Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Studies on the binding specificity of actinomycin D (ACTD) to tetranucleotide sequences of the form -XGCY- have been extended to include the non-self-complementary sequences. ACTD binding characteristics are investigated by equilibrium, kinetic, and thermal denaturation for decameric duplexes d(ATA-XGCY-ATA)-d(TAT-Y'GCX'-TAT), where X and Y are complementary to X' and Y', respectively, but not to each other. The results indicate that when X = G or Y = C, the oligomers exhibit significantly weaker ACTD binding affinities, smaller melting temperature increases upon drug binding, and faster SDS-induced ACTD dissociation rates than the other sequences. Estimated binding constants at 18.5 degrees C for decameric duplexes containing -AGCA-/-TGCT-, -AGCG-/-CGCT-, or -CGCA-/-TGCG- are in the range of 4-9 microM-1, whereas for the ones containing -GGCT-/-AGCC-, -GGCA-/-TGCC-, or -GGCG-/-CGCC- they range from 0.6 to 2 microM-1. In contrast to the characteristic SDS-induced ACTD dissociation times of 600-1000 s for the stronger binding sites, the sequences containing X = G or Y = C exhibit at least an order of magnitude faster dissociation kinetics. These observations are further supported by the induced CD results and fluorescence measurements with 7-amino-ACTD. The findings from these non-self-complementary -XGCY- tetranucleotide sequences are consistent with those found earlier for the self-complementary counterparts, and they together clearly demonstrate that a base sequence alteration adjacent to the GC site can have a profound effect on the ACTD binding as well as dissociation characteristics, likely a consequence of subtle conformational alterations near the binding site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding specificities of actinomycin D to non-self-complementary -XGCY-tetranucleotide sequences. 162 64

Activation of human normal serum with tetanus/antitetanus immune complexes (TAT-IC) resulted in increased binding of 125I-labeled interleukin-1 beta (IL-1 beta) to serum factors, as opposed to untreated serum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography showed labeling of two large molecular mass factors of an apparent molecular weight (Mr) of 200,000 and 400,000, respectively. These complexes could be dissociated by reduction. No complexes were formed when reducing compounds were added to serum-TAT-IC-125I-IL-1 beta mixtures. Complex formation was largely prevented by alkylating compounds. Molecular sieve chromatography of TAT-IC-activated serum confirmed that 125I-IL-1 beta became bound to high Mr serum proteins. Fractions containing high molecular 125I-IL-1 serum protein complexes partially retained IL-1-like activity since they induced proliferation of an IL-1-dependent murine T helper (D10G4) cell lineage. The 125I-IL-1 beta binding factors could be immunoprecipitated from TAT-IC-activated serum 125I-IL-1 beta solutions by antisera to alpha 2-macroglobulin (alpha 2M) or to the third complement component (C3). SDS-PAGE of the immunoprecipitates showed radioactive bands corresponding to the expected Mr resulting from complex formation between 125I-IL-1 beta and these two proteins. Treatment of purified plasma alpha 2M and C3 with trypsin or activation with methylamine, which causes cleavage of the internal thiol ester and the appearance of free thiol groups in these proteins, mediated binding of 125I-IL-1 beta to alpha 2M and C3b. The results suggest that cleavage of the internal thiol ester in C3 and alpha 2M makes these plasma proteins susceptible to binding of 125I-IL-1 beta and that free thiol groups do play a role in the formation of 125I-IL-1 beta plasma protein complexes. Activated C3 and alpha 2M may function as IL-1 beta carrier proteins in biologic fluids, in addition to their other physiologic roles.
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PMID:Binding of recombinant interleukin-1 beta to the third complement component and alpha 2-macroglobulin after activation of serum by immune complexes. 169 30

