Purification, cloning and autoproteolytic processing of an aspartic proteinase from Centaurea calcitrapa.

Plant aspartic proteinases (APs) have been isolated from several seed and leaf sources but the only well characterized enzymes from flowers are cardosins and cyprosins from cardoon, Cynara cardunculus L. Here we report a full-length cDNA clone encoding an AP named cenprosin from the flowers of Centaurea calcitrapa L., a thistle related to cardoon. As found for all eukaryotic APs, the deduced primary sequence consists of a signal sequence, a propart and a mature enzyme. In addition, an internal sequence region of 104 residues typical only of plant APs (a plant-specific insert) is present in the primary structure. Northern analysis revealed that the strongest expression is in fresh flowers. The enzyme is also expressed in fairly high amounts in seeds and in leaves, a feature not detected for cardoon APs. The corresponding enzyme was purified in its precursor form from fresh flowers using ammonium-sulfate precipitation followed by ion-exchange and hydrophobic-interaction chromatography. The processing of the precursor into its mature form was studied in vitro. The enzyme underwent autocatalytic processing at pH 3.0 resulting in two chains of 16 and 30 kDa. When dried flowers were used as a starting material for purification, only 16- and 30-kDa chains were obtained, suggesting that autoproteolytic activation of procenprosin in vivo occurs mainly during drying of the flowers. This may indicate a specific degradative role for the enzyme during senescence of the flowers.     

Regulation of particulate guanylate cyclase by atriopeptins: relation between peptide structure, receptor binding, and enzyme kinetics

Structural analogs of atriopeptins (APs) were compared for their ability to activate particulate guanylate cyclase and bind to specific receptors in rat adrenal membranes. All analogs tested increase Vmax without altering the concentration of substrate required for half-maximum activity or the positive coperativity exhibited by the enzyme. Maximum velocities (pmoles of cGMP produced per min per mg protein) achieved in the absence and presence of APs were 128.3 +/- 6.6 and 283.8 +/- 20.6 using Mn2+-GTP, and 53.7 +/- 3.7 and 149.9 +/- 7.6 using Mg2+-GTP as the substrate, respectively. Although all APs were equally efficacious in activating the enzyme, their rank potency was ANF (8-33) = AP III = AP II greater than AP I when either divalent cation was used as the cofactor. The EC50 for activation of guanylate cyclase by AP I was about 10(-7) M, while that for the other peptides was about 10(-8) M, using either divalent cation cofactor. 125I-labeled ANF bound to rat adrenal membranes with a KD of 5.10(-10) M. Although all APs were equally efficacious in competing with labeled ANF for receptor binding, their rank potency was identical to that for enzyme activation. The Ki for AP I was about 10(-8) M, while that for the other peptides was about 10(-10) M. These data suggest that the carboxy terminal Phe-Arg present in the AP analogs except AP I and critical for biological and receptor-binding activity are also important in coupling receptor-ligand interaction with guanylate cyclase activation. The correlation between the rank order potency for receptor binding, enzyme activation, and the reported physiological actions of APs support the suggestion of a functional coupling between these proteins.     

Structural and Biochemical Characterization of a Halophilic Archaeal Alkaline Phosphatase

Phosphate is an essential component of all cells that must be taken up from the environment. Prokaryotes commonly secrete alkaline phosphatases (APs) to recruit phosphate from organic compounds by hydrolysis. In this study, the AP from Halobacterium salinarum, an archaeon that lives in a saturated salt environment, has been functionally and structurally characterized. The core fold and the active-site architecture of the H. salinarum enzyme are similar to other AP structures. These generally form dimers composed of dominant β-sheet structures sandwiched by α-helices and have well-accessible active sites. The surface of the enzyme is predicted to be highly negatively charged, like other proteins of extreme halophiles. In addition to the conserved core, most APs contain a crown domain that strongly varies within species. In the H. salinarum AP, the crown domain is made of an acyl-carrier-protein-like fold. Different from other APs, it is not involved in dimer formation. We compare the archaeal AP with its bacterial and eukaryotic counterparts, and we focus on the role of crown domains in enhancing protein stability, regulating enzyme function, and guiding phosphoesters into the active-site funnel.     

