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2.
We have established seven murine hybridoma cell lines which produce monoclonal antibodies (mAbs) against Pseudomonas aeruginosa elastase. The seven mAbs recognized at least six different epitopes on the elastase molecule. All mAbs inhibited both enzymatic activities of elastase and protease, in which elastin fluorescein and hide powder azure were used as substrates, respectively. One of them, mAb E-4D3, strongly neutralized enzymatic activities of peptidase in which furylacryloyl-glycyl-leucinamide was used as a substrate, as well as of elastase and protease. In contrast, the other six mAbs did not neutralize peptidase activity at all. The Ki value for furylacryloyl-glycl-leucinamide of E-4D3, as well as its Fab fragment, was comparable to those for metalloprotease inhibitors such as phosphoramidon and Zincov inhibitor. The binding of mAb E-4D3 was inhibited by phosphoramidon and Zincov inhibitor, but not by metal chelators such as EDTA and o-phenanthroline. A line of evidence suggests that mAb E-4D3 directly interacts with active site and highly neutralizes enzymatic activity of P. aeruginosa elastase. Data of Western blotting and ELISA suggest that mAb E-4D3 is likely to recognize an elastase molecule in a conformation-dependent manner as an epitope. In contrast, the neutralizing activity of the other mAbs against elastase and protease seems to be caused by a low accessibility of an enzyme to insoluble and high-molecular-mass substrates through the binding and steric hindrance of the mAbs to an enzyme. 相似文献
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4.
Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H 总被引:2,自引:0,他引:2
To cleave RNA molecules using RNase H in a site-specific manner, a short deoxyoligonucleotide (3-5mer) joining with 2'-O-methyl oligonucleotide(s) was designed as a DNA splint to be used. Model experiments were carried out using ribooligonucleotide substrates (9 and 18 mer). It was found that the use of this type of splints (9 mer) causes a unique cleavage by RNase H. For example, when 3'm (GA)d(AGAA)m(GGU)5' was used as a hybridization strand, 32pUCUUUCUUCUUCCAGGAU was cleaved specifically between U11 and C12 to yield 32pUCUUUCUUCUU. This method will have a variety of applications for the study of RNA. 相似文献
5.
Identification of I:A mismatch base-pairing structure in DNA 总被引:7,自引:0,他引:7
S Uesugi Y Oda M Ikehara Y Kawase E Ohtsuka 《The Journal of biological chemistry》1987,262(15):6965-6968
Deoxyoligonucleotides containing deoxyinosine residues at positions corresponding to ambiguous nucleotides derived from an amino acid sequence have been successfully used as hybridization probes. It is assumed that the hypoxanthine residue can make base pairs with multiple bases. In order to obtain direct evidence for I:A base-pairing, a self-complementary deoxyoligonucleotide, d(G-G-I-A-C-C), was synthesized and its properties were examined by NMR spectroscopy. Three hydrogen-bonded imino proton resonances are observed at low temperatures in H2O suggesting the formation of a self-duplex with complete base pairing. Nuclear Overhauser effect (NOE) experiments showed that a signal at 15.1 ppm originated from the imino proton (H1) of the dI residue (I3) which is hydrogen-bonded to the dA residue (A4). Both the I3 and A4 residues were assumed to have taken an anti glycosidic conformation since irradiating the H1 of I3 gave NOEs both to its own H2 and to that of A4, an NOE also being observed between the H2 protons of I3 and A4. Comparison of the 31P NMR spectra of d(G-G-I-A-C-C) and d(G-G-I-C-C-C) showed the backbone structure of d(G-G-I-A-C-C) to have been disturbed by the presence of purine:purine base pairs in the middle of the hexamer duplex. 相似文献
6.
The in vivo stability, maturation and aminoacylation of anticodon-substituted Escherichia coli initiator methionine tRNAs 总被引:1,自引:0,他引:1
H Grosjean S De Henau T Doi A Yamane E Ohtsuka M Ikehara N Beauchemin K Nicoghosian R Cedergren 《European journal of biochemistry》1987,166(2):325-332
We have constructed eight anticodon-modified Escherichia coli initiator methionine (fMet) tRNAs by insertion of synthetic ribotrinucleotides between two fragments ('half molecules') derived from the initiator tRNA. The trinucleotides, namely CAU (the normal anticodon), CAA, CAC, CAG, GAA, GAC, GAG and GAU, were joined to the 5' and 3' tRNA fragments with T4 RNA ligase. The strategy of reconstruction permitted the insertion of radioactive 32P label between nucleotides 36 and 37. tRNAs were microinjected into the cytoplasm of Xenopus laevis oocytes, and the following properties were evaluated: the stability of these eubacterial tRNA variants in the eukaryotic oocytes; the enzymatic modification of the adenosine at position 37 (3' adjacent to the anticodon) and aminoacylation of the chimeric tRNAs by endogenous oocyte aminoacyl-tRNA synthetases. In contrast to other variants, the two RNAs having CAU and GAU anticodons were stable and underwent quantitative modification at A-37. These results show that the enzyme responsible for the modification of A-37 to N-[N-(9-beta-D-ribofuranosylpurine-6-yl)carbamoyl]threonine (t6A) is present in the cytoplasm of oocytes and is very sensitive to the anticodon environment of the tRNA. Also, these same GAU and CAU anticodon-containing tRNAs are fully aminoacylated with the heterologous oocyte aminoacyl-tRNA synthetases in vivo. During the course of this work we developed a generally applicable assay for the aminoacylation of femtomole amounts of labelled tRNAs. 相似文献
7.
