BBA - General Subjects (v.1569, #1-3)

Ascorbic acid: much more than just an antioxidant by Oreste Arrigoni; Mario C. De Tullio (1-9).
Vitamin C (ascorbic acid (AA)) is very popular for its antioxidant properties. Consequently, many other important aspects of this multifaceted molecule are often underestimated or even ignored. In the present paper, we have tried to bring to the foreground some of these aspects, including the peculiarities of the AA biosynthetic pathway in different organisms, the remarkable function of AA as a co-substrate of many important dioxygenases, the role of AA-regenerating enzymes and the known pathways of AA catabolism, as well as the intriguing function of AA in gene expression.
Keywords: Ascorbic acid; Dehydroascorbic acid; Oxoglutarate-dependent dioxygenase;

Interactions of wheat-germ agglutinin with GlcNAcβ1,6Gal sequence by Michiro Muraki; Miyuki Ishimura; Kazuaki Harata (10-20).
The interactions of wheat-germ agglutinin (WGA) with the GlcNAcβ1,6Gal sequence, a characteristic component of branched poly-N-acetyllactosaminoglycans, were investigated using isothermal titration calorimetry and X-ray crystallography. GlcNAcβ1,6Gal exhibited an affinity greater than GlcNAcβ1,4GlcNAc to all WGA isolectins, whereas Galβ1,6GlcNAc showed much less affinity than GlcNAcβ1,4GlcNAc. X-ray structural analyses of the glutaraldehyde-crosslinked WGA isolectin 3 crystals in complex with GlcNAcβ1,6Gal, GlcNAcβ1,4GlcNAc and GlcNAcβ1,6Galβ1,4Glc were performed at 2.4, 2.2 and 2.2 Å resolution, respectively. In spite of different glycosidic linkages, GlcNAcβ1,6Gal and GlcNAcβ1,4GlcNAc exhibited basically similar binding modes to each other, in contact with side chains of two aromatic residues, Tyr64 and His66. However, the conformations of the ligands in the two primary binding sites were not always identical. GlcNAcβ1,6Gal showed more extensive variation in the parameters defining the glycosidic linkage structure compared to GlcNAcβ1,4GlcNAc, demonstrating large conformational flexibility of the former ligand in the interaction with WGA. The difference in the ligand binding conformation was accompanied by alterations of the side chain conformation of the amino acid residues involved in the interactions. The hydrogen bond between Ser62 and the non-reducing end GlcNAc was always observed regardless of the ligand type, indicating the key role of this interaction. In addition to the hydrogen bonding and van der Waals interactions, CH–π interactions involving Tyr64, His66 and Tyr73 are suggested to play an essential role in determining the ligand binding conformation in all complexes. One of the GlcNAcβ1,6Gal ligands had no crystal packing contact with another WGA molecule, therefore the conformation might be more relevant to the interaction mode in solution.
Keywords: Wheat-germ agglutinin; β1,6-Glycosidic linkage; Isothermal titration calorimetry; X-ray structure; Hevein domain; CH–π interaction;

Real-time kinetics of ligand–ligate interaction has predominantly been studied by either fluorescence or surface plasmon resonance based methods. Almost all such studies are based on association between the ligand and the ligate. This paper reports our analysis of dissociation data of monoclonal antibody-antigen (hCG) system using radio-iodinated hCG as a probe and nitrocellulose as a solid support to immobilize mAb. The data was analyzed quantitatively for a one-step and a two-step model. The data fits well into the two-step model. We also found that a fraction of what is bound is non-dissociable (tight-binding portion (TBP)). The TBP was neither an artifact of immobilization nor does it interfere with analysis. It was present when the reaction was carried out in homogeneous solution in liquid phase. The rate constants obtained from the two methods were comparable. The work reported here shows that real-time kinetics of other ligand–ligate interaction can be studied using nitrocellulose as a solid support.
Keywords: Real time kinetics; Dissociation; Antigen–antibody; Human chorionic gonadotropin;

