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P Zavodszky

Publications and source records attributed to P Zavodszky.

8 recordsLinked to original sources
PubMed2005

Cloning, sequencing, structural and molecular biological characterization of placental protein 20 (PP20)/human thiamin pyrophosphokinase (hTPK).

Full-length cDNAs of placental protein 20 (PP20) were cloned by screening a human placental cDNA library, which encode a 243 amino acid protein, identical to human thiamin pyrophosphokinase (hTPK) as confirmed by protein sequence analysis. Genomic alignment showed that the PP20/hTPK gene contains 9 exons. It is abundantly expressed in placenta, as numerous EST clones were identified. As thiamine metabolism deficiencies have been seen in placental infarcts previously, these indicate that PP20/hTPK may have a role in placental diseases. Analysis of the 1kb promoter region showed numerous putative transcription factor binding sites, which might be responsible for the ubiquitous PP20/hTPK expression. This may also be in accordance with the presence of the protein in tissues responsible for the regulation of the exquisite balance between cell division, differentiation and survival. TPK activity of the purified and recombinant protein was proved by mass spectrometry with electrospray ionization. By Western blot, PP20/hTPK was found in all human normal and tumorous adult and fetal tissues in nearly equal amounts, but not in sera. By immunohistochemical and immunofluorescent confocal imaging methods, diffuse labelling in the cytoplasm of the syncytiotrophoblasts and weak staining of the trophoblasts were observed, and the amount of PP20/hTPK decreased from the first trimester to the end of gestation. A 3D model of PP20/hTPK was computed (PDB No.: 1OLY) by homology modelling. A high degree of structural homology showed that the thiamin binding site was highly similar to that of the mouse enzyme, but highly different from the bacterial ones. Comparison of the catalytic centre sequences revealed differences, raising the possibility of designing new drugs which specifically inhibit bacterial and fungal enzymes without affecting PP20/hTPK and offering the possibility for safe antimicrobial therapy during pregnancy.

Adult↗
PubMed1997

The human complement C1 complex has a picomolar dissociation constant at room temperature.

Periodic sampling of serum or reconstituted C1 initially diluted 1/2000 and 1/4000 (that is, to 0.1 and 0.05 nM) into a recombinant C1s-containing solution showed a gradual decline of hemolytic activity until equilibrium was approached, consistent with a simple dissociation, reassociation equilibrium, presumably C1 <--> C1q + C1r2C1s2. The presence of excess (5 nM) recombinant C1s minimized further dissociation of the C1r2C1s2, allowing the first step to be studied independently of the dissociation of C1r2C1s2 <--> C1r2 + 2 C1s. Reassociation experiments were also performed, starting with the dissociated C1 diluted to the same concentrations and following the regain of hemolytic activity to approximately the same values, showing that the same equilibrium had been achieved from both directions. Analysis of the kinetic data yielded forward and reverse rate constants and the equilibrium constant, for which values of approximately 72 and 3 pM were estimated at 0 and 23 degrees C, respectively. The effects of temperature, ionic strength, Ca2+ ion concentration, and activation of the zymogen on the equilibrium constants were explored; extreme sensitivity to temperature, ionic strength, and activation were found. At 23 and 30 degrees C, slow activation of C1 was also evident. Highly purified, reconstituted C1 yielded approximately the same values for the kinetic and equilibrium parameters as serum C1, suggesting that the structure of the reconstituted complex was similar to or identical with that of the serum C1 complex.

Calcium↗
PubMed1992

Invertebrate aspartyl/asparaginyl beta-hydroxylase: potential modification of endogenous epidermal growth factor-like modules.

