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1.
N Yamano Y Kawata H Kojima K Yoda M Yamasaki 《Bioscience, biotechnology, and biochemistry》1992,56(7):1017-1026
Biotinylation of fusion proteins in E. coli was studied using a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. As the biotinylation sequence, we examined two sequences: one was of amino acid residues [84-123] of 1.3S, a partial sequence containing a region from a conserved tetrapeptide (Ala-Met-Bct-Met) around the biotinyl lysine (Bct) to the carboxyl terminal; the other was of an almost entire sequence [18-123]. We constructed recombinant plasmids for fusion proteins of beta-galactosidase, of chloramphenicol acetyltransferase, and of alkaline phosphatase. We found the biotinylation in the [18-123] sequence fused to alkaline phosphatase. 相似文献
2.
Keisuke Yamano Masatomo Tagawa Evelyn Grace de Jesus Tetsuya Hirano Satoshi Miwa Yasuo Inui 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1991,161(4):371-375
Summary To clarify the hormonal regulation of metamorphosis of the conger eel (Conger myriaster), changes in whole body concentrations of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), and cortisol during metamorphosis were examined, as well as the changes in the histological activity of the thyroid gland. In larvae before metamorphosis, T4 and T3 levels were less than 5 and 0.15 ng·g-1 respectively. Levels of T4 increased to about 30 ng·g-1 during early metamorphosis, and decreased subsequently. Levels of T3 increased gradually in early metamorphosis, and then increased abruptly to about 2.0 ng·g-1 in late metamorphosis. Before metamorphosis, cortisol levels of the leptocephali less than 11 cm in total length were greater than 200 ng·g-1. Cortisol levels decreased rapidly in larger premetamorphic leptocephali, and low levels were maintained throughout the metamorphic period. Histological observation revealed an activation of the thyroid gland in early metamorphosis; thyroid follicle epithelial cells became columnar and their nuclei larger. Active uptake of colloid by these cells and intensive vascularization of the gland were also observed. By the end of metamorphosis, follicle epithelial cells became squamous, indicating a low level of glandular activity. These results suggest that thyroid hormone plays an important role in regulation of conger eel metamorphosis.Abbreviations
AL
anal length
-
TL
total length
-
T
3
triiodothyronine
-
T
4
thyroxine 相似文献
3.
Molecular cloning and sequence analysis of full-length cDNA for rabbit liver NADPH-cytochrome P-450 reductase mRNA 总被引:4,自引:0,他引:4
M Katagiri H Murakami Y Yabusaki T Sugiyama M Okamoto T Yamano H Ohkawa 《Journal of biochemistry》1986,100(4):945-954
The nucleotide sequence of the mRNA for NADPH-cytochrome P-450 reductase from rabbit liver was determined from a full-length cDNA clone (pFP105). The clone contains 2,269 nucleotides complementary to rabbit liver reductase mRNA. The single open reading frame of 2,037 nucleotides codes for a 679-amino acid polypeptide with a calculated molecular weight of 76,583 daltons. The cloned cDNA contains the complete 3'-noncoding region of 193 nucleotides, including 68 nucleotides of poly(A), and 39 nucleotides of the 5'-noncoding region. The nucleotide sequence in the coding region of cDNA of rabbit reductase (pFP105) showed 85% homology to that of rat reductase (Porter, T.D. & Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U.S. 82, 973-977, and Murakami, H. et al. (1986) DNA 5, 1-10). Rabbit reductase has one more amino acid residue than the rat enzyme, and the amino acid compositions of the two enzymes are similar. The amino acid sequence of the rabbit enzyme showed 91% identity with that of the rat enzyme. The segment related to binding of FMN and FAD was well conserved among rabbit, rat, and pig reductases. The sequence related to AMP moiety-binding was also conserved among these species, and was found in the amino acid sequence of NADH-cytochrome b5 reductase, another flavoenzyme in the microsomal electron transport system. 相似文献
4.
T Ohnishi A Wada Y Nonaka T Sugiyama T Yamano M Okamoto 《Journal of biochemistry》1986,100(4):1065-1076
Bovine adrenocortical calmodulin was purified and its general properties were examined. The latter were similar to those of bovine brain calmodulin. When added to a cytochrome P-450(11)beta-reconstituted system in the presence of dilauroylphosphatidylcholine, calmodulin decreased the rate of aldosterone production from corticosterone from 0.8 to 0.1 nmol/(min X nmol P-450), while it increased the rate of 18-hydroxycorticosterone production from 1.8 to 4.6 nmol/(min X nmol P-450). This effect of calmodulin on steroid production was maximum at a concentration of 1 microM, when 1 microM cytochrome P-450(11)beta was used. The effect was dependent on the presence of Ca2+, and maximal response was observed at less than 1 microM Ca2+. There was essentially no difference in the effect when bovine brain calmodulin was used. Calmodulin induced a change in the activity of cytochrome P-450(11)beta in the presence of a wide concentration range of corticosterone as a substrate. As for 18-hydroxycorticosterone production, calmodulin increased both the maximal activity and the apparent Km for corticosterone, but it decreased the apparent Km for adrenodoxin. Adrenodoxin at a concentration of less than 20 microM did not fully abolish the effect of calmodulin. A small type I difference spectrum appeared when calmodulin was added to cytochrome P-450(11)beta. The difference spectrum increased significantly in the presence of both Ca2+ and adrenodoxin. These results suggest that calmodulin interacts with cytochrome P-450(11)beta in the presence of adrenodoxin and then modulates the activity of aldosterone synthesis catalyzed by cytochrome P-450(11) beta. 相似文献
5.
