Acidic pH Optimum Research Articles

Amino acid residues crucial in pH regulation and proteolytic activation of N-acylethanolamine-hydrolyzing acid amidase

N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme which hydrolyzes bioactive N-acylethanolamines, including anandamide and N-palmitoylethanolamine. NAAA shows acidic pH optimum in terms of both catalytic activity and maturation by specific proteolysis. However, molecular mechanism involved in this characteristic pH dependency remained unclear. Here we report the important role of Glu-195 of human NAAA by analyzing the mutants E195A and E195Q overexpressed in human embryonic kidney 293 cells. Concanamycin A, raising lysosomal pH, inhibited maturation of the wild-type, but not of the Glu-195 mutants. The purified precursors of the mutants, but not the wild-type, were proteolytically cleaved at pH 7.4 during 24-h incubation. Furthermore, when assayed for N-palmitoylethanolamine-hydrolyzing activity at different pH, the mutants did not exhibit a sharp peak around pH 4.5 in the pH-dependent activity profile. Mutants of other seven glutamic acid residues did not show such an abnormality. These results suggested a unique role of Glu-195 in the pH-dependent activity and proteolytic maturation. Moreover, Arg-142, Asp-145, and Asn-287 as well as previously identified Cys-126 were shown to be essential for the proteolytic activation. Since these residues were predicted to be catalytically important, the results strongly suggested that the proteolysis occurs through an autocatalytic mechanism.

O1-04-01: Novel drugs simultaneously modulate both beta- and gamma-secretase cleavage of the amyloid precursor protein

The beta-secretase BACE1 catalyzes the first step in Abeta generation. However, suitable inhibitors for clinical development have been difficult to obtain. Thus, it is essential to explore alternative ways to reduce beta-secretase activity. To identify new mechanisms and compounds modulating beta-secretase cleavage of APP, a small molecule library was screened for compounds altering APP alpha- and beta-secretase cleavage. We identified the calcium blocker bepridil as a novel inhibitor of the beta-secretase cleavage of APP, using a variety of different cell lines including primary neurons. The IC50 was 6 μM, which corresponds to the concentration found in plasma of patients treated with bepridil. Bepridil did not affect alpha-secretase cleavage or total APP levels in the cell lysate. Surprisingly, bepridil did not directly inhibit BACE1, as determined using in vitro assays. Mechanistically, we found that bepridil reduces beta-secretase cleavage independent of its calcium-blocking activity. Instead, our data support a model in which bepridil increases the endosomal pH. This reduces the activity of the BACE1 protease, which has an acidic pH optimum and cleaves APP mostly in the acidic environment of the endosome. Surprisingly, bepridil also modulated gamma-secretase cleavage of APP and increased the ratio of Abeta42/38, similar to the so-called inverse NSAIDs. Due to the simultaneous inhibition of beta-secretase, treatment with bepridil led to a strong decrease in total levels of Abeta38, a slightly less strong decrease in Abeta40 and only mild effects on Abeta42. Chemical modification is used to invert bepridil's specificity for gamma-secretase, while retaining the inhibitory effect on beta-secretase. Our results show that it is possible to efficiently inhibit beta-secretase cleavage using compounds, which raise the endosomal pH at physiologically attainable and non-toxic concentrations. Moreover, it is possible to identify “2-in-1 modulators”, which simultaneously modulate beta- and gamma-secretase cleavage of APP. This opens the possibility of developing compounds targeting beta- and gamma-secretase with a single drug.

DNA-hydrolysing activity of immunoglobulin G from bulk bovine colostrum

This paper represents the first report of DNA-hydrolysing activity of IgG fraction from bulk bovine colostrum. Ultrafiltration, Sephadex G-150 chromatography, protein G affinity chromatography and dialysis were used to obtain the IgG fraction, which was isolated and finally purified to homogeneity by SDS-PAGE. pBR322 DNA in the incubated mixture with the IgG fraction, which was subjected to electrophoresis in 0.8% agarose gel, was found to be hydrolysed. This result was confirmed by a hyperchromicity assay using calf thymus DNA as the substrate. The DNase specific activity of the IgG preparations from bulk bovine colostrum was 12.2–24.1 U/mg (checked by RID) in this work. The DNase activity of bovine colostral IgG had an acidic pH optimum (pH 3.9–4.0), which was very different from the DNase (7.0–7.2) of the milk of healthy mothers (Kanyshkova, Buneva, Nevinsky, & Neustroev, 1997). The DNA-hydrolysing activity of bovine colostral IgG showed no significant requirement for divalent metal ions. These data taken together showed that bovine colostral IgG possessed DNA hydrolytic activity and shared biochemical properties with DNase II. We also found that the abzyme activity of IgG was not simply proportional to the IgG content.

