scholarly journals Human diadenosine triphosphate hydrolase: preliminary characterisation and comparison with the Fhit protein, a human tumour suppressor.

2000 ◽  
Vol 47 (2) ◽  
pp. 435-441 ◽  
Author(s):  
A C Asensio ◽  
C R Rodríguez-Ferrer ◽  
S Oaknin ◽  
P Rotllán
Keyword(s):  
Protein A ◽  
Gel Filtration ◽  
Human Platelets ◽  
Tumour Suppressor ◽  
Human Tumour ◽  
Fhit Protein ◽  
Diadenosine Triphosphate

Human platelets diadenosine triphosphatase was characterised and compared with the Fhit protein, a human tumour suppressor with diadenosine triphosphatase activity. Both enzymes exhibit similar Km, are similarly activated by Mg2+, Ca2+ and Mn2+, and inhibited by Zn2+ and suramin. However, they are differentially inhibited by Fhit antibodies and exhibit differences in gel-filtration behaviour.

Functional characterization of the recombinant human tumour suppressor 101F6 protein, a cytochrome b561 homologue

2012 ◽  
Vol 153 (2) ◽  
pp. 233-242 ◽  
Author(s):  
Mariam C. Recuenco ◽  
Md. Motiur Rahman ◽  
Yoichi Sakamoto ◽  
Fusako Takeuchi ◽  
Hiroshi Hori ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Protein A ◽  
Tumour Suppressor ◽  
Human Tumour ◽  
Cytochrome B561

scholarly journals Tanshinones induce tumor cell apoptosis via directly targeting FHIT

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Xianglian Zhou ◽  
Yuting Pan ◽  
Yue Wang ◽  
Bojun Wang ◽  
Yu Yan ◽  
...  
Keyword(s):  
Salvia Miltiorrhiza ◽  
Water Soluble ◽  
Tanshinone Iia ◽  
Binding Affinities ◽  
Tumor Cell Apoptosis ◽  
Anti Cancer ◽  
Sodium Tanshinone Iia Sulfonate ◽  
Direct Binding ◽  
Fhit Protein ◽  
Diadenosine Triphosphate

AbstractThe liposoluble tanshinones are bioactive components in Salvia miltiorrhiza and are widely investigated as anti-cancer agents, while the molecular mechanism is to be clarified. In the present study, we identified that the human fragile histidine triad (FHIT) protein is a direct binding protein of sodium tanshinone IIA sulfonate (STS), a water-soluble derivative of Tanshinone IIA (TSA), with a Kd value of 268.4 ± 42.59 nM. We also found that STS inhibited the diadenosine triphosphate (Ap3A) hydrolase activity of FHIT through competing for the substrate-binding site with an IC50 value of 2.2 ± 0.05 µM. Notably, near 100 times lower binding affinities were determined between STS and other HIT proteins, including GALT, DCPS, and phosphodiesterase ENPP1, while no direct binding was detected with HINT1. Moreover, TSA, Tanshinone I (TanI), and Cryptotanshinone (CST) exhibited similar inhibitory activity as STS. Finally, we demonstrated that depletion of FHIT significantly blocked TSA’s pro-apoptotic function in colorectal cancer HCT116 cells. Taken together, our study sheds new light on the molecular basis of the anti-cancer effects of the tanshinone compounds.

scholarly journals Presence in human eosinophils of a lysophospholipase similar to that found in the pancreas

1995 ◽  
Vol 309 (1) ◽  
pp. 141-144 ◽  
Author(s):  
F W Holtsberg ◽  
L E Ozgur ◽  
D E Garsetti ◽  
J Myers ◽  
R W Egan ◽  
...  
Keyword(s):  
Synthetic Peptide ◽  
Protein A ◽  
Pancreatic Enzyme ◽  
Gel Filtration ◽  
Pancreatic Tissue ◽  
Supernatant Fraction ◽  
Polyadenylated Rna ◽  
Rabbit Polyclonal Antibody ◽  
Dna Fragment ◽  
Lysophospholipase Activity

