Inhibitory effect of copper(II) on zinc(II)-induced aggregation of amyloid beta-peptide

Aggregation of amyloid beta-peptide (Abeta), a key pathological event in Alzheimer's disease, has been shown in vitro to be profoundly promoted by Zn(II). This fact suggests that some factors in the normal brain protect Abeta from the Zn(II)-induced aggregation. We demonstrate for the first time that Cu(II) effectively inhibits the Abeta aggregation by competing with Zn(II) for histidine residues. The Raman spectrum of a metal-Abeta complex in the presence of both Zn(II) and Cu(II) shows that the cross-linking of Abeta through binding of Zn(II) to the N(tau) atom of histidine is prevented by chelation of Cu(II) by the N(pi) atom of histidine and nearby amide nitrogens. The inhibitory effect is strongest at a Cu/Abeta molar ratio of around 4. Above this ratio, Cu(II) itself promotes the Abeta aggregation by binding to the phenolate oxygen of Tyr10. These results emphasize the importance of regulation of Cu(II) levels to inhibit Abeta aggregation, and are consistent with an altered metal homeostasis in Alzheimer's disease.     

The effect of temperature on the agglutinability by lectins of liposomes prepared from total lipids of erythrocytes

Multilamellar liposomes prepared from total lipids of red blood cells are agglutinable by the addition of soybean lectin. At 5 °C the rate of agglutination is significantly slower than at 37 °C, in contrast to erythrocyte ghosts and ghosts sonicated to 1 μ vesicles. The slower lateral mobilities of the lectin glycolipid receptor in the lipid liposomes due to increased microviscosity of the bilayer at the lower temperature, might be one explanation of our agglutination results. However, the opposite temperature dependence seen with ghosts argues for a possible protein modulation of the agglutination reaction.     

Formation of toxic fibrils of Alzheimer's amyloid beta-protein-(1-40) by monosialoganglioside GM1, a neuronal membrane component

A pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid beta-protein (Abeta) in fibrillar form on neuronal cells. However, the role of Abeta fibrils in neuronal dysfunction is highly controversial. This study demonstrates that monosialoganglioside GM1 (GM1) released from damaged neurons catalyzes the formation of Abeta fibrils, the toxicity and the cell affinity of which are much stronger than those of Abeta fibrils formed in phosphate-buffered saline. Abeta-(1-40) was incubated with equimolar GM1 at 37 degrees C. After a lag period of 6-12 h, amyloid fibrils were formed, as confirmed by circular dichroism, thioflavin-T fluorescence, size-exclusion chromatography, and transmission electron microscopy. The fibrils showed significant cytotoxicity against PC12 cells differentiated with nerve growth factor. Trisialoganglioside GT1b also facilitated the fibrillization, although the effect was weaker than that of GM1. Our study suggests an exacerbation mechanism of AD and an importance of polymorphisms in Abeta fibrils during the pathogenesis of the disease.     

Inhibition of high affinity choline uptake in the rat brain by neurotoxins: effect of monosialoganglioside GM1.

Mustard derivatives of ethyl-choline and hemicholinium-3 have been suggested as possible specific cholinergic neurotoxins. In this study a structural analog of hemicholinium-3, a,a'-bis[di(2-chloroethyl)amino]-4,4'-2-biacetophenone (toxin 7), was added to synaptosomes prepared from the cortex, striatum or hippocampus of rat brain. Synaptosomal high affinity choline uptake (HACU) was significantly decreased in a dose-dependent manner by addition of toxin 7, while synaptosomal uptake of GABA or dopamine was not changed. Incubation of cortical synaptosomes with the monosialoganglioside GM1 prevented the decrease in HACU seen following administration of toxin 7. This preventative effect of GM1 was greater if GM1 was added prior to or concomitant with toxin 7, than if GM1 was added following toxin 7. Two newly synthesized hemicholinium-3 analogs, 4-[3'-di(2-chloroethyl)aminopropionyl]biphenyl (toxin 5) and 4-[3'-di(2-bromoethyl)aminopropionyl]biphenyl (toxin 6) caused a large decrease in HACU when added to cortical synaptosomes, this decrease was significantly greater than that seen with the same dose of toxin 7 or ethyl-choline aziridinium (AF64A). Ultrastructural changes in the synaptosomal membrane following incubation with toxin 7 or toxin 7 with GM1 were examined by electron microscopy. Development of a compound which is both a potent neurotoxin, and is specific for cholinergic neurons will allow new insights into the normal function of the cholinergic system in the CNS and provide animal models of disease states in which cholinergic degeneration is an important element.     