Homology Modeling and Site-Directed Mutagenesis To Identify Selective Inhibitors of Endothelin-Converting Enzyme-2

Endothelin-converting enzyme-2 (ECE-2), a member of M13 family of zinc metallopeptidases, has previously been shown to process a number of neuropeptides including those derived from prodynorphin, proenkephalin, proSAAS, and amyloid precursor protein. ECE-2, unlike ECE-1, exhibits restricted neuroendocrine distribution and acidic pH optimum; it is consistent with a role in the regulation of neuropeptide levels in vivo. Here, we report the generation of a three-dimensional (3D) molecular model of ECE-2 using the crystal structure of neprilysin (EC 3.4.24.11) as a template. On the basis of the predictions made from the molecular model, we mutated and tested two residues, Trp 148 and Tyr 563, in the catalytic site. The mutation of Tyr 563 was found to significantly affect the catalytic activity and inhibitor binding. The molecular model was used to virtually screen a small molecule library of 13 000 compounds. Among the top-scoring compounds three were found to inhibit ECE-2 with high affinity and exhibited specificity for ECE-2 compared to neprilysin. Thus, the model provides a new useful tool to probe the active site of ECE-2 and design additional selective inhibitors of this enzyme.

Arylsulfatase G, a Novel Lysosomal Sulfatase

The sulfatases constitute a conserved family of enzymes that specifically hydrolyze sulfate esters in a wide variety of substrates such as glycosaminoglycans, steroid sulfates, or sulfolipids. By modifying the sulfation state of their substrates, sulfatases play a key role in the control of physiological processes, including cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked with various severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase G (ASG), was initially described as an enzyme lacking in vitro arylsulfatase activity and localizing to the endoplasmic reticulum. Contrary to these results, we demonstrate here that ASG does indeed have arylsulfatase activity toward different pseudosubstrates like p-nitrocatechol sulfate and 4-methylumbelliferyl sulfate. The activity of ASG depends on the Cys-84 residue that is predicted to be post-translationally converted to the critical active site C(alpha)-formylglycine. Phosphate acts as a strong, competitive ASG inhibitor. ASG is active as an unprocessed 63-kDa monomer and shows an acidic pH optimum as typically seen for lysosomal sulfatases. In transfected cells, ASG accumulates within lysosomes as indicated by indirect immunofluorescence microscopy. Furthermore, ASG is a glycoprotein that binds specifically to mannose 6-phosphate receptors, corroborating its lysosomal localization. ARSG mRNA expression was found to be tissue-specific with highest expression in liver, kidney, and pancreas, suggesting a metabolic role of ASG that might be associated with a so far non-classified lysosomal storage disorder.

A single histidine residue modulates enzymatic activity in acidic mammalian chitinase

Mammals express two active chitinases, chitotriosidase and AMCase. Only AMCase displays an extremely acidic pH optimum, consistent with its observed presence in the gastro-intestinal tract. A structural model of AMCase reveals the presence of a conserved histidine residue in the active site. Mutational analyses and molecular dynamics simulations show that His187 is responsible for the acidic optimum and suggest pH dependent modulation of the reaction mechanism that is unique to AMCases. Concluding, His187 is a crucial structural component of the active site of AMCase and this unique feature may serve as a lead for the development of specific inhibitors.

Modifying the red cell surface: towards an ABO‐universal blood supply

Eliminating the risk for ABO-incompatible transfusion errors and simplifying logistics by creating a universal blood inventory is a challenging idea. Goldstein and co-workers pioneered the field of enzymatic conversion of blood group A and B red blood cells (RBCs) to O (ECO). Using alpha-galactosidase from coffee beans to produce B-ECO RBCs, proof of principle for this revolutionary concept was achieved in clinical trials. However, because this enzyme has poor kinetic properties and low pH optimum the process was not economically viable. Conversion of group A RBCs was only achieved with the weak A2 subgroup with related enzymes having acidic pH optima. More recently, the identification of entirely new families of bacterial exoglycosidases with remarkably improved kinetic properties for cleaving A and B antigens has reinvigorated the field. Enzymatic conversion of groups A, B and AB RBCs with these novel enzymes resulting in ECO RBCs typing as O can now be achieved with low enzyme protein consumption, short incubation times and at neutral pH. Presently, clinical trials evaluating safety and efficacy of ECO RBCs are ongoing. Here, we review the status of the ECO technology, its impact and potential for introduction into clinical component preparation laboratories.

Digestive peptidases in Tenebrio molitor and possibility of use to treat celiac disease

AbstractDigestion in Tenebrio molitor larvae occurs in the midgut, where there is a sharp pH gradient from 5.6 in the anterior midgut (AM) to 7.9 in the posterior midgut (PM). Accordingly, digestive enzymes are compartmentalized to the AM or PM. Enzymes in the AM are soluble and have acidic or neutral pH optima, while PM enzymes have alkaline pH optima. The main peptidases in the AM are cysteine endopeptidases presented by two to six subfractions of anionic proteins. The major activity belongs to cathepsin L, which has been purified and characterized. Serine post‐proline cleaving peptidase with pH optimum 5.3 was also found in the AM. Typical serine digestive endopeptidases, trypsin‐like and chymotrypsin‐like, are compartmentalized to the PM. Trypsin‐like activity is due to one cationic and three anionic proteinases. Chymotrypsin‐like activity consists of one cationic and four anionic proteinases, four with an extended binding site. The major cationic trypsin and chymotrypsin have been purified and thoroughly characterized. The predicted amino acid sequences are available for purified cathepsin L, trypsin and chymotrypsin. Additional sequences for putative digestive cathepsins L, trypsins and chymotrypsins are available, implying multigene families for these enzymes. Exopeptidases are found in the PM and are presented by a single membrane aminopeptidase N‐like peptidase and carboxypeptidase A, although multiple cDNAs for carboxypeptidase A were found in the AM, but not in the PM. The possibility of the use of two endopeptidases from the AM – cathepsin L and post‐proline cleaving peptidase – in the treatment of celiac disease is discussed.

The crystal structure of a lysozyme c from housefly Musca domestica, the first structure of a digestive lysozyme

Lysozymes from family 22 of glycoside hydrolases are usually part of the defense system against bacteria. However in ruminant artiodactyls and saprophagous insects, lysozymes are involved in the digestion of bacteria. Here, we report the first crystallographic structure of a digestive lysozyme in its native and complexed forms, the structure of lysozyme 1 from Musca domestica larvae midgut (MdL1). Structural and biochemical data presented for MdL1 are analyzed in light of digestive lysozymes’ traits. The structural core is similar, but a careful analysis of a structural alignment generated with other lysozymes c reveals that significant differences occur in coil regions. The loop from MdL1 defined by residues 98–100 has one deletion previous to residue Gln100, which leads to a less exposed conformation and might justify the resistance to proteolysis observed for MdL1. In addition, Gln100 is directly involved in a few hydrogen bonds to the ligand in a yet unobserved substrate binding mode. The pKas of the MdL1 catalytic residues (Glu32 and Asp50) are lower (6.40 and 3.09, respectively) than those from Gallus gallus egg lysozyme (GgL, hen egg white lysozyme-HEWL) (6.61 and 3.85, respectively). A unique feature of MdL1 is a hydrogen bond between Thr107 Oγ and Glu32 carboxylate group, which combined with the presence of Ser106 contributes to decrease the pKa of Glu32. Furthermore, in MdL1 the presence of Asn46 preventing the occurrence of an electrostatic repulsion with Asp50 and the increment in the solvent exposition of Asp50 due to Pro42 insertion contribute to reduce the pKa of Asp50. These structural elements affecting the pKas of the catalytic residues should contribute to the acidic pH optimum presented by MdL1.

