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Biomedical subjects

S Takemori

Publications and source records attributed to S Takemori.

At least 91 records · Page 5Linked to original sources
PubMed1988

Relationship between zonal distribution of microsomal cytochrome P-450s (P-450(17)alpha,lyase and P-450C21) and steroidogenic activities in guinea-pig adrenal cortex.

In an attempt to elucidate the functional role of the three zones of the adrenal cortex, the localization of microsomal cytochrome P-450s (P450(17)alpha,lyase and P-450C21) and their relation to steroidogenesis in the guinea-pig adrenal cortex were studied. The intracellular localization and zonal distribution of P-450(17)alpha,lyase and P-450C21 were examined by the direct peroxidase-labelled antibody technique. The cytochromes were localized immunocytochemically on the smooth-surfaced endoplasmic reticulum. P-450(17)alpha,lyase was found to be distributed in the zona fasciculata, especially in the externa, and less in the zona reticularis. The zona glomerulosa was negative for immunohistochemical staining for P-450(17)alpha,lyase. In contrast, P-450C21 was distributed in all three zones in the adrenal cortex. The cytochrome P-450 contents and their steroidogenic activities were determined with microsomes prepared from the outer zone (zonae glomerulosa and fasciculata) and from the inner zone (zona reticularis) of guinea-pig adrenals. Using an enzyme-linked immunosorbent assay, the ratio of P-450C21 content in the inner zone to that in the outer zone microsomes was estimated to be 1.24. 21-Hydroxylase activity was higher in the inner zone microsomes, which was in good agreement with the relative P-450C21 contents. The ratio of P-450(17)alpha,lyase content in the inner zone to that in the outer zone microsomes was estimated to be 0.75. 17 alpha-Hydroxylase and C17-20-lyase activities were five- to sixfold greater in the outer than in the inner zone microsomes, which could not be explained by the P-450(17)alpha,lyase contents in the two zones.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Glands↗
PubMed1987

Two modes of binding of adrenal NADPH-cytochrome P-450 reductase to liposomal membranes.

NADPH-cytochrome P-450 reductase, purified from bovine adrenocortical microsomes, was shown to bind in two different modes to liposomal membranes composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. As demonstrated by Ficoll density gradient centrifugation and HPLC gel filtration, the cholate dialysis method made the reductase bind tightly to the liposomal membranes, while the incubation with the preformed vesicles made the reductase bind loosely to the membranes. From the experiments of electron transfer to P-450C21 residing at the other vesicles, the loosely bound reductase was found to be transferable between the vesicles, whereas the tightly bound reductase was not readily transferred. The rates of the binding and the release of the loosely bound reductase to and from the membranes were measured with the stopped-flow method by observing the reduction of P-450C21 embedded in the vesicles. These kinetic studies showed that the rate-limiting step of the reductase transfer between the vesicles was the release of the reductase from the membranes. The reductase in both binding modes well supported the steroid 21-hydroxylase activity.

Adrenal Cortex↗
PubMed1986

Structural analysis of cloned cDNA for mRNA of microsomal cytochrome P-450(C21) which catalyzes steroid 21-hydroxylation in bovine adrenal cortex.

We have isolated cDNA clones of the mRNA for cytochrome P-450 that catalyzes the steroid C-21 hydroxylation (P-450(C21)), which specifically catalyzes 21-hydroxylation of steroids in the microsomes of bovine adrenal cortex by using synthetic oligonucleotides as probes. Sequence determination of the cloned cDNA showed that it contains 2157 nucleotides and a poly(A) chain and that a single open reading frame of 1488 nucleotides codes for a polypeptide of 496 amino acids with a molecular weight of 56,113. The deduced amino acid composition is in agreement with that determined by direct amino acid analysis of purified P-450(C21) and the predicted primary structure contained amino acid sequences of N-terminal region and two internal tryptic fragments of the protein so far analyzed. Comparing the amino acid sequence with those of other forms of P-450 reveals that a conserved amino acid sequence containing a putative heme-binding cysteine is present in the equivalent position, proximate to the COOH terminus of the molecules and that P-450(C21) is phylogenically situated in an intermediate position between steroidogenic mitochondrial cytochrome P-450 which catalyzes the side-chain cleavage of cholesterol (P-450(SCC)) and drug-metabolizing microsomal P-450s. However, the amino acid sequence of P-450(C21) is much closer to that of drug-metabolizing P-450s than to that of P-450(SCC).

Adrenal Cortex↗
PubMed1986

Studies on the interaction of steroid substrates with adrenal microsomal cytochrome P-450 (P-450C21) in liposome membranes.