A new solid-phase synthesis of oligoribonucleotides by the phosphoro-p-anisidate method using tetrahydrofuranyl protection of 2''-hydroxyl groups. 总被引:3,自引:3,他引:0
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Six nonaribonucleotides containing the 5'-splice site, one complementary nonamer and an octadecamer containing the 3'-splice site have been synthesized on a polymer support using the phosphoro-p-anisidate method. A 5'-linked 2'-O-tetrahydrofuranyl-N-protected nucleoside 3'-(o-chlorophenyl)phosphoro-p-anisidate was used as the starting nucleotide, and the chain elongated in the 3'-direction by removing the p-anisidate protecting group with isoamyl nitrite under neutral conditions. The octadecamer has been synthesized using dinucleotide blocks and a 3'-terminal trinucleotide. 相似文献
8.
Takashi Ooba Hideyuki Hayashi Sachiko Karaki Manabu Tanabe Kyoichi Kano Masafumi Takiguchi 《Immunogenetics》1989,30(2):76-80
The primary structure ofHLA-B51 andHLA-Bw52 suggested thatHLA-B51 was derived fromHLA-Bw52 by the combination of a genetic exchange withHLA-B8 and a point mutation. To investigate the evolution of theHLA-B5 cross reactive group, theHLA-B35 gene was cloned and the primary structure was determined.HLA-B35 is identical toHLA-Bw58 except in the α1 domain. The α1 domain ofHLA-B35 except Bw4/Bw6-associated amino acids is identical to that ofHLA-B51
*, which was suspected to be an intermediate gene betweenHLA-B51 andHLA-Bw52. These data suggest thatHLA-B35 has evolved fromHLA-Bw58 in two steps; an in vivo replacement of the α1 domain withHLA-B51 and genetic exchange with one of theHLA-Bw6 genes. These three genes andHLA-Bw58 are postulated to share a common ancestor. 相似文献
9.
M Ozawa T Ohtsuka N Okamura S Ishibashi 《Archives of biochemistry and biophysics》1989,273(2):491-496
Treatment of guinea pig polymorphonuclear leukocytes (PMNL) with various fatty acids elicited superoxide anion (O2-) production and an increase in intracellular Ca2+ [( Ca2+]i). Both responses, however, were seldom observed when PMNL were treated at lower concentrations. But, simultaneous addition of 1-oleoyl-2-acetylglycerol (OAG), a protein kinase C activator, caused an increase in O2- production even at the lower concentrations of fatty acids. In contrast to the synergism in O2- production, [Ca2+]i remained at almost the basal level irrespective of the presence of OAG. Among saturated fatty acids, those with carbon numbers of 14 to 18 were most effective in stimulating O2- production in combination with OAG. Unsaturated fatty acids with a carbon number of 18 were almost equally effective irrespective of the number of double bonds. 相似文献
10.
J M Ha Y Ito G Kawai T Miyazawa K Miura E Ohtsuka S Noguchi S Nishimura S Yokoyama 《Biochemistry》1989,28(21):8411-8416
1H NMR spectra of a GDP/GTP-binding domain of human c-Ha-ras gene product (residues 1-171) in which glutamine-61 was replaced by leucine [ras(L61/1-171) protein] were analyzed. By one-dimensional and two-dimensional homonuclear Hartmann-Hahn spectroscopy and nuclear Overhauser effect (NOE) spectroscopy of the complex of the ras(L61/1-171) protein and GDP, the ribose H1', H2', H3', and H4' proton resonances of the bound GDP were identified. The guanine H8 proton resonance of the bound GDP was identified by substituting [8-2H]GDP for GDP. The dependences of the H1' and H8 proton resonance intensities on the duration of irradiation of the H1', H2', H3', and H8 protons were measured. By numerical simulation of these time-dependent NOE profiles, the conformation of the protein-bound GDP was elucidated; the guanosine moiety takes the anti form about the N-glycosidic bond with a dihedral angle of chi = -124 +/- 2 degrees and the ribose ring takes the C2'-endo form. Such an analysis of the conformation of a guanine nucleotide as bound to a GTP-binding protein will be useful for further studies on the molecular mechanism of the conformational activation of ras proteins on ligand substitution of GDP with GTP. 相似文献