d-Alanine in the islets of Langerhans of rat pancreas

Relatively high levels of d-alanine (d-Ala), an endogenous d-amino acid, have been found in the endocrine systems of several animals, especially in the anterior pituitary; however, its functional importance remains largely unknown. We observed d-Ala in islets of Langerhans isolated from rat pancreas in significantly higher levels than in the anterior/intermediate pituitary; specifically, 180 ± 60 fmol d-Ala per islet (300 ± 100 nmol/g islet), and 10 ± 2.5 nmol/g of wet tissue in pituitary. Additionally, 12 ± 5% of the free Ala in the islets was in the d form, almost an order of magnitude higher than the percentage of d-Ala found in the pituitary. Surprisingly, glucose stimulation of the islets resulted in d-Ala release of 0.6 ± 0.5 fmol per islet. As d-Ala is stored in islets and released in response to changes in extracellular glucose, d-Ala may have a hormonal role.     

The amidating enzyme in pituitary will accept a peptide with C-terminal D-alanine as substrate

A series of tripeptides which terminated in d-alanine, d-serine, d-leucine or l-alanine was synthesized and the peptides tested for their ability to act as substrates for an amidating enzyme present in porcine pituitary. The peptides were allowed to compete with a radiolabelled substrate 125I d-Tyr Phe Gly in the presence of a rate limiting concentration of amidating enzyme and the degree of conversion to 125I d-Tyr Phe amide was determined by ion exchange chromatography. An accelerated procedure was developed for investigating the rates of reaction. The results showed that d-Tyr Phe d-Ala has a significant affinity for the amidating enzyme; no affinity could be demonstrated with d-Tyr Phe 1-Ala, d-Tyr Phe d-Ser or d-Tyr Phe d-Leu. Direct evidence that d-Tyr Phe d-Ala can undergo amidation was obtained by incubating the 125I labelled tripeptide with the pituitary enzyme. Amidation took place readily with d-Tyr Phe d-Ala but not with the other tripeptides; thus, while the enzyme is unable to catalyse the conversion of a peptide terminating in 1-alanine, it can accept a peptide terminating in d-alanine. The results indicate that the amidating enzyme has a highly compact substrate binding site.     

Characterization of the peptidoglycan of vancomycin-susceptible Enterococcus faecium

Vancomycin and other antibacterial glycopeptide analogues target the cell wall and affect the enzymatic processes involved with cell-wall biosynthesis. Understanding the structure and organization of the peptidoglycan is the first step in establishing the mode of action of these glycopeptides. We have used solid-state NMR to determine the relative concentrations of stem-links (64%), bridge-links (61%), and cross-links (49%) in the cell walls of vancomycin-susceptible Enterococcus faecium (ATTC 49624). Furthermore, we have determined that in vivo only 7% of the peptidoglycan stems terminate in d-Ala- d-Ala, the well-known vancomycin-binding site. Presumably, d-Ala- d-Ala is cleaved from uncross-linked stems in mature peptidoglycan by an active carboxypeptidase. We believe that most of the few pentapeptide stems ending in d-Ala- d-Ala occur in the template and nascent peptidoglycan strands that are crucial for cell-wall biosynthesis.     

Using surface plasmon resonance to directly measure slow binding of low-molecular mass inhibitors to a VanX chip

VanX, a d,d-dipeptidase, is one of five gene products responsible for vancomycin resistance in pathogenic bacteria and is an attractive drug target in circumventing clinical drug resistance. Our previous combinatorial search of VanX substrates in a dipeptide library of d-X(1)-d-X(2) (19(2)=361) forms has led to the discovery of three new compounds (d-Ala-d-Phe, d-Ala-d-Tyr, and d-Ala-d-Trp) having higher k(cat)/K(M) values than those of its natural substrate, d-Ala-d-Ala. Based on structures of newly identified substrates, two representative transition state analogs of substrates, d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) dipeptide phosphonates, used as VanX inhibitor were rationally designed and chemically synthesized. In the synthesis, eight synthetic steps in total were employed for preparing each VanX inhibitor, and their overall isolated yields were 21 and 11% for 6a and 6b, respectively. Binding interactions of d-Ala(P,O)d-Phe (6a) and d-Ala(P,O)d-Ala (6b) with VanX were confirmed unambiguously and measured quantitatively by surface plasmon resonance. The result reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX (for 6a, k(on)=1.18 x 10(3)M(-1)s(-1), k(off)=2.31 x 10(-3) s(-1), K(D)=1.96 microM, chi(2)=0.0737; for 6b, k(on)=1.09 x 10(3)M(-1)s(-1), k(off)=1.80 x 10(-2)s(-1), K(D)=16.5 microM, chi(2)=0.0599). This suggests that only a fraction of the conformers of the inhibitors in solution adopts a conformation best suited for binding interaction with VanX and that the VanX-inhibitor complex may concomitantly undergo a conformational isomerization from an initial but fast weak-binding adduct to slowly convert to a tight-binding complex with a more stable bound geometry. Moreover, in comparison with 6b, an additional aromatic interaction of 6a with the Phe79 residue in the active site of the enzyme, through an energetically favorable face-to-face offset stacked orientation, may account for its higher affinity than 6b to VanX.     

