Use of a simple method for the Epstein-Barr virus transformation of lymphocytes from members of large families of Réunion Island

A simple method for the preparation of lymphoblastoid cell lines from small amounts (100 microliter) of frozen whole blood is described. A success score greater than 90% was obtained for EBV transformations using blood samples which had been collected several months before the infection. Due to the simplicity of the technique, up to 80 samples could be processed per day. This technique was used to prepared 242 permanent cell lines from 13 large families from Réunion Island showing blood group H deficiency. These cell lines are now available for genetic studies.     

Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k

The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF-hand calcium-binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ -binding sites. The Ca2+-loaded forms of the wild-type and mutant calbindins are too stable to be significantly denatured by heating at 90 degrees C or by adding 8 M urea. For the Ca2+-free (apo) forms thermal unfolding appears to be only half complete at 90 degrees C, while denaturation is complete in 7-8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu-17----Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state. According to X-ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr-13) is hydrogen-bonded to the carboxyl group of Glu-35, thus linking the two alpha helices flanking the N-terminal Ca2+ site. The pK of ionization of the Tyr-13 hydroxyl group was over 13 for calcium forms of the wild-type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.     

Intramitochondrial factors controlling hepatic fatty acid oxidation at weaning in the rat

Fatty acid oxidation was studied in isolated liver mitochondria of rats during the suckling-weaning transition. The oxidation rate of oleyl-CoA and palmitoylcarnitine was reduced 2.5-fold in rats weaned on a high-carbohydrate diet compared to suckling rats, when acetyl-CoA produced by beta-oxidation was directed towards ketone-body synthesis. Weaning on a high-fat diet minimized this change. Channeling of acetyl-CoA towards citrate synthesis doubled the oxidation rate of both substrates in HC-weaned rats. Thus, in addition to changes in carnitine palmitoyltransferase I activity, the beta-hydroxymethylglutaryl-CoA synthase pathway is also involved in the decreased fatty acid oxidation at weaning. This was confirmed by measurement of beta-hydroxymethylglutaryl-CoA synthase pathway activity.     

Absence of 7-acetyl taxol binding to unassembled brain tubulin

The effect of taxol on microtubule proteins at 0 degrees C is controversial. In order to determine if taxol is unable to bind to unassembled tubulin, as has been hypothesized, the binding of [3H]acetyl taxol has been studied using equilibrium microdialysis. Ac-taxol bound to microtubules at 37 degrees C and the binding remained stable when the temperature was lowered to 0 degrees C. Ac-taxol bound also at 0 degrees C to microtubules stabilized with rhazinilam. In contrast, there was no binding of Ac-taxol to unassembled tubulin, either free tubulin at 0 degrees C or tubulin, complexed with several microtubule poisons, at 0 and 37 degrees C.     

Simultaneous purification and characterization of aspartate aminotransferase isoenzymes from chicken liver

Cytosolic and mitochondrial isoenzymes of aspartate aminotransferase (EC 2.6.1.1) were purified to homogeneity from chicken liver, without previous fractionation of the subcellular components. The procedure includes initial heat treatment and ammonium sulfate fractionation. The two isoenzymes can then be separated by a DEAE-Sepharose chromatography using a linear gradient of L-aspartate (reaction substrate). The separated fractions can be further purified by a parallel step with HA-Ultrogel prior to octyl-Sepharose (c-AAT) and CM-Sepharose (m-AAT) chromatographies. Michaelis constants, pI values, inhibition by adipate and subforms generation with time were studied for both isoenzymes.     

Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein

Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.     

The error in the cryptic stereospecificity of methylmalonyl-CoA mutase. The use of carba-(dethia)-coenzyme A substrate analogues gives new insight into the enzyme mechanism

A preparation containing 80.0 +/- 0.5% (2RS)-methylmalonyl-carba-(dethia)-CoA and 20.0 +/- 0.5% propionyl-carba-(dethia)-CoA was reacted in buffered deuterium oxide with catalytic amounts of coenzyme B12, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase. The rearrangement of the methylmalonyl-carba-(dethia)-CoA to succinyl-carba-(dethia)-CoA was monitored by recording 500-MHz 1H-NMR spectra in short time intervals. After reaching equilibrium (approximately equal to 28 min) the products showed chemical stability for about 17 h, i.e. succinyl species did not undergo the spontaneous hydrolysis encountered with normal succinyl-CoA. In the pre-equilibrium stage only about 66% of the produced succinyl-CH2CoA was the expected monodeuterated species. The remainder was 15.5% unlabelled and 18.3% 3,3-dideuterated. After reaching equilibrium a continuous deuterium incorporation (washing-in) from the solvent to the products was observed and quantified. The time course of the appearance of unlabelled, mono-, di- and trideuterated succinyl-CH2CoA species was determined by assigning and integrating the isotope-shifted 1H signals from the various species. Furthermore, mutase catalyses slow deuterium incorporation into first the methylene and then the methyl group of propionyl-CH2CoA. On the basis of these data it was concluded that methylmalonyl-CoA mutase and epimerase are responsible for continuous deuterium incorporation and multiple incorporation occurs when the backward reaction (succinyl-CH2CoA----methylmalonyl-CH2CoA) becomes important. To account for all of the results obtained with dethia and natural substrates we propose a new mutase mechanism whereby the enzyme can retain full stereospecificity at C-3 of succinyl while an internal 1,2-H shift to give a C-2 succinyl radical is responsible for partial scrambling of diastereotopic protons at C-3. This mechanism successfully predicts the observed deuterium disproportionation in succinyl species and the order of appearance of di- and trideuterated products via the washing-in process.     

Acetylcholinesterase from Drosophila melanogaster. Identification of two subunits encoded by the same gene

Purified acetylcholinesterase from Drosophila melanogaster is composed of a 55 kDa and a 16 kDa noncovalently associated subunit. Cleavage of disulfide bonds reveals that two 55 kDa polypeptides are linked together in native dimeric AChE. Western blots with two antibodies directed against the N- and C-termini of the predicted AChE primary sequence show that the 55 and 16 kDa polypeptides originate from proteolysis of the same precursor encoded by the Ace locus.     

The R-banding pattern of the Chinese hamster Don cell line

Chinese hamster cells (Don line) were treated in vivo with 5-BrdU and 33258-Hoechst fluorochrome for obtaining the partial inhibition of condensation that causes the R-banding pattern. Untreated chromosomes were stained by a standard G-banding method. Statistical measurements show significant differences in the band numbers between the two treatments. The Don cell line in the authors' laboratory presents some karyotypical differences from Don cell lines studied by other authors.