Biomedical subjects
Publications and source records attributed to K Takeyasu.
DNA-cytochrome c complexes adsorbed on carbon-coated mica surfaces were directly imaged by atomic force microscopy in air using commercially available cantilevers, with a routine resolution of 6 nm. Images of M13 phage DNA and M13-DNA polymerase complex are also shown.
To assess the role of cis-acting elements within the smooth muscle alpha-actin gene in smooth muscle cells (SMC), we transfected chicken smooth muscle alpha-actin promoter-chloramphenicol acetyltransferase gene fusion plasmids into SMC derived from rat and chicken aortas. In marked contrast to effects in chicken skeletal myoblasts and fibroblasts, p122CAT (positions -122 to +19), containing two conserved CArG elements, elicited a modest increase in chloramphenicol acetyltransferase reporter activity in chicken SMC. Addition of upstream sequences between -122 and -151 (p151CAT) increased activity in adult chicken SMC. Addition of sequence between positions -151 and -257 (p257CAT) resulted in a 7-fold increase in chloramphenicol acetyltransferase activity over that of p151CAT in rat SMC, but not in chicken SMC. A genomic clone encoding the rat smooth muscle alpha-actin gene was isolated, and the 5'-flanking region was partially characterized. Comparison of primary sequence between rat and chicken promoters showed a conserved E box motif at position -214 in the chicken gene and at position -213 in the rat gene. Results of these studies demonstrate that regions upstream of the conserved CArG elements exert potent regulatory effects on transcription and that SMC require different cis-acting elements than other cell types to transcriptionally regulate this gene.
Ferritin molecules were imaged directly in air by scanning tunneling microscopy (STM). The lateral dimensions were close to the values determined by electron microscopy, and the vertical dimension was much reduced. Several clusters of partially naked ferritin cores displayed a hexagonal structure of lattice constant 4.9 +/- 0.5 A. It is thus shown that the STM can be used to image thin ionic crystals at high resolution.
Proposed models for the catalytic subunit of the E1E2-ATPases (ion pumps) predict that the first four transmembrane domains (M1 - M4) reside in the NH2 terminal one-third of the molecule, and the remainder (M5 - M10) in the COOH terminal one-third. The amino-acid sequences for the 5'-(p-fluorosulfonyl)-benzoyl-adenosine (FSBA) binding region residing just before M5 segment are very well conserved among distinct ion pumps. Taking advantage of these models, we have constructed a set of chicken chimeric ion pumps between the (Na++ K+)-ATPase alpha-subunit and the Ca(2+)-ATPase using the FSBA-binding site as an exchange junction, thereby preserving overall topological structure as E1E2 ATPases. From various functional assays on these chimeric ion pumps, including ouabain-inhibitable ATPase activity, Ca2+ binding, Ca2+ uptake, and subunit assembly based on immuno-coprecipitation, the following conclusions were obtained: (a) A (Na++ K+)-ATPase inhibitor, ouabain, binds to the regions before M4 in the alpha-subunit and exerts its inhibitory effect. (b) The regions after M5 of the (Na++ K+)-ATPase alpha-subunit bind the beta-subunit, even when these regions are incorporated into the corresponding domains in the Ca(2+)-ATPase. (c) The corresponding domains of the Ca(2+)-ATPase, the regions after M5, bind 45Ca even when it is incorporated into the corresponding position of the (Na++ K+)-ATPase alpha-subunit.
Chimeric ion-pumps, consisting of the N-terminal 2/3 of the alpha 1-subunit of the ouabain-sensitive chicken Na+,K(+)-ATPase and the C-terminal 1/3 of the sarcoplasmic reticulum Ca2(+)-ATPase, were expressed in ouabain-insensitive mouse L cells. These chimeric molecules exhibited ouabain-sensitive ATPase activity very similar to that of the wild-type chicken Na+, K(+)-ATPase. This ATPase activity could be stimulated by adding Ca2+ to the assay system. These results suggest that the sites for ouabain-inhibition are restricted to the N-terminal 2/3 of the Na-pump, and the C-terminal 1/3 of the Ca-pump interacts with Ca2+.