An invertebrate alpha-ketoglutarate-dependent aspartyl/asparaginyl beta-hydroxylase, which posttranslationally hydroxylates specific aspartyl or asparaginyl residues within epidermal growth factor-like modules, was identified, partially purified and characterized. Preparations derived from two insect cell lines catalyzed the hydroxylation of the expected asparaginyl residue within a synthetic epidermal growth factor-like module. This activity was found to be similar to that of the purified mammalian aspartyl/asparaginyl beta-hydroxylase with respect to cofactor requirements, stereochemistry and substrate sequence specificity. Furthermore, recombinant human C1r, expressed in an insect cell-derived baculovirus expression system, was also found to be hydroxylated at the expected asparaginyl residue. Thus, these results establish the potential for invertebrate aspartyl/asparaginyl hydroxylation. Since several invertebrate proteins known to be required for proper embryonic development contain a putative consensus sequence that may be required for hydroxylation, the studies presented here provide the basis for further investigations concerned with identifying hydroxylated invertebrate proteins and determining their physiologic function.

Amino Acid Sequence↗
PubMed1992

Recombinant human complement subcomponent C1s lacking beta-hydroxyasparagine, sialic acid, and one of its two carbohydrate chains still reassembles with C1q and C1r to form a functional C1 complex.

In contrast to the human serum protein which is approximately one-half erythro-beta-hydroxyasparagine at asparagine 134 [Theilens et al. (1990) Biochemistry 29, 3570-3578], recombinant C1s expressed by insect cells after infection with recombinant baculovirus entirely lacks posttranslational modification at asparagine 134. It is also incompletely glycosylated, lacking, at least, sialic acid. Site-directed mutagenesis of one of the two sites of carbohydrate attachment (Asn 159 to Gln 159) yields a faster migrating recombinant C1s still abundantly secreted. Furthermore, the mutated protein displays good hemolytic activity when reassembled with C1q and either human serum or recombinant C1r, demonstrating that these posttranslational modifications are not critical for any of the multiple interactions between C1s and C1q, C1r, C2, and C4 required for reassembly of the C1 complex, activation, and initiation of the classical complement pathway. The 4.0S recombinant C1s dimerizes to yield 5.6S C1s2 in the presence of Ca2+ and forms the 9.1S C1s-C1r-C1r-C1s tetramer upon the addition of human serum C1r and the 15.6S C1 complex upon the addition of C1q to the tetramer. The recombinant C1s and human serum C1s have identical N-terminal amino acid sequences, indicating proper recognition by the insect signal peptidase. The recombinant C1s is secreted and isolated as the unactivated zymogen, and it may be activated by human serum C1r which cleaves at Arg422-Ile423 to yield the characteristic heavy and light chains. A very tight complex is formed between C1-inhibitor and the light chain of recombinant C1s.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Spontaneous activation of serum C1 in vitro. Role of C1 inhibitor.

The temperature and ionic strength dependence of the spontaneous activation of C1 were determined for normal human serum, and the free energy, enthalpy, and entropy of spontaneous activation were calculated. The half-life of C1 in human serum was approximately 15 h at 37 degrees C. This half-life was markedly extended by dilution with C1-depleted serum, and an extrapolated upper limit of 40 to 50 h was reached at infinite dilution. Thus, the spontaneous activation of C1 in serum appeared to involve a dilution-sensitive reaction as well as a dilution-insensitive, first order reaction. A reaction mechanism was developed combining: 1) first order spontaneous activation of C1; 2) second order, C1-catalyzed activation of C1; and 3) second order inactivation of C1 by C1-inhibitor. A steady state equation was derived from this reaction mechanism, which provided a reasonable fit to the experimental data. The equation predicts that when the C1-inhibitor concentration decreases so that the steady state condition is lost, the concentration of C1 builds up quickly, and activation of most of the C1 occurs rapidly.

Complement Activation↗
PubMed1989

Spontaneous activation of reconstituted and serum C1 and the role of C1-inhibitor.

Evidence will be presented that first order, spontaneous activation of solution C1 at 37 degrees C under physiological conditions is a very slow process with a half-life of the order of one day and perhaps considerably longer. In addition, negative evidence will be presented showing that the formation of functionally significant levels of a complex between C1-Inhibitor and unactivated C1 does not occur. Such a complex had been previously postulated to explain the strong inhibition of the spontaneous activation of C1 which was observed upon the addition of C1-Inhibitor. Rather, we shall demonstrate that C1 catalytically activates C1, and that a critical role for C1-inhibitor is to complex with C1 to eliminate this autocatalytic reaction.

Catalysis↗