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At least 1,081 records · Page 60Linked to original sources
PubMed1993

Lysines 72, 80 and 213 and aspartic acid 210 of the Lactococcus lactis LacR repressor are involved in the response to the inducer tagatose-6-phosphate leading to induction of lac operon expression.

Site-directed mutagenesis of the Lactococcus lactis lacR gene was performed to identify residues in the LacR repressor that are involved in the induction of lacABCDFEGX operon expression by tagatose-6-phosphate. A putative inducer binding domain located near the C-terminus was previously postulated based on homology studies with the Escherichia coli DeoR family of repressors, which all have a phosphorylated sugar as inducer. Residues within this domain and lysine residues that are charge conserved in the DeoR family were changed into alanine or arginine. The production of the LacR mutants K72A, K80A, K80R, D210A, K213A and K213R in the LacR-deficient L.lactis strain NZ3015 resulted in repressed phospho-beta-galactosidase (LacG) activities and decreased growth rates on lactose. Gel mobility shift assays showed that the complex between a DNA fragment carrying the lac operators and LacR mutants K72A, K80A, K213A and D210A did not dissociate in the presence of tagatose-6-phosphate, in contrast to wild type LacR. Other mutations (K62A/K63A, K72R, K73A, K73R, T212A, F214R, R216R and R216K) exhibited no gross effects on inducer response. The results strongly suggest that the lysines at positions 72, 80 and 213 and aspartic acid at position 210 are involved in the induction of lac operon expression by tagatose-6-phosphate.

Amino Acid Sequence↗
PubMed1993

(2R), (1'R) and (2R), (1'S)-2-amino-3-(1,2-dicarboxyethylthio)propanoic acids from Amanita pantherina. Antagonists of N-methyl-D-aspartic acid (NMDA) receptors.

An antagonist of NMDA-sensitive glutamate receptors in rat brain membrane and rat spinal motoneurones was isolated from Amanita pantherina and identified as a diastereoisomeric mixture of 2-amino-3-(1,2-dicarboxyethylthio)propanoic acids. The mixture was separated and the absolute configurations of the components were determined as (2R), (1'R) and (2R), (1'S), by analysis of the optical properties.

Amanita↗
PubMed1995

Structure-function analysis of the mammalian DNA polymerase beta active site: role of aspartic acid 256, arginine 254, and arginine 258 in nucleotidyl transfer.

The crystal structure of the catalytic domain of rat DNA polymerase beta revealed that Asp256 is located in proximity to the previously identified active site residues Asp190 and Asp192. We have prepared and kinetically characterized the nucleotidyl transfer activity of wild type and several mutant forms of human and rat pol beta. Herein we report steady-state kinetic determinations of KmdTTP, Km(dT)16, and kcat for mutants in residue Asp256 and two neighboring residues, Arg254 and Arg258, all centrally located on strand beta 7 in the pol beta structure. Mutation of Asp256 to alanine abolished the enzymatic activity of pol beta. Conservative replacement with glutamic acid (D256E) led to a 320-fold reduction of kcat compared to wild type. Replacement of Arg254 with an alanine (R254A) resulted in a 50-fold reduction of kcat compared to wild type. The Km(dT)16 of D256E and R254A increased about 18-fold relative to wild type. Replacement of Arg254 with a lysine resulted in a 15-fold decrease in kcat, and a 5-fold increase in the Km(dT)16. These kinetic observations support a role of Asp256 and Arg254 in the positioning of divalent metal ions and substrates in precise geometrical orientation for efficient catalysis. The mutation of Arg258 to alanine (R258A) resulted in a 10-fold increase in KmdTTP and a 65-fold increase in Km(dT)16 but resulted in no change of kcat. These observations are discussed in the context of the three-dimensional structures of the catalytic domain of pol beta and the ternary complex of pol beta, ddCTP, and DNA.

Amino Acid Sequence↗
PubMed2002

Aspartic acid 564 in the third cytoplasmic loop of the luteinizing hormone/choriogonadotropin receptor is crucial for phosphorylation-independent interaction with arrestin2.