We have obtained atomic resolution images of poly-alpha-D-glucose by scanning tunneling microscopy. The oxygen atoms near the scanning tip were imaged, but the carbon and hydrogen atoms were not visible. The measured inter-atomic distances are consistent with the molecular structure of poly-alpha-D-glucose deduced from chemical and X-ray diffraction studies. The results also demonstrate that is is feasible to image surface atomic structures of a relatively thick non-conducting specimen, suggesting that the technique may be applied to the study of other macromolecules of biological importance.
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Encoding DNA for alpha 2- and alpha 3-isoforms of the alpha-subunit of the chicken Na(+)-K(+)-ATPase have been cloned, and their nucleotide sequences and deduced amino acid sequences are reported. Comparisons between these data and comparable data for the rat alpha-subunit isoforms make possible an assessment of alpha-subunit isoform diversity among vertebrates. There is approximately twice as much amino acid sequence difference between alpha-isoforms within a single species as there is difference between corresponding alpha-isoforms of bird and mammal. These data are consistent with triplication of the alpha-subunit gene and evolution of substantially different alpha-subunit isoforms before the separation of avian and mammalian lineages over 200 million years ago and then retention of the majority of these structural differences through subsequent evolution. The implications of this conversation of isoform-specific structural features are discussed in terms of transport functions and bioregulation of the Na(+)-K(+)-ATPase.
The (Na+ + K+)-ATPase (sodium pump) is an ouabain-sensitive, electrogenic ion pump responsible for maintaining the balance of sodium and potassium ions in almost all animal cells. Robust, ouabain-sensitive rubidium uptake, indicative of the sodium pump, was found in tissue-cultured Drosophila cells, and both larvae and adults die when fed a diet containing ouabain. A monoclonal antibody to the avian sodium pump alpha-subunit was found to cross-react with the Drosophila sodium pump alpha-subunit. Immunofluorescence microscopy was used to obtain a semi-quantitative view of the expression of the sodium pump in Drosophila tissues: high levels of the sodium pump were detected in malpighian tubules, indirect flight muscles and tubular muscles, and throughout the nervous system. The cDNA encoding this sodium pump alpha-subunit in Drosophila melanogaster was cloned, sequenced and expressed in mouse L cells. At the amino acid level, its deduced sequence of 1038 residues (the first such sequence for an invertebrate) is approximately 80% similar to alpha-subunit sequences reported for vertebrates. Only one gene was found in Drosophila, located on the third chromosome at position 93B. A restriction site polymorphism has been found, and several mutations exist that may involve the alpha-subunit gene.
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cDNA encoding the alpha-subunit of the (Na+ + K+)-ATPase was cloned from a chicken kidney cDNA library and the nucleotide sequence determined. The deduced amino acid sequence showed 92% sequence homology with the alpha-subunit of the sheep kidney (Na+ + K+)-ATPase, and high cross-species homologies were found among nucleotide sequences both in the 5'- and 3'-untranslated regions of the "kidney-type" alpha-subunit mRNAs. The cDNA was subcloned into a shuttle vector derived from pSV2CAT and was stably incorporated into mouse Ltk- cells. Expression of the avian alpha-sub-unit could be activated by culture of the cells in 10 mM butyrate. Cells expressing avian alpha-subunits displayed high-affinity ouabain binding (KD = 2.6 +/- 0.7 x 10(-7) M) and ouabain-sensitive 86Rb+ uptake, characteristic of avian cells.