Degradation of non-esterified and esterified xanthophylls by free radicals by Antonio Pérez-Gálvez; M.Isabel Mı́nguez-Mosquera (31-34).
β-Carotene and other xanthophylls present in pepper fruit as both free and esterified forms were oxidized using a free radical initiator (2,2′-azo-bis-isobutyronitrile). Capsorubin was degraded most slowly, followed by zeaxanthin, capsanthin, and β-carotene. The presence of keto groups at the ends of the polyene chain could be a structural factor contributing to this difference in reactivity. It was also shown that whereas capsanthin and its esters and capsorubin and its esters were degraded at the same rate, zeaxanthin esters responded differently to the oxidation process, and were degraded more quickly than free zeaxanthin. The presence of unsaturated fatty acids (mainly linoleic) that esterify zeaxanthin help to accelerate the degradation of this xanthophyll and decreasing its antioxidant action. The antioxidant capacity of capsorubin and capsanthin (both in free and esterified form) exclusive to the genus capsicum should be taken into account.
Keywords: Antioxidant; β-Carotene; Capsanthin; Capsorubin; Ester; Free radical; Oxidation; Unsaturated fatty acid; Zeaxanthin;

Kinetic analysis and mechanistic aspects of autoxidation of catechins by Manabu Mochizuki; Shin-ichi Yamazaki; Kenji Kano; Tokuji Ikeda (35-44).
A peroxidase-based bioelectrochemical sensor of hydrogen peroxide (H2O2) and a Clark-type oxygen electrode were applied to continuous monitoring and kinetic analysis of the autoxidation of catechins. Four major catechins in green tea, (−)-epicatechin, (−)-epicatechin gallate, (−)-epigallocatechin, and (−)-epigallocatechin gallate, were used as model compounds. It was found that dioxygen (O2) is quantitatively reduced to H2O2. The initial rate of autoxidation is suppressed by superoxide dismutase and H+, but is independent of buffer capacity. Based on these results, a mechanism of autoxidation is proposed; the initial step is the one-electron oxidation of the B ring of catechins by O2 to generate a superoxide anion (O •− 2) and a semiquinone radical, as supported in part by electron spin resonance measurements. O •− 2 works as a stronger one-electron oxidant than O2 against catechins and is reduced to H2O2. The semiquinone radical is more susceptible to oxidation with O2 than fully reduced catechins. The autoxidation rate increases with pH. This behavior can be interpreted in terms of the increase in the stability of O •− 2 and the semiquinone radical with increasing pH, rather than the acid dissociation of phenolic groups. Cupric ion enhances autoxidation; most probably it functions as a catalyst of the initial oxidation step of catechins. The product cuprous ion can trigger a Fenton reaction to generate hydroxyl radical. On the other hand, borate ion suppresses autoxidation drastically, due to the strong complex formation with catechins. The biological significance of autoxidation and its effectors are also discussed.
Keywords: Catechin; Autoxidation; Superoxide dismutase; Cupric ion; Borate; H2O2 sensor;

Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting β cell line by João C Miguel; Yasser H.A Abdel-Wahab; Paulo C.F Mathias; Peter R Flatt (45-50).
Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal β cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1–m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 μM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 μM of methoctramine (M2 antagonist) increased ACh (100 μM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release.
Keywords: Muscarinic receptor; Insulin; β cell; Acetylcholine;

