Biomedical subjects

T Papamarcaki

Publications and source records attributed to T Papamarcaki.

10 recordsLinked to original sources

PubMed1998

Prothymosin alpha modulates the interaction of histone H1 with chromatin.

Prothymosin alpha (ProTalpha) is an abundant acidic nuclear protein that may be involved in cell proliferation. In our search for its cellular partners, we have recently found that ProTalpha binds to linker histone H1. We now provide further evidence for the physiological relevance of this interaction by immunoisolation of a histone H1-ProTalpha complex from NIH 3T3 cell extracts. A detailed analysis of the interaction between the two proteins suggests contacts between the acidic region of ProTalpha and histone H1. In the context of a physiological chromatin reconstitution reaction, the presence of ProTalpha does not affect incorporation of an amount of histone H1 sufficient to increase the nucleosome repeat length by 20 bp, but prevents association of all further H1. Consistent with this finding, a fraction of histone H1 is released when H1-containing chromatin is challenged with ProTalpha. These results imply at least two different interaction modes of H1 with chromatin, which can be distinguished by their sensitivity to ProTalpha. The properties of ProTalpha suggest a role in fine tuning the stoichiometry and/or mode of interaction of H1 with chromatin.

3T3 Cells↗

PubMed1996

Selective interaction between parathymosin and histone H1.

We have studied the molecular associations of parathymosin, an acidic polypeptide with a wide tissue distribution, by means of three approaches; ligand blotting; native electrophoresis; and immunoprecipitation. We report here that parathymosin binds specifically to the linker histone H1. This binding is enhanced by Zn2+ and is dependent on the concentration of parathymosin. Poly(glutamic acid) is able to compete fully with parathymosin for binding to histone H1, suggesting that this interaction is mediated by the acidic domain of the protein. Moreover, we demonstrate that parathymosin interacts with the globular domain of histone H1 under native conditions. Based on these data, we postulate that parathymosin may belong to a group of nuclear acidic proteins that affect histone H1 function.

Amino Acid Sequence↗

PubMed1996

Human placental ATP diphosphohydrolase is a highly N-glycosylated plasma membrane enzyme.

Human placental ATP diphosphohydrolase (ATP-DPH), has been previously characterized as an azide-sensitive, Ca(2+)- or Mg(2+)-dependent triphospho- and diphosphonucleosidase which migrates as an 82 kDa protein band on SDS-PAGE (Christoforidis, S. et al. (1995) Eur. J. Biochem. 234, 66-74). In this paper we have studied the subcellular localization of placental ATP-DPH by differential centrifugation and flotation experiments. Using specific enzymatic markers it was found that ATP-DPH is localized on plasma membrane. ATP-DPH was found to be a highly N-glycosylated protein which is a common post-translational modification of plasma membrane proteins. Extensive incubation of the native pure enzyme with N-glycosidase F resulted in the elimination of the 82 kDa form and the concurrent formation of a deglycosylated product of 57.5 kDa and four other intermediate products, indicating the presence of at least five N-glycosylation sites within the ATP-DPH molecule. The partially deglycosylated sample retained its activity in solution and in native gel electrophoresis and activity staining.

Amidohydrolases↗

PubMed1995

Purification and properties of human placental ATP diphosphohydrolase.

ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the particulate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mol. Cell. Biochem. 97, 1-8]. In the present study we have purified to homogeneity and characterized this activity. A 260-fold purification has been obtained by solubilization of the particulate fraction and subsequent chromatography on DEAE Sepharose CL-6B and 5'-AMP Sepharose 4B. The preparation has been shown to be free of alkaline phosphatase even though the placental extract is rich in this activity. The purified enzyme is a glycoprotein and migrates as a single broad band of 82 kDa on SDS/PAGE. The same band is obtained after photoaffinity labeling of the enzyme with 8-azido-[alpha-32P]ATP. The enzyme has a broad substrate specificity, hydrolyzing triphosphonucleosides and diphosphonucleosides but not monophosphonucleosides or other phosphate esters. The activity is dependent on the addition of divalent cations Ca2+ or Mg2+. The Km values for ATP and ADP were determined to be 10 microM and 20 microM, respectively. Maximum activity was found at pH 7.0-7.5 with ATP as substrate, and pH 7.5-8.0 with ADP. The enzymic activity is inhibited by NaN3, NaF, adenosine 5'-[beta,gamma-imido]triphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate. Protein sequence analysis showed ATP-DPH to be N-terminally blocked. Partial internal amino acid sequence information was obtained after chymotryptic cleavage and identified a unique sequence with no significant similarity to known proteins. ATP-DPH activity has been reported to be implicated in the prevention of platelet aggregation, hydrolysing ADP to AMP and thus preventing blood clotting.

Affinity Labels↗

PubMed1994

Prothymosin alpha binds to histone H1 in vitro.

Previous studies have shown that prothymosin alpha (ProT alpha) is a nuclear acidic protein implicated in cell proliferation. To identify proteins that interact with ProT alpha we have used ligand-blotting assays. We report here that purified ProT alpha binds specifically to histone H1 in a dose dependent manner. Polyglutamic acid, an analog of the central acidic domain of ProT alpha, strongly inhibits the above interaction, suggesting that the binding of ProT alpha to histone H1 is mediated through its acidic domain.

Amino Acid Sequence↗

PubMed1993

Transient arrest of 3T3 cells in mitosis and inhibition of nuclear lamin reassembly around chromatin induced by anti-vimentin antibodies.