Cytochrome P-450 (P-450C21), purified from bovine adrenocortical microsomes, was incorporated into the single bilayer liposomes of egg yolk phosphatidylcholine by gel filtration, using a high pressure liquid chromatography system. Interaction of the steroid substrates, 17 alpha-hydroxyprogesterone and progesterone, with P-450C21 in the liposomes was studied in the equilibrium state by measuring substrate-induced spectral change. The apparent dissociation constant of the P-450C21-substrate complex increased with phosphatidylcholine concentration in the system, showing the substrate to be partitioned between the aqueous and lipid phases. Partition coefficients, determined by equilibrium dialysis and the Hummel-Dreyer method, were 3500 for progesterone and 2000 for 17 alpha-hydroxyprogesterone at 25 degrees C. The binding process of the substrates to P-450C21 in the liposomes and their dissociation were measured by a stopped flow method. The apparent rate of substrate binding to P-450C21 in the liposomes was not effected by substrate partitioning, indicating partitioning to occur much more quickly than substrate binding to P-450C21. Absorption changes observed in the stopped flow experiments were analyzed at a rapid equilibrium of partitioning. Based on these results, the substrate binding site of P-450C21 was concluded to face the lipid phase of the liposome membranes.

17-alpha-Hydroxyprogesterone↗
PubMed1986

Graphical analysis of two-dimensional eye movements.

Horizontal and vertical monocular movements were recorded by electronystagmography using silver plate electrodes or by the magnetic search coil system. Both horizontal and vertical monocular movements were simultaneously measured with the use of a computer-controlled testing system. The data was composed by the computer and presented as a two-dimensional graphical plot of the actual eye movement trajectories. There are three factors in eye movements, that is, slow component of eye movements--pursuit; quick component--saccades; and fixation. The two-dimensional plot of pursuit monocular movements in normal subjects indicated that the eye did not move in a perfectly straight horizontal or vertical line. The plot appeared to be a series of smooth and snaky movements. Saccades showed quick jumping movement to reach a target and fixation. During fixation, eye position was not restricted to the point of a target and the eye moved around the target. Disturbance of pursuit is known as saccadic or ataxic. A two-dimensional plot of pathological pursuit showed a series of smaller saccades and fixation. Disturbance of saccades is shown as overshoot or undershoot. Also, a two-dimensional plot of pathological saccades was made of smaller saccades and of fixation. Failure of fixation showed larger and more irregular movements around the target. Thus, the two-dimensional plot of monocular movements clearly showed normal or pathological pursuit, saccades, and fixation.

Brain Diseases↗
PubMed1985

Studies on cytochrome P-450 (P-450 17 alpha,lyase) from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome b5.

We have reported (Kominami, S., Shinzawa K. and Takemori, S. (1982) Biochem. Biophys. Res. Commun. 109, 916-921) that a cytochrome P-450 purified from guinea pig adrenal microsomes shows 17 alpha-hydroxylase and C-17,20-lyase activities in a reconstituted system with NADPH-cytochrome P-450 reductase. The homogeneity of the purified cytochrome P-450 was examined with the following methods: isoelectric focusing, immunoelectrophoresis and affinity chromatography on cytochrome b5-immobilized Sepharose. It was found that progesterone competitively inhibited C-17,20-lyase reaction and that progesterone was converted into androstenedione by 17 alpha-hydroxylation followed by the lyase reaction. These results indicate that the dual activities are carried out by a single enzyme (P-450 17 alpha,lyase). P-450 17 alpha,lyase had the maximum activity at pH 6.1 both for 17 alpha-hydroxylation (6.0 nmol/min per nmol of P-450) and the lyase reaction (11.0 nmol/min per nmol of P-450). Upon addition of cytochrome b5 to the reconstituted system, the optimal pH for 17 alpha-hydroxylation was shifted to 7.0 and that of the lyase reaction to 6.6. The maximum activities at these optimal pH values were almost the same in the presence or absence of cytochrome b5. With the addition of cytochrome b5, both the activities were stimulated above pH 6.3-6.5 and were suppressed below pH 6.3-6.5. These results indicate that cytochrome b5 plays some important role in controlling the dual activities of P-450 17alpha,lyase.

Adrenal Glands↗
PubMed1985

Vestibular training after sudden loss of vestibular functions.

12 cases of unilateral labyrinthectomy, 3 cases of VIIIth nerve section, 22 cases of streptomycin sulfate infusion into the middle ear cavity and 8 cases of bilateral vestibular a-functions underwent vestibular training. Our training is very useful for regaining equilibrium and for evaluating the effects of training on equilibrium by recording the gravity center movements.

