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J Takada

Publications and source records attributed to J Takada.

At least 73 records · Page 4Linked to original sources
PubMed1992

K(+)-site-directed pyridine derivative, AU-1421, activates hydrolysis of the K(+)-sensitive phosphoenzyme of sarcoplasmic reticulum Ca(2+)-ATPase and inactivates that of K(+)-transporting ATPases.

(Z)-5-Methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, AU-1421, interacted at 0 degree C with the K(+)-sensitive phosphoenzymes of three transport ATPases, Ca(2+)-, H+/K(+)- and Na+/K(+)-ATPase. In the case of Ca(2+)-ATPase, AU-1421 at about 80 microM stimulated 6-fold the rate of splitting of the phosphoenzyme, on which K+ simply functions as an accelerator from one side of the membrane. Probably AU-1421 also simply interacts with the K(+)-binding site of the phosphoenzyme that is easily accessible from the aqueous phase. In the cases of H(+)/K(+)- and Na(+)/K(+)-ATPases, AU-1421 stabilized the phosphoenzymes which accept K+ as the translocating ion. The rate constants of dephosphorylation for H(+)/K(+)-ATPase and Na(+)/K(+)-ATPase were decreased to half by AU-1421 at about 5 and 10 microM, respectively. Presumably after binding of AU-1421 to a K(+)-recognition site of the phosphoenzyme, local motion of the peptide region near the binding site that serves to move the bound ion into the ion-transport pathway (occlusion center) might be inhibited. Thus AU-1421 may be able to distinguish two modes of K+ action on the K(+)-sensitive phosphoenzymes.

Adenosine Triphosphatases↗
PubMed1992

Biochemical properties of endothelin converting enzyme in renal epithelial cell lines.

This is the first report clearly demonstrating the presence of endothelin (ET) converting enzyme (ECE) in non-vascular cells (renal epithelial cell lines, MDCK and LLC-PK1). ECEs derived from these epithelial cells were very similar to the endothelial ECE in the following biochemical properties: 1) The optimum pH was 7.0; 2) the Km value for big ET-1 was approximately 30 microM; 3) the enzyme was potently inhibited by EDTA, o-phenanthroline and phosphoramidon; and 4) the enzyme did not convert big ET-2 or big ET-3. These data suggest that phosphoramidon-sensitive ECE is involved in the processing of big ET-1 to ET-1 in the renal tubule.

Animals↗
PubMed1992

Usefulness of a novel monoclonal antibody against human osteocalcin in immunohistochemical diagnosis.

A novel monoclonal antibody against human osteocalcin, recently established in our laboratory, was shown by immunoblotting and immunohistochemistry to react specifically with human osteoblasts. In the present study, the antibody was applied to the immunohistochemical diagnosis of human bone tumours, especially osteoblastic tumours. The antibody reacted with all 27 osteosarcomas. No positive reaction was found either in chondrosarcoma, giant cell tumours of bone, soft tissue tumours or epithelial tumours. A positive reaction was found preferentially in the cytoplasm of most of the osteosarcoma cells, but not in the extracellular matrix. Since the antibody reacted with formalin-fixed and paraffin-embedded tissues, it will be a useful tool for routine immunohistochemical diagnosis of osteoblastic lesions.

Animals↗
PubMed1991

Importance of the C-terminal region of big endothelin-1 for specific conversion by phosphoramidon-sensitive endothelin converting enzyme.

Based on our previous findings that phosphoramidon-sensitive endothelin (ET) converting enzyme (ECE) converts human big ET-1 but does not big ET-3, we investigated structural requirement for substrate peptide. We prepared shorter peptides of big ET-1 and measured hydrolysis of the Trp-Val bond of these peptides. Relative hydrolysis ratios of big ET-1(1-38), (1-37), (16-37), (1-31) and (17-26) were 1, 1.15, 3.71, 0.01 and 0, respectively. In addition, big ET-2 and big ET-3 were not significantly converted by ECE. These results suggest that the carboxyl-terminal sequence at residues 32-37 of big ET-1 is important for conversion, whereas the amino-terminal disulfide loop structure appears to interfere with access of ECE to big ET-1.

Amino Acid Sequence↗
PubMed1991

Rapid inhibition of the K(+)-sensitive phosphoenzyme of Na+/K(+)-ATPase by (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine.