Accelerated release of exosome-associated GM1 ganglioside (GM1) by endocytic pathway abnormality: another putative pathway for GM1-induced amyloid fibril formation

Exosomes are extracellularly released small vesicles that are derived from multivesicular bodies formed via the endocytic pathway. We treated pheochromocytoma PC12 cells with chloroquine, an acidotropic agent, which potently perturbs membrane trafficking from endosomes to lysosomes. Chloroquine treatment increased the level of GM1 ganglioside in cell media only when the cells were exposed to KCl for depolarization, which is known to enhance exosome release from neurons. In the sucrose-density-gradient fractionation of cell media, GM1 ganglioside was exclusively recovered with Alix, a specific marker of exosomes, in the fractions with the density corrresponding to that of exosomes. Notably, amyloid-β assembly was markedly accelerated when incubated with the exosome fraction prepared from the culture media of PC12 cells treated with chloroquine and KCl. Furthermore, amyloid-β assembly was significantly suppressed by the co-incubation with an antibody specific to GM1-bound amyloid-β, an endogenous seed for amyloid formation of Alzheimer's disease. Together with our previous finding that chloroquine treatment induces the accumulation of GM1 ganglioside in early endosomes, results of this study suggest that endocytic pathway abnormality accelerates the release of exosome-associated GM1 ganglioside following its accumulation in early endosomes. Furthermore, this study also suggests that extracellular amyloid fibril formation is induced by not only GM1 gangliosides accumulated on the surface of the cells but also those released in association with exosomes.     

Interactions of amyloid beta-peptide (1-40) with ganglioside-containing membranes

Interactions between amyloid beta-peptides (Abeta) and neuronal membranes have been postulated to play an important role in the neuropathology of Alzheimer's disease. To gain insight into the molecular details of this association, we investigated the interactions of Abeta (1-40) with ganglioside-containing membranes by circular dichroism (CD) and Fourier transform infrared-polarized attenuated total reflection (FTIR-PATR) spectroscopy. The CD study revealed that at physiological ionic strength Abeta (1-40) specifically binds to ganglioside-containing membranes inducing a two-state, unordered --> beta-sheet transition above a threshold intramembrane ganglioside concentration, which depends on the host lipid bilayers used. Furthermore, differences in the number and position of sialic acid residues of the carbohydrate backbone significantly affected the conformational transition of the peptide. FTIR-PATR spectroscopy experiments demonstrated that Abeta (1-40) forms an antiparallel beta-sheet, the plane of which lies parallel to the membrane surface, inducing dehydration of lipid interfacial groups and perturbation of acyl chain orientation. These results suggest that Abeta (1-40) imposes negative curvature strain on ganglioside-containing lipid bilayers, disturbing the structure and function of the membranes.     

Studies on osmotic stability of liposomes prepared with bacterial membrane lipids by carboxyfluorescein release.

The authors measured the osmotic stability of liposomes prepared with membrane lipids of bacteria, using the osmotic-shock release of entrapped carboxyfluorescein as an indicator. The sub-second physical changes of liposomes suspended in a solution of low osmotic pressure were examined by stopped flow spectrophotometry. The entrapped carboxyfluorescein was released when the liposomes burst on inflow of excess water. Liposomes prepared with the lipids of a stable Staphylococcus aureus L-form strain were more resistant to low osmotic pressure than those prepared from the wild strain of S. aureus, and liposomes prepared from Mycoplasma orale were even more resistant. Cardiolipin enhanced the lipid membrane stability in S. aureus and cholesterol in M. orale. The stability of lipid membranes to low osmotic pressure could be precisely determined by the present method.     