Characterization of a monomeric Escherichia coli alkaline phosphatase formed upon a single amino acid substitution

Alkaline phosphatase (AP) from Escherichia coli as well as APs from many other organisms exist in a dimeric quaternary structure. Each monomer contains an active site located 32 A away from the active site in the second subunit. Indirect evidence has previously suggested that the monomeric form of AP is inactive. Molecular modeling studies indicated that destabilization of the dimeric interface should occur if Thr-59, located near the 2-fold axis of symmetry, were replaced by a sterically large and charged residue such as arginine. The T59R enzyme was constructed and characterized by sucrose-density gradient sedimentation, size-exclusion chromatography, and circular dichroism (CD) and compared with the previously constructed T59A enzyme. The T59A enzyme was found to exist as a dimer, whereas the T59R enzyme was found to exist as a monomer. The T59A, T59R, and wild-type APs exhibited almost identical secondary structures as judged by CD. The T59R monomeric AP has a melting temperature (Tm) of 43 degrees C, whereas the wild-type AP dimer has a Tm of 97 degrees C. The catalytic activity of the T59R enzyme was reduced by 104-fold, whereas the T59A enzyme exhibited an activity similar to that of the wild-type enzyme. The T59A and wild-type enzymes contained similar levels of zinc and magnesium, whereas the T59R enzyme has almost undetectable amounts of tightly bound metals. These results suggest that a significant conformational change occurs upon dimerization, which enhances thermal stability, metal binding, and catalysis.     

Amino acid sequence of the cold-active alkaline phosphatase from Atlantic cod (Gadus morhua)

Atlantic cod is a marine fish that lives at low temperatures of 0-10 degrees C and contains a cold-adapted alkaline phosphatase (AP). Preparations of AP from either the lower part of the intestines or the pyloric caeca area were subjected to proteolytic digestion, mass spectrometry and amino acid sequencing by Edman degradation. The primary structure exhibits greatest similarity to human tissue non-specific AP (80%), and approximately 30% similarity to AP from Escherichia coli. The key residues required for catalysis are conserved in the cod AP, except for the third metal binding site, where cod AP has the same variable residues as mammalian APs (His153 and His328 by E. coli AP numbering). General comparison of the amino acid composition with mammalian APs showed that cod AP contains fewer Cys, Leu, Met and Ser, but proportionally more Asn, Asp, Ile, Lys, Trp and Tyr residues. Three N-linked glycosylation sites were found. The glycan structure was determined as complex biantennary in type with fucose and sialic acid attached, although a trace of complex tri-antennary structure was also observed. A three-dimensional model was obtained by homology modelling using the human placental AP scaffold. Cod AP has fewer charged and hydrophobic residues, but more polar residues at the intersubunit surface. The N-terminal helix arm that embraces the second subunit in dimeric APs may be more flexible due to a replaced Pro at its base. One disulfide bridge was found instead of the two present in most other APs. This may invoke greater movement in the structure that together with weaker subunit contacts leads to improved catalytic efficiency.     

Simultaneous Retention of Thermostability and Specific Activity in Chimeric Human Alkaline Phosphatases

Alkaline phosphatases (APs) are a family of dimeric metalloenzymes that has been utilized in many areas due to its ability to hydrolyze a variety of phosphomonoesters. While mammalian APs have higher specific activity than prokaryotic APs, they are generally less thermostable. To cultivate the possibility to confer mammalian APs with higher thermostability as well as high activity, we focused on human AP isozymes. Among the four isozymes of human APs, placental AP (PLAP) retains the highest thermostability, while intestinal AP (IAP) has the highest specific activity. Since the two APs display high homology, a series of chimeric enzymes were made in a secreted form to analyze their properties. Surprisingly, chimeric APs with IAP residues at the N-terminal and PLAP residues at the C-terminal regions showed higher specific activity than PLAP, while keeping thermostability as high as PLAP. Especially, one showed similar specific activity to IAP, while showing slower inactivation than PLAP after incubation at 75 °C. Interestingly, the mutant also showed higher resistance to uncompetitive inhibitors Phe and Leu than their parent enzymes, possibly due to increased hydrophilicity of the active site entrance residues. The obtained chimera will be useful as a novel reporter in various assays including gene hybridization.     

The influence of glutamic and aminoacetic acids on the excitability of the liverwort Conocephalum conicum

Intracellular microelectrode measurements revealed that a resting potential (RP), an action potential (AP) and a calcium component of AP (named voltage transient, VT) can be influenced by glutamic acid (Glu) and aminoacetic acid (glycine, Gly) in the liverwort Conocephalum conicum. In the continuous presence of 5mM Glu or 5mM Gly, the RP hyperpolarized constantly and the plants became desensitized to the excitatory amino acids (Glu or Gly). Under such circumstances, the amplitudes of APs evoked by stimuli other than Glu or Gly grew, as did their calcium components (VTs). The sudden application of 1-15 mM Glu or Gly to a thallus not yet desensitized resulted in an excitation, i.e. a single AP or AP series. Aspartate (Asp) could not substitute for Glu in any way. Simultaneous action of both amino acids acted synergically to trigger APs. The same phenomenon was observed when glycine solution was enriched with N-methyl-D-aspartic acid (NMDA). Gly-induced APs were totally hindered by 1mM D-amino-5-phosphonopentanoic acid (AP5)--an inhibitor of ionotropic glutamate receptors of the NMDA kind. Glu-induced APs could be totally suppressed by 1mM AP5 as well as by 1mM 6,7-dinitroquinoxaline-2,3-dione (DNQX)--an inhibitor of AMPA/KA receptors. DNQX also completely blocked the calcium component of Glu-evoked APs. After DNQX treatment, the only response to Glu was a membrane potential hyperpolarization (like the Glu response in a desensitized plant). It was concluded that the Glu-induced depolarization and hyperpolarization are separate phenomena. The stimulatory effects of both Glu and Gly on liverwort excitability may be the consequences of an activation of a variety of ionotropic Glu receptor subtypes.     

Action potentials in fast- and slow-twitch mammalian muscles during reinnervation and development

Action potentials (APs) were recorded from the extrajunctional membrane of surface fibers of the fast-twitch extensor digitorum longus (extensor) and the slow-twitch soleus muscles of adult rats. APs of the extensor muscle had a significantly faster rate of rise and fall, as well as a shorter duration, than those of the soleus. In addition, the overshoot of APs and the resting membrane potential was greater for the extensor. Whereas the soleus produced only one AP regardless of the stimulus duration, the number of extensor responses was directly proportional to the stimulus duration. This repetitive activity was greatly reduced by a concentration of tetrodotoxin (TTX) as low as 5 X 10(11) g/ml. Within 8 d after crush of the nerves to these two muscles, all differences in AP properties disappeared and both muscles became partially resistant to TTX. Reinnervation brought about a redifferentiation so that differences in AP were again significant at 22 d after nerve crush. However, the rate of rise of extensor APs did not attain normal values even as late as 60 d after nerve crush. APs were found to be the same for extensor and soleus muscles from 12-d-old rats. At 18 d after birth, rate of rise was equivalent to that of adult muscle for the soleus although 50--60 d were required before this parameter was fully mature for the extensor. Nevertheless, APs of the extensor and soleus were clearly differentiated within 25 d after birth. Differences in fast and slow muscle APs are discussed with regard to differences in ion gradients and sarcolemmal conductance.     

Developmental changes in the antigenicity and sugar-chain heterogeneity of rabbit alkaline phosphatases

1. Rabbit alkaline phosphatases (APs) clearly fused with the anti-human AP antibodies. In particular, fetal liver and kidney APs reacted slightly less with the anti-intestinal AP antibody as did adult enzymes, suggesting that intestinal AP-like isozyme is expressed at earlier stages of gestation in rabbit liver and kidney. 2. Immunohistochemical data indicated that intestinal AP-like isozyme in the kidney was mainly localized in the distal convoluted tubules and slightly in the proximal straight tubules, whereas liver/bone/kidney AP-like enzyme was found more in the glomeruli and interstitial capillary walls as a major component. 3. The sugar-chain heterogeneity of adult and fetal rabbit APs displayed organ-specificity as did of rat and human APs. Moreover, in fetal development, the expression of high-mannose type or hybrid type sugar chains precedes the expression of complex type sugar chains in fetal development.     

