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  • 1
    ISSN: 1573-6881
    Keywords: Chloroplast ATPase ; reaction rate ; HPLC ; Mg2+
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F1 ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima,K m values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg2+. Although the equations giving the reaction rate are similar in the two cases, the calculated values ofK m are widely different. The value obtained from the classical mechanism does not agree withK D , the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na+ is more easily explained as a competition between this ion and the activating Mg2+, than by the classical mechanism.
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  • 2
    ISSN: 1619-7089
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract In order to study “in vivo” the distribution kinetics of (-)-nicotine in animals, this molecule was labeled with carbon-11. Two synthesis methods are described. Immediately after intravenous administration of (-)-nicotine-methyl-11C in rabbits the gamma-camera shows a strong radioactivity build-up in the brain and kidneys. The biological interest of this carbon-11 labeled molecule is discussed.
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  • 3
    ISSN: 1573-5079
    Keywords: activation of catalytic properties ; binding change mechanism ; regulatory sites ; role of magnesium ; tentoxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The binding of nucleotides on isolated subunits as well as on reconstituted CF1 core complex is reviewed. Nucleotide interaction with CF1 and consequent ATPase activity are always associated with the presence of Mg2+. The metal binding site studies using Electron Paramagnetic Resonance (EPR) and pulsed EPR conclude that the metal binding occurs prior to any nucleotide addition. The addition of nucleotide does not modify the enzyme's metal binding site but brings on additional ligands with the phosphates of the nucleotides. The ATPase and nucleotide binding experiments with CF1 are also better interpreted by the hypothesis that Mg2+ is an activator rather than an inhibitor of the enzyme and that the actual substrate of CF1-ATPase is ATP rather than MgATP. The dual role of tentoxin as an inhibitor at low concentration (10-8-10-7 M) and activator at higher concentrations (10-6 M) of the enzymatic activity of CF1, is due to the presence of two different binding sites on CF1. The synthesis of a new cyclic analogue of tentoxin with alanine changed for a serine has shown that it was possible to dissociate the two roles. The serine tentoxin analogue has the same inhibition effect on CF1 but is no longer an activator. The binding of nucleotides may influence the stability, produce structural changes and, over long distance, cause movements of CF1. All these effects of nucleotide or metal binding and activation or inhibition of CF1 may help also to elucidate the role played by the catalytic and non catalytic sites. These questions are reviewed and analyzed with respect to the current views on the catalytic mechanism.
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  • 4
    ISSN: 1573-6881
    Keywords: F1-ATPase, enzymatic activity ; nucleotide binding, cooperativity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The fundamental question of the cooperativity between the enzymatic sites of F1-ATPase is examined in the light of new measurements of the enzymatic rate of ATP hydrolysis by CF1, the enzyme isolated from spinach chloroplasts. The experimental data, obtained with a chromatographic method, fit a model that involves two kinds of independent enzymatic sites working with metal-free ATP, with no need of cooperativity between the sites. Binding measurements between ADP or ATP and CF1 by the chromatographic method of Hummel and Dreyer (1962) also support this conclusion. The present data and interpretation are in agreement with those reported recently (Reynafarje and Pedersen, 1996) which show that the first order rate constant of ATP hydrolysis by MF1, the analogous enzyme from mitochondria, is virtually constant under experimental conditions involving either unisite or multisite hydrolysis of ATP. The present data and interpretation are discussed together with those reported previously, in particular with regard to the methods that were used to support the commonly accepted opposite viewpoint.
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  • 5
    ISSN: 1619-7089
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A methylated derivative of serotonin, O-methyl-bufotenine has been labeled with 11C on the two methyl groups of the amine function. In order to avoid the cyclization which occurs during the Eschweiler-Clarke synthesis, we adopted a milder methylation procedure, based on Borch's method using [11C]formaldehyde and sodium cyanoborohydride. Several tens of millicuries of injectable product could be obtained in 50 min in a perfectly pure state and having a specific radioactivity of 50 to 100mCi/μmol. The distribution study of O-methyl-bufotenine in the mouse and rabbit showed an accumulation of significant quantities of the compound in the brain, kidneys, lungs and liver. The study of the rapid kinetics of this hallucinogenic molecule is compatible with labeling by 11C, having a period of 20 min. The use of O-methyl-[11C]bufotenine to detect serotonin receptors in vivo in mental diseases, is considered.
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  • 6
    ISSN: 1432-2307
    Keywords: Gastrin ; Gastrinomas ; Ultrastructure ; Immunogold technique
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Localisation of C-terminal gastrin immunoreactivity has been studied, using the immunogold staining procedure, on ultrathin sections of 6 human gastrinomas conventionally processed for electron microscopy. The specific labelling, whose density depended on the mean diameter of the gold marker, was restricted to endocrine secretory granules. However, in poorly differentiated cells from malignant tumours, a number of granules remained unreactive. The labelling pattern depended also on the functional state of each cell. The immunoreactive granules showed various morphological features. A moderate number of gold particles was demonstrated over the floccular content of the infrequent diagnostic G-type granules. Non-diagnostic round granules of varying size and electron density were prevalent in most cells; their usually strong immunostaining allowed immediate recognition of cell specificity. Dense granules which were large in size and angular in shape and present in one case, were also intensely labelled. In the same tumour, unequal labelling occurred over polymorphous, often elongated granules, of varying size. Granules of different types, including intermediate forms, could be found in the same cell, indicating a spectrum of granule maturation towards well-defined types of the fetal or adult normal tissues. The present methodology would help to identify gastrin-producing cells in prospective or retrospective electron microscopy studies of multihormonal endocrine tumours.