Leucopyrokinin (LPK) analog [d-Ala(5)]-[2-8]-LPK inhibits LPK-induced analgesia in rats

It has been previously found in our laboratory that insect neuropeptide leucopyrokinin and [2-8]-leucopyrokinin, a truncated analog without the first aminoacid of leucopyrokinin peptide chain exert an antinociceptive effect in rats. The present study confirmed our previous results, and moreover it has been found that [d-Ala(5)]-[2-8]-leucopyrokinin, an analog of leucopyrokinin antagonized the antinociceptive effect of leucopyrokinin and of [2-8]-leucopyrokinin. We conclude that this synthetic analog is a probable leucopyrokinin antagonist.     

Genes homologous to glycopeptide resistance vanA are widespread in soil microbial communities

The occurrence of d-Ala : d-Lac ligase genes homologous to glycopeptide resistance vanA was studied in samples of agricultural (n=9) and garden (n=3) soil by culture-independent methods. Cloning and sequencing of nested degenerate PCR products obtained from soil DNA revealed the occurrence of d-Ala : d-Ala ligase genes unrelated to vanA. In order to enhance detection of vanA-homologous genes, a third PCR step was added using primers targeting vanA in soil Paenibacillus. Sequencing of 25 clones obtained by this method allowed recovery of 23 novel sequences having 86-100% identity with vanA in enterococci. Such sequences were recovered from all agricultural samples as well as from two garden samples with no history of organic fertilization. The results indicated that soil is a rich and assorted reservoir of genes closely related to those conferring glycopeptide resistance in clinical bacteria.     

A liquid chromatography–tandem mass spectrometry assay for d-Ala-d-Lac: A key intermediate for vancomycin resistance in vancomycin-resistant enterococci

Vancomycin exerts its antibacterial activity by binding to d-Ala-d-Ala in bacterial cell wall precursors. Vancomycin resistance in vancomycin-resistant enterococci (VRE) is due to an alternative cell wall biosynthesis pathway in which d-Ala-d-Ala is replaced, most commonly by d-Ala-d-Lac. In this study, we extend our recently developed Marfey’s derivatization-based liquid chromatography–tandem mass spectrometry (LC–MS/MS) assay for l-Ala, d-Ala, and d-Ala-d-Ala to d-Ala-d-Lac and apply it to the quantitation of these metabolites in VRE. The first step in this effort was the development of an effective washing method for removing medium components from VRE cells. Mar-d-Ala-d-Lac was well resolved chromatographically from Mar-d-Ala-d-Ala, a prerequisite for MS/MS quantitation of d-Ala-d-Ala and d-Ala-d-Lac. Mar-d-Ala-d-Lac gave similar detection parameters, sensitivity, and linearity as Mar-d-Ala-d-Ala. l-Ala, d-Ala, d-Ala-d-Ala, and d-Ala-d-Lac levels in VRE were then determined in the presence of variable vancomycin levels. Exposure to vancomycin resulted in a dramatic reduction of d-Ala-d-Ala, with a response midpoint at approximately 0.06 μg/ml vancomycin and with a broad response profile up to 128 μg/ml vancomycin. In contrast, d-Ala-d-Lac was present in the absence of vancomycin, with its level constant up to 128 μg/ml vancomycin. This method will be useful for the discovery, characterization, and refinement of new agents targeting vancomycin resistance in VRE.     