The supernatant fraction from lysed human eosinophils, when separated by gel-filtration chromatography, contains a protein with lysophospholipase activity of approximate molecular mass 74 kDa. This mass differs substantially from the 17 kDa of a previously cloned eosinophil lysophospholipase (Charcot-Leyden crystal protein), but is similar to that reported for a pancreatic enzyme. We have therefore further characterized this pancreatic-like lysophospholipase in human eosinophils. A rabbit polyclonal antibody was produced against a synthetic peptide consisting of amino acids 325-349 from the 74 kDa rat pancreatic lysophospholipase. Western-blot analysis of eosinophil extracts indicate that this antibody recognizes a single 74 kDa band in these preparations. Incubation of the supernatant fraction from sonified eosinophils with this antibody, followed by precipitation of antibody-antigen complexes with Protein A, removes the majority of the lysophospholipase activity. Indirect immunofluorescence examination with this antibody indicates this protein to be localized to granules of eosinophils and not in other leucocytes. Moreover, reverse transcriptase PCR of polyadenylated RNA from eosinophils and from rat pancreatic tissue with primers to rat pancreatic lysophospholipase resulted in readily detectable 1 kb DNA products in both samples. Sequencing revealed this DNA fragment to be identical with the human pancreatic lysophospholipase cDNA sequence. Taken together, these data indicate that eosinophils contain a lysophospholipase that is similar to the human pancreatic enzyme.

scholarly journals Characterization of a glutathione S-transferase and a related glutathione-binding protein from gill of the blue mussel, Mytilus edulis

1995 ◽  
Vol 305 (1) ◽  
pp. 145-150 ◽  
Author(s):  
P J Fitzpatrick ◽  
T O B Krag ◽  
P Højrup ◽  
D Sheehan
Keyword(s):  
Amino Acid ◽  
Mytilus Edulis ◽  
Blue Mussel ◽  
Binding Protein ◽  
Protein A ◽  
Gel Filtration ◽  
Cumene Hydroperoxide ◽  
Gill Tissue ◽  
Glutathione S Transferase ◽  
Sequencing Studies

The major isoenzyme of glutathione S-transferase (GST 1) was purified to homogeneity from cytosolic extracts of Mytilus edulis gill tissue by GSH-agarose affinity chromatography followed by Mono Q ion-exchange f.p.l.c. This enzyme was particularly active with 1-chloro-2,4-dinitrobenzene, ethacrynic acid and cumene hydroperoxide as substrates. Immunoblotting and amino acid sequencing studies indicate that the enzyme belongs to the Pi class of GSTs. A related protein which binds to GSH-agarose was also purified. This GSH-binding protein did not immunoblot with GST antisera and showed no detectable catalytic activity with GST substrates although its N-terminal sequence was similar to Mu-class GSTs. Gel-filtration chromatography indicated that GST 1 is a dimer and the GSH-binding protein a monomer. Mass spectrometry and SDS/PAGE indicate subunit molecular masses of 24 kDa (GST 1) and 25 kDa (GSH-binding protein), respectively. Both proteins have amino acid compositions typical of GSTs.

Human High Molecular Weight Kininogen - A Secreted Platelet Coagulant Protein

1981 ◽  
Author(s):  
A H Schmaier ◽  
J Kuchibhotla ◽  
R W Colman
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Human Platelets ◽  
Metabolic Inhibitors ◽  
Ionophore A23187 ◽  
High Molecular Weight Kininogen ◽  
Contact Phase ◽  
Platelet Factor ◽  
Coagulant Activity

Platelets have been shown to contain a number of secret- able coagulant proteins, which participate as substrates or cofactors in plasma coagulation reactions. Since we have previously demonstrated that high molecular weight kininogen (HMWK) is immunochemically present in platelet extracts, we posited that HMWK is secreted during activation of platelets. Fresh normal platelets were washed by a combination of albumin-gradient and gel-filtration procedures. In 11 experiments the supernates of freeze-thaw lysates of normal human platelets contained a mean of 5.7 Units (range 3.16 to 8.14) of HMWK coagulant activity/3 × 1011 platelets. This coagulant activity was neutralized by a goat antiki- ninogen antibody. Using a 125I-HMWK tracer in PRP, the supernate of washed activated platelets contained 0.082% radioactivity as the starting PRP, suggesting that 14% of the total HMWK coagulant activity could be accounted for by plasma contamination. In four experiments, ionophore A23187 (15μM) induced a net secretion of 39% of the total platelet HMWK (range 16 to 49%). Platelet HMWK secretion by A23187 was concentration dependent (1 to 15 μM) . At A23187 (15μM) platelets released 75% 14C-5HT (range 61 to 99%) and 81% low affinity platelet Factor 4 (range 60 to 99%). Ninety-five percent of A23187-induced secretion of HMWK could be blocked by platelet pretreatment with metabolic inhibitors. LDH determinations indicated that only 5% (range 0 to 10%) of total secreted platelet HMWK could be attributed to lysis. Collagen and PGH2 also caused secretion of platelet HMWK coagulant activity. This study indicates that human platelets contain functional HMWK which may be secreted locally to modulate the reactions of the contact phase of plasma proteolysis.

Fluoride, An Internally Acting Platelet Activator, Causes Phosphorylation Of Platelet Phosphatidic Acid And 20K And 47K Proteins

1981 ◽  
Author(s):  
E H Mürer ◽  
E Siojo ◽  
J L Daniel
Keyword(s):  
Phosphatidic Acid ◽  
Platelet Activation ◽  
Great Increase ◽  
Platelet Rich Plasma ◽  
Gel Filtration ◽  
Human Platelets ◽  
Silica Gels ◽  
Late Step ◽  
Aluminum Plates ◽  
Chloroform Methanol

The effects of fluoride, which is transported into platelets in order to induce secretion, are compared with known effects of thrombin, which acts via external sites. Thus, the changes related to transmission of signal through the platelet membrane will not be common to the two activators, only those changes which are subsequent to the internal triggering of platelet activation. Human platelets were prepared by collection in EDTA and washing in saline-EDTA or by gel filtration of citrated platelet-rich plasma. The two methods gave similar results. Platelets prelabeled in plasma with 32P and them separated were incubated at 37°C with 10 mM fluoride at pH 7.4, and samples removed at intervals. (1) The protein was precipitated with HC104, then solubilized by sonication with SDS buffer and the protein bands separated by acrylamide slab gel electrophoresis. The 20K and 47K bands showed 100 to 200% increase in label, with maximum at 8 min incubation (50% secretion) and a great increase seen already at 3 min incubation, where little secretion is observed. (2) Samples were extracted with chloroform-methanol, evaporated to dryness under N2, redissolved in chloroform and applied on thinlayer silica gels on aluminum plates. Two different systems for separating phosphatidic acid (PA) were used. No significant increase in 32P radioactivity was seen in PA the first 3 min. The label at 20 min was 3x that at 8 min. Thus the labeling related to contractile events, a late step in secretion, precedes the labeling of PA, suggesting that the major part of this labeling is not related to the initial phase of platelet activation.

Depletion of Dense Granule Nucleotides during Storage of Human Platelets

1990 ◽  
Vol 63 (02) ◽  
pp. 275-278 ◽  
Author(s):  
D de Korte ◽  
C W N Gouwerok ◽  
R Fijnheer ◽  
R N I Pietersz ◽  
D Roos
Keyword(s):  
Specific Activity ◽  
Adenine Nucleotide ◽  
Human Platelets ◽  
Dense Granule ◽  
Serotonin Content ◽  
Storage Pool ◽  
Diadenosine Triphosphate ◽  
Metabolic Pool ◽  
And Storage ◽  
Hydrolysis Of

SummaryThe energy metabolism of human platelets was studied during storage of platelet concentrates. The platelets were prepared from buffy coats in PVC/DEHP bags and stored for 7 days at room temperature at a concentration of 1.0 × 109/ml with horizontal agitation. The total amount of ATP and ADP decreased with 40% during this storage. This decrease correlated with the disc-tosphere transformation associated with the loss of platelet viability. During storage, the ability to incorporate 3H-adenosine into metabolic ATP and ADP (45 min at 37° C) decreased with 50%. Via measurement of the specific activity of actin-bound ADP and the amount of incorporated radioactivity into total ATP and ADP, we calculated the content of the metabolic and storage pools of ATP and ADP. The results indicate that the decrease in adenine nucleotide levels during storage was mainly caused by a depletion of ATP and ADP from the storage pool, whereas the metabolic pool remained nearly intact. After 7 days, the ATP : ADP ratio of the storage pool had decreased from 1.0 to 0.3, indicating hydrolysis of ATP.Diadenosine-triphosphate and diadenosine-tetraphosphate (present in the storage pool) decreased with only 30%, and the serotonin content remained nearly constant. Therefore, it is unlikely that the storage pool was completely secreted. Probably, the storage pool of nucleotides serves as an internal supply for maintaining the contents of the metabolic pool of ATP and ADP during storage of platelets.

Aspirin Preferentially Inhibits Arachidonic Acid Metabolism In Collagen Vs. Thrombin-Stimulated Platelets

1981 ◽  
Author(s):  
D Deykin ◽  
R Vaillancourt
Keyword(s):  
High Performance Liquid Chromatography ◽  
Arachidonic Acid ◽  
Liquid Chromatography ◽  
High Performance ◽  
Acid Metabolism ◽  
Gel Filtration ◽  
Human Platelets ◽  
Arachidonic Acid Metabolism ◽  
Selective Effect ◽  
Oxygenase Activity

The purpose of this study was to compare the effect of aspirin on the release of metabolites of arachidonic acid from thrombin and collagen stimulated platelets. Human platelets were incubated with tritium-labeled arachidonic acid and then isolated by gel filtration. The labeled platelets were stimulated with varied doses of either thrombin or collagen for 15 minutes. The platelets were then pelleted and the released metabolites of arachidonic acid were separated by high-performance liquid chromatography. In experiments with aspirin, the aspirin was added 5 minutes before either thrombin or collagen. The total release of radioactivity was comparable at 15 μg/ml of collagen and 1.0 units/ml of thrombin (approximately 10% of the total) and at 100 μg/ml of collagen and 5 units/ml of thrombin (approximately 30%). Aspirin (25 μg/ml) preferentially inhibited collagen-stimulated release of radioactivity (62% inhibition of release with 15 μg/ml of collagen vs. 25% inhibition of release with 1.0 units/ml of thrombin; 54% inhibition of release with 100 μg/ml of collagen vs. 8% inhibition of release with 5.0 units/ml of thrombin). At all concentrations of collagen or thrombin, cyclo-oxygenase activity was markedly reduced by aspirin. The selective effect of aspirin on collagen reflects primarily preferential suppression of HETE formation. We conclude that aspirin inhibits the formation of both lipoxygenase and cyclooxygenase-derived products in collagen-stimulated platelets.

Effect of Phospholipase C on Human Platelets

1975 ◽  
Author(s):  
A.-B. Otnœss
Keyword(s):  
Bacillus Cereus ◽  
Phospholipase C ◽  
Electron Micrographs ◽  
Gel Filtration ◽  
Outer Part ◽  
Human Platelets ◽  
Scanning Electron Micrographs ◽  
Platelet Factor ◽  
Asymmetrical Distribution ◽  
Scanning Electron

The effect on human platelets of phospholipase C (Bacillus cereus) has been studied. Platelets prepared by gel filtration lost 20-30% of their phospholipids when incubated with phospholipase C for 20 min. Phosphatidylethanolamine (PE) was reduced by about 50%, whereas phosphatidylcholine and phosphatidylserine were reduced each by about 20%. Sphingomyelin was not reduced.These data suggest an asymmetrical distribution of phospholipids in the platelet membrane, PE being more accessible and therefore probably mainly located in the outer part of the membrane.The loss of phospholipids was not accompanied by aggregation, nor did the platelets lose their ability to aggregate with thrombin or ADP. Data on release of serotonin, platelet factor 3 and 4 and scanning electron micrographs of treated platelets will be given.

scholarly journals Molecular Distinction of Circulating Pregnancy-Associated Plasma Protein A in Myocardial Infarction and Pregnancy

2005 ◽  
Vol 51 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Qiu-Ping Qin ◽  
Saara Kokkala ◽  
Juha Lund ◽  
Natalia Tamm ◽  
Liisa-Maria Voipio-Pulkki ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Pregnant Women ◽  
Molecular Mass ◽  
Plasma Protein ◽  
Specific Antibody ◽  
Protein A ◽  
Gel Filtration ◽  
Single Peak ◽  
Serum Samples ◽  
A Subunit

Abstract Background: In the blood of pregnant women, pregnancy-associated plasma protein A (PAPP-A) is present as a covalent complex with the proform of eosinophil major basic protein (proMBP). Recently, increased serum concentrations of PAPP-A have been found in acute coronary syndromes (ACS). The aim of this study was to investigate whether the circulating PAPP-A in ACS is the same as that in pregnancy. Methods: We developed two time-resolved immunofluorometric assays based on a relative epitope map constructed by the use of 17 monoclonal antibodies. One assay, which measured total PAPP-A, used two PAPP-A subunit-specific antibodies. The other assay, which measured PAPP-A/proMBP complex, used one proMBP subunit-specific antibody and one PAPP-A subunit-specific antibody. Serum samples from four patients with myocardial infarction (MI), three pregnant women in their first trimester, and one in her third trimester were fractionated by gel filtration on a Superose™ 6 precision column. The two assays were used to analyze fractions obtained by gel filtration as well as serum samples serially collected from four other MI patients. Results: Pregnancy-related PAPP-A was eluted as a single peak with a molecular mass of ∼700 kDa, whereas ACS-related PAPP-A was also eluted as a single peak but with a molecular mass of ∼530 kDa. Pregnancy-related PAPP-A was detected equally by the two assays, whereas increased ACS-related PAPP-A was detected only by the assay for total PAPP-A. Conclusions: Our results provide the first evidence that circulating ACS-related PAPP-A is different from circulating pregnancy-related PAPP-A in that it is not complexed with proMBP. These findings provide a solid foundation for the design of immunoassays to accurately measure atherosclerosis-associated plasma protein A in the circulation.

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