Previous studies have indicated that cyst fluid of ovarian tumors contains 2 trypsinogen isoenzymes, called tumor-associated trypsinogen-I (TAT-I) and trypsinogen-2 (TAT-2), the levels of which correlate with the degree of malignancy of the tumors. In addition, these cyst fluids contain large amounts of tumor-associated trypsin inhibitor (TATI), which is also expressed in many other human tumors. In the present study we examined the production of TAT-I, TAT-2 and TATI in 9 established tumor-cell lines. TAT-2 was produced by 5 cell lines. Its concentration in the conditioned medium of COLO 205 colon adenocarcinoma cells, K-562 erythroleukemia cells and fibrosarcoma cell lines HT 1080, 8387 and A 9733 was 460 micrograms/l, 9.8 micrograms/l, 21 micrograms/l, 8.8 micrograms/l and 0.24 micrograms/l, respectively. TAT-I was detectable in the conditioned medium of COLO 205 and HT 1080 cells at concentrations of 64 micrograms/l and 0.5 micrograms/l, respectively. TATI was detected only in the media of COLO 205 cells at a concentration of 23 micrograms/l. TAT-2 zymogen was purified from the conditioned medium of COLO 205 and HT 1080 cells by immunoaffinity chromatography. According to its aminoterminal amino acid sequence, a molecular mass of 28 kDa by SDS-PAGE, elution pattern in ion-exchange chromatography and ability to be activated by enteropeptidase, the zymogen is identical to that previously isolated from cyst fluid of ovarian tumors. In addition, we found that TAT-2 secretion could be down-regulated by dexamethasone in HT 1080 cells but not in COLO 205 cells. The abundant production of TAT-2 isoenzyme in different cancer cell lines suggests that it could contribute to the increased proteolytic activity of many human tumors.
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PMID:Human colon carcinoma, fibrosarcoma and leukemia cell lines produce tumor-associated trypsinogen. 199 87

Binding of actinomycin D (ACTD) to self-complementary decamers d(ATA-XGCY-TAT), where XGCY = TGCA, AGCT, CGCG, and GGCC, has been investigated by equilibrium, kinetic, and thermal denaturation studies. The results indicate that despite the presence of a GC dinucleotide sequence, -GGCC- exhibits a much weaker binding affinity toward ACTD than the other three tetranucleotide sequences. Binding constants estimated from Scatchard plots indicate that binding to the -GGCC- site is at least an order of magnitude weaker than binding to -CGCG- and -AGCT-, which in turn is only slightly weaker than binding to the -TGCA- sequence. At 18.5 degrees C and 1% SDS, ACTD dissociates from d-(ATA-TGCA-TAT) with a slow characteristic time of 3300 s, roughly 4 times slower than dissociation from those containing -CGCG- and -AGCT- sequences and more than 2 orders of magnitude slower than that from -GGCC-. An 18.2 degrees C increase in the melting temperature is observed for the -TGCA-containing decamer upon binding of the ACTD, whereas increases of 10.3, 6.7, and 2.0 degrees C are observed for the -CGCG-, -AGCT-, and -GGCC-containing decamers, respectively. The effects observed by changing the adjacent base pair (sequence) may occur as a result of differential stacking and/or peptide ring-DNA groove interactions. Base sequence alterations adjacent to the ACTD binding site may result in differences in the minor groove environment and/or subtle conformational alterations at the intercalation site.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Binding specificities of actinomycin D to self-complementary tetranucleotide sequences -XGCY-. 321 42