We have microinjected anti-vimentin antibodies into nocodazole-arrested, prometaphasic 3T3 cells. Exit of injected and control cells from mitosis has been assessed at various time points after release from nocodazole by examining the distribution of nuclear lamins, the degree of chromatin condensation and the appearance of daughter cells. It is shown here that 1 h after release from nocodazole, the vast majority of cells injected with control antibodies enter interphase, whereas most of the cells injected with antivimentin antibodies remain prometaphasic. Microinjection of vimentin-free (MCF-7) cells with anti-vimentin antibodies does not affect the normal completion of mitosis in the majority of the cells. The arresting effect on 3T3 cells is dependent on the concentration of the microinjected anti-vimentin antibodies and can also be detected when Fabs are injected instead of intact IgG. Sampling at later time points and monitoring of individual microinjected cells indicate that the antibody-induced effect lasts for 1.5 to 4 h, i.e., up to 400% longer than the normal mitotic cycle of 3T3 cells. However, 4 h after release from nocodazole, most of the antibody-arrested cells recover and divide successfully. Electron microscopy shows that the IFs of the microinjected cells are thicker than normal and tend to anastomose. Interestingly, the nuclei of some of the cells that escape the antibody-induced arrest and successfully divide appear segmented or lobulated. Using a mitotic cell-free system, we further demonstrate that anti-vimentin antibodies interfere with nuclear lamin assembly around chromatin particles. In agreement to the in vivo results, the inhibitory effect of the antibodies appears to subside with time. Taken together, these data suggest that the remodelling of the vimentin network which normally takes place during mitosis is an important rearrangement of the cytoplasm required for efficient nuclear reassembly at the end of cell division.

3T3 Cells↗

PubMed1991

The "lamin B-fold". Anti-idiotypic antibodies reveal a structural complementarity between nuclear lamin B and cytoplasmic intermediate filament epitopes.

Previous studies have shown that nuclear lamin B binds specifically to the C-terminal domains of type III intermediate filament (IF) proteins under in vitro conditions. To further explore such site-specific interactions, we have used a two-step anti-idiotypic antibody approach. First, a monoclonal antibody disrupting the cytoplasmic IF network organization of living cells (mAb7A3) (Matteoni, R., and Kreis, T. E. (1987) J. Cell Biol. 105, 1253-1265) was characterized. Epitope mapping demonstrated that this antibody recognized a site located in the C-terminal domains of vimentin and peripherin (type III IF proteins). mAb7A3 was able to inhibit more than 80% of the in vitro binding of nuclear lamin B to PI, a synthetic peptide modeled after the C-terminal domain of peripherin that comprises a lamin B-binding site (Djabali, K., Portier, M. M., Gros, F., Blobel, G., and Georgatos, S. D. (1991) Cell 64, 109-121). In a second step, animals were immunized with mAb7A3 and the resulting anti-idiotypic sera were screened. Two of these antisera reacted specifically with nuclear lamin B but not with type A lamins or cytoplasmic IF proteins. The anti-lamin B activity of one of the antisera was isolated by affinity chromatography using a lamin B-agarose matrix. The reaction of these affinity-purified antibodies with lamin B was inhibited by mAb7A3. Furthermore, the anti-lamin B antibodies reacted with Fab fragments of mAb7A3 and abolished binding of lamin B to PI. From these data we conclude that anti-idiotypic antibodies against the paratope of mAb7A3 recognize specific epitopes of the lamin B molecule that have shapes complementary to the one of the C-terminal domain of type III IF proteins. We speculate that these (regional) conformations, which we term the "lamin B-fold," may also occur in non-lamin proteins that mediate the anchorage of IFs to various membranous organelles.

Animals↗

PubMed1991

Demonstration of fructose 1,6-bisphosphatase in human term placenta.

A proteolysed form of fructose 1,6-bisphosphatase (Fru-P2ase) has been detected and characterized in human term placenta. The extract was found to contain very low levels of activity with an alkaline pH optimum. Western blotting demonstrated a polypeptide of Mr 26,000, instead of the subunit of Mr 36,000 observed in native mammalian Fru-P2ases.

Blotting, Western↗

PubMed1991

A potential role for the COOH-terminal domain in the lateral packing of type III intermediate filaments.

To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti-idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness.

Animals↗

PubMed1990

Identification of ATP diphosphohydrolase activity in human term placenta using a novel assay for AMP.

Human term placenta contains an ATP diphosphohydrolase activity which hydrolyses ATP to ADP and inorganic phosphate and ADP to AMP and a second mole of inorganic phosphate. The activity has a pH optimum between 8.0 and 8.5. Magnesium or calcium ions are required for maximum activity. Other nucleoside phosphates, p-nitrophenyl phosphate or sodium pyrophosphate, are not hydrolysed. The activity is not due to ATPases, or to myokinase, as determined by the use of inhibitors. NaF and NaN3 were found to inhibit strongly the activity thus identifying it as an ATP diphosphohydrolase. A sensitive enzymatic assay for measurement of AMP, one of the products of the reaction, was established, based on the strong inhibition of muscle fructose 1,6-biphosphatase by AMP. The range of the assay was 0.05-0.8 microM AMP. ATP diphosphohydrolase was found to have a rate of AMP production from ADP twice the rate from ATP. Under the same conditions, the assay for Pi release, on the other hand, gave velocities similar to each other for the two substrates. The activity appears to be identical to the ADP-hydrolysing activity in placenta reported by others.

Adenosine Monophosphate↗