LEP100, a membrane glycoprotein that has the unique property of shuttling from lysosomes to endosomes to plasma membrane and back, was purified from chicken brain. Its NH2-terminal amino acid sequence was determined, and an oligonucleotide encoding part of this sequence was used to clone the encoding cDNA. The deduced amino acid sequence consists of 414 residues of which the NH2-terminal 18 constitute a signal peptide. The sequence includes 17 sites for N-glycosylation in the NH2-terminal 75% of the polypeptide chain followed by a region lacking N-linked oligosaccharides, a single possible membrane-spanning segment, and a cytoplasmic domain of 11 residues, including three potential phosphorylation sites. Eight cysteine residues are spaced in a regular pattern through the lumenal (extracellular) domain, while a 32-residue sequence rich in proline, serine, and threonine occurs at its midpoint. Expression of the cDNA in mouse L cells resulted in targeting of LEP100 primarily to the mouse lysosomes.
A cDNA encoding the beta-subunit of the (Na+ + K+)-ATPase was cloned from a chicken brain cDNA library, and its nucleotide sequence was determined. High cross-species sequence homologies were found both in coding and noncoding regions. The cDNA was subcloned into a shuttle vector derived from pSV2CAT and was stably incorporated into mouse Ltk-cells. The avian beta-subunit was expressed on the cell surface (1-8 X 10(5) molecules/cell) complexed with alpha-subunits of the murine (Na+ + K+)-ATPase. In the hybrid system there was rapid assembly of subunits, post-translational N-glycosylations of the beta-subunit at its three Asn-X-Ser (or Thr) positions, and modification of high mannose oligosaccharides to complex type. Avian beta-subunits expressed in the mouse cells had an apparent molecular weight of about 55,000 as compared with 47,000 in avian cells, due to post-translational modifications, presumably differences in complex oligosaccharides. Despite the high number of interspecies hybrid (Na+ + K+)-ATPase molecules, the cells had none of the high affinity ouabain binding sites (KD = 2 X 10(-7) M) characteristic of avian cells, consistent with the view that the ouabain binding site is located largely or exclusively on the alpha-subunit and is not greatly affected by alpha-beta interaction.
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Evidence for a voltage-dependent regulatory (inhibitory) site on the nicotinic acetylcholine receptor to which acetylcholine binds was obtained in membrane vesicles prepared from the Torpedo californica electric organ. Two rate coefficients, JA and alpha, which pertain to the receptor-controlled ion flux, were measured. A 1000-fold concentration range of acetylcholine was used in a transmembrane voltage (Vm) range from 0 to -48 mV under a voltage-clamped condition at pH 7.4, 1 degrees C. The following observations were made. (i) At low acetylcholine concentrations, the value of JA, the rate coefficient for ion translocation by the active (nondesensitized) state of the receptor, increased with increasing concentration. (ii) JA decreased at high acetylcholine concentrations. (iii) In contrast, alpha, the rate coefficient for receptor desensitization, did not show such a decrease. (iv) When the transmembrane potential of the vesicle membrane was changed to more negative values, the value of KR (the dissociation constant for binding of acetylcholine to the regulatory site) decreased by a factor of approximately 9 for a 25 mV change in Vm, while KI (the dissociation constant for binding of acetylcholine to the receptor site that controls channel opening) did not show such a change and has a value of 80 microM. When Vm is -48 mV, KR has a value of 8 microM. (v) The effect of a transmembrane voltage on the regulatory site was reversible and occurred within the time resolution (5 ms) of the quench-flow technique used in the measurements.(ABSTRACT TRUNCATED AT 250 WORDS)
Does the acetylcholine receptor have a specific regulatory (inhibitory) site for the natural receptor ligand acetylcholine? This paper deals with this question. The inhibition of acetylcholine-receptor function by diverse organic cations including local anesthetics such as procaine has been well documented. Evidence indicates that these compounds are noncompetitive inhibitors, enter the open-channel form of the receptor, and block it and that the extent of this blockage depends on the transmembrane voltage of the cell. Recently we reported that in the electroplax of Electrophorus electricus the receptor-controlled transmembrane ion flux is inhibited by acetylcholine in a voltage-dependent, noncompetitive manner. We report here that the Torpedo californica receptor also has an inhibitory site for acetylcholine. The question of whether acetylcholine, which is an organic cation, binds to the same site as other organic cations such as the noncompetitive inhibitor procaine is important and is addressed. The results reported here of chemical kinetic investigations, with receptor-rich E. electricus and T. californica membrane vesicles, indicate that the inhibition of receptor function by acetylcholine and by a local anesthetic, procaine, involves two different receptor sites. The existence of a specific inhibitory site for the natural receptor-ligand acetylcholine suggests that this site can play an important role in the modulation of receptor function and in the regulation of transmission of signals between cells.