Activation of biliverdin-IXα reductase by inorganic phosphate and related anions

The effect of pH on the initial-rate kinetic behaviour of BVR-A (biliverdin-IXalpha reductase) exhibits an alkaline optimum with NADPH as cofactor, but a neutral optimum with NADH as cofactor. This has been described as dual cofactor and dual pH dependent behaviour; however, no mechanism has been described to explain this phenomenon. We present evidence that the apparent peak of activity observed at neutral pH with phosphate buffer and NADH as cofactor is an anion-dependent activation, where inorganic phosphate apparently mimics the role played by the 2'-phosphate of NADPH in stabilizing the interaction between NADH and the enzyme. The enzymes from mouse, rat and human all exhibit this behaviour. This behaviour is not seen with BVR-A from Xenopus tropicalis or the ancient cyanobacterial enzyme from Synechocystis PCC 6803, which, in addition to being refractory to activation by inorganic phosphate, are also differentiated by an acid pH optimum with both nicotinamide nucleotides.

It's elementary: Enhancing Fe 3+ reduction improves rice yields

Most of the world relies on plants as their dietary source of iron (1, 2). Unfortunately, plants are not a good source of this essential nutrient for two reasons: iron deficiency often limits plant growth, and the iron that accumulates in plants is not readily available. The first problem stems not from the abundance of iron in soil but rather from its solubility, with pH and oxygen controlling the amount of free iron found in solution. When faced with an iron shortage, plants either use a reduction strategy (strategy I) and take up Fe2+ or use a chelation strategy (strategy II) and take up Fe3+ (Fig. 1). However, not all plants are proficient at mounting these iron-deficiency responses. Rice, one of the world's most important food crops, is much more susceptible to iron deficiency than other grasses, presumably because it releases low quantities of phytosiderophores that act as Fe3+ chelators (3). Although rice plants are also capable of taking up Fe2+ (4), rice does not have an inducible ferric chelate reductase activity, as do plants that use the reduction strategy to generate Fe2+ from the more abundant Fe3+ found in aerobic soils. In this issue of PNAS, Ishimaru et al. (5) ask whether introducing this activity into rice would improve its iron uptake ability. They report an impressive 7.9-fold increase in grain yield for transgenic rice plants engineered to express a ferric chelate reductase when these plants are grown in calcareous soil. Importantly, the ferric chelate reductase that they introduced had been selected for better performance at high pH because the native enzyme has an acidic pH optimum (6). Because one-third of the world's soils are alkaline, the results presented here could have a major impact on rice production, allowing rice to grow in soils now considered marginal and increasing crop biomass in soils now in cultivation. Furthermore, all plants, except the grasses, use the reduction strategy to mobilize iron from the rhizosphere, and ferric chelate reductase activity has been shown to be the rate-limiting step for iron uptake (7). Thus, all plants would benefit from increased expression of a ferric chelate reductase that functions better at alkaline pH.

β-Glucuronidase activity in seedlings of the parasitic angiosperm Cusctua pentagona: developmental impact of the β-glucuronidase inhibitor saccharic acid 1,4-lactone.

Endogenous plant β-glucuronidase (β-GUS) activity was detected in germinating seeds, seedlings, stems, flowers and haustoria of the parasitic angiosperm Cuscuta pentagona Engelm. In vitro characterisation of this activity showed it to have an acidic pH optimum, similar to previously characterised plant activities, and a sensitivity to the β-GUS inhibitor saccharic acid 1,4-lactone (SAL). Application of SAL to seeds immediately after chemical scarification resulted in a significant developmental delay and, frequently, in the total arrest of seedling growth. In contrast, application of SAL subsequent to the emergence of the radicle did not produce a significant effect on the development of the seedling. Thus, the distribution of activity and the developmentally contingent potency of SAL in inhibiting growth suggest a role for β-GUS at an early stage of seed germination or seedling growth. Further, the inability of the inhibitor to prevent subsequent shoot elongation suggests that at least some plant growth processes do not require this activity, or that it is required only at minimal levels and is unaffected by the application of SAL.

Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin‐ and NPY‐containing chromaffin granules

Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.