Human plasma high density lipoproteins (HDL) have been labeled with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide (NEM-TEMPO). The spin-labeled HDL exhibited an ESR spectrum containing signals of both strongly immobilized and weakly immobilized components by the reaction with a high concentration of NEM-TEMPO, while an ESR spectrum containing only signals of a strongly immobilized component range between 4 degrees C and 37 degrees C, the signal height of the strongly immobilized component exhibited reversible temperature-dependent changes, whereas that of the weakly immobilized component changed irreversibly at temperatures above 25 degrees C. The activation energy of the irreversible change was estimated to be 26 kcal per mol. The strongly immobilized component was derived from NEM-TEMPO which modified apolipoprotein A-I covalently, while the weakly immobilized component was derived from NEM-TEMPO noncovalently bound to HDL. The rate of binding of NEM-TEMPO to either the strongly binding or weakly binding sites and the number of the strongly binding sites in apolipoprotein A-I were estimated to be 125 M-1.day-1 and 1.78, respectively. The binding of NEM-TEMPO to the strongly binding sites was suppressed greatly by pretreatment of HDL with 2,4,6-trinitrobenzene sulfonic acid (TNBS). The slow reaction and suppression with TNBS suggest that NEM-TEMPO binds to some amino acid residue, probably a lysine residue, in apoprotein A-I. The strongly immobilized and weakly immobilized components were reduced almost completely by ascorbate at the same rate, 0.048 min-1 at pH 7.4 and at 4 degrees C. 相似文献
6.
Interpretation of the stokes radius of macromolecules determined by gel filtration chromatography 总被引:4,自引:0,他引:4
From a comparison of the gel chromatographic properties of large randomly-coiled polypeptides in 6 M guanidine hydrochloride and of large globular proteins, we found that the distribution coefficient was more closely correlated with the intrinsic viscosity-based Stokes radius than with the translational frictional coefficient-based Stokes radius. This means that the effect of the hydrodynamic flow of dissolved molecules during gel chromatography should be considered. The ratio of transport of solute by bulk flow as compared with that by net diffusion (i.e., Brownian motion) is large under some conditions. On the other hand, we consider that the distribution coefficient obtained in static equilibrium experiments should be determined by the translational frictional coefficient-based Stokes radius, since the solvent does not flow. On this basis, we discuss the meaning of the Stokes radius and the separation mechanism of macromolecules by gel filtration. 相似文献
7.
8.
9.
K Ohyama Y Yamano S Chaki T Kondo T Inagami 《Biochemical and biophysical research communications》1992,189(2):677-683
To delineate domains essential for G-protein coupling in angiotensin II type 1 receptor (AT1), we mutated the receptor cDNA in the putative cytosolic regions and determined consequent changes in the effect of GTP analogs on angiotensin II (Ang II) binding and in inositol trisphosphate production in response to Ang II. Polar residues in targeted areas were replaced by small neutral residues. Mutations in the second cytosolic loop, carboxy terminal region of the third cytosolic loop or deletional mutation in the carboxyl terminal tail simultaneously abolished both the GTP-induced shift to the low affinity form and Ang II-induced stimulation of inositol trisphosphate production. These results suggest that polar residues in the second cytosolic loop, the carboxy terminal region of the third cytosolic loop, and the carboxy terminal cytosolic tail are important for G-protein coupling of AT1 receptor. 相似文献
10.
Y Yamano K Ohyama S Chaki D F Guo T Inagami 《Biochemical and biophysical research communications》1992,187(3):1426-1431
To determine the specific mechanism of ligand binding to angiotensin (Ang II) receptor AT1, mutagenized rat receptor cDNAs were expressed transiently in COS-7 cells and the effect of the mutations on the binding to peptidic and non-peptidic ligands was analyzed by Scatchard plots. Mutation of Lys199 to Gln in the intramembrane domain strongly reduced the affinity to both [125I] Ang II and [125I]-1Sar, 8Ile-Ang II whereas mutation of two other Lys had little effect, indicating involvement of Lys199 in binding ligands. Replacement of each of four Cys in the extracellular domain markedly reduced binding affinity, indicating the importance of two putative disulfide bridges in the formation of active receptor conformation. Substitution of Asp for Asn in N-glycosylation had no effect on ligand binding or expression of the receptor. These studies indicate mutated receptors are expressed in the plasma membrane and are amenable for further detailed studies. 相似文献