Gamma-glutamylcysteine synthetase-glutathione synthetase: domain structure and identification of residues important in substrate and glutathione binding

In most organisms, glutathione (GSH) is synthesized by the sequential action of distinct enzymes, gamma-glutamylcysteine synthetase (gamma-GCS) and GSH synthetase (GS). In Streptococcus agalactiae, GSH synthesis is catalyzed by a single enzyme, gamma-glutamylcysteine synthetase-glutathione synthetase (gamma-GCS-GS). The N-terminal sequence of gamma-GCS-GS is similar to Escherichia coli gamma-GCS, but the C-terminal sequence is an ATP-grasp domain more similar to d-Ala, d-Ala ligase than to any known GS. In the present studies, C-terminally and N-terminally truncated constructs were characterized in order to define the limits of the gamma-GCS and GS domains, respectively. Although WT gamma-GCS-GS is nearly uninhibited by GSH (K(i) approximately 140 mM), shorter gamma-GCS domain constructs were unexpectedly found to be strongly inhibited (K(i) approximately 15 mM), reproducing a physiologically important regulation seen in monofunctional gamma-GCS enzymes. Because studies with E. coli gamma-GCS implicate a flexible loop region in GSH binding, chimeras of S. agalactiae gamma-GCS-GS were made containing gamma-GCS domain flexible loop sequences from Enterococcus faecalis and Pasteurella multocida gamma-GCS-GS, isoforms that are inhibited by GSH. Inhibition remained S. agalactiae-like (i.e., very weak). C-Terminal constructs of gamma-GCS-GS have GS activity (0.01-0.04% of WT), but proper folding and significant GS activity required a covalently linked gamma-GCS domain. In addition, site-directed mutants in the middle region of the gamma-GCS-GS sequence established that GS activity depends on residues in a region that is also part of the gamma-GCS domain. Our results provide new insights into the structure of gamma-GCS-GS and suggest gamma-GCS-GS evolved from a monomeric gamma-GCS that became C-terminally fused to a multimeric ATP-grasp protein.     

Effect of d-amino acids on collagen fibrillar assembly and stability: Experimental and modelling studies

The effect of selected d-amino acids (d-AAs) on collagen with 1-ethyl-3-(3-dimethylamino propyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) initiated crosslinking is evaluated by using experimental and modelling tools. The experimental results suggest that d-Lysine (d-Lys) plays a pivotal role in the self-assembly and conformation of collagen fibrils than d-Alanine (d-Ala) and d-Glutamic acid (d-Glu). The SDS-PAGE, absorption spectrum and viscosity measurements indicate significant differences in the d-Lys crosslinked collagen when compared to other systems. The CD spectra show an increase in the peak intensity at 220 nm in the presence of d-Lys, which could be due to increase in propensity of the structure to form a triple helix. Modelling studies indicated that d-Lys bind with collagen-like peptide (CLP) through multiple H-bonding and hydrophobic interactions. d-Lys has the lowest binding energy (−4.2 kcal/mol, indicating strongest interactions) when compared to d-Ala and d-Glu (−3.6 and −3.7 kcal/mol, respectively). Orientational changes in the collagenase on CLP-d-Lys are observed which may decrease its accessibility to degradation and stabilise CLP against the action of the former. d-Lys has the lowest binding energy and improved fibrillar-assembly and staggered alignment without the undesired structural stiffness and aggregations. The information derived from the present study could help in designing heterochiral collagen-based biomaterial.     

Effect of the A118G polymorphism on binding affinity, potency and agonist-mediated endocytosis, desensitization, and resensitization of the human mu-opioid receptor

The most prevalent single-nucleotide polymorphism (SNP) A118G in the human mu-opioid receptor gene predicts an amino acid change from an asparagine residue to an aspartatic residue in amino acid position 40. This N40D mutation, which has been implicated in the development of opioid addiction, was previously reported to result in an increased beta-endorphin binding affinity and a decreased potency of morphine-6-glucuronide. Therefore, in the present study we have investigated whether this mutation might affect the binding affinity, potency, and/or the agonist-induced desensitization, internalization and resensitization of the human mu-opioid receptor stably expressed in human embryonic kidney 293 cells. With the exception of a reduced expression level of N40D compared to human mu-opioid receptor (hMOR) in HEK293 cells, our analyses revealed no marked functional differences between N40D and wild-type receptor. Morphine, morphine-6-glucuronide and beta-endorphin revealed similar binding affinities and potencies for both receptors. Both the N40D-variant receptor and hMOR exhibited robust receptor internalization in the presence of the opioid peptide [d-Ala(2),N-MePhe(4),Glyol(5)]enkephalin (DAMGO) and beta-endorphin but not in response to morphine or morphine-6-glucuronide. After prolonged treatment with morphine, morphine-6-glucuronide or beta-endorphin both receptors showed similiar desensitization time courses. In addition, the receptor resensitization rates were nearly identical for both receptor types.     