In vivo electroporation (EP) is gaining momentum for drug and gene delivery. In particular, DNA transfer by EP to muscle tissue can lead to highly efficient long-term gene expression. We characterized a vascular effect of in vivo EP and its consequences for drug and gene delivery. Pulses of 10–20 000 μs and 0.1–1.6 kV/cm were applied over hind- and forelimb of mice and perfusion was examined by dye injection. The role of a sympathetically mediated vasoconstrictory reflex was investigated by pretreatment with reserpine. Expression of a transferred gene (luciferase), permeabilization (determined using 51Cr-EDTA), membrane resealing and effects on perfusion were compared to assess the significance of the vascular effects. Above the permeabilization threshold, a sympathetically mediated Raynaud-like phenomenon with perfusion delays of 1–2 min was observed. Resolution of this phase followed kinetics of membrane resealing. Above a second threshold, irreversible permeabilization led to long perfusion delays. These vascular reactions (1) affect kinetics of drug delivery, (2) predict efficient DNA transfer, which is optimal during short perfusion delays, and (3) might explain electrocardiographic ST segment depressions after defibrillation as being caused by vascular effects of EP of cardiac muscle.
Keywords: Electroporation; Blood flow; Non-viral gene delivery; Drug delivery; Muscle; Cardiac ischemia;

Tissue-specific regulation of Ca2+ channel protein expression by sex hormones by Gustavo Helguera; Riccardo Olcese; Min Song; Ligia Toro; Enrico Stefani (59-66).
The L-type Ca2+ channel pore-forming α subunit, α1C can be detected in brain and heart as two proteins with molecular masses of ∼240 kDa and ∼190 kDa known as α1C-long and α1C-short, respectively. In brain, the α1C-short is thought to be the product of a ∼50 kDa C-terminus calpain-mediated proteolytic deletion. We now show that uterine smooth muscle also possesses α1C-long and α1C-short isoforms, and that the relative expression of these two forms is regulated by sex hormones in a tissue-specific manner. Protein expression of α1C L-type Ca2+ channels was examined in uterine smooth muscle, brain and heart, comparing non-pregnant (NP) estrus vs. late-pregnant (21 days) rats. The two forms of α1C were detected in all studied tissues. In late-pregnant uterus, α1C-long doubled the expression of α1C-short; in NP uterus the opposite occurred. However, these changes were restricted to the uterine muscle, with no changes in brain and heart. To investigate the mechanism of such regulation, ovariectomized rats were treated with sex hormones, progesterone (P4) and/or 17β-estradiol (estrogen, E2). P4 treatment, which yielded P4 plasma levels of 5±1 ng/ml and a high P4/E2 ratio (3±1.5×103) similar to the ratio in late-pregnant uterus (1.5±0.3 ×103), facilitated α1C-long expression. In contrast, E2 or E2+P4 treatment that increased E2 plasma levels to 60±8 pg/ml and 75±24 pg/ml, produced low P4/E2 ratios of 0.03±0.006 and 0.2±0.1, respectively. These low P4/E2 ratios also found in NP rats at estrus (0.3±0.1) favored the expression of α1C-short form in myometrium. Neither hormone treatment altered α1C expression in brain or heart. Our results indicate that expression of α1C isoforms depends on P4/E2 ratios. Plasma P4/E2 ratios <1×103 favor the expression of the α1C-short; whereas ratios >1×103 facilitate the expression of the α1C-long form. This regulation is tissue-specific for myometrium since it did not occur in heart and brain tissues.
Keywords: L-type Ca2+ channel; Protein expression; Myometrium; Heart; Brain; Pregnancy; Estrogen; Progesterone; Rat;