Strikingly different effects of cholesterol and 7-ketocholesterol on lipid bilayer-mediated aggregation of amyloid beta (1-42)

Oxidized cholesterol has been widely reported to contribute to the pathogenesis of Alzheimer's disease (AD). However, the mechanism by which they affect the disease is not fully understood. Herein, we aimed to investigate the effect of 7-ketocholesterol (7keto) on membrane-mediated aggregation of amyloid beta (Aβ-42), one of the critical pathogenic events in AD. We have shown that when cholesterol is present in lipid vesicles, kinetics of Aβ nuclei formation is moderately hindered while that of fibril growth was considerably accelerated. The partial substitution of cholesterol with 7keto slightly enhanced the formation of Aβ-42 nuclei and remarkably decreased fibril elongation, thus maintaining the peptide in protofibrillar aggregates, which are reportedly the most toxic species. These findings add in understanding of how cholesterol and its oxidation can affect Aβ-induced cytotoxicity.     

神经节苷脂GM1对体外缺糖缺氧/再灌注大鼠海马脑片保护作用的研究

目的 :探讨神经节苷脂GM 1对体外缺糖 /缺氧再灌注 (OGD/Rep)大鼠海马脑片的保护作用。 方法 :采用测定脑片OGD/Rep后光通透度改变和 2 ,3 ,5 三苯基氯化四氮唑 (TTC)染色。结果 :①在 0 (对照 )、0 .1、1.0、10 μmol/LGM1四个处理组中 ,1μmol/LGM1组脑片光通透度峰值明显低于对照组和 0 .1μmol/LGM1组 (P <0 .0 1,ANO VA) ,10 μmol/LGM 1组脑片的峰值明显低于其他组 (P <0 .0 1)。脑片OGD后光通透度到达峰值的时间在四组间有显著性差异 (P <0 .0 5 ,Kruskal Wallistest) ,1μmol/LGM1组较对照组有显著差异 (P <0 .0 1,Mann WhitneyUtest)。②GM1与OGD/RP后大鼠海马脑片TTC染色呈现一定的剂量反应关系。在 0 (对照 )、0 .0 1、0 .1、1.0、10μmol/LGM1五组中 ,1μmol/LGM 1组脑片TTC染色最深 (P <0 .0 5vs对照、0 .0 1和 0 .1μmol/L组 ,ANOVA) ,10 μmol/LGM 1组次之 (P <0 .0 5vs对照和 0 .0 1μmol/L组 ,ANOVA)。 结论 :GM 1可以有效的保护体外大鼠海马脑片缺糖 /缺氧再灌注损伤。     

Physical properties of liposomes and proteoliposomes prepared from Escherichia coli polar lipids

Reconstituted proteoliposomes serve as experimental systems for the study of membrane enzymes. Osmotic shifts and other changes in the solution environment may influence the structures and membrane properties of phospholipid vesicles (including liposomes, proteoliposomes and biological membrane vesicles) and hence the activities of membrane-associated proteins. Polar lipid extracts from Escherichia coli are commonly used in membrane protein reconstitution. The solution environment influenced the phase transition temperature and the diameter of liposomes and proteoliposomes prepared from E. coli polar lipid by extrusion. Liposomes prepared from E. coli polar lipids differed from dioleoylphosphatidylglycerol liposomes in Young's elastic modulus, yield point for solute leakage and structural response to osmotic shifts, the latter indicated by static light scattering spectroscopy. At high concentrations, NaCl caused aggregation of E. coli lipid liposomes that precluded detailed interpretation of light scattering data. Proteoliposomes and liposomes prepared from E. coli polar lipids were similar in size, yield point for solute leakage and structural response to osmotic shifts imposed with sucrose as osmolyte. These results will facilitate studies of bacterial enzymes implicated in osmosensing and of other enzymes that are reconstituted in E. coli lipid vesicles.     

Cytosolic and nuclear aggregation of the amyloid beta-peptide following its expression in the endoplasmic reticulum

Misfolded secretory and membrane proteins are known to be exported from the endoplasmic reticulum (ER) to the cytosol where they are degraded by proteasomes. When the amount of exported misfolded proteins exceeds the capacity of this degradation mechanism the proteins accumulate in the form of pericentriolar aggregates called aggresomes. Here, we show that the amyloid beta-peptide (Abeta) forms cytosolic aggregates after its export from the ER. These aggregates share several constituents with aggresomes. However, Abeta aggregates are distinct from aggresomes in that they do not accumulate around the centrosome but are distributed randomly around the nucleus. In addition to these cytosolic aggregates, Abeta forms intranuclear aggregates which have as yet not been found for proteins exported from the ER. These findings show that proteins exported from the ER to the cytosol which escape degradation by the proteasome are not necessarily incorporated into aggresomes. We conclude that several distinct aggregation pathways may exist for proteins exported from the ER to the cytosol.     

Effect of epidermal acylglucosylceramides and acylceramides on the morphology of liposomes prepared from stratum corneum lipids

Epidermal acylglucosylceramides (AGC) and acylceramides (AC) cause aggregation and stacking of stratum corneum lipid liposomes formed from a lipid mixture containing epidermal ceramides (40%), cholesterol (25%), palmitic acid (25%), and cholesteryl sulfate (10%). This demonstrates the ability of these sphingolipids to hold adjacent bilayers in close apposition and their roles in the assembly of lamellar structures in the epidermis. However, AGC and AC in their hydrogenated form also caused aggregation and stacking of the stratum corneum lipid liposomes. This throws into doubt the proposed structural specificity of linoleate in the function of AGC and AC as molecular rivets in the assembly of the epidermal lamellar granules and the stratum corneum intercellular lamellae, respectively.     

Surface distribution of monosialoganglioside GM1 on human blood cells and the effect of exogenous GM1 and neuraminidase on cholera toxin surface labeling. A quantitative immunocytochemical study

The cholera toxin-colloidal gold-labeled IgG-F(ab')2 anticholera toxin ultrastructural immunocytochemical procedure was used for the localization of GM1 monosialoganglioside on the surface of human blood cells. The number of gold particles per micron of cell surface was counted and the data subjected to statistical analysis. Cholera toxin (CT) binding characteristics assessed in several subjects showed consistent labeling patterns for the various hemic cells, although some quantitative differences were noted in surface labeling densities between subjects. Neutrophils were invariably the most heavily labeled of the hemic cells, while lymphocytes, erythrocytes, and platelets exhibited only limited CT labeling. Exposure of hemic cells to neuraminidase induced a major increase in surface CT labeling that proved to be directly related to cell type and differed in many respects with the CT labeling pattern noted in nonenzyme treated cells. Newly exposed CT binding sites attributed to "masked" GM1 and/or to neuraminidase-transformed GD1a or GT1 gangliosides, showed that the number of new binding sites were nearly twice as abundant on platelet and monocyte sufaces as on the surfaces of neutrophil, lymphocyte, and erythrocyte populations. However, ratios of new CT binding sites to those normally available for CT binding were approximately 10:1 for erythrocytes, approximately 3--7:1 for lymphocytes, monocytes, and platelets, and approximately 1:1 for the neutrophil group. Exogenous GM1 was incorporated into the cell surface of the hemic cells in a differential manner. Platelets showed a dramatic increase in surface CT labeling, viz. approximately 12- to 20-fold, compared to that of other hemic cells; however, neutrophil and erythrocyte GM1 uptake was limited. Our studies have demonstrated that distinct differences exist in the extent of surface CT labeling of the various types of blood cells. They further indicated that the ability of the cell surface to incorporate exogenous GM1 may represent a differential expression of the physiochemical properties of the surface of the individual cell types.     

Metabolic fate and effect on cholesterol removal of liposomes prepared from 1,3-di-O-octadecenylglycero-2-phosphocholine studied in vivo and in vitro

Available methodology was adapted to synthesize a labeled diether analog of 2-phosphatidylcholine (1,3-di-O-9'-cis-[9',10' (n)-3H]octadecenylglycero-2-phosphocholine [( 3H]DOE-2-PC). Unilamellar liposomes prepared by sonication from this phospholipid were injected into rats and, 4 h later, 65-78% of injected label was recovered in the liver. Thereafter, liver radioactivity disappeared with a half-life of 2-3 days. The radioactivity lost from the liver was recovered in the feces and in bile. Analysis of liver radioactivity showed that at all time intervals examined (4 h to 3 days after injection), 90% of the label remained as phospholipid. These findings provide evidence that this structural isomer is not readily metabolized, but is fairly rapidly eliminated from the liver. Of the 10% recovered as neutral lipid, 70% comigrated with diacylglycerol and 30% with triacylglycerol. Similar results were obtained when human hepatoma G2 cells in culture were incubated with [3H]DOE-2-PC liposomes. Following incubation of liposomes with liver homogenates, up to 10% conversion of [3H]DOE-2PC to neutral lipid occurred at pH 4.6, but not at pH 7.4. These data show that conversion of [3H]DOE-2-PC to dialkenylglycerol is catalyzed by a lysosomal enzyme. In separate experiments with cultured cells, sonicated dispersions of DOE-2-PC were mixed with high-density apolipoprotein and were shown to enhance markedly cellular cholesterol efflux. This novel diether phospholipid fulfills some of the criteria required of liposomes for their ability to remove cholesterol from the periphery as well as for drug delivery to the liver, i.e., stability in the circulation, marked hepatic uptake, slow metabolism, and elimination from the body.     