Baroreceptor reflex regulation in anesthetized transgenic rats with low glia-derived angiotensinogen

Endogenous angiotensin (ANG) II and ANG-(1-7) act at the nucleus tractus solitarius (NTS) to differentially modulate neural control of the circulation. The role of these peptides endogenous to NTS on cardiovascular reflex function was investigated in transgenic rats with low brain angiotensinogen (Aogen) due to glial overexpression of an antisense to Aogen (ASrAOGEN) and in Sprague-Dawley (SD) rats. Arterial baroreceptor reflex sensitivity (BRS) for control of heart rate (HR) in response to increases in mean arterial pressure (MAP) was tested before and after bilateral microinjection of the angiotensin type 1 (AT(1)) receptor blocker candesartan or the ANG-(1-7) receptor blocker (d-Ala(7))-ANG-(1-7) into the NTS of urethane-chloralose-anesthetized ASrAOGEN and SD rats. Baseline MAP was higher in ASrAOGEN than in SD rats under anesthesia (P < 0.01). Injection of candesartan or (d-Ala(7))-ANG-(1-7) decreased MAP (P < 0.01) and HR (P < 0.05) in ASrAOGEN, but not SD, rats. The BRS at baseline was similar in ASrAOGEN and SD rats. Candesartan increased BRS by 41% in SD rats (P < 0.01) but was without effect in ASrAOGEN rats. In contrast, the reduction in BRS after (d-Ala(7))-ANG-(1-7) administration was comparable in SD (31%) and ASrAOGEN rats (34%). These findings indicate that the absence of glia-derived Aogen is associated with 1) an increase in MAP under anesthesia mediated via AT(1) and ANG-(1-7) receptors within the NTS, 2) the absence of an endogenous ANG II contribution to tonic inhibition of BRS, and 3) a continued contribution of endogenous ANG-(1-7) to tonic enhancement of BRS.     

Crystal structure of a peptidoglycan synthesis regulatory factor (PBP3) from Streptococcus pneumoniae

Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. The single d-Ala,d-Ala (d,d)-carboxypeptidase in Streptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 A resolution. PBP3 folds into an NH(2)-terminal, d,d-carboxypeptidase-like domain, and a COOH-terminal, elongated beta-rich region. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel beta-sheet surrounded by alpha-helices. As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. PBP3 performs substrate acylation in a highly efficient manner (k(cat)/K(m) = 50,500 M(-1) x s(-1)), an event that may be linked to role in control of pneumococcal peptidoglycan reticulation. A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to "skid" on the surface of the membrane, trimming pentapeptides during the cell growth and division processes.     

d-Amino acid oxidase deficiency is caused by a large deletion in the Dao gene in LEA rats

d-Amino acids, enantiomers of l-amino acids, are increasingly recognized as physiologically active molecules as well as potential biomarkers for diseases. d-Amino acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids and is present in a wide variety of organisms from yeasts to humans. Previous studies indicated that LEA rats lacked DAO activity, and levels of d-Ser and d-Ala were markedly increased in their tissues, suggesting a mutated locus responsible for the lack of Dao activity (ldao) existed in the LEA genome. Sequence analysis identified deletion breakpoints located in intron 4–5 of the Dao gene and intron 1–2 of the Svop gene, resulting in a 54.1-kb deletion which encompassed exons 5–12 of the Dao gene and exons 2–16 of the Svop gene. We developed a novel congenic rat strain, F344-Dao^ldao, harboring the Dao^ldao mutation from LEA rats delivered onto the F344 genetic background. Compared to the parental F344 strain, in F344-Dao^ldao rats d-Ala was markedly increased in both cerebrum and cerebellum, while d-Ser content was increased in cerebellum but not cerebrum. d-Ala, d-Ser, d-Pro and d-Leu levels were also elevated in F344-Dao^ldao plasma. F344-Dao^ldao rats represent a novel model system that will aid in elucidating the physiological functions of d-amino acids in vivo. (203 words).     