Cytosolic glycerol-3-phosphate dehydrogenase (cG3PDH) occupies the branch point between the glycolytic pathway and triglyceride biosynthesis. However, the regulatory mechanism of the cG3PDH activity has remained obscure. Here we report that cG3PDH is efficiently inhibited by modification of the thiol group through a redox mechanism. In this study, we found that sodium selenite and nitric oxide (NO) donors such as S-nitroso-N-acetylpenicillamine and 3-morpholinosydnonimine inhibited cG3PDH activity, and that similar effects could be achieved with selenium metabolites such as selenocysteine and selenomethionine. Furthermore, we found that reducing agents, such as dithiothreitol and β-mercaptoethanol, restored the cG3PDH activity suppressed by selenite and NO both in vitro and in cultured cells. Buthionine sulfoximine depleted levels of both reduced glutathione and the oxidized form but had no effect on the suppression of cG3PDH activity by selenite in cultured cells. Moreover, thiol-reactive agents, such as N-ethylmaleimide and o-iodosobenzoic acid, blocked the enzyme activity of cG3PDH through the modification of redox-sensitive cysteine residues in cG3PDH. The inhibitor of NO synthase, L-N G-nitro-arginine, restored the cG3PDH activity inhibited by NO in cultured cells, whereas the inhibitor of guanylyl cyclase, 1H-[1,2,4] oxadiazole[4,3-α] quinoxalin-1-one (ODQ), has no effect. NO directly inhibits cG3PDH activity not via a cGMP-dependent mechanism. Finally, using site-directed mutagenesis, we found that Cys102 of cG3PDH was sensitive to both selenite and NO. From the results, we suggest that cG3PDH is a target of cellular redox regulation.
Keywords: Cytosolic glycerol-3-phosphate dehydrogenase; Sodium selenite; S-Nitroso-N-acetylpenicillamine; Thiol; Reducing agent; Redox mechanism;

Evaluation of glycated glucagon-like peptide-1(7-36)amide in intestinal tissue of normal and diabetic animal models by Mark H Mooney; Yasser H.A Abdel-Wahab; Aine M McKillop; Finbarr P.M O’Harte; Peter R Flatt (75-80).
Glucagon-like peptide-1(7-36)amide (tGLP-1) is an important insulin-releasing hormone of the enteroinsular axis which is secreted by endocrine L-cells of the small intestine following nutrient ingestion. The present study has evaluated tGLP-1 in the intestines of normal and diabetic animal models and estimated the proportion present in glycated form. Total immunoreactive tGLP-1 levels in the intestines of hyperglycaemic hydrocortisone-treated rats, streptozotocin-treated mice and ob/ob mice were similar to age-matched controls. Affinity chromatographic separation of glycated and non-glycated proteins in intestinal extracts followed by radioimmunoassay using a fully cross-reacting anti-serum demonstrated the presence of glycated tGLP-1 within the intestinal extracts of all control animals (approximately 19% of total tGLP-1 content). Chemically induced and spontaneous animal models of diabetes were found to possess significantly greater levels of glycated tGLP-1 than controls, corresponding to between 24–71% of the total content. These observations suggest that glycated tGLP-1 may be of physiological significance given that such N-terminal modification confers resistance to DPP IV inactivation and degradation, extending the very short half-life (<3 min) and bioactivity of the native peptide.
Keywords: Glucagon-like peptide-1; Diabetes; Glycation;

Glutathione transferase isoenzymes from frog (Xenopus laevis) liver and embryo by Stefania Angelucci; Paolo Sacchetta; Antonella De Luca; Pasquale Moio; Fernanda Amicarelli; Carmine Di Ilio (81-85).
The expression of glutathione transferase isoenzymes has been investigated in embryo and adult liver of the frog Xenopus laevis. By analysing the GST isoenzymes recovered from GSH-affinity chromatography in terms of electrophoretic mobility, HPLC elution profile, immunological reactivity, N-terminal amino acid sequence and mass spectrometry molecular mass no significant difference in the GST subunit composition between embryos and liver was found. In both tissues the same three subunits, showing similarity to mu, alpha and sigma class GSTs, are present. These results, together with those previously reported for toad (Bufo bufo), strongly support the notion that the transition from an aquatic environment to a terrestrial atmosphere containing high oxygen concentration has accompanied specific GST gene expression.
Keywords: Glutathione transferase; Amphibia; Mass spectrometry; N-Terminal sequence; Xenopus laevis;

