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.     

Mycobacteria in the light of modern genetics development

It is generally assumed that genetic research of mycobacteria is delayed as compared with other, more commonly used, bacterial models, particularly in the field of genetic transfers. In the field of mutagenesis the problems have been studied to such an extent that replication maps of the chromosome of M. phlei and M. tuberculosis H37 Rv have already been constructed and a new model of the cell cycle of bacteria exhibiting a slow growth rate has been worked out. When the problems of mycobacterial genetics are looked upon in the light of gene manipulations it may be concluded that mycobacteria belong to a few models whose genes are used for cloning and that problems of practical significance will be studied by means of the most modern approaches.     

Effect of detergents on aspartate ammonia-lyase activity inEscherichia alcalescens

The effect of twelve detergents on aspartate ammonia-lyase activity of Escherichia alcalescens used for the production of L-aspartic acid was tested. Best permeabilization was found with Triton X-100, Slovafol 910 and Corona, a mixed commercial preparation. In contrast to Triton X-100 and Slovafol 910, a much narrower range of suitable concentrations was observed with Corona.     

Pigmentation changes in Brevibecteria induced by mutagenic treatment

Inducible pigmentation changes were observed in pigmented strains of Brevibacterium sp. M27 and B. flavum treated with N-methyl-N'-nitro-N-nitrosoguanidine. The highest frequency of induction was reached already at a survival of 30-40% with the maximal yield of 6-10%. As compared with the initial yellow colour, three new pigmentation types, viz. white, pink and orange, were observed. The yellow pigmented parent strains are most resistant to the lethal effects of UV radiation. By selecting pigmented mutants of all types on media containing antibiotics it was possible to obtain strains that were resistant either to tetracycline or to streptomycin. Auxotrophic pigmented mutants were also isolated. In multiple mutant strains of Brevibacterium sp. M27 a number of strainsexhibited a changed L-lysine production. In some strains the production was variable, whereasother strains did not produce L-lysine at all and stains with a limited production of other amino acids were also detected.