The interaction of antipsychotic drugs with lipids and subsequent lipid reorganization investigated using biophysical methods

The interaction of antipsychotic drugs (AP) with lipids and the subsequent lipid reorganization on model membranes was assessed using a combination of several complementary biophysical approaches (calorimetry, plasmon resonance, fluorescence microscopy, X-ray diffraction and molecular modeling). The effect of haloperidol (HAL), risperidone (RIS), and 9-OH-risperidone (9-OH-RIS) was examined on single lipid and mixtures comprising lipids of biological origin. All APs interact with lipids and induced membrane reorganization. APs showed higher affinity for sphingomyelin than for phosphatidylcholine. Cholesterol increased AP affinity for the lipid bilayer and led to the following AP ranking regarding affinity and structural changes: RIS >9-OH-RIS >HAL. Liquid-ordered domain formation and bilayer thickness were differentially altered by AP addition. Docking calculations helped understanding the observed differences between the APs and offer a representation of their conformation in the lipid bilayer. Present results indicate that AP drugs may change membrane compartmentalization which could differentially modulate the signaling cascade of the dopamine D2 receptor for which APs are ligands.     

The 1.4 A crystal structure of the large and cold-active Vibrio sp. alkaline phosphatase

Alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 is among the AP variants with the highest known k(cat) value. Here the structure of the enzyme at 1.4 A resolution is reported and compared to APs from E. coli, human placenta, shrimp and the Antarctic bacterium strain TAB5. The Vibrio AP is a dimer although its monomers are without the long N-terminal helix that embraces the other subunit in many other APs. The long insertion loop, previously noted as a special feature of the Vibrio AP, serves a similar function. The surface does not have the high negative charge density as observed in shrimp AP, but a positively charged patch is observed around the active site that may be favourable for substrate binding. The dimer interface has a similar number of non-covalent interactions as other APs and the "crown"-domain is the largest observed in known APs. Part of it slopes over the catalytic site suggesting that the substrates may be small molecules. The catalytic serines are refined with multiple conformations in both monomers. One of the ligands to the catalytic zinc ion in binding site M1 is directly connected to the crown-domain and is closest to the dimer interface. Subtle movements in metal ligands may help in the release of the product and/or facilitate prior dephosphorylation of the covalent intermediate. Intersubunit interactions may be a major factor for promoting active site geometries that lead to the high catalytic activity of Vibrio AP at low temperatures.     

Characterization of acid phosphatase and phosphorylcholine hydrolase in adult Haemonchus contortus

An acid phosphatase (AP) and a phosphorylcholine hydrolase (PCH) were detected in excretory-secretory (ESP) products from adult Haemonchus contortus. The AP had a pH optimum of 4.5 and was inhibited by tartaric acid and sodium fluoride, but not by o-phenanthroline. The AP hydrolyzed paranitrophenol (pnp)-phosphate and to a lesser extent pnp-phenyl-phosphonate but did not hydrolyze diester substrates. Purified AP consisted of heterodimers with relative molecular weight (Mr) of 41.9 and 48.7 kDa and had a native molecular weight of 98 kDa by size-exclusion chromatography (SEC). The PCH had a pH optimum of about 9.5 and was inhibited by EDTA and o-phenanthroline but not by the specific phospholipase inhibitor D609. The specific activity of PCH in the ESP was approximately 25-fold less than that of AP. PCH also hydrolyzed 5'-thymidine monophosphate-pnp at a rate about 40% lower than pnp-phosphorylcholine but did not hydrolyze 3'-thymidine monophosphate-pnp. Partial purification of PCH suggests an Mr of 50.2 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an Mr of 102 kDa by SEC. Both AP and PHC were secreted in vitro in a time-dependent manner and had their highest concentrations in the intestine. The results indicate that H. contortus adults secrete significant amounts of AP that might be a digestive enzyme. PCH is also an intestinal enzyme and is secreted in lesser amounts than AP. The PCH is probably not a phospholipase C but has some characteristics of a type I phosphodiesterase.     