Spatiotemporal dynamics of contact activation factors of blood coagulation by Andrei Yu Kondratovich; Alexandra V Pokhilko; Fazoil I Ataullakhanov (86-104).
A new in vitro model is proposed for studying the spatiotemporal distributions of activated clotting factors, in which clotting is activated in a thin layer of nonstirred plasma supplemented with a fluorogenic substrate and is monitored by fluorescence from its cleavage product. Analysis of the spatiotemporal dynamics of factor XIa and kallikrein in glass-activated human plasma provides evidence that both contact factors remain restricted to the glass surface and possibly a narrow boundary zone (<0.1 mm). The kinetics of factor XIa and kallikrein studied by a new method (in nonstirred plasma) coincided with those studied fluorimetrically with full stirring: their concentrations rapidly rose for the first few minutes after activation and then slowly declined. Factor XI and prekallikrein activation is likely to be restricted by the limited number of sites available for binding to the surface. The maximum concentration of the active factors was estimated at 2×108 molecules per mm2 at the glass surface (irrespective of stirring). At the plastic surface, this value was 15–30 times lower.
Keywords: Contact activation system; Blood coagulation; Spatiotemporal dynamics; Factor XIa; Kallikrein; Citrated plasma;

Prolonged blood glucose reduction in mrp-2 deficient rats (GY/TR) by the glucose-6-phosphate translocase inhibitor S 3025 by Andreas W. Herling; Dietmar Schwab; Hans-Joerg Burger; Jochen Maas; Roland Hammerl; Dietmar Schmidt; Sabine Strohschein; Horst Hemmerle; Gerrit Schubert; Stefan Petry; Werner Kramer (105-110).
Chlorogenic acid derivatives are potent inhibitors of hepatic glucose production by inhibition of the glucose-6-phosphate translocase component of the hepatic glucose-6-phosphatase system. The pharmacological proof of concept was clearly demonstrated during i.v. infusion of potent derivatives (S 4048, S 3483) in rats. However, the blood glucose lowering effect of S 4048 after bolus i.v. injection lasted only 60–90 min. Plasma clearance of S 4048 was very high, and the parent compound was rapidly and efficiently excreted into the bile of Wistar and GY/TR rats, indicating that mrp-2 was not involved in this hepatobiliary elimination process. About 72% of the total administered radioactivity appeared in the bile within 20 min after i.v. bolus injection of the radiolabeled analogue [3H]S 1743 in a Wistar rat. However, in GY/TR rats the dicarboxylic analogue of S 4048, S 3025, was cleared from the plasma less rapidly than its parent compound and its biliary elimination was comparatively low. In contrast, S 3025 exhibited comparable pharmacokinetics and biliary elimination profile as S 4048 in Wistar rats, suggesting that biliary elimination of S 3025 is facilitated by mrp-2, functionally absent in GY/TR rats. Targeting to mrp-2 resulted in a significantly prolonged reduction of blood glucose levels in GY/TR rats after i.v. bolus administration of S 3025.
Keywords: Glucose-6-phosphate translocase; S 4048; mrp-2; Blood glucose; Hepatobiliary elimination;

Perferryl complex of nitric oxide synthase: role in secondary free radical formation by Supatra Porasuphatana; Pei Tsai; Sovitj Pou; Gerald M Rosen (111-116).
Neuronal nitric oxide synthase (NOS I) has been shown to generate nitric oxide (NO) and superoxide (O2 ⋅−) during enzymatic cycling, the ratio of each free radical is dependent upon the concentration of l-arginine. Using spin trapping and electron paramagnetic resonance (EPR) spectroscopy, we recently reported that NOS I can oxidize ethanol (EtOH) to α-hydroxyethyl radical (CH3 CHOH). We speculated that the perferryl complex of NOS, (NOS–[Fe5+O]3+) was responsible for the generation of CH3 CHOH. Using potassium monopersulfate (KHSO5) to oxidize the heme of NOS I to NOS–[Fe5+O]3+, we were able to demonstrate that this perferryl complex can oxidize l-arginine to l-citrulline and NO. Even in the absence of l-arginine, EtOH was oxidized to CH3 CHOH by NOS–[Fe5+O]3+. Sodium cyanide (NaCN), a heme blocker, inhibited the formation of CH3 CHOH by NOS.
Keywords: Nitric oxide synthase (NOS); α-hydroxyethyl radical (CH3 CHOH); Free radical formation; Oxidation; Sodium cyanide (NaCN); Inhibition;

