A new phospholipase C-beta from Xenopus oocyte: Cloning and characterization.
Item
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Title
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A new phospholipase C-beta from Xenopus oocyte: Cloning and characterization.
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Identifier
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AAI9521295
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identifier
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9521295
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Creator
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Ma, Hai-Wen.
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Contributor
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Adviser: Ravi Iyengar
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Date
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1995
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular | Biology, Animal Physiology
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Abstract
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Xenopus oocytes exhibit a receptor-evoked Cl{dollar}\sp-{dollar} current that is mediated through the activation of phospholipase C (PLC) and a subsequent increase in {dollar}\rm\lbrack Ca\sp{lcub}2+{rcub}\rbrack\sb{lcub}i{rcub}.{dollar} A major component of the PLC response in oocytes is PTX-sensitive. However, the identity of the PLC(s) mediating this effect is unknown.;A new form of PLC (PLC-{dollar}X\beta){dollar} was isolated from a Xenopus oocyte cDNA library. PLC-{dollar}X\beta{dollar} is most similar in sequence and overall structure to the {dollar}\beta{dollar}-class of mammalian PLCs. Northern blot analysis demonstrates that PLC-{dollar}X\beta{dollar} mRNA is expressed in Xenopus oocytes as 5 Kb transcripts. Immunoblot analysis demonstrates that PLC-{dollar}X\beta{dollar} is ubiquitously expressed as a 140 kDa protein in all the nine tissues examined.;Injection into oocytes of antisense, but not sense, oligonucleotides to a specific region of PLC-{dollar}X\beta{dollar} results in degradation of its mRNA and a significant reduction of Cl{dollar}\sp-{dollar} currents evoked by endogenous angiotensin receptors and expressed {dollar}\rm\alpha\sb{lcub}1b{rcub}{dollar}-adrenergic receptors and M{dollar}\sb1{dollar}-muscarinic receptors. All of the receptor responses are at least partially PTX-sensitive in oocytes. Cl{dollar}\sp-{dollar} current responses to subsequent injection of IP{dollar}\sb3{dollar} are indistinguishable in oocytes injected with antisense or sense oligonucleotides. Inhibition of the M{dollar}\sb1{dollar}-muscarinic response produced by antisense oligonucleotides was nonadditive with that produced by pertussis toxin. These results indicate that PLC-{dollar}X\beta{dollar} is involved in the PTX-sensitive phosphoinositide metabolism pathway in Xenopus oocytes.;G{dollar}\rm\sb{lcub}o{rcub}{dollar} has been implicated as the signal transducer in the PTX-sensitive PLC pathway in Xenopus oocytes. G{dollar}\rm\sb{lcub}o{rcub}{dollar} is directly involved in the {dollar}\rm\alpha\sb{lcub}1b{rcub}{dollar}-adrenergic receptor-mediated pathway (Blitzer et al., 1993). More recently, PLC-{dollar}X\beta{dollar} and {dollar}\rm\alpha\sb{lcub}o{rcub},\ but\ not\ \alpha\sb{lcub}q{rcub},{dollar} are shown to be involved in the PTX-sensitive response evoked by {dollar}\rm5HT\sb{lcub}1c{rcub}{dollar} receptor. On the other hand, {dollar}\rm\alpha\sb{lcub}q{rcub},{dollar} but not PLC-{dollar}X\beta{dollar} and {dollar}\rm\alpha\sb{lcub}o{rcub},{dollar} is involved in the PTX-insensitive response evoked by TRH receptor (Quick et al., 1994). Taken together, these results indicate that PLC-{dollar}X\beta{dollar} is regulated by {dollar}\rm G\sb{lcub}o{rcub}{dollar} in oocytes and represents a new effector in the PTