Modulation of coupling in the Escherichia coli ATP synthase by ADP and Pi: Role of the ε subunit C-terminal domain

The ε-subunit of ATP-synthase is an endogenous inhibitor of the hydrolysis activity of the complex and its α-helical C-terminal domain (εCTD) undergoes drastic changes among at least two different conformations. Even though this domain is not essential for ATP synthesis activity, there is evidence for its involvement in the coupling mechanism of the pump. Recently, it was proposed that coupling of the ATP synthase can vary as a function of ADP and P_i concentration. In the present work, we have explored the possible role of the εCTD in this ADP- and P_i-dependent coupling, by examining an εCTD-lacking mutant of Escherichia coli. We show that the loss of P_i-dependent coupling can be observed also in the εCTD-less mutant, but the effects of P_i on both proton pumping and ATP hydrolysis were much weaker in the mutant than in the wild-type. We also show that the εCTD strongly influences the binding of ADP to a very tight binding site (half-maximal effect ≈ 1 nM); binding at this site induces higher coupling in EF_OF₁ and increases responses to P_i. It is proposed that one physiological role of the εCTD is to regulate the kinetics and affinity of ADP/P_i binding, promoting ADP/P_i-dependent coupling.     

Stimulation of unprimed macrophages with immune complexes triggers a low output of nitric oxide by calcium-dependent neuronal nitric-oxide synthase

Immune complexes composed of IgG-opsonized pathogens, particles, or proteins are phagocytosed by macrophages through Fcγ receptors (FcγRs). Macrophages primed with IFNγ or other pro-inflammatory mediators respond to FcγR engagement by secreting high levels of cytokines and nitric oxide (NO). We found that unprimed macrophages produced lower levels of NO, which required efficient calcium (Ca(2+)) flux as demonstrated by using macrophages lacking selenoprotein K, which is required for FcγR-induced Ca(2+) flux. Thus, we further investigated the signaling pathways involved in low output NO and its functional significance. Evaluation of inducible, endothelial, and neuronal nitric-oxide synthases (iNOS, eNOS, and nNOS) revealed that FcγR stimulation in unprimed macrophages caused a marked Ca(2+)-dependent increase in both total and phosphorylated nNOS and slightly elevated levels of phosphorylated eNOS. Also activated were three MAP kinases, ERK, JNK, and p38, of which ERK activation was highly dependent on Ca(2+) flux. Inhibition of ERK reduced both nNOS activation and NO secretion. Finally, Transwell experiments showed that FcγR-induced NO functioned to increase the phagocytic capacity of other macrophages and required both NOS and ERK activity. The production of NO by macrophages is conventionally attributed to iNOS, but we have revealed an iNOS-independent receptor/enzyme system in unprimed macrophages that produces low output NO. Under these conditions, FcγR engagement relies on Ca(2+)-dependent ERK phosphorylation, which in turn increases nNOS and, to a lesser extent, eNOS, both of which produce low levels of NO that function to promote phagocytosis.     

Dopamine D2 receptor-mediated Akt/PKB signalling: initiation by the D2S receptor and role in quinpirole-induced behavioural activation

The short and long isoforms of the dopamine D2 receptor (D2S and D2L respectively) are highly expressed in the striatum. Functional D2 receptors activate an intracellular signalling pathway that includes a cAMP-independent route involving Akt/GSK3 (glycogen synthase kinase 3). To investigate the Akt/GSK3 response to the seldom-studied D2S receptor, we established a rat D2S receptor-expressing cell line [HEK (human embryonic kidney)-293/rD2S]. We found that in HEK-293/rD2S cells, the D2/D3 agonists bromocriptine and quinpirole significantly induced Akt and GSK3 phosphorylation, as well as ERK1/2 (extracellular-signal-regulated kinase 1/2) activation. The D2S receptor-induced Akt signals were profoundly inhibited by the internalization blockers monodansyl cadaverine and concanavalin A. Activation of the D2S receptor in HEK-293/rD2S cells appeared to trigger Akt/phospho-Akt translocation to the cell membrane. In addition to our cell culture experiments, we studied D2 receptor-dependent Akt in vivo by systemic administration of the D2/D3 agonist quinpirole. The results show that quinpirole evoked Akt-Ser⁴⁷³ phosphorylation in the ventral striatum. Furthermore, intra-accumbens administration of wortmannin, a PI3K (phosphoinositide 3-kinase) inhibitor, significantly suppressed the quinpirole-evoked behavioural activation. Overall, we demonstrate that activation of the dopamine D2S receptor stimulates Akt/GSK3 signalling. In addition, in vivo Akt activity in the ventral striatum appears to play an important role in systemic D2/D3 agonist-induced behavioural activation.     

Phosphorylation and Inactivation of Brain Glycogen Synthase by a Multifunctional Calmodulin-Dependent Protein Kinase

Glycogen synthase was partially purified from canine brain to about 70% purity. The purified enzyme showed differences from the properties of the skeletal muscle enzyme with respect to molecular weights of the holoenzyme and subunit and phosphopeptide mapping. The multifunctional calmodulin-dependent protein kinase from the brain phosphorylated brain glycogen synthase with concomitant inactivation of the enzyme. Although about 1.3 mol of phosphate/mol subunit was maximally incorporated into glycogen synthase, 0.4 mol of phosphate/mol subunit was sufficient for the maximal inactivation of the enzyme. The results indicate that brain glycogen synthase is regulated in a calmodulin-dependent manner similarly to the skeletal muscle enzyme, but that the brain enzyme is different from the skeletal muscle enzyme.     