Membrane-bound Na+,K(+)-ATPase (0.1 mg/ml) was incubated with the K(+)-site-directed probe (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421) (Takada, J. et al. (1990) Biochim. Biophys. Acta 1037, 373-379) at 37 degrees C for 30 min in the absence of ligands, then the Na(+)-dependent phosphorylation level was examined in the presence of 10 microM [32P]ATP at 0 degrees C. The level was decreased to 50% and 0% by about 50 microM and 100 microM AU-1421, respectively. Addition of 1 mM K+ during the treatment with AU-1421 resulted in complete maintenance of the phosphorylation level. When the preincubation was performed at 0 degrees C for 10 s, even 100 microM AU-1421 did not impair the phosphorylation. In contrast to the non-phospho form of the enzyme, the K(+)-sensitive phosphoenzyme formed from ATP was immediately inhibited by the addition of AU-1421 at 0 degrees C. The reactivity of the inhibited phosphoenzyme was restored by the addition of K+. About 1 mM K+ gave the same maximal reactivity in the presence of various fixed concentrations (8-41 microM) of AU-1421, but the apparent affinity for K+ decreased simply with the increase of AU-1421 concentration. From this simple competitive relationship, the apparent Ki value of AU-1421 for the phosphoenzyme was calculated to be 7.2 microM. Compared to the non-phospho form of the enzyme, the phospho form appears to be rather susceptible to AU-1421, probably because the K(+)-site of the phosphoenzyme is exposed to the extracellular aqueous phase.

Adenosine Triphosphate↗
PubMed1991

Phosphoramidon-sensitive endothelin-converting enzyme in the cytosol of cultured bovine endothelial cells.

Neutral metalloproteases with endothelin-1 (ET-1) converting activity were detected in membranous and cytosolic fractions of cultured endothelial cells (EC) from bovine carotid artery in a ratio of 5:1, respectively. The cytosolic enzyme specifically and quantitatively converts big ET-1 to ET-1 (Km = 10.7 microM), but does not convert big ET-3. Like the membranous enzyme, the cytosolic enzyme is only active at pH 6.5-7.5, and is competitively inhibited by phosphoramidon (Ki = 0.79 microM). The apparent molecular weight of the cytosolic enzyme is about 540 kD, which is 5-6 times greater than that of the membranous enzyme. These results indicate the presence of two types of phosphoramidon-sensitive neutral ET-converting enzyme in vascular EC.

Animals↗
PubMed1991

Production of monoclonal antibodies specific for human bone gamma-carboxyglutamic acid containing protein.

Murine monoclonal antibodies specific for human bone gamma-carboxyglutamic acid containing protein (BGP) were produced against BGP purified from young adult human long bones. The amino acid composition of purified protein corresponds with that of human BGP, and Western blot analysis revealed that the antibodies reacted most intensely with the cytoplasm of osteoblasts and less intensely with the cytoplasm of osteocytes, but did not react with any other cells, such as chondrocytes, or osteoclasts. Because of their ability to react with routinely processed tissue sections and their marked reactivity with human osteoblastic cells, the antibodies are expected to be a useful tool for studying the process of ossification in human bones and for the immunohistochemical diagnosis of human osteogenic tumours.

Amino Acids↗
PubMed1991

Endothelin-converting enzyme and its in vitro and in vivo inhibition.

We investigated endothelin (ET)-converting enzyme and its localization in the vasculature. The membrane and cytosol fractions of cultured endothelial cells of bovine carotid artery contain phosphoramidon-sensitive ET-converting enzymes, and their molecular weights are about 100 and 540 kDa, respectively. The specific conversion of big ET-1 by these enzymes proceeds at pH 7.0 +/- 0.5, and it is inhibited by EDTA, o-phenanthroline, and phosphoramidon. Big ET-3 is converted by the membrane enzyme at a rate about one-tenth that of big ET-1, but it is not converted by the cytosol enzyme. Big ET-1 (but not ET-1)-induced hypertension in rats was remarkably suppressed by pretreatment with phosphoramidon, and big ET-1 (but not ET-1)-induced contraction of isolated coronary arteries, either with or without the endothelium, was substantially suppressed by phosphoramidon. These results suggest an essential role of phosphoramidon-sensitive enzyme(s) in the vascular conversion of big ET-1, and the existence of such enzymes also in nonendothelial cells. We found three converting enzymes operating at different optimal pH values in noncultured vascular smooth muscle cells; two pepstatin-sensitive, cytosolic acid proteinases and a phosphoramidon-sensitive neutral enzyme(s) in the membrane and cytosol. All of these findings strongly suggest the importance of phosphoramidon-sensitive neutral enzymes in the vascular conversion of big ET-1.