Evidence for a mu-delta opioid receptor complex in CHO cells co-expressing mu and delta opioid peptide receptors

Based on non-competitive binding interactions we suggested that mu and delta receptors associate as a mu/delta receptor complex in rat brain. We hypothesized that the same non-competitive binding interactions observed in rat brain will be seen in CHO cells that co-express mu and delta receptors, but not in cells that express just mu or delta receptors. We used CHO cells expressing the cloned human mu receptor, cloned human delta receptor, or cloned mouse delta/human mu ("dimer cell"). Cell membranes were prepared from intact cells pretreated with 100nM SUPERFIT. [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding assays followed published procedures. SUPERFIT, a delta-selective irreversible ligand, decreased [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to delta receptors by approximately 75% and to mu receptors by approximately 50% in dimer cells. SUPERFIT treatment did not decrease [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to mu cells. The IC(50) values observed in SUPERFIT-treated dimer cells were: [d-Pen(2),d-Pen(5)]enkephalin (1820nM) and morphine (171nM). Saturation binding experiments with SUPERFIT-treated dimer cells showed that [d-Pen(2),d-Pen(5)]enkephalin (5000nM) was a competitive inhibitor. In contrast, morphine (1000nM) lowered the B(max) from 1944fmol/mg to 1276fmol/mg protein (35% decrease). Both [d-Pen(2),d-Pen(5)]enkephalin and morphine competitively inhibited [(3)H][d-Ala(2),d-Leu(5)]enkephalin binding to SUPERFIT-treated mu cells. The results indicate that the mu-delta opioid receptor complex defined on the basis of non-competitive binding interactions in rat brain over 20 years ago likely occurs as a consequence of the formation of mu-delta heterodimers. SUPERFIT-treated dimer cells may provide a useful model to study the properties of mu-delta heterodimers.     

Enhanced mu-opioid responses in the spinal cord of mice lacking protein kinase Cgamma isoform

The protein kinase C (PKC)gamma isoform is a major pool of the PKC family in the mammalian spinal cord. PKCgamma is distributed strategically in the superficial layers of the dorsal horn and, thus, may serve as an important biochemical substrate in sensory signal processing including pain. Here we report that mu-opioid receptor-mediated analgesia/antinociception and activation of G-proteins in the spinal cord are enhanced in PKCgamma knockout mice. In contrast, delta- and kappa-opioidergic and ORL-1 receptor-mediated activation of G-proteins in PKCgamma knockout mice was not altered significantly relative to the wild-type mice. Deletion of PKCgamma had no significant effect on the mRNA product of spinal mu-opioid receptors but caused an increase of maximal binding of the mu-opioid receptor agonist [3H][d-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin in spinal cord membranes obtained from PKCgamma knockout mice. These findings suggest that deletion of PKCgamma genes protects the functional mu-opioid receptors from degradation by phosphorylation. More importantly the present data provide direct evidence that PKCgamma constitutes an essential pathway through which phosphorylation of mu-opioid receptors occurs.     

Functional role,cellular source,and tissue distribution of rat elastase-2, an angiotensin II-forming enzyme

We recently described a chymostatin-sensitive elastase-2 as the major angiotensin (ANG) II-forming enzyme in the perfusate of the rat mesenteric arterial bed (MAB) with the same cDNA sequence as rat pancreatic elastase-2. The role of this enzyme in generating ANG II was examined in the rat isolated and perfused MAB. The vasoconstrictor effect elicited by ANG I and the renin substrate tetradecapeptide was only partially inhibited by captopril but abolished by the combination of captopril and chymostatin or N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK; inhibitor originally developed for human elastase-2). The effect induced by [Pro11,d-Ala12]-ANG I, an ANG I-converting enzyme (ACE)-resistant biologically inactive precursor of ANG II, was blocked by chymostatin or Ac-AAPL-CK. It was also demonstrated that cultured rat mesenteric endothelial cells synthesize elastase-2 and that mRNA for this enzyme can be detected in different rat tissues such as the pancreas, MAB, lung, heart, kidney, liver, and spleen. In conclusion, the demonstration of a functional alternative pathway to ACE for ANG II generation in the rat MAB and the fact that cultured MAB endothelial cells are capable of producing and secreting elastase-2 represent strong evidence of a physiological role for this enzyme in the rat vasculature.     