Arrestin2 binding to the active but unphosphorylated luteinizing hormone/choriogonadotropin receptor (LH/CG R) in ovarian follicles is triggered by activation of ADP-ribosylation factor 6 (ARF6) and leads to uncoupling of this receptor from cAMP signaling. We sought to determine how arrestin2 binds to LH/CG R, if binding is of high affinity, and if the receptor also binds arrestin3. Desensitization of intact LH/CG R was equally sensitive to ectopic constructs of arrestin2 that bind other G protein-coupled receptors (GPCRs) either in a phosphorylation-independent or -dependent manner. Intact LH/CG R was not desensitized by ectopic arrestin3 constructs. Surface plasmon resonance studies showed that arrestin2 bound a synthetic third intracellular (3i) LH/CG R loop peptide with picomolar affinity; arrestin3 bound with millimolar affinity. To determine whether Asp-564 in the 3i loop mimicked the phosphorylated residue of other GPCRs, human embryonic kidney (HEK) cells were transfected with wild-type (WT) and D564G LH/CG R. An agonist-stimulated ARF6-dependent arrestin2 undocking pathway to drive desensitization of WT receptor was recapitulated in HEK cell membranes, and ectopic arrestin2 promoted desensitization of WT LH/CG R. However, D564G LH/CG R in HEK cells was not desensitized, and synthetic 3i D564G peptide did not bind arrestin2. Synthetic 3i loop peptides containing D564E, D564V, or D564N also did not bind arrestin2. We conclude that the ARF6-mediated mechanism to release a pool of membrane-delimited arrestin to bind GPCRs may be a widespread mechanism to deliver arrestin to GPCRs for receptor desensitization. Unlike other GPCRs that additionally require receptor phosphorylation, LH/CG R activation is sufficient to expose a conformation in which Asp-564 in the 3i loop confers high affinity binding selectively to arrestin2.

ADP-Ribosylation Factor 6↗
PubMed1991

Role of the invariant aspartic acid 99 of human choriogonadotropin beta in receptor binding and biological activity.

The four human glycoprotein hormones are heterodimers that contain a common alpha subunit and a hormone-specific beta subunit. Within this hormone family, 23 amino acid sequences from 11 mammalian species are available. There are 19 invariant amino acid residues in the beta subunits, 12 of which are Cys that form six disulfide bonds. Of the remaining seven conserved amino acid residues, we have investigated the role of an Asp which occurs at position 99 in human choriogonadotropin beta (hCG beta). Site-directed mutagenesis was used to replace hCG beta Asp99 with three residues, Glu, Asn, and Arg, and to prepare an inversion double mutant protein, Arg94----Asp and Asp99----Arg. The cDNAs were placed in a eukaryotic expression vector, and the plasmids were transiently transfected into Chinese hamster ovary cells containing a stably integrated gene for bovine alpha. Radioimmunoassays demonstrated that the mutant forms of hCG beta were capable of subunit assembly to the same extent as hCG beta wild type. The heterologous heterodimers were assayed in vitro using transformed mouse Leydig cells (MA-10) by competitive inhibition of 125I-hCG binding and stimulation of progesterone production. The gonadotropins containing Glu and Asn were active, although the potency was less than that associated with the hCG beta wild type-containing gonadotropin. In contrast, the Arg99-containing mutant protein and the inversion mutant protein Asp94/Arg99 were devoid of activity. Thus, in hCG beta Asp99 can be substituted with certain residues without total loss of function, although replacement with a positively charged residue leads to an inactive heterodimer. The primary role of Asp99 in hCG beta seems to involve, either directly or indirectly, receptor recognition.

Amino Acid Sequence↗
PubMed1990

Gangliosides attenuate the delayed neurotoxicity of aspartic acid in vitro.

The neurotoxic effects of L-aspartate were evaluated in rat cerebellar granule cell cultures. Acute (15 min) exposure to L-aspartate produced a time-dependent, delayed degeneration of neuronal cell bodies and neurites (LD50 about 40 microM) over 24 h. Aspartate neurotoxicity was prevented by competitive and non-competitive N-methyl-D-aspartate (NMDA) antagonists, but not by non-NMDA antagonists, suggesting a major involvement of NMDA receptors in this neuronal injury. Gangliosides, including GM1, were also effective in attenuating the cytotoxicity of L-aspartate. The neurotoxic potential of L-aspartate may thus contribute to pathologies involving the action of endogenous excitatory amino acids.

Animals↗