Differential Enzymatic Characteristics and Tissue-Specific Expression of Human TPST-1 and TPST-2

Protein tyrosine sulfation is emerging as a widespread post-translational modification in multicellular eukaryotes. The responsible enzyme, named tyrosylprotein sulfotransferase (TPST), catalyzes the sulfate transfer from 3'-phosphoadenosine 5'-phosphosulfate to tyrosine residues of proteins. Two distinct TPSTs, designated TPST-1 and TPST-2, had previously been identified. In the present study, we cloned human TPST-1 and TPST-2 expressed and characterized the recombinant enzymes using peptide substrates. These enzymes displayed distinct acidic pH optima and stimulatory effects of Mn(2+). Additionally, the activity of TPST-2, but not TPST-1, was stimulated in the presence of Mg(2+). Compared with TPST-2, TPST-1 displayed considerably lower K(m) and V(max) for the majority of the tested peptide substrates, implying their differential substrate specificity. Quantitative real-time PCR analysis showed that although the two TPSTs were co-expressed in all 20 human tissues examined, the levels of expression of TPST-1 and TPST-2 varied significantly among different tissues. These latter findings may imply distinct physiological functions of TPST-1 and TPST-2.

A role for falcilysin in transit peptide degradation in the Plasmodium falciparum apicoplast

Falcilysin (FLN) is a zinc metalloprotease thought to degrade globin peptides in the acidic vacuole of the human malaria parasite Plasmodium falciparum. The enzyme has been found to have acidic or neutral pH optima on different peptides and to have additional distribution outside the food vacuole. These data suggested that FLN has an additional function in the parasite. To further probe the functions of FLN, we created a transgenic parasite clone expressing a chromosomally encoded FLN-GFP fusion. Unexpectedly, FLN was found in the apicoplast, an essential chloroplast-like organelle. Nuclear encoded apicoplast proteins are targeted to the organelle by a bipartite N-terminal sequence comprised of a signal sequence followed by a positively charged transit peptide domain. Free transit peptides are thought to be toxic to the plastid and need to be rapidly degraded after proteolytic release from proproteins. We hypothesized that FLN may participate in transit peptide degradation in the apicoplast based on its preference for basic residues at neutral pH and on phylogenetic comparison with other M16 family metalloproteases. In vitro cleavage by FLN of the transit peptide from the apicoplast-resident acyl carrier protein supports this idea. The importance of FLN for parasite development is suggested by our inability to truncate the chromosomal FLN open reading frame. Our work indicates that FLN is an attractive target for antimalarial development.

Positional specificity of lysosomal phospholipase A2

Lysosomal phospholipase A(2) (Lpla2) is highly expressed in alveolar macrophages and may mediate the phospholipid metabolism of surfactant. Studies on the properties of this phospholipase are consistent with the presence of both phospholipase A(1) and phospholipase A(2) activities. These activities were studied through the production of O-acyl compounds, produced by the transacylase activity of Lpla2. Liposomes containing POPC and N-acetylsphingosine (NAS) were incubated with the soluble fraction obtained from MDCK cells stably transfected with the mouse Lpla2 gene. Two 1-O-acyl-NASs, 1-O-palmitoyl-NAS and 1-O-oleoyl-NAS, were produced by Lpla2. The formation rate of 1-O-oleoyl-NAS was 2.5-fold that of 1-O-palmitoyl-NAS. When 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) was used, the formation rate of 1-O-oleoyl-NAS was 5-fold higher than that of 1-O-palmitoyl-NAS. Thus, Lpla2 can act on acyl groups at both sn-1 and sn-2 positions of POPC and OPPC. When 1-palmitoyl-2-unsaturated acyl-sn-glycero-3-phosphocholines were used as acyl donors, the transacylation of the acyl group from the sn-2 position to NAS was preferred to that of the palmitoyl group from the sn-1 position. An exception was observed for 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC), for which the formation rate of 1-O-palmitoyl-NAS from PAPC was 4-fold greater than that of 1-O-arachidonoyl-NAS. Thus, Lpla2 has broad positional specificity for the sn-1 and sn-2 acyl groups in phosphatidylcholine and phosphatidylethanolamine.