Animals↗
PubMed1991

Environmental differences of a pyridine derivative (AU-1421)-access sites between (H+,K+)-ATPase and (Na+,K+)-ATPase.

We investigated the environmental differences of a pyridine derivative-access sites between hog gastric (H+,K+)-ATPase and dog kidney (Na+,K+)-ATPase. The environmental differences are inferred from the marked contrasts in inhibition properties of (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine hydrochloride (AU-1421) between the two adenosine triphosphatases (ATPases). AU-1421 is a hydrophobic pyridine derivative which inhibits (H+,K+)-ATPase and (Na+,K+)-ATPase competitively with respect to K+. AU-1421 inhibition of (Na+,K+)-ATPase activity is preincubation time-dependent, pH-independent (pH 6.4-8.4), and hardly reversible by dialysis. These features are opposite to those found with the gastric (H+,K+)-ATPase. Furthermore, in the presence of dilute sodium dodecyl sulfate (30-120 microM), the inhibitory potency of AU-1421 for (Na+,K+)-ATPase is increased, whereas the inhibitory potency for (H+,K+)-ATPase is decreased. These results favor proposals that protonated species of AU-1421 interact with its access-site of (H+,K+)-ATPase at the exterior surface of the enzyme molecule by ionic interaction, whereas AU-1421 interacts with its access-site of (NA+,K+)-ATPase at the interior domain of the enzyme molecule predominantly by hydrophobic interaction. The differences indicate the existence of a different feature of K(+)-access sites between the two ATPases.

Adenosine Triphosphatases↗
PubMed1991

Changes in mouse brain monoamine oxidase activity in the first, second and fourth generations after manganese administration.

The present study was conducted to explore whether or not manganese effect on brain monoamine oxidase (EC 1.4.3.4) is subject to hereditary genetic amplification. Mice of both sexes were given manganese through four generations, and the enzyme activity was measured in the cerebral cortex, cerebellum, hypothalamus and hippocampus of each of the generations except for the third, whose activity we were not in a position to measure. Intrinsic enzyme activity was highest in the cerebellum, and was followed by those in the cerebral cortex and hypothalamus. The activity in the hippocampus was the lowest. Manganese administration greatly stimulated the activity in the cerebellum. However, as generation succeeded, the level of susceptibility to manganese gradually declined. Manganese concentration in pooled suborgan fractions proved to be, in every case, higher in the cerebral cortex, cerebellum and hippocampus and lower in the hypothalamus. No indication was found that the manganese effect is genetically inherited.

Activation Analysis↗
PubMed1991

[Normal and abnormal aging of cardiovascular system].

Studies of normal aging in the cardiovascular system in humans are affected by the study population. Besides intrinsic biological aging, extrinsic factors including overt or latent cardiovascular diseases as well as life style variables such as physical activity, diet, alcohol and smoking may influence the age-related changes of cardiovascular function. We have recruited "normal" elderly subjects from community-dwelling volunteers by extensive health screening procedures including treadmill maximum exercise tests. Some of their cardiovascular functions, such as various cardiovascular regulatory functions, were altered compared to normal young subjects, while others such as resting hemodynamics were not. Interrelationships among various autonomic functions in the elderly were not recognized. Although general effects of life styles on circulatory regulatory functions were not clearly indicated, variables such as sodium intake or body mass index appeared to affect some of the sympathetic nervous functions. Furthermore, hypertension in the elderly had much less impact on cardiovascular functions than is generally expected, based on the results from young or middle-aged subjects. To identify factors which either modify (accelerate) or do not affect the aging of the cardiovascular functions is important not only to achieve a good aging process but also to establish therapeutic goals in elderly subjects.

Aged↗
PubMed1990

(Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), calcium ions and ethylenediamine as the K(+)-site directed probe for Na+/K(+)-ATPase.

Recently, we have shown that a hydrophobic amine (AU-1421) produces an irreversible inactivation of Na+/K(+)-ATPase activity. This inactivation was prevented by K+ and its congeners. In this study, we examined the possibility of Ca2+ or ethylenediamine as a probe of the K+ occlusion center of Na+/K(+)-ATPase. The inactivation by AU-1421 was prevented by Ca2+ with an apparent high affinity (approximately 0.1 mM). Ca2+ protection was cancelled by high concentrations of ATP, ADP or Mg2+. Ca2+ and K+ were similar in these respects. Kinetic analyses of the above data indicated the presence of two AU-1421 occlusion sites on the enzyme, either one of which is susceptible to Ca2+ occlusion. Ethylenediamine also prevented the inactivation by AU-1421 or by C12E8 solubilization of the enzyme, suggesting that ethylenediamine, like K+, stabilized the enzyme. However, an apparent affinity of ethylenediamine (approximately 1.4 mM) was one order of magnitude lower than that of K+ (approximately 0.2 mM), and the protective manner did not show a simple competition. In addition, ethylenediamine binding was unaffected by ATP or ADP at a low affinity site, and antagonized K+ binding. From these results we concluded that ethylenediamine does not act like K+ or Ca2+ in protecting AU-1421 inactivation, since it can't stabilize the enzyme conformation as an E2 (K(+)-bound form).

