Microheterogeneity of beta-2 glycoprotein I: implications for binding to anionic phospholipids.
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Considerable interest is currently focused on the interactions of beta-2 glycoprotein I (beta2GPI) and anti-phospholipid antibodies with anionic phospholipids in an attempt to understand the association between these antibodies and clinical diseases such as thrombosis. The interactions of beta2GPI and anionic phospholipids have only been characterized partially, and the physiological role of this glycoprotein remains uncertain. In this study we have explored in detail the physical and phospholipid-binding characteristics of a number of beta2GPI preparations. We have found (i) that perchloric acid-purification methods are damaging to beta2GPI during purification, (ii) that the dissociation constants of the various preparations for phosphatidylserine vary between 0. 1-2 microM and are considerably weaker than previously reported, (iii) that considerable differences in affinity of the various beta2GPI preparations for anionic phospholipids are obtained when comparing anionic phospholipids immobilized to a solid-phase versus phospholipid assembled in unilamellar vesicles, (iv) that the integrity of the fifth domain of beta2GPI is important for binding immobilized anionic phospholipid but not especially important in binding vesicular anionic phospholipid, and (v) that beta2GPI preparations with differing isoelectric species content bind anionic phospholipids differently, suggesting that varying glycosylation and/or protein polymorphisms impact upon phospholipid binding. These results highlight the importance of assessing the determinants of the interaction of beta2GPI with anionic phospholipids assembled in unilamellar vesicles. (+info)
Analysis of long-range interactions in a model denatured state of staphylococcal nuclease based on correlated changes in backbone dynamics.
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An expanded, highly dynamic denatured state of staphylococcal nuclease exhibits a native-like topology in the apparent absence of tight packing and fixed hydrogen bonds (Gillespie JR, Shortle D, 1997, J Mol Biol 268:158-169, 170-184). To address the physical basis of the long-range spatial ordering of this molecule, we probe the effects of perturbations of the sequence and solution conditions on the local chain dynamics of a denatured 101-residue fragment that is missing the first three beta strands. Structural interactions between chain segments are inferred from correlated changes in the motional behavior of residues monitored by 15N NMR relaxation measurements. Restoration of the sequence corresponding to the first three beta strands significantly increases the average order of all chain segments that form the five strand beta barrel including loops but has no effect on the carboxy terminal 30 residues. Addition of the denaturing salt sodium perchlorate enhances ordering over the entire sequence of this fragment. Analysis of seven different substitution mutants points to a complex set of interactions between the hydrophobic segment corresponding to beta strand 5 and the remainder of the chain. General patterns in the data suggest there is a hierarchy of native-like interactions that occur transiently in the denatured state and are consistent with the overall topology of the denatured state ensemble being determined by many coupled local interactions rather than a few highly specific long-range interactions. (+info)
Hofmeister effects of anions on the kinetics of partial reactions of the Na+,K+-ATPase.
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The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na+,K+-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (+/- 0.6) microM and 143 (+/- 17) microM in 130 mM NaCl solution to values of 42 (+/- 3) microM and 660 (+/- 100) microM in 130 mM NaClO4 (pH 7.4, 24 degrees C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8-10 mM in NaCl to 2.5-3.5 mM in NaClO4 solution. The rate of equilibration of the reaction, E1P(Na+)3 left arrow over right arrow E2P + 3Na+, decreased from 393 (+/- 51) s-1 in NaCl solution to 114 (+/- 15) s-1 in NaClO4. This decrease is attributed predominantly to an inhibition of the E1P(Na+)3 --> E2P(Na+)3 transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na+,K+-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E1P(Na+)3 --> E2P(Na+)3 is a major charge translocating step of the pump cycle. (+info)
Perchloric acid-soluble proteins from goat liver inhibit chemical carcinogenesis of Syrian hamster cheek-pouch carcinoma.
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Chemically induced Syrian hamster cheek-pouch squamous cell carcinoma is very similar to the corresponding human tumour. This paper describes a blind study in which inhibition of dimethylbenzanthracene-induced cheek-pouch tumours by a goat liver extract denominated UK101 was investigated. Less than 40% of animals treated with UK101 developed tumours compared with 100% of the controls. Intermediate results (80%) were noted in a positive control group treated with Calmette-Guerin bacillus. Immunocytochemical testing of cheek-pouch mucosa by Mib5 showed significantly less proliferating cells in UK101 animals than in the controls. The effect of UK101 was completely reversed when dexamethasone was added in a third control group. A significant difference in complement-mediated cytotoxicity was noted in the sera of UK101-tested and control animals. These findings suggest that an immune mechanism is responsible for the inhibition of hamster cheek-pouch carcinoma by UK101. (+info)
Thyroid gland function and growth in IGF binding protein-1 transgenic mice.