Crystal Structure of Scytalidoglutamic Peptidase with its First Potent Inhibitor Provides Insights into Substrate Specificity and Catalysis

Scytalidoglutamic peptidase (SGP) from Scytalidium lignicolum is the founding member of the newly discovered\\ family of peptidases, G1, so far found exclusively in fungi. The crystal structure of SGP revealed a previously undescribed fold for peptidases and a unique catalytic dyad of residues Gln53 and Glu136. Surprisingly, the β-sandwich structure of SGP is strikingly similar to members of the carbohydrate-binding concanavalin A-like lectins/glucanases superfamily. By analogy with the active sites of aspartic peptidases, a mechanism employing nucleophillic attack by a water molecule activated by the general base functionality of Glu136 has been proposed. Here, we report the first crystal structures of SGP in complex with two transition state peptide analogs designed to mimic the tetrahedral intermediate of the proteolytic reaction. Of these two analogs, the one containing a central S-hydroxyl group is a potent sub-nanomolar inhibitor of SGP. The inhibitor binds non-covalently to the concave surface of the upper β-sheet and enables delineation of the S4 to S3′ substrate specificity pockets of the enzyme. Structural differences in these pockets account for the unique substrate preferences of SGP among peptidases having an acidic pH optimum. Inhibitor binding is accompanied by a structuring of the region comprising residues Tyr71–Gly80 from being mostly disordered in the apoenzyme and leading to positioning of crucial active site residues for establishing enzyme–inhibitor contacts. In addition, conformational rearrangements are seen in a disulfide bridged surface loop (Cys141–Cys148), which moves inwards, partially closing the open substrate binding cleft of the native enzyme. The non-hydrolysable scissile bond analog of the inhibitor is located in the active site forming close contacts with Gln53 and Glu136. The nucleophilic water molecule is displaced and a unique mode of binding is observed with the S-OH of the inhibitor occupying the oxyanion binding site of the proposed tetrahedral intermediate. Details of the enzyme–inhibitor interactions and mechanistic interpretations are discussed.

Mode of De-esterification of Alkaline and Acidic Pectin Methyl Esterases at Different pH Conditions

Highly esterified citrus pectin was de-esterified at pH 4.5 and 8.0 by a fungal pectin methyl esterase (PME) that was shown to have an acidic isoelectric pH (pI) and an acidic pH optimum and by a plant PME that was characterized by an alkaline pI and an alkaline pH optimum. Interchain and intrachain de-esterification patterns were studied by digestion of the pectin products with endo-polygalacturonase and subsequent analysis using size exclusion and anion-exchange chromatography. No effect of pH was observed on the de-esterification mode of either of the two enzymes. Acidic, fungal PME converted pectin according to a multiple-chain mechanism, with a limited degree of multiple attack at the intrachain level, both at pH 4.5 and at pH 8.0. A multiple-attack mechanism, with a high degree of multiple attack, was more appropriate to describe the action mode of alkaline, plant PME, both at pH 4.5 and at pH 8.0.

Gamma-aminobutyrate: defense against invertebrate pests?

Gamma-aminobutyrate (GABA) is a ubiquitous four-carbon, non-protein amino acid. In plants, stress-induced GABA accumulation is well documented. However, the role(s) of GABA accumulation is contentious. In this Opinion article, we argue that wounding due to herbivory and crawling by insect larvae causes rapid GABA accumulation via the disruption of cellular compartmentation and the release of the acidic vacuolar contents to the cytosol. The activity of glutamate decarboxylase, the cytosolic enzyme responsible for GABA synthesis, has an acidic pH optimum. Subsequent GABA ingestion has a plant defense function by directly acting on GABA-regulated invertebrate neuromuscular junctions. Plants with an enhanced GABA-producing capacity reduce herbivory by invertebrate pests. These findings suggest that GABA accumulation is a rapidly deployed, local resistance mechanism that constitutes a first line of defense in deterring herbivory.

Phagosome Neutrality in Host Defense

Activation of proteases in the phagosomes of neutrophils occurs by neutralization of the phagosomal pH by NADPH oxidase. In this issue, Savina et al. (2006) show that dendritic cell phagosomes also recruit NADPH oxidase but with different results. Here, the neutralization of phagosomal pH reduces protease activity, which preserves antigens for crosspresentation on class I MHC molecules.