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51.
Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes.
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K Yanai N Takaya N Kojima H Horiuchi A Ohta M Takagi 《Journal of bacteriology》1992,174(22):7398-7406
Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal. 相似文献
52.
Three anonymous chromosome 17 DNA markers, D17Tu36, D17Tu43, and D17Le66B, differentiate between house mouse species and/or between t chromosomes. The D17Tu36 probe, which maps near the Fu locus and to the In(17)4 on t chromosomes, identifies at least 15 haplotypes, each haplotype characterized by a particular combination of DNA fragments obtained after digestion with the Taq I restriction endonuclease. Ten of these haplotypes occur in Mus domesticus, while the remaining five occur in M. musculus. In each of these two species, one haplotype is borne by t chromosomes while the other haplotypes are present on non-t chromosomes. The D17Tu43 probe, which maps near the D17Leh122 locus and to the In(17)3 on t chromosomes, also identifies at least 15 haplotypes in Taq I DNA digests, of which nine occur in M. domesticus and six in M. musculus. One of the nine M. domesticus haplotypes is borne by t chromosomes, the other haplotypes are borne by non-t chromosomes; two of the six M. musculus haplotypes are borne by t chromosomes and the remaining four by non-t chromosomes. Some of the D17Tu43 haplotypes are widely distributed in a given species, while others appear to be population-specific. Exceptions to species-specificity are found only in a few mice captured near the M. domesticus-M. musculus hybrid zone or in t chromosomes that appear to be of hybrid origin. The D17Leh66B probe, which maps to the In(17)2, distinguishes three haplotypes of M. domesticus-derived t chromosomes and one haplotype of M. musculus-derived t chromosomes. Because of these characteristics, the three markers are well suited for the study of mouse population genetics in general and of t chromosome population genetics in particular. A preliminary survey of wild M. domesticus and M. musculus populations has not uncovered any evidence of widespread introgression of genes from one species to the other; possible minor introgressions were found only in the vicinity of the hybrid zone. Typing of inbred strains has revealed the contribution of only M. domesticus DNA to the chromosome 17 of the laboratory mouse. 相似文献
53.
H Shirataki K Kaibuchi T Yamaguchi K Wada H Horiuchi Y Takai 《The Journal of biological chemistry》1992,267(16):10946-10949
The smg-25A/rab3A protein (smg p25A) is a small GTP-binding protein implicated in intracellular vesicle traffic, particularly in neurotransmitter release from the presynapse. In the present study, we attempted to identify a target protein in bovine brain crude membranes that might be interacted with the GTP-bound form of smg p25A. When the guanosine-5'-(3-O-thio)triphosphate (GTP gamma S)-bound form of radioiodinated smg p25A and the crude membrane fraction of bovine brain were incubated with a cross-linker, disuccinimidyl suberate, and the sample was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography, one radioactive band with a M(r) of about 110,000 was detected. This radioactive band appeared to be composed of radioiodinated smg p25A and a molecule with a M(r) of about 86,000. This molecule, tentatively termed here smg p25A target, was extracted from the membranes by a detergent and highly purified by column chromatographies and sucrose density gradient ultracentrifugation. The purified smg p25A target was sensitive to heat boiling and tryptic digestion, indicating that smg p25A target is a protein molecule. The M(r) of the purified smg p25A target was estimated to be about 85,000-86,000 from SDS-PAGE and to be about 100,000 from the S value. The cross-linking of radioiodinated smg p25A with the purified smg p25A target was inhibited by the GTP gamma S-bound form of non-radioactive smg p25A with an IC50 of about 8 nM. The GDP-bound form of smg p25A was much less effective. Other small GTP-binding proteins, such as c-Ki-ras p21, rhoA p21, smg p21B, and rab11 p24 were ineffective. These results indicate that a protein with a M(r) of about 85,000-100,000 is a target for smg p25A. 相似文献
54.
M Hidaka T Kobayashi Y Ishimi M Seki T Enomoto M Abdel-Monem T Horiuchi 《The Journal of biological chemistry》1992,267(8):5361-5365
DNA replication terminus (ter)-binding protein (TBP) in Escherichia coli binds specifically to the terminus (ter) site, and the resulting complex severely blocks DNA replication in an unique orientation by inhibiting the action of helicases. To generalize the intrinsic nature of the orientated ter-TBP complex against various helicases, we tested the potential of the complex to inhibit the action of three helicases, DNA helicase I, simian virus 40 (SV40) large tumor (T) antigen, and helicase B, derived from F plasmid, SV40, and mouse FM3A cell, respectively. The complex impeded the unwinding activities of all tested helicases in a specific orientation, with the same polarity observed in case of blockage of a replication fork, and, as a result, there was a block of SV40 DNA replication in both crude and purified enzyme systems in vitro. As the specificity in polarity of inhibition extends to heterologous systems, there may be common structure/mechanism features in helicases. 相似文献
55.