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  • 7
    ISSN: 1432-2307
    Keywords: Insulin ; Insulinomas ; Ultrastructure ; Immunogold technique
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Localisation of insulin-like immunoreactivity has been studied using the immunogold staining procedure on thin sections of 6 human insulinomas, conventionally processed for electron microscopy. The labelling was restricted to the secretory granules. Depending on their morphology, these either resembled B-cell granules of human adult pancreas or belonged to the atypical (non-diagnostic) group. Within the former group, those with a crystalloid core or an amorphous dense or moderately dense core were strongly immunoreactive, whereas others, filled with a pale material, were poorly labelled. Most granules of this type were stored together within the heavily granulated cells of 3 insulinomas, presenting the classical features of clinical and biological behaviour and a typical light microscopic staining pattern. In contrast, the non-diagnostic granules, characterized by their smaller size, a very dense core and a thin halo, were mainly found within the poorly granulated cells making up the other tumours, and showed a very uneven labelling. Strongly labelled granules were found in one insulinoma that also belonged to the classical type; these were stored together with a few diagnostic granules within the same cells. Only poorly labelled atypical granules were present in two cases revealing a number of unusual features; these included moderate elevation of insulinaemia, uncertain tumour histology, as well as weak immunostaining for insulin/proinsulin and variable argyrophilia of the tumour in paraffin sections. These findings suggest that human insulinomas differ not only in storage capacity but also in their degree of granule maturation. This may involve some deficiency of either the prohormone conversion or the subsequent processing of the cleavage products.
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  • 8
    ISSN: 1432-2307
    Keywords: Parathyroid ; Storage granule ; Parathormone ; Chromogranine A ; Immunoelectron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Parathyroid hormone (PTH) and chromogranin A/secretory protein-I (SP-I) have been localized on immunoelectron microscopy in double-fixed tissues from adenomatous and secondary hyperplastic parathyroid glands. Storage organelles, identified on the basis of their consistent labelling, included two morphologically distinct varieties of granules/vesicles; the mature granules and the progranules. The former consisted of dense, mostly rounded, medium to large-sized bodies which were strongly labelled and predominant in the proximity of the cell membrane. The other variety of body included a spectrum of small pale vesicles/granules which were mainly located in the Golgi area. Because their morphology and their labelling pattern varied other bodies were assumed to be engaged in degradation or cleavage of the secretory proteins. These bodies comprised crinophagic structures, that is to say multivesicular bodies and large Golgi-related vesicles, as well as a number of atypical solid bodies. Whereas most of the granulated cells stored a mature or a maturing population of vesicles/granules, the process of maturation appeared to be either absent or incomplete in a number of cells from some glands. The major defects were frequently associated with an unusual labelling pattern of the Golgi area and selectively affected groups of cells from all the transitional oxyphil cell adenomas. The minor defects concerned individual cells of different types present in both categories of glands. The present data suggest that in hyperfunctioning glands, the type of hormone processing depends on the capacity of each cell in progranule maturation and that the maturation capacity may decrease dramatically in adenomatous or chronically hyperstimulated cells of the transitional oxyphil type.
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  • 9
    ISSN: 1432-2307
    Keywords: Medullary thyroid carcinoma ; Calcitonin ; Amyloid fibrils
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Using 3 polyclonal antisera directed against synthetic human calcitonin, we investigated at the electron microscope level the intra-or-extracellular fibrillar/filamentous aggregates found in 4 amyloid-rich medullary thyroid carcinomas (MTC) and in a number of other endocrine polypeptide tumours with or without demonstrable amyloid deposition. The antisera were applied by the immunogold procedure on ultrathin sections of glutaraldehyde-fixed, usually osmium-postfixed, tissues. In MTC cases, a strong labelling was present over two types of aggregates: one composed of rigid, criss-crossing fibrils 7–10 nm in diameter, suggestive of amyloid, and the other consisting of loosely arranged fibrils, 4–7 nm in width, often wavy or poorly defined. In both cases, the labelling was closely associated with that part of the sectioned fibril exposed to the antiserum. Amorphous material was sometimes present adjacent to the latter aggregates, but did not bind the calcitonin antibodies. In contrast, no labelling occurred over the amyloid deposits found in two non-calcitonin-producing endocrine tumours of the pancreas, nor over the cytoskeletal filaments stored in various endocrine polypeptide tumours. The specific value of the labelling for calcitonin-like immunoreactivity was assessed by control tests, such as absorption of the antiserum by excess calcitonin and comparative use of normal serum and antisera directed against human IgG and P component. No immunoreactivity of the MTC amyloid fibrils was found using antibodies directed against katacalcin and human prealbumin. We conclude that in tumour tissues conventionally processed for electron microscopy, MTC amyloid fibrils of varying morphology can be selectively and specifically labelled for calcitonin-like immunoreactivity.
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