Isolation and characterization of matrix proteoglycans from human nasal cartilage by Achilleas D Theocharis; Nikos K Karamanos; Nikoletta Papageorgakopoulou; Constantinos P Tsiganos; Dimitrios A Theocharis (117-126).
The content, types and the fine structures of proteoglycans (PGs) present in human normal nasal cartilage (HNNC) were investigated and compared with those in human scoliotic nasal cartilage (HSNC). Three PG types were identified in both HNNC and HSNC; the large-sized high buoyant density aggrecan, which is the predominant PG population, and the small-sized low buoyant density biglycan and decorin. HSNC contained a significantly higher amount of keratan sulfate (KS)-rich aggrecan (30%) of smaller hydrodynamic size as compared to HNNC. The average molecular sizes (M rs) of aggecan-derived chondroitin sulfate (CS) chains in both HNNC and HSNC were identical (18 kDa), but they significantly differ in disaccharide composition, since CS isolated from HSNC contained higher proportions of 6-sulfated disaccharides as compared to those from HNNC. Scoliotic tissue contained also higher amounts (67%) of the small PGs, biglycan and decorin as compared to HNNC. It is worth noticing that both normal and scoliotic human nasal cartilage contain also non-glycanated forms of decorin and biglycan. Dermatan sulfate (DS) was the predominant glycosaminoglycan (GAG) present on biglycan and decorin in both tissues. The small PGs-derived CS chains in both normal and scoliotic cartilage had the same M r (20 kDa), whereas DS chains from scoliotic cartilage were of greater M r (32 kDa) than those from normal cartilage (24 kDa). Furthermore, scoliotic tissue-derived DS chains contained higher amounts of iduronate (20%) as compared to those of normal cartilage (12%). Disaccharide analysis of small PGs showed that both HNNC and HSNC were rich in 4-sulfated disaccharides and in each case, the small size PGs contained a considerably higher proportion of 4-sulfated disaccharides than the aggrecan of the same tissue. The higher amounts of matrix PGs identified in scoliotic tissue as well as the differences in fine chemical composition of their GAG chains may reflect the modified architecture and functional failure of scoliotic tissue.
Keywords: Human; Nasal cartilage; Proteoglycan; Decorin; Biglycan; Scoliosis;

Binding of CaMKII to the giant muscle protein projectin: stimulation of CaMKII activity by projectin by Michael Fährmann; Martin Erfmann; Gernot Beinbrech (127-134).
Projectin is an integral high molecular mass protein of insect flight muscle binding to myosin and paramyosin. Yet, the role of projectin in insect flight muscle is not well understood. In this study we provide evidence for the interaction of projectin with the calcium sensor Ca2+/calmodulin-dependent protein kinase II (CaMKII). Two CaMKII variants of 52 and 60 kDa, respectively, of locust flight muscle cells were shown by an anti-CaMKII antibody. Both variants were associated to myofibrils. The 52 kDa variant was also shown abundant in the cytosol. The cytosolic CaMKII variant was co-precipitated in vitro with externally added purified projectin in a dose-dependent manner. To specify the binding properties of CaMKII to projectin we used both purified projectin from the flight muscle of locust and CaMKII from rat forebrain, a naturally rich source of CaMKII. CaMKII is highly conserved even between insects and mammals. The binding of CaMKII to native projectin was demonstrated in vitro by the solid phase enzyme assay, immunoprecipitation, and ‘overlay’ binding. One mol projectin bound maximally 1.38±0.02 mol CaMKII in vitro with a K d of 3.08±0.09 nM. Application of in vitro autophosphorylated CaMKII revealed a decreased stoichiometry of binding to projectin (0.86±0.04 mol mol−1) accompanied by a lower affinity (K d of 5.54±0.73) compared to non-autophosphorylated CaMKII. Furthermore, the CaMKII phosphotransferase activity was stimulated up to 2-fold by projectin. Even in the presence of calmodulin projectin enhanced the CaMKII activity moderately. Our data suggest that projectin represents a subcellular compartment for CaMKII to achieve its specificity, and activity in insect flight muscle cells.
Keywords: Ca2+/calmodulin-dependent protein kinase; Calmodulin; Insect flight muscle; Projectin; Protein binding; Protein kinase;