Adult↗
PubMed1985

Recovery of vestibulospinal balance function after unilateral labyrinthectomy in patients with Ménière's disease.

The influence of unilateral labyrinthectomy on vestibulospinal balance function in patients with Ménière's disease is studied with a gravicorder. Our data disclosed that vestibulospinal balance recovery was better when tested in the light, including the total length and area. Moreover, this quantitative test which measures changes in the center of gravity in standing subjects revealed that total length recovery was almost achieved within 5 weeks when measured in the light but required 12 months when measured in the dark; total area was almost in the normal range within 1 week when measured in the light but required 5 weeks when measured in the dark.

Adult↗
PubMed1984

Interaction between cytochrome P-450 (P-450C21) and NADPH-cytochrome P-450 reductase from adrenocortical microsomes in a reconstituted system.

The interaction between P-450C21 and NADPH-cytochrome P-450 reductase, both purified from bovine adrenocortical microsomes, has been investigated in a reconstituted system with a nonionic detergent, Emulgen 913, by kinetic analysis and gel filtrations. Steady state kinetic data in progesterone 21-hydroxylation showed formation of an equimolar complex between the two enzyme proteins at low Emulgen concentration. Steady state kinetic studies on the electron transfer from NADPH to P-450C21 via the reductase showed that a stable complex formation between the two enzyme proteins was not involved in the steady state electron transfer at high Emulgen concentration. In stopped flow experiments, a time course of the P-450C21 reduction showed biphasic kinetics composed of fast and slow phases. The dependence of kinetic parameters on Emulgen concentration indicates that the fast phase corresponds to the electron transfer within the complex and the slow phase to the electron transfer through a random collision between P-450C21 and the reductase. The stable complex formation between P-450C21 and the reductase has been clearly demonstrated by gel filtration. The stable complex was composed of several molecules of the two enzyme proteins at an equimolar ratio, which was active for progesterone 21-hydroxylation and had a tendency to dissociate at high Emulgen concentration.

Adrenal Cortex↗
PubMed1984

Saccades--random or constant stimuli and predictive adaptability.

The influence of the predictive adaptability on saccades was studied in 20 normal subjects ranging in age from 18 to 43 years. The average velocity of random and constant saccades showed almost the same results. The maximum velocity of constant saccades was slightly faster than that of the random saccades. The latency of the random saccades was shorter than that of the constant ones. Thus, the predictive adaptability of the stimuli has little influence on the maximum velocity and on the latency of saccades in normal subjects.

Adaptation, Ocular↗
PubMed1983

Immunochemical studies on cytochrome P-450 in adrenal microsomes.

An antibody was prepared against electrophoretically homogeneous cytochrome P-450C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450C21 and guinea pig adrenal microsomes against anti-cytochrome P-450C21 IgG. Anti-cytochrome P-450C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450C21 IgG did not inhibit the activities of 17 alpha-hydroxylase and C17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450C21 catalyzes the 17 alpha-hydroxylation and C17,20 bond cleavage. The stimulation of 17 alpha-hydroxylation and C17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.

Adrenal Cortex↗
PubMed1983

Contributions of cytoplasmic free and membrane-bound ribosomes to the synthesis of mitochondrial cytochrome P-450(SCC) and P-450(11 beta) and microsomal cytochrome P-450(C-21) in bovine adrenal cortex.

Rabbit antibodies against cytochrome P-450 (SCC), P-450 (11 beta), and P-450 (C-21) from bovine adrenal cortex were prepared, and it was confirmed that these three cytochrome P-450 species are immunologically distinct from one another. Cytoplasmic sites of synthesis of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) in bovine adrenal cortex were determined by examining the presence of their nascent peptides on isolated free and bound ribosomes. Nascent peptides were released in vitro from ribosomes by [3H]puromycin in a high salt buffer in the presence of a detergent, and the nascent peptides of P-450 (SCC), P-450 (11 beta), and P-450 (C-21) were isolated by immunoprecipitation. The nascent peptides of these three cytochrome P-450 species were found in both free and bound ribosomal fractions, suggesting that they share common sites of synthesis in the cytoplasm. However, the nascent peptides of mitochondrial P-450 (SCC) and P-450 (11 beta) were more concentrated in the free ribosomal fraction, whereas those of microsomal P-450 (C-21) were more abundant in the bound ribosomal fraction. The nascent peptides of the three cytochrome P-450 species were released from the membrane-bound ribosomes of rough microsomes into the cytoplasmic surface of microsomal vesicles by puromycin treatment.

Adrenal Cortex↗