Suberyldicholine-induced ion translocation in the millisecond time region in acetylcholine receptor rich membrane vesicles prepared from the electric organ of Electrophorus electricus was investigated in eel Ringer's solution, pH 7.0, 1 degree C. A quench-flow technique with a time resolution of 5 ms was used to measure the transmembrane flux of a radioactive tracer ion (86Rb+). JA, the rate coefficient for ion flux mediated by the active form of the receptor, and alpha, the rate coefficient for the inactivation of the ion flux, increase with increasing suberyldicholine concentrations and reach a plateau value at about 15 microM. At higher suberyldicholine concentrations (greater than 50 microM), a concentration-dependent decrease in the ion flux rate was observed without a corresponding decrease in the rate of receptor inactivation. This regulatory effect was not observed with acetylcholine or carbamoylcholine. The minimal kinetic scheme previously presented for acetylcholine and carbamoylcholine, modified by the inclusion of an additional regulatory ligand-binding site for suberyldicholine and characterized by a single dissociation constant, KR, is consistent with the results obtained over a 10 000-fold concentration range of this ligand. Rate and equilibrium constants pertaining to this scheme were elucidated. Suberyldicholine binds to the regulatory site (KR = 500 microM) approximately 100-fold less well than to its activating sites, and the binding to the regulatory site has no effect on the inactivation (desensitization) rate coefficient alpha [alpha(max) = 5.7 s-1], which is comparable to that observed with acetylcholine. The maximum influx rate coefficient [JA(max) = 18.5 s-1] is approximately twice that obtained when carbamoylcholine is the activating ligand and somewhat higher than when acetylcholine is used.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetylcholine receptor mediated ion translocation in membrane vesicles prepared from the Electrophorus electricus electroplax was investigated under voltage clamp conditions by using a quench-flow technique that allows the translocation to be measured in the millisecond to second time region. Two rate coefficients were measured over a 500-fold concentration range of acetylcholine, at a transmembrane voltage, Vm, of -45 mV, at pH 7.0, 1 degrees C. JA is the rate coefficient for ion translocation by the active state of the receptor in the absence of inactivation (desensitization), and alpha is the rate coefficient for the inactivation of the receptor by acetylcholine. (1) The values of JA and alpha increase with increasing acetylcholine concentration up to 300 microM. At higher concentrations, a concentration-dependent decrease in the ion flux rate was observed without a concomitant change in the inactivation rate. This inhibitory effect has not been reported previously and was not observed with acetylcholine or carbamoylcholine in the absence of a transmembrane voltage. (2) The value of the maximum influx rate coefficient, 26 s-1, is approximately twice that observed at 0 mV [JA(max) = 15 s-1]. This is consistent with previous interpretations that related JA(max) values to the channel-opening equilibrium constant, 1/phi, and with the relation of 1/phi to the mean lifetime of the open receptor channel in muscle cells, which is dependent on Vm. (3) The maximum observed inactivation rate coefficient [alpha(max) = 8.5 s-1] is somewhat larger than that observed at 0 mV [alpha(max) = 5 s-1].(ABSTRACT TRUNCATED AT 250 WORDS)