Differential response of fast and slow myosin ATPase from skeletal muscle to F-actin and to phalloidin F-actin by Giorgio Trombetta; Raffella Adami; Orietta Cintio; Enrico Grazi (135-138).
Fast muscle myosin responds in similar way to F-actin and to phalloidin F-actin. It is activated 7.5 fold at infinite F-actin concentration and 6.8 fold at infinite phalloidin F-actin. The actomyosin dissociation constants are 0.89±0.34 μM with F-actin and 0.90±0.71 μM with phalloidin F-actin. Slow muscle myosin responds differently to F-actin and to phalloidin F-actin. It is activated 3.76 fold at infinite F-actin concentration and only 2.27 fold at infinite phalloidin F-actin concentration. The actomyosin dissociation constants are 1.95±1.27 μM with F-actin and 0.27±0.16 μM with phalloidin F-actin. At first glance this means that substitution of F-actin with phalloidin F-actin magnifies the difference between fast muscle and slow muscle myosins. Furthermore the change of the dissociation constants may affect the contractile force of the attached crossbridge.
Keywords: Fast; Slow myosin; Phalloidin F-actin;

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I–hydrophobin I by Anna Collén; Josefine Persson; Markus Linder; Tiina Nakari-Setälä; Merja Penttilä; Folke Tjerneld; Ulf Sivars (139-150).
Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase Icore–hydrophobin I (EGIcore–HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGIcore–HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGIcore–HFBI was quantitatively back-extracted (K EGIcore–HFBI=150, yield=99%) into a water phase. In this second step, ethylene oxide–propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55°C was performed. Total recovery of EGIcore–HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGIcore–HFBI into a water phase.
Keywords: Aqueous two-phase system; Detergent removal; Extraction; Fusion protein; Hydrophobin; Protein purification;

A facilitated electron transfer of copper–zinc superoxide dismutase (SOD) based on a cysteine-bridged SOD electrode by Yang Tian; Mieko Shioda; Shinjiro Kasahara; Takeyoshi Okajima; Lanqun Mao; Toru Hisabori; Takeo Ohsaka (151-158).
The direct electrochemical redox reaction of bovine erythrocyte copper–zinc superoxide dismutase (Cu2Zn2SOD) was clearly observed at a gold electrode modified with a self-assembled monolayer (SAM) of cysteine in phosphate buffer solution containing SOD, although its reaction could not be observed at the bare electrode. In this case, SOD was found to be stably confined on the SAM of cysteine and the redox response could be observed even when the cysteine-SAM electrode used in the SOD solution was transferred to the pure electrolyte solution containing no SOD, suggesting the permanent binding of SOD via the SAM of cysteine on the electrode surface. The electrode reaction of the SOD confined on the cysteine-SAM electrode was found to be quasi-reversible with the formal potential of 65±3 mV vs. Ag/AgCl and its kinetic parameters were estimated: the electron transfer rate constant k s is 1.2±0.2 s−1 and the anodic (α a) and cathodic (α c) transfer coefficients are 0.39±0.02 and 0.61±0.02, respectively. The assignment of the redox peak of SOD at the cysteine-SAM modified electrode could be sufficiently carried out using the native SOD (Cu2Zn2SOD), its Cu- or Zn-free derivatives (E2Zn2SOD and Cu2E2SOD, E designates an empty site) and the SOD reconstituted from E2Zn2SOD and Cu2+. The Cu complex moiety, the active site for the enzymatic dismutation of the superoxide ion, was characterized to be also the electroactive site of SOD. In addition, we found that the SOD confined on the electrode can be expected to possess its inherent enzymatic activity for dismutation of the superoxide ion.
Keywords: Superoxide dismutase (SOD); Self-assembled monolayer (SAM); Cysteine; Redox reaction; Electron transfer; Copper; Zinc;

