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K Motohashi

Publications and source records attributed to K Motohashi.

At least 19 recordsLinked to original sources
PubMed1996

A novel factor required for the assembly of the DnaK and DnaJ chaperones of Thermus thermophilus.

We previously reported the isolation of T.DnaK.DnaJ chaperone complex from Thermus thermophilus. Here, we show that a novel factor is necessary for the assembly of T.DnaK and T.DnaJ into the complex. A dnaK gene cluster of T. thermophilus contained five genes, dnaK-grpE-dnaJ-orf4-clpB. Interestingly, T.DnaJ lacks the whole "cysteine-rich region" that has been postulated to be necessary to bind unfolded proteins. The orf4 gene encodes a novel 78-amino acid protein. Curiously, T.DnaK and T.DnaJ expressed in Escherichia coli did not form the complex. Careful reexamination of the T.DnaK.DnaJ complex revealed the presence of a small protein in the complex, which turned out to be a product of orf4. As expected, expression of three genes, dnaK-dnaJ-orf4, resulted in production of a T.DnaK.DnaJ complex in E. coli that was indistinguishable from the authentic complex in its ability to interact with nucleotide and denatured protein. The product of orf4 was also required for in vitro reconstitution of the complex and named T.DafA (T.DnaK.DnaJ assembly factor A). The complex comprises three copies each of T.DnaK, T.DnaJ, and T.DafA. Even though a definite homolog of T.DafA has not been found in the data base, this finding raises a possibility that interaction between DnaK and DnaJ chaperones in other organisms is also mediated by a small protein yet unnoticed.

Amino Acid Sequence↗
PubMed1994

Isolation of the stable hexameric DnaK.DnaJ complex from Thermus thermophilus.

A DnaK homolog (T.DnaK) has been purified as a stable complex with a DnaJ homolog (T.DnaJ) from a thermophilic bacterium, Thermus thermophilus. This complex has an approximate molecular size of 300 kDa and appears to contain three copies of each of T.DnaK and T.DnaJ molecules. Consistently, trigonal ring structures with a diameter (trigonal apex-to-apex) of about 11 nm were observed with electron microscopy. The complex has no endogenously bound AT(D)P and is stable in the presence of Mg-AT(D)P. It possesses a weak ATPase activity and retains about 3 mol of ADP/mole of the complex when incubated with Mg-ATP. This complex is able to interact with the reduced carboxymethylated alpha-lactalbumin which we used as a model unfolded protein.

Adenosine Triphosphatases↗
PubMed1994

Micromachined electroporation system for transgenic fish.

Luciferase gene was introduced into fertilized eggs of medaka by a localized electric field between thin film electrodes formed on a glass plate. Miniaturization of the electrodes enabled us to apply a localized electric field to the animal pole of the fertilized egg. Biochemical luminescence and an electrophoresis pattern showed expression and integration of the gene, respectively. The presented system had a higher ratio of gene introduction than the conventional electroporation method.

Animals↗
PubMed1993

Purification from liver microsomes from untreated cynomolgus monkeys of cytochrome P450 closely related to human cytochrome P450 2B6.

A cytochrome P450 (P450) (referred to as P450CMLa) was purified and characterized from hepatic microsomes from untreated cynomolgus monkeys (Macaca irus). The final preparation was electrophoretically homogeneous and its estimated minimum molecular mass was 49.5 kDa. The amino-terminal amino acid sequence of the protein (first 34 residues) closely resembled that of the protein encoded by the 2B6 cDNA from humans (94%). This protein was cross-reactive with antibodies raised against P450 2B1 (P450 b), P450 2B11 (P450 PBD-2), and P450GP-1, which were purified from hepatic microsomes from phenobarbital-pretreated rats, beagle dogs, and guinea pigs, respectively. Also, the antibody raised against P450CMLa was able to cross-react with P450 2B1, P450 2B11, and P450GP-1. P450CMLa was capable of catalyzing benzphetamine N-demethylation and testosterone 16 beta-hydroxylation in a reconstituted system. Anti-P450CMLa antibody inhibited the activity of testosterone 16 beta-hydroxylase but not the activities of testosterone 2 beta- and 6 beta-hydroxylases in liver microsomes from cynomolgus monkeys. The content of P450CMLa, as estimated by immunoblot analysis, was 70 pmol/mg (about 5% of total P450). The protein immunoreactive with the anti-P450CMLa antibody was also present in liver microsomes from Japanese monkeys, baboons, common marmosets, and common squirrel monkeys. In liver microsomes from common squirrel monkeys, the content of protein immunoreactive with the anti-P450CMLa antibody and the activity of testosterone 16 beta-hydroxylase were effectively increased by pretreatment with phenobarbital. The antibody against P450CMLa strongly inhibited the activity of testosterone 16 beta-hydroxylase in liver microsomes not only from untreated cynomolgus monkeys but also from phenobarbital- and pregnenolone 16 alpha-carbonitrile-pretreated common squirrel monkeys. These results indicated that the P450CMLa purified here is very similar to the forms of P450 classified into the 2B subfamily, in its amino-terminal amino acid sequence, catalytic activities, and immunochemical properties.

Amino Acid Sequence↗
PubMed1984

Purification and properties of cytochrome P-450 from untreated monkey liver microsomes.

Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogeneous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448(1) and MC P-448(2] catalyzed 7-ethoxycoumarin O-deethylation activities.

Animals↗
PubMed1983

The bioavailability of flufenamic acid and its dissolution rate from capsules.

The bioavailabilities of five commercially available flufenamic acid (FA) capsules were studied in humans and beagle dogs. The dissolution rates of these capsules were determined by several methods. Experiments on in vitro/in vivo and humans/dogs correlations were performed to evaluate the dissolution test methods and the values of beagle dogs as models for predicting bioavailability of weak acid drugs in humans. Significant differences in the rates and extents of bioavailability of the different capsules were observed both in humans and dogs, but results in humans differed from those in dogs. The dissolution rates, determined by dissolution methods involving pretreatment with acidic solutions, correlated significantly with bioavailabilities in humans and dogs; however, those obtained by the rotating basket and paddle methods without any surface active agents did not correlate with in vivo data.

Adult↗
PubMed1980

Isolation and characterization of a composite plasmid Rms201 mutant temperature sensitive for replication.

A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37 degrees C. When Escherichia coli ML1410(Rms201ts14)(+) was grown at temperatures between 40 and 42 degrees C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)(+) in L-broth was shifted to 42 from 30 degrees C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42 degrees C. At 30 degrees C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42 degrees C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42 degrees C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42 degrees C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 x 10(6) and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 x 10(6). These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.

Ampicillin↗