine alkaloids in L. angustifolus ranged from 11.4 to 19.6 microg mg(-1) dw (bitter cultivars), from 0.18 to 0.47 microg mg(-1) dw (sweet), in L. albus from 0.58 microg mg(-1) dw (sweet) to 29.6 microg mg(-1) dw (bitter) and in L. luteus from 0.59 (sweet) to 14.7 microg mg(-1) dw (bitter). Total biogenic polyamine content ranged in L. angustifolius from 2,773.9 to 3,180.2 pmol mg(-1) dw (bitter) and from 315.0 to 599.0 pmol mg(-1) dw (sweet), in L. albus from 432.6 pmol mg(-1) dw (sweet) to 1,832.0 pmol mg(-1) dw (bitter) and in L. luteus from 506.9 pmol mg(-1) dw (sweet) to 2,091.8 pmol mg(-1) dw (bitter). Total basic amino acids varied in L. angustifolus from 1,034.3 to 1,704.6 pmol mg(-1) dw (bitter) and from 1,761.9 to 2,101.9 pmol mg(-1) dw (sweet), in L. albus from 696.9 pmol mg(-1) dw (bitter) to 1,269.2 pmol mg(-1) dw (sweet) and in L. luteus from 927.6 pmol mg(-1) dw (bitter) to 1,598.3 pmol mg(-1) dw (sweet). We found a close dependence between alkaloid content and level of biogenic polyamines and basic amino acids in all three lupin species tested. All bitter lupin seeds also contain high level of biogenic polyamines but a low content of basic amino acids. The reverse relationship in sweet lupin seeds was found. The findings demonstrate that lupin nitrogen basic compounds are in steady equilibrium and that change of content in one compound leads to corresponding change in the content of another.

Alkaloids↗
PubMed1989

Site-specific mutagenesis identifies amino acid residues critical in prohormone processing.

Peptide hormones are generally synthesized as inactive higher mol. wt precursors. Processing of the prohormone into biologically active peptides by specific proteolytic cleavages occurs most often at pairs of basic amino acids but also at single arginine residues. To study the role of protein secondary structure in this process, we used site-directed mutagenesis to modify the predicted secondary structure around the cleavage sites of human prosomatostatin and monitored the processing of the precursor after introduction of the mutated cDNAs in Neuro2A cells. Amino acid substitutions were introduced that affected the possibility of forming beta-turn structures in the immediate vicinity of the somatostatin-28 (S-28) and somatostatin-14 (S-14) cleavage sites. Infection of Neuro2A cells with a retrovirus carrying a human somatostatin cDNA resulted in the expression of prosomatostatin and its processing into S-28 and S-14, indicating that these cells have the necessary enzymes to process prohormone at both single and paired amino acid residues. Disruption of the different beta-turns had various effects on prosomatostatin processing: substitution of Ala for Pro-5 drastically decreased prosomatostatin processing and replacement of Pro-9 by Ala led to the accumulation of the intermediate maturation product [Arg-2Lys-1]-S-14. In contrast, substitution of Ala for Asn-12, Gly+2 and Cys+3 respectively had only very little effect on the proteolytic processing of prosomatostatin. Our results show that amino acids other than the basic amino acid residues are required to define the cleavage sites for prohormone proteolytic processing and suggest that higher orders of protein structure are involved in substrate recognition by the endoproteases.

Acyltransferases↗
PubMed1995

Oligomeric structure of a renal cystine transporter: implications in cystinuria.

Homologous proteins (NBAT) which mediate sodium-independent transport of neutral as well as basic amino acids and cystine when expressed in Xenopus oocytes were recently cloned from mammalian kidneys. Mutations in human NBAT have been implicated in cystinuria. Here, we show that rat kidney and jejunal brush border membrane NBAT (85 kDa) is found in association with a 50 kDa protein. The association involves one or more interprotein disulfide bonds. Rabbit kidney brush border membranes and membranes of NBAT cRNA-injected Xenopus oocytes also contain such heterodimers. Our data suggest that the heterodimer is the minimal functional unit of NBAT-mediated amino acid transport and that the NBAT-associated 50 kDa protein could play a role in cystinuria.

Amino Acid Transport Systems, Basic↗
PubMed1998

Progressive C-terminal deletions of the renal cystine transporter, NBAT, reveal a novel bimodal pattern of functional expression.

Nearly identical proteins (denoted NAA-Tr, rBAT, D2, NBAT), cloned from mammalian kidneys, induce a largely sodium-independent high-affinity transport system for cystine, basic amino acids, and some neutral amino acids in Xenopus oocytes (system b0,+-like). Mutations in the human NBAT gene have been found in several type I cystinurics. In kidney, NBAT is associated with a second, smaller protein (approximately 45 kDa), and this heterodimer has been proposed to be the minimal functional unit of the renal cystine transporter (Wang, Y., and Tate, S. S. (1995) FEBS Lett. 368, 389-392). To delineate regions minimally required for functional expression in oocytes, we constructed a series of C-terminal truncated mutants of rat kidney NBAT (wild-type (WT), 683 amino acids). Expression of these mutants in oocytes yielded an unusual bimodal pattern for the induction of amino acid transport activity. Thus, initial C-terminal truncations aborted elicitation of transport activity. The next mutant in the series, Delta588-683, exhibited most of the transport-inducing potential inherent in the WT/NBAT. Further deletions again attenuated transport activity. Although both the WT/NBAT and the truncated mutant, Delta588-683, induce qualitatively similar transport systems, the two forms of the protein exhibit contrasting sensitivities toward a point mutation in which the cysteine residue at position 111 was mutated to serine. This mutation did not greatly affect induction of transport by the WT/NBAT; however, the Delta588-683 mutant was inactivated by this mutation. Our data further suggest that cysteine 111 is probably the site of disulfide linkage with an approximately 45-kDa oocyte protein producing a complex equivalent to that seen in kidney membranes.

Amino Acid Transport Systems, Basic↗
PubMed2001

Identification of genes encoding amino acid permeases by inactivation of selected ORFs from the Synechocystis genomic sequence.

Genes encoding elements of four amino acid permeases were identified by insertional inactivation of ORFs from the genomic sequence of the cyanobacterium Synechocystis sp. strain PCC 6803 whose putative products are homologous to amino acid permease proteins from other bacteria. A transport system for neutral amino acids and histidine and a transport system for basic amino acids and glutamine were identified as ABC-type transporters, whereas Na(+)-dependent transport of glutamate was found to be mediated by at least two systems, the secondary permease GltS and a TRAP-type transporter. Except for GltS, substrate specificities of the identified permeases do not match those of previously characterized systems homologous to these permeases.

Amino Acid Transport Systems↗