Radiolabeled antithrombin III (ATIII) was incubated at 37 degrees C with purified vitronectin (VN) or fibrinogen-deficient plasma before thrombin was added to initiate complex formation. Incorporation of radiolabeled ATIII was detected using polyacrylamide gel electrophoresis (PAGE) and autoradiography. The PAGE conditions appeared to be crucial for the detection of VN.TAT complexes. In the absence of SDS, ternary complexes formed instantaneously, whereas in the presence of SDS, only 50% of the TAT was associated with VN after a 60-min incubation. Formation of ternary complexes could be confirmed by gel filtration of the plasma to which thrombin was added. Furthermore, TAT in patient plasmas (disseminated intravascular coagulation and sepsis) was found to bind to heparin-Sepharose, indicating that this endogenously formed TAT was also associated with VN. The amino-terminal region of VN and the thrombin moiety of the TAT complex were found to be responsible for their interaction, which was stabilized by disulfide bridges. These results indicate that in normal plasma all TAT is complexed with VN. This association alters the conformational state of plasma VN, which appears to be responsible for the clearance of thrombin complexes from the circulation.
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PMID:Ternary vitronectin-thrombin-antithrombin III complexes in human plasma. Detection and mode of association. 767 52

A slow migrating variant of human serum albumin, present in lower amount than the normal protein, has been detected by routine clinical electrophoresis at pH 8.6 in two members of a family living in Asola (Lombardia, Italy). Ion-exchange chromatography of serum samples failed to separate the normal protein from the variant. Analysis of the albumin peak by SDS/PAGE revealed that the variant had a lower apparent molecular mass than its normal counterpart. However, the abnormal band was not detectable when the separation was performed under reducing conditions or when both albumins were carboxymethylated. Isoelectric-focusing analysis of CNBr fragments localized the mutation to fragment CNBr 3 (residues 124-298). This fragment was isolated on a preparative scale and subjected to tryptic digestion. Sequence determination of the abnormal tryptic peptide revealed that the variant arises from a Tyr140--> Cys substitution. This result was confirmed by DNA sequence analysis, which showed a single transition of TAT-->TGT at nucleotide position 5074. Despite the presence of an additional cysteine residue, several lines of evidence indicated that albumin Asola has no free -SH group; therefore, we propose the formation of a new S-S bond between Cys140 and Cys34, the only free sulphydryl group present in the normal protein. The relatively low level of the variant in serum and its abnormal mobility on cellulose acetate electrophoresis and SDS/PAGE are probably caused by a gross conformational change of the molecule induced by the new S-S bridge.
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PMID:A genetic variant of albumin (albumin Asola; Tyr140-->Cys) with no free -SH group but with an additional disulfide bridge. 788 97

Immunological infertility is thought to be caused by the binding of antibodies to 'fertility-related' antigen(s) on the sperm membrane. We compared antibody profiles in sera from 20 ASA(+) and ASA(-) men, using a sperm membrane extract as an antigen. Antigens were separated by SDS-PAGE under reducing conditions. The patients were classed as ASA(+) by the MAR (> 50%), d-IBT (> 20%) and TAT (> 1:64). The results showed that immunoreactive bands in both the ASA(+) and ASA(-) groups were heterogeneous and included bands covering the whole molecular weight range. Statistical analysis showed significantly more patients in the ASA(+) group having immunoreactive bands at molecular weights of 32 Kd (P = 0.006) and 79 Kd (P = 0.02) when compared to the ASA(-) group. In the ASA(-) group significantly more patients had reactive bands at 81 Kd (P = 0.01) when compared to the ASA(+) group. The 32 Kd antigen reacted only with sera from ASA(+) patients. We conclude that differences exist between the ASA(+) and ASA(-) groups when this extraction method is used and that the isolation and purification of the 32 Kd protein may justify further investigation.
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PMID:Analysis of human sperm membrane antigens reacting with sera from antisperm antibody positive and negative patients by western blotting. 851 54