Action Potential Energy Efficiency Varies Among Neuron Types in Vertebrates and Invertebrates

The initiation and propagation of action potentials (APs) places high demands on the energetic resources of neural tissue. Each AP forces ATP-driven ion pumps to work harder to restore the ionic concentration gradients, thus consuming more energy. Here, we ask whether the ionic currents underlying the AP can be predicted theoretically from the principle of minimum energy consumption. A long-held supposition that APs are energetically wasteful, based on theoretical analysis of the squid giant axon AP, has recently been overturned by studies that measured the currents contributing to the AP in several mammalian neurons. In the single compartment models studied here, AP energy consumption varies greatly among vertebrate and invertebrate neurons, with several mammalian neuron models using close to the capacitive minimum of energy needed. Strikingly, energy consumption can increase by more than ten-fold simply by changing the overlap of the Na⁺ and K⁺ currents during the AP without changing the APs shape. As a consequence, the height and width of the AP are poor predictors of energy consumption. In the Hodgkin–Huxley model of the squid axon, optimizing the kinetics or number of Na⁺ and K⁺ channels can whittle down the number of ATP molecules needed for each AP by a factor of four. In contrast to the squid AP, the temporal profile of the currents underlying APs of some mammalian neurons are nearly perfectly matched to the optimized properties of ionic conductances so as to minimize the ATP cost.     

Engineered disulfide bonds increase active-site local stability and reduce catalytic activity of a cold-adapted alkaline phosphatase

Alkaline phosphatase is an extracellular enzyme that is membrane-bound in eukaryotes but resides in the periplasmic space of bacteria. It normally carries four cysteine residues that form two disulfide bonds, for instance in the APs of Escherichia coli and vertebrates. An AP variant from a Vibrio sp. has only one cysteine residue. This cysteine is second next to the nucleophilic serine in the active site. We have individually modified seven residues to cysteine that are on two loops predicted to be within a 5 A radius. Four of them formed a disulfide bond to the endogenous cysteine. Thermal stability was monitored by circular dichroism and activity measurements. Global stability was similar to the wild-type enzyme. However, a significant increase in heat-stability was observed for the disulfide-containing variants using activity as a measure, together with a large reduction in catalytic rates (k(cat)) and a general decrease in Km values. The results suggest that a high degree of mobility near the active site and in the helix carrying the endogenous cysteine is essential for full catalytic efficiency in the cold-adapted AP.     

Variance-based signal conditioning technique: Comparison to a wavelet-based technique to improve spike detection in multiunit intrafascicular recordings

Detection of single unit action potentials (APs) from peripheral nerve recordings is complicated by low signal-to-noise ratio (SNR) due to the activity of nearby muscles, interference from more distant nerve fibers, and thermal noise from the neural interface. In this study, we propose a novel signal conditioning technique for multiunit signals (i.e. a signal comprised of multiple units coming from different nerve fibers), based on the variance to be applied prior to detection of APs. The proposed technique was tested on experimental and simulated intrafascicular recordings; and was compared to a wavelet-based conditioning (also applied before AP detection). The outputs of both conditioning schemes were sent to an AP detection algorithm that used a simple threshold (equal to the standard deviation of the signal). The overall performance of the detection phase was superior when using the wavelet-based conditioned signal especially for SNR ≤ 2 dB. However, when using the variance-based conditioned signal, the AP detection phase resulted in lower number of false positives for SNR > 2 dB. The novel variance-based method improves the SNR by attenuating the background noise between APs and can be applied as pre-conditioning processing for AP detection.     

Modulation of action potential by [Ca2+]i in modeled rat atrial and guinea pig ventricular myocytes

We simulated mechanisms that increase Ca2+ transients with two models: the Luo-Rudy II model for guinea pig (GP) ventricle (GP model) representing long action potential (AP) myocytes and the rat atrial (RA) model exemplifying myocytes with short APs. The interventions were activation of stretch-gated cationic channels, increase of intracellular Na+ concentration ([Na+]i), simulated bet-adrenoceptor stimulation, and Ca2+ accumulation into the sarcoplasmic reticulum (SR). In the RA model, interventions caused an increase of AP duration. In the GP model, AP duration decreased except in the simulated beta-stimulation where it lengthened APs as in the RA model. We conclude that the changes in the APs are significantly contributed by the increase of the Ca2+ transient itself. The AP duration is controlled differently in cardiac myocytes with short and long AP durations. With short APs, an increase of the Ca2+ transient promotes an inward current via Na+/Ca2+-exchanger lengthening the AP. This effect is similar regardless of the mechanism causing the increase of the Ca2+ transient. With long APs the Ca2+ transient increase decreases the AP duration via inactivation of the L-type Ca2+ current. However, L-type current increase (as with beta-stimulation) increases the AP duration despite the simultaneous Ca2+ transient augmentation. The results explain the dispersion of AP changes in myocytes with short and long APs during interventions increasing the Ca2+ transients.     