The mechanism for the inhibition of actin-activated ATPase of smooth muscle heavy meromyosin by calponin. 总被引:4,自引:0,他引:4
Calponin, an actin-binding protein, inhibited the acto-heavy meromyosin (HMM) MgATPase and lowered the binding of HMM to actin. The amount of calponin bound to actin or tropomyosin-actin was the same when the ATPase was inhibited 80-90%. While the KATPase was diminished only less than 2-fold in the presence of calponin, the Vmax was decreased 6-fold and 2-fold with actin and tropomyosin-actin, respectively. A comparison of the kinetic constants for the ATP hydrolysis obtained in the presence of actin-calponin and tropomyosin-actin-calponin revealed that the tropomyosin augmented the Vmax 5-fold from the inhibited level, but there was no effect on the KATPase. 相似文献
56.
Projection of Monte Carlo and molecular dynamics trajectories onto the normal mode axes: human lysozyme 总被引:3,自引:0,他引:3
A method is presented to describe the internal motions of proteins obtained from molecular dynamics or Monte Carlo simulations as motions of normal mode variables. This method calculates normal mode variables by projecting trajectories of these simulations onto the axes of normal modes and expresses the trajectories as a linear combination of normal mode variables. This method is applied to the result of the molecular dynamics and the Monte Carlo simulations of human lysozyme. The motion of the lowest frequency mode extracted from the simulations represents the hinge bending motion very faithfully. Analysis of the obtained motions of the normal mode variables provides an explanation of the anharmonic aspects of protein dynamics as due first to the anharmonicity of the actual potential energy surface near a minimum and second to trans-minimum conformational changes. 相似文献
57.
Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-13C]Cys. The13C signals were assigned by the15N,13C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys71-Cys101 disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met73-Val74, is more accessible to solvent than the other disulfide bond. Cys35-Cys50, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys71 and Cys101 regenerates at a much faster rate than that between Cys35 and Cys50.Nomenclature of the various states of SSI that are observed in the present study
Fully oxidized-folded
native or intact (without GuHCl or DTT)
-
half reduced-folded
(Cys71-Cys101 reduced; DTT without GuHCl)
-
inversely half reduced-folded
(Cys35-Cys50 reduced; a reoxidation intermediate from fully reduced-folded state)
-
fully reduced-unfolded
(reduced by DTT in the presence of GuHCl)
-
fully reduced-folded
(an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture) 相似文献
58.
Cloning and nucleotide sequence of the ispA gene responsible for farnesyl diphosphate synthase activity in Escherichia coli 总被引:7,自引:0,他引:7
The molecular cloning and the determination of the nucleotide sequence of the ispA gene responsible for farnesyl diphosphate (FPP) synthase [EC 2.5.1.1] activity in Escherichia coli are described. E. coli ispA strains have temperature-sensitive FPP synthase, and the defective gene is located at about min 10 on the chromosome. The wild-type ispA gene was subcloned from a lambda phage clone containing the chromosomal fragment around min 10, picked up from the aligned genomic library of Kohara et al. [Kohara, Y., Akiyama, K., & Isono, K. (1987) Cell 50, 495-508]. The cloned gene was identified as the ispA gene by the recovery and amplification of FPP synthase activity in an ispA strain. A 1,452-nucleotide sequence of the cloned fragment was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected molecular weights of 8,951 and 32,158, respectively. A part of the deduced amino acid sequence of ORF-2 showed similarity to the sequences of eucaryotic FPP synthases and of crtE product of a photosynthetic bacterium. The plasmid carrying ORF-2 downstream of the lac promoter complemented the defect of FPP synthase activity of the ispA mutant, showing that the product encoded by ORF-2 is the ispA product. The maxicell analysis indicated that a protein of molecular weight 36,000, approximately consistent with the molecular weight of the deduced ORF-2-encoded protein, is the gene product. 相似文献
59.
Structural organization of the bovine adrenodoxin gene 总被引:1,自引:0,他引:1
Y Sagara H Sawae A Kimura Y Sagara-Nakano K Morohashi K Miyoshi T Horiuchi 《Journal of biochemistry》1990,107(1):77-83
60.
Identification of 2Fe-2S cysteine ligands in putidaredoxin 总被引:2,自引:0,他引:2
N C Gerber T Horiuchi H Koga S G Sligar 《Biochemical and biophysical research communications》1990,169(3):1016-1020
The iron-sulfur center of putidaredoxin is coordinated by four cysteine sulfhydrals. In order to determine which of the six cysteine residues in the protein coordinate the Fe-S center, we have individually mutated cysteine residues 73, 85 and 86 into serines. Of these mutant proteins, only C85S and C73S express holo-protein as evidence by SDS-PAGE and EPR spectroscopy. This leads us to the conclusion that residues 39,45,48, and 86 are the cysteines that coordinate the iron-sulfur center in putidaredoxin. 相似文献