The roles of ATP and calcium in morphological changes and cytotoxicity induced by 1,4-benzoquinone in platelets by Sun-Ku Lee; Seung-Min Chung; Moo-Yoel Lee; Joo-Young Lee; Ok-Nam Bae; Jin-Ho Chung (159-166).
To understand the mechanism of 1,4-benzoquinone-induced cytotoxicity in platelets, the roles of ATP and calcium in platelet toxicity and morphological changes were investigated. Using scanning electron microscopy, morphological changes including membrane blebbing were observed in rat platelets 5 min after exposure to 1,4-benzoquinone, which were significantly different from shape changes (pseudopod formation) observed in response to physiological agonists. Benzoquinone-induced membrane blebbing of platelets was associated with rapid depletion of intracellular ATP and was independent of the presence of extracellular calcium. Benzoquinone-induced platelet lysis observed between 20 and 30 min was dependent on extracellular calcium and associated with increased cytosolic calcium. Cytotoxicity induced by 1,4-benzoquinone was inhibited by antagonists of calmodulin, suggesting that calmodulin could play an important role in platelet toxicity. These results suggested that the progression of events for benzoquinone-induced cytotoxicity in platelets was as follows: 1,4-benzoquinone depletes intracellular ATP; membrane blebbing occurs; calcium homeostasis is disrupted, activation of calmodulin-dependent processes results; finally cytotoxicity occurs.
Keywords: Adenosine 5′-triphosphate; Calcium; Morphological change; Platelet; Cytotoxicity; 1,4-benzoquinone;

Single chain antibodies specific for fatty acids derived from a semi-synthetic phage display library by Ari Gargir; Itzhak Ofek; Shiri Meron-Sudai; Meital Gal Tanamy; Panagiotis S Kabouridis; Ahuva Nissim (167-173).
The biological activities of many acylated molecules are lipid dependent. Lipids, however, are poorly immunogenic or non-immunogenic. We employed a phage display semi-synthetic human antibody library to isolate anti-lipid antibodies. Selection was done against methyl palmitate, a 16 carbon aliphatic chain, and a major component of bacterial glycolipids and lipoproteins in animal cells. The selected single chain variable fragment (scFv) bound specifically to a 16 carbon aliphatic chain and to a lesser extent to a 14 or 18 carbon aliphatic chain and poorly to either 12, 22 or 8 carbon aliphatic chains. Furthermore, the scFv prevented micelle formation of lipoteichoic acid from Gram-positive bacteria; inhibited lipopolysaccharide-induced tumor necrosis factor α release in mononuclear cells; bound to hydrophobic bacterial surfaces, especially those of Gram-positive bacteria, and bound to Lck, a mammalian palmitated lipoprotein. Our data suggest that the phage antibody library can be successfully employed to obtain human anti-aliphatic scFv human antibody fragment with potential therapeutic applications in neutralizing the deleterious effects of bacterial toxins as well as in structure–function analysis of lipoproteins in animal cells.
Keywords: Phage display; Single chain variable fragment; Methyl palmitate; Lipoteichoic acid;

Author Index (174-175).