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OBJECTIVE: IGF-I, IGF-I receptor and IGF-binding proteins (IGFBPs) are expressed in thyroid tissue and are associated with the function and growth of the thyroid. This study investigated the in vivo and in vitro effects of increased IGFBP-1 levels on the function and growth of the thyroid gland. DESIGN: Transgenic mice which constitutively overexpress IGFBP-1 were used. These mice have a phenotype consistent with partial inhibition of IGF-I action. METHODS: Thyroid growth, morphology and hormonogenesis were determined in transgenic mice treated with goitrogens, sodium perchlorate and methimazole. In vitro cell proliferation in thyroid follicles was assessed in response to IGF-I and TSH. RESULTS: Thyroid weight was increased in transgenic mice, relative to their body mass, whereas serum tri-iodothyronine (T(3)), thyroxine and T(3)-binding capacity were reduced, compared with wild-type. While an inverse relationship between T(3) and TSH was observed in both groups of goitrogen-treated mice, the slope of the line of best fit was less steep in transgenic mice compared with wild-type mice. Thyroid growth was less marked in transgenic than wild-type mice in response to goitrogens, although TSH levels were higher in goitrogen-treated transgenics. In vitro proliferative response of isolated thyroid follicles to IGF-I, but not to TSH, was reduced in transgenic, compared with wild-type mice. CONCLUSIONS: The results of this study suggest that, while overexpression of IGFBP-1 attenuates IGF-I action in vitro, it enhances thyroid growth in vivo, presumably as a result of perturbations in thyroid function at multiple levels. (+info)
Differential effects of caffeine and perchlorate on excitation-contraction coupling in mammalian skeletal muscle.
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1. Enzymatically dissociated single muscle fibres of the rat were studied under voltage clamp conditions in a double Vaseline gap experimental chamber. Intramembrane charge movement and changes in intracellular calcium concentration ([Ca2+]i) were measured and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. This enabled the determination of SR permeability and thus the estimation of the transfer function between intramembrane charge movement and SR permeability. 2. Perchlorate (3 mM) shifted the membrane potential dependence of intramembrane charge movement to more negative voltages without any effect on the steepness or on the maximal available charge. The drug increased SR permeability at every membrane potential but did not alter the peak-to-steady level ratio. It also increased the slope of the transfer function, indicating a more efficient coupling between the voltage sensors and the ryanodine receptors. 3. Caffeine (1 mM), on the other hand, increased SR permeability without altering the voltage dependence of intramembrane charge movement. It neither prolonged the depolarization-induced increase in [Ca2+]i at short pulse durations nor altered the time to peak of Rrel. The augmentation of SR permeability by the drug was more pronounced during the peak caffeine response than during its steady level. This was manifested in a leftward shift of the transfer function rather than an increase in its slope. 4. These observations indicate that perchlorate and caffeine alter the coupling between the voltage sensors and SR calcium release channels in mammalian skeletal muscle. They do not, however, share a common mechanism for enhancing the depolarization-induced release of calcium from the SR. (+info)
Purification and characterization of (per)chlorate reductase from the chlorate-respiring strain GR-1.
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Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an alpha(3)beta(3) composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The K(m) values for perchlorate and chlorate were 27 and <5 microM, respectively. Besides perchlorate and chlorate, nitrate, iodate, and bromate were also reduced at considerable rates. The resemblance of the enzyme to nitrate reductases, formate dehydrogenases, and selenate reductase is discussed. (+info)
Ubiquity and diversity of dissimilatory (per)chlorate-reducing bacteria.
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Environmental contamination with compounds containing oxyanions of chlorine, such as perchlorate or chlorate [(per)chlorate] or chlorine dioxide, has been a constantly growing problem over the last 100 years. Although the fact that microbes reduce these compounds has been recognized for more than 50 years, only six organisms which can obtain energy for growth by this metabolic process have been described. As part of a study to investigate the diversity and ubiquity of microorganisms involved in the microbial reduction of (per)chlorate, we enumerated the (per)chlorate-reducing bacteria (ClRB) in very diverse environments, including pristine and hydrocarbon-contaminated soils, aquatic sediments, paper mill waste sludges, and farm animal waste lagoons. In all of the environments tested, the acetate-oxidizing ClRB represented a significant population, whose size ranged from 2.31 x 10(3) to 2.4 x 10(6) cells per g of sample. In addition, we isolated 13 ClRB from these environments. All of these organisms could grow anaerobically by coupling complete oxidation of acetate to reduction of (per)chlorate. Chloride was the sole end product of this reductive metabolism. All of the isolates could also use oxygen as a sole electron acceptor, and most, but not all, could use nitrate. The alternative electron donors included simple volatile fatty acids, such as propionate, butyrate, or valerate, as well as simple organic acids, such as lactate or pyruvate. Oxidized-minus-reduced difference spectra of washed whole-cell suspensions of the isolates had absorbance maxima close to 425, 525, and 550 nm, which are characteristic of type c cytochromes. In addition, washed cell suspensions of all of the ClRB isolates could dismutate chlorite, an intermediate in the reductive metabolism of (per)chlorate, into chloride and molecular oxygen. Chlorite dismutation was a result of the activity of a single enzyme which in pure form had a specific activity of approximately 1,928 micromol of chlorite per mg of protein per min. Analyses of the 16S ribosomal DNA sequences of the organisms indicated that they all belonged to the alpha, beta, or gamma subclass of the Proteobacteria. Several were closely related to members of previously described genera that are not recognized for the ability to reduce (per)chlorate, such as the genera Pseudomonas and Azospirllum. However, many were not closely related to any previously described organism and represented new genera within the Proteobacteria. The results of this study significantly increase the limited number of microbial isolates that are known to be capable of dissimilatory (per)chlorate reduction and demonstrate the hitherto unrecognized phylogenetic diversity and ubiquity of the microorganisms that exhibit this type of metabolism. (+info)