鸭肝脂肪酸合成酶的胍变性与失活

报道了鸭肝脂肪酸俣成酶在胍变性过程中构变性过程中构象变化和活性变化的关系,首次验证了邹承鲁提出的酶活性部位构象的理论适用于多功能复合酶,同时该酶变性及复性均可测定出多个阶段,且证明有无活性稳态酶存在,在低浓度盐酸胍溶液中该酶的全反应活性和其中两个还原部位单独活性被同步可逆抑制,随着胍浓度增高,出现不可逆失活且程度和速度均迅速提高,在０．５４ｍｏｌ／Ｌ胍中该酶全反应活性在１．５分种内已有一半不可逆失     

Localization of nitric oxide synthase in canine ileocolonic and pyloric sphincters

The distribution of neurons containing NADPH-diaphorase (NADPH-d) activity and nitric oxide synthase-like immunoreactivity (NOS-LI) in the canine pyloric and ileocolonic sphincters was studied. Cells within the myenteric and submucosal ganglia were positive for NADPH-d. These cells generally had the morphology of Dogiel type-I enteric neurons, however, there was some diversity in the morphology of NADPH-d-positive neurons in the myenteric plexus of the pylorus. Intramuscular ganglia were observed in both sphincters, and NADPH-d was found in a sub-population of neurons within these ganglia. Dual staining with an antiserum raised against nitric oxide synthase (NOS) demonstrated that almost all cells with NOS-LI were also NADPH-d positive. Varicose fibers within ganglia and within the circular and longitudinal muscle layers also possed NOS-LI and NADPH-d activity. Dual staining with anti-VIP antibodies showed that some of the NADPH-d-positive cells in the myenteric and submucosal ganglia also contained VIP-LI, but all VIP-LI-positive cells did not express NADPH-d activity. These data are consistent with recent physiological studies suggesting that nitric oxide serves as an inhibitory neurotransmitter in the pyloric and ileocolonic sphincters. The data also suggest that VIP is expressed in a sub-population of NADPH-d-positive neurons and may therefore act as a co-transmitter in enteric inhibitory neurotransmission to these specialized muscular regions.     

Chlorsulfuron resistance in Daucus carota cell lines and plants:Involvement of gene amplification

Daucus carota L. cell lines stably resistant to the herbicide chlorsulfuron (CS) have been isolated according to a stepwise selection. Studies carried out during different selection steps show that the specific activity of the target enzyme acetohydroxyacid synthase (AHAS) increases along with CS resistance. Southern hybridization analysis performed with aBrassica napus AHAS probe in a CS highly-resistant cell line reveals the presence of a greatly amplifiedEcoRI fragment of genomic DNA. This indicates that AHAS overproduction induced by stepwise selection is due to gene amplification. Regenerants from some resistant cell lines maintained the CS-resistant trait at the whole plant level.     

Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment

A 13 kb DNA fragment was isolated from a grapevine (Vitis var. Optima) genomic library by hybridizing with elicitor-induced stilbene synthase cDNA as a probe. After fragmentation with Eco RI, subcloning and sequencing, two full-size stilbene synthase genes (Vst1 and Vst2) and the 3 end of a third stilbene synthase gene (Vst3) were located within the 13 kb fragment. Vst1 and Vst2, differing only slightly in the coding region, are distinguished in the intron size and in the structure of the promoter region. The 5 flanking region of gene Vst1 contains a TATAA box at nucleotide –48. The substantial structural differences found for the promoters of the two genes are paralleled by a striking difference in the expression of the two genes in elicitor-treated cells. Moreover, the accumulation upon elicitation of six different stilbene synthase mRNAs was studied and found to differ by two orders of magnitude.     

Phenotypic changes associated with RNA interference silencing of chalcone synthase in apple (Malus × domestica)

We have identified in apple (Malus × domestica) three chalcone synthase (CHS) genes. In order to understand the functional redundancy of this gene family RNA interference knockout lines were generated where all three of these genes were down‐regulated. These lines had no detectable anthocyanins and radically reduced concentrations of dihydrochalcones and flavonoids. Surprisingly, down‐regulation of CHS also led to major changes in plant development, resulting in plants with shortened internode lengths, smaller leaves and a greatly reduced growth rate. Microscopic analysis revealed that these phenotypic changes extended down to the cellular level, with CHS‐silenced lines showing aberrant cellular organisation in the leaves. Fruit collected from one CHS‐silenced line was smaller than the ‘Royal Gala’ controls, lacked flavonoids in the skin and flesh and also had changes in cell morphology. Auxin transport experiments showed increased rates of auxin transport in a CHS‐silenced line compared with the ‘Royal Gala’ control. As flavonoids are well known to be key modulators of auxin transport, we hypothesise that the removal of almost all flavonoids from the plant by CHS silencing creates a vastly altered environment for auxin transport to occur and results in the observed changes in growth and development.     

Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target

1. Download high-res image (140KB)
2. Download full-size image