Constitutively active mu-opioid receptors inhibit adenylyl cyclase activity in intact cells and activate G-proteins differently than the agonist [D-Ala2,N-MePhe4,Gly-ol5]enkephalin

The most convincing evidence demonstrating constitutive activation of mu-opioid receptors is the observation that putative inverse agonists decrease basal G-protein activity in membrane preparations. However, it is not clear whether constitutively active receptors in isolated membranes have any physiological relevance in intact cells. GH3 cells expressing mu-opioid receptors (GH3MOR) exhibit higher basal G-protein activity and lower basal cAMP levels than wild-type GH3 cells, indicative of constitutively active receptors. This study determined whether alkylation of mu-opioid receptors by the irreversible antagonist beta-funaltrexamine would decrease spontaneous receptor activity in intact cells, revealing constitutive activity. GH3MOR cells were pretreated with increasing concentrations of beta-funaltrexamine followed by functional testing after removal of unbound drug. beta-Funaltrexamine pretreatment produced a concentration-dependent decrease in mu-opioid receptor binding with an IC50 of 0.98 nm and an Emax of 77%. Similar concentrations of beta-funaltrexamine pretreatment produced a half-maximal reduction in basal [35S]GTPgammaS binding, a decrease in basal photolabeling of G-proteins with azidoanilido-[alpha-32P]GTP, and an increase in basal adenylyl cyclase activity in intact cells. Therefore, mu-opioid receptors are constitutively active in intact cells, producing stimulation of G-proteins and inhibition of adenylyl cyclase. Importantly, photolabeling of Galpha-subunits with azidoanilido-[alpha-32P]GTP demonstrated that constitutively active mu-opioid receptors activate individual G-proteins differently than the agonist [d-Ala2,N-MePhe4,Gly-ol5]enkephalin.     

Balance between two transpeptidation mechanisms determines the expression of beta-lactam resistance in Enterococcus faecium

The d,d-transpeptidase activity of high molecular weight penicillin-binding proteins (PBPs) is essential to maintain cell wall integrity as it catalyzes the final cross-linking step of bacterial peptidoglycan synthesis. We investigated a novel beta-lactam resistance mechanism involving by-pass of the essential PBPs by l,d-transpeptidation in Enterococcus faecium. Determination of the peptidoglycan structure by reverse phase high performance liquid chromatography coupled to mass spectrometry revealed that stepwise selection for ampicillin resistance led to the gradual replacement of the usual cross-links generated by the PBPs (d-Ala(4) --> d-Asx-Lys(3)) by cross-links resulting from l,d-transpeptidation (l-Lys(3) --> d-Asx-Lys(3)). This was associated with no modification of the level of production of the PBPs or of their affinity for beta-lactams, indicating that altered PBP activity was not required for ampicillin resistance. A beta-lactam-insensitive l,d-transpeptidase was detected in membrane preparations of the parental susceptible strain. Acquisition of resistance was not because of variation of this activity. Instead, selection led to production of a beta-lactam-insensitive d,d-carboxypeptidase that cleaved the C-terminal d-Ala residue of pentapeptide stems in vitro and caused massive accumulation of cytoplasmic precursors containing a tetrapeptide stem in vivo. The parallel dramatic increase in the proportion of l-Lys(3) --> d-Asx-Lys(3) cross-links showed that the enzyme was activating the resistance pathway by generating the substrate for the l,d-transpeptidase.     

Endogenous RGS protein action modulates mu-opioid signaling through Galphao. Effects on adenylyl cyclase,extracellular signal-regulated kinases,and intracellular calcium pathways

RGS (regulators of G protein signaling) proteins are GTPase-activating proteins for the Galpha subunits of heterotrimeric G proteins and act to regulate signaling by rapidly cycling G protein. RGS proteins may integrate receptors and signaling pathways by physical or kinetic scaffolding mechanisms. To determine whether this results in enhancement and/or selectivity of agonist signaling, we have prepared C6 cells stably expressing the mu-opioid receptor and either pertussis toxin-insensitive or RGS- and pertussis toxin-insensitive Galpha(o). We have compared the activation of G protein, inhibition of adenylyl cyclase, stimulation of intracellular calcium release, and activation of the ERK1/2 MAPK pathway between cells expressing mutant Galpha(o) that is either RGS-insensitive or RGS-sensitive. The mu-receptor agonist [d-Ala(2),MePhe(4),Gly(5)-ol]enkephalin and partial agonist morphine were much more potent and/or had an increased maximal effect in inhibiting adenylyl cyclase and in activating MAPK in cells expressing RGS-insensitive Galpha(o). In contrast, mu-opioid agonist increases in intracellular calcium were less affected. The results are consistent with the hypothesis that the GTPase-activating protein activity of RGS proteins provides a control that limits agonist action through effector pathways and may contribute to selectivity of activation of intracellular signaling pathways.