The critical role of methionine 35 in Alzheimer's amyloid beta-peptide (1-42)-induced oxidative stress and neurotoxicity

Amyloid beta-peptide (1-42) [Abeta(1-42)] has been proposed to play a central role in the pathogenesis of Alzheimer's disease, a neurodegenerative disorder associated with cognitive decline and aging. AD brain is under extensive oxidative stress, and Abeta(1-42) has been shown to induce protein oxidation, lipid peroxidation, and reactive oxygen species formation in neurons and synaptosomes, all of which are inhibited by the antioxidant vitamin E. Additional studies have shown that Abeta(1-42) induces oxidative stress when expressed in vivo in Caenorhabditis elegans, but when methionine 35 is replaced by cysteine, the oxidative stress is attenuated. This finding coupled with in vitro studies using mutant peptides have demonstrated a critical role for methionine 35 in the oxidative stress and neurotoxic properties of Abeta(1-42). In this review, we discuss the role of methionine 35 in the oxidative stress and neurotoxicity induced by Abeta(1-42) and the implications of these findings in the pathogenesis of AD.     

Protective effect of the xanthate, D609, on Alzheimer's amyloid beta-peptide (1-42)-induced oxidative stress in primary neuronal cells

Tricyclodecan-9-yl-xanthogenate (D609) is an inhibitor of phosphatidylcholine-specific phospholipase C, and this agent also has been reported to protect rodents against oxidative damage induced by ionizing radiation. Previously, we showed that D609 mimics glutathione (GSH) functions and that a disulfide is formed upon oxidation of D609 and the resulting dixanthate is a substrate for GSH reductase, regenerating D609. Considerable attention has been focused on increasing the intracellular GSH levels in many diseases, including Alzheimer's disease (AD). Amyloid β-peptide [Aβ(1-42)], elevated in AD brain, is associated with oxidative stress and toxicity. The present study aimed to investigate the protective effects of D609 on Aβ(1-42)-induced oxidative cell toxicity in cultured neurons. Decreased cell survival in neuronal cultures treated with Aβ(1-42) correlated with increased free radical production measured by dichlorofluorescein fluorescence and an increase in protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (4-hydroxy-2-nonenal) formation. Pretreatment of primary hippocampal cultures with D609 significantly attenuated Aβ(1-42)-induced cytotoxicity, intracellular ROS accumulation, protein oxidation, lipid peroxidation and apoptosis. Methylated D609, with the thiol functionality no longer able to form the disulfide upon oxidation, did not protect neuronal cells against Aβ(1-42)-induced oxidative stress. Our results suggest that D609 exerts protective effects against Aβ(1-42) toxicity by modulating oxidative stress. These results may be of importance for the treatment of AD and other oxidative stress-related diseases.     

Amyloid beta-peptide1-40 increases neuronal membrane fluidity: role of cholesterol and brain region

There is increasing evidence of an interaction between cholesterol dynamics and Alzheimer's disease (AD), and amyloid beta-peptide may play an important role in this interaction. Abeta destabilizes brain membranes and this action of Abeta may be dependent on the amount of membrane cholesterol. We tested this hypothesis by examining effects of Abeta1-40 on the annular fluidity (i.e., lipid environment adjacent to proteins) and bulk fluidity of rat synaptic plasma membranes (SPM) of the cerebral cortex, cerebellum, and hippocampus using the fluorescent probe pyrene and energy transfer. Amounts of cholesterol and phospholipid of SPM from each brain region were determined. SPM of the cerebellum were significantly more fluid as compared with SPM of the cerebral cortex and hippocampus. Abeta significantly increased (P < or = 0.01) annular and bulk fluidity in SPM of cerebral cortex and hippocampus. In contrast, Abeta had no effect on annular fluidity and bulk fluidity of SPM of cerebellum. The amounts of cholesterol in SPM of cerebral cortex and hippocampus were significantly higher (P < or = 0.05) than amount of cholesterol in SPM of cerebellum. There was significantly less (P < or = 0.05) total phospholipid in cerebellar SPM as compared with SPM of cerebral cortex. Neuronal membranes enriched in cholesterol may promote accumulation of Abeta by hydrophobic interaction, and such an interpretation is consistent with recent studies showing that soluble Abeta can act as a seed for fibrillogenesis in the presence of cholesterol.     