Animals↗
PubMed1990

Reversible inhibition of sodium and potassium-dependent adenosine triphosphatase by the pyridine derivative, AU-1421 during turnover cycle.

A novel pyridine derivative, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidonopyridine hydrochloride, AU-1421, was found to produce reversible inhibition of the dog kidney sodium and potassium ion-dependent adenosine triphosphatase [(Na,K)-ATPase] with I50 values of about 50 microM. The reversible inhibition was observed when the enzyme was added directly to the enzyme assay media in the presence of saturating concentrations of the enzyme ligands, Na+, K+, Mg2+ and ATP ("turnover conditions"). In the present study, we focused on the reversible inhibition without preincubation of the enzyme with AU-1421. This inhibition was competitive with respect to K+. The K(+)-pNPPase activity of the same preparation was also inhibited by AU-1421 with I50 values of about 90 microM, and this manner was also competitive with respect to K+. ATP enhanced the AU-1421 inhibition of (Na,K)-ATPase, suggesting that AU-1421 also bound to the enzyme-substrate complex. AU-1421 inhibition of (Na,K)-ATPase was not antagonized by ouabain, suggesting the difference of the binding sites between AU-1421 and ouabain. It is therefore proposed that AU-1421 reversibly interacts at or near the K+ site during turnover conditions.

4-Nitrophenylphosphatase↗
PubMed1990

Conversion of big endothelin-1 by membrane-bound metalloendopeptidase in cultured bovine endothelial cells.

We propose a candidate for the "putative" endothelin (ET) converting enzyme in the cultured endothelial cells (ECs) of bovine carotid artery. The enzyme is membrane-bound, soluble in 0.5% Triton X-100, and capable of converting human big ET-1 to ET-1 by a specific cleavage between Trp21 and Val22. The conversion reached 90% after a 5-hr incubation in the presence of DFP, PCMS and pepstatin A, but it was inhibited by EDTA, omicron-phenanthroline or phosphoramidon. The enzyme is very sensitive to pH, and active only between pH 6.6 and pH 7.6. Conversion of big ET-3 by this enzyme was only 1/9 that of big ET-1. From these results, ET-1 converting enzyme in the bovine EC is most likely to be a membrane-bound, neutral metalloendopeptidase, which is much less susceptible to big ET-3.

Amino Acid Sequence↗
PubMed1990

A novel hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine, as a probe of the K+ occlusion center of Na+/K(+)-ATPase.

A hydrophobic amine, (Z)-5-methyl-2-[2-(1-naphthyl)ethenyl]-4-piperidinopyridine (AU-1421), was examined as a probe of the K+ occlusion center of Na+/K(+)-ATPase. Treatment of the enzyme with AU-1421 at 37 degrees C and pH 7.0 produced irreversible inactivation of the enzyme. This inactivation was prevented, with simple competitive kinetics, by K+ or its congeners in the order of Tl+ greater than Rb+ greater than NH+4 greater than Cs+. The concentrations of these cations required for the protection, were consistent with the affinities for transport and ATPase activity. The apparent binding constant for K+ was calculated to be 0.03 mM, from the competition with AU-1421. This protection was cancelled by a high concentration of ATP or ADP. A high concentration of Na+ (Kd = 6.5-6.9 mM), as a substitute for K+, also prevented the inactivation by AU-1421. Thus, the enzyme was protected from AU-1421 when the occlusion center was occupied by a monovalent cation, irrespective of the enzyme conformation, E1 (Na(+)-bound form) or E2 (K(+)-bound form). On the other hand, the enzyme was most sensitive to AU-1421 in the presence of low concentration of Na+ (0.4-0.8 mM) or a high concentration of ATP. Tris, imidazole or choline, which favors the E1 state, also accelerated the inactivation by AU-1421. These suggest that AU-1421 reacts with the occlusion center through the E1 state.

Adenosine Diphosphate↗