Reaction mechanism of alkaline phosphatase based on crystal structures. Two-metal ion catalysis

Alkaline phosphatase (AP) is a widely distributed non-specific phosphomonoesterase that functions through formation of a covalent phosphoseryl intermediate (E-P). The enzyme also catalyzes phosphoryl transfer reaction to various alcohols. Escherichia coli AP is a homodimer with 449 residues per monomer. It is a metalloenzyme with two Zn2+ and one Mg2+ at each active site. The crystal structure of native E. coli AP complexed with inorganic phosphate (Pi), which is a strong competitive inhibitor as well as a substrate for the reverse reaction, has been refined at 2.0 A resolution. Some parts of the molecular have been retraced, starting from the previous 2.8 A study. The active site has been modified substantially and is described in this paper. The changes in the active site region suggest the need to reinterpret earlier spectral data, and suggestions are made. Also presented are the structures of the Cd-substituted enzyme complexed with inorganic phosphate at 2.5 A resolution, and the phosphate-free native enzyme at 2.8 A resolution. At pH 7.5, where the X-ray data were collected, the Cd-substituted enzyme is predominantly the covalent phosphoenzyme (E-P) while the native Zn/Mg enzyme exists in predominantly noncovalent (E.P) form. Implication of these results for the catalytic mechanism of the enzyme is discussed. APs from other sources are believed to function in a similar manner.     

Activity and localisation of the lysosomal marker enzymes acid phosphatase, N-acetyl-beta-D-glucosaminidase, and beta-galactosidase in the earthworms Eisenia fetida and E. veneta

The present study describes the activity and localisation of three putative lysosomal marker enzymes, acid phosphatase (AP), N-acetyl-beta-D-glucosaminidase (beta-NAG), and beta-galactosidase (beta-Gal), in whole individuals and in distinct parts of the earthworms, Eisenia veneta and Eisenia fetida. Activities of AP and beta-NAG were high in the two species with most of the activity located to the anterior and mid-parts of the worms. The activity of beta-Gal was low in all body regions. We found interspecies difference in the AP activity as E. veneta had significantly higher activity of AP than E. fetida in posterior and mid-parts, as well as in whole individuals. Of the three enzymes tested, AP was the only enzyme located to lysosomes, yielding high latency all over the worms with especially high latency in the coelomic fluids and posterior regions. The lysosomal APs in E. veneta and E. fetida may be utilised as a new biomarker for xenobiotic-induced lysosomal membrane damage in earthworms.     

The purification and properties of nucleoside phosphotransferase from mucosa of chicken intestine

Nucleoside phosphotransferase (nucleotide: 3'-deoxynucleoside 5'-phosphotransferase, EC 2.7.1.77) has been purified from chicken intestine mucosa to apparent homogeneity. The enzyme is represented by a multisubunit protein at different degrees of association. It can dissociate into a component with a marked fall in catalytic activity. The associated forms are similar to the enzyme previously purified from chick embryo as regards: substrate specificity both with respect to nucleoside monophosphate donors and to deoxyribonucleoside acceptors; sigmoidicity in the rate curve with a variable phosphate donor; instability to heat, dilution and lowering of pH; the activating and protecting effect of nucleotides, particularly the diphosphate forms. The dissociated form displays lower Vmax and higher S0.5 than the associated ones; and the Hill constants are always about 1. With this form, nucleotides show only a modest activating effect and do not protect. Mg2+, Mn2+ or Co2+ are required for catalytic activity, whereas the protective effect of nucleotides is independent of divalent metals. Inorganic phosphate stabilizes associated forms of the enzyme, but inhibits its activity by competing with nucleotide effectors. The enzyme behaves also as a phosphohydrolase, particularly with respect to deoxyribonucleoside monophosphates; deoxyuridine and deoxythymidine inhibit hydrolytic activity.