Perturbation effect of eight calcium channel blockers on liposomal membranes prepared from rat brain total lipids.

EPR spectroscopy of phosphatidylcholine or stearic acid labeled at the doxyl group at the 16-carbon position was used to compare the perturbation effect of eight calcium channel blockers (CB) on overall dynamics/disorder of the hydrophobic part of liposome membranes prepared from rat brain total lipids at the drug/lipid molar ratio of 1/2. Nifedipine (NIF), nimodipine, niludipine and nitrendipine had a minor effect on the dynamics/disorder of the liposome membranes, whereas the disordering effect of verapamil (VER), mepamil, gallopamil and diltiazem was more pronounced. Concentration dependence of the overall disordering effect of VER on liposomal membranes was found at the VER/lipid ratio greater than 0.02 and for the tranquilizer thioridazine greater than 0.005. VER exerted a disordering effect at the hydrophobic part of synaptosomal membranes at concentrations greater than or equal to 0.32 mmol/l, whereas NIF did not exhibit a disordering effect even at concentrations of 10-20 mmol/l.     

The effect of macromolecular crowding on protein aggregation and amyloid fibril formation

Macromolecular crowding is expected to have several significant effects on protein aggregation; the major effects will be those due to excluded volume and increased viscosity. In this report we summarize data demonstrating that macromolecular crowding may lead to a dramatic acceleration in the rate of protein aggregation and formation of amyloid fibrils, using the protein alpha-synuclein. The aggregation of alpha-synuclein has been implicated as a critical factor in development of Parkinson's disease. Various types of polymers, from neutral polyethylene glycols and polysaccharides (Ficolls, dextrans) to inert proteins, are shown to accelerate alpha-synuclein fibrillation. The stimulation of fibrillation increases with increasing length of polymer, as well as increasing polymer concentration. At lower polymer concentrations (typically up to approximately 100 mg/ml) the major effect is ascribed to excluded volume, whereas at higher polymer concentrations evidence of opposing viscosity effects become apparent. Pesticides and metals, which are linked to increased risk of Parkinson's disease by epidemiological studies, are shown to accelerate alpha-synuclein fibrillation under conditions of molecular crowding.     

pH-dependent aggregate forms and conformation of Alzheimer amyloid beta-peptide (12-24)

The conformational transition to a beta-structure and the aggregation process of Alzheimer amyloid beta-peptide (12-24) [abbreviated as A beta(12-24)] were studied. The influence of sample dissolution methods for the aggregate structure was examined by electron microscopy (EM). The difference in the width of the aggregate of A beta(12-24) depended on the pH immediately after sample dissolution. Two types of sample dissolution methods, F and R, were employed. For dissolution method F, the peptide sample was immediately dissolved in water and then adjusted to pH 2.2 by adding buffer, while for dissolution method R, the peptide was directly dissolved in the buffer solution. In the latter case, the starting pH was 3.0. Slight fibrils (10-12 nm in diameter) were observed with method F, and wider ribbon-like aggregates (17-20 nm in diameter) with method R, despite the same pH range. A difference between methods F and R was also detected in the CD spectra, especially at pHs near 5.0. The CD intensity of the 214 nm band with method R changed with pH, with the highest value at pH 3.7, whereas that with method F was unchanged at pHs below 5.0. The temperature-dependent CD results showed that a thermostable aggregate of A beta(12-24) occurs at higher pHs than 3.0. NMR analysis showed that deprotonation of the C-terminal carboxylate group in A beta(12-24) triggered the aggregate formation, and the transition from a random coil to a beta-conformation in the C-terminal region of V18-V24 was detected on analysis of the (3)Ja(N) coupling constant in the pH range of 2.2 to 3.0.