Internalization of the ternary vitronectin-thrombin-antithrombin (VN-TAT) complex by human umbilical vein endothelial cells was investigated. Radiolabeled VN-TAT was bound to the cell surface at 4 degrees C, and internalization was initiated by increasing the temperature to 37 degrees C. After 30 min about half of the VN-TAT complex disappeared from the cell surface and accumulated in the subendothelial matrix. Translocation of VN-TAT complex from the luminal to the basolateral side was confirmed by electron microscopic evaluation of cross-sections of endothelial cells incubated with gold-conjugated VN-TAT complex. Furthermore, cells cultured in VN-TAT deficient serum, incubated with purified VN-TAT, and subsequently assayed for fluorescent staining using a monoclonal antibody directed against thrombin-modified antithrombin and a polyclonal antibody against vitronectin showed co-localization of both antibodies in punctates. Punctates were randomly distributed in both the xy and xz plane of endothelial cells as evidenced by confocal laser scanning microscopy. Trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis showed that VN-TAT was not degraded during translocation and inhibition of the microfilament system reduced release of VN-TAT to the matrix, indicating that transcytosis was responsible for translocation. These findings emphasize that VN-TAT complex is taken up by endothelial cells, not only leading to the removal of inactivated thrombin from the circulation but also to deposition of VN into the subendothelial matrix.
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PMID:Internalization of vitronectin-thrombin-antithrombin complex by endothelial cells leads to deposition of the complex into the subendothelial matrix. 853 May 13

A point mutation in the plastome-encoded psaB gene of the mutant en:alba-1 of Antirrhinum majus L. was identified by an analysis of chloroplast DNA with a modified PCR-SSCP technique. Application of this technique is indicated when a gene or a group of genes is known in which the point mutation is located. Analysis of primary photosynthetic reactions in the yellowish white plastome mutant indicated a dysfunction of photosystem (PS) I. The peak wavelength of PS I-dependent chlorophyll (Chl) fluorescence emission at 77 K was shifted by 4 nm to 730 nm, as compared to fluorescence from wild-type. There were no redox transients of the reaction center Chl P700 upon illumination of leaves with continuous far-red light or with rate-saturating flashes of white light. The PS I reaction center proteins PsaA and PsaB are not detectable by SDS-PAGE in mutant plastids. Hence, plastome encoded PS I genes were regarded as putative sites of mutation. In order to identify plastome mutations we developed a modified SSCP (single-strand conformation polymorphism) procedure using a large PCR fragment which can be cleaved with various restriction enzymes. When DNA from wild-type and en:alba-1 was submitted to SSCP analysis, a single stranded HinfI fragment of a PCR product of the psaB gene showed differences in electrophoretic mobility. Sequence analysis revealed that the observed SSCP was caused by a single base substitution at codon 136 (TAT-->TAG) of the psaB gene. The point mutation produces a new stop codon that leads to a truncated PsaB protein. The results presented indicate that the mutation prevents the assembly of a functional PS I complex. The applicability to other plastome mutants of the new method for detection of point mutations is discussed.
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PMID:Detection of point mutations in chloroplast genes of Antirrhinum majus L. I. Identification of a point mutation in the psaB gene of a photosystem I plastome mutant. 854 19

Congenital hypofibrinogenemia, fibrinogen Tottori II, caused by a nonsense mutation in the fibrinogen Bbeta chain gene, was found in a 68-year-old Japanese female. The plasma fibrinogen level was 99.2 mg dL(-1) as determined by the thrombin time method. No overt molecular abnormalities were observed in purified patient fibrinogen by SDS-PAGE analysis. After sequencing all exons and exon-intron boundaries of three fibrinogen genes, we found a heterozygous single point mutation of T-->G at position 3356 of the patient fibrinogen Bbeta chain gene. This nucleotide mutation results in a nonsense mutation (TAT sequence for Bbeta 41Tyr to TAG sequence for a translation termination signal). The mutation was confirmed by polymerase chain reaction-restriction fragment length polymorphism analysis, since this nucleotide mutation results in a new NheI recognition sequence at this position. These data indicated that the nonsense mutation of the fibrinogen Bbeta chain gene caused a truncated fibrinogen Bbeta chain, which may not be assembled in the fibrinogen molecule.
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PMID:Hypofibrinogenemia caused by a nonsense mutation in the fibrinogen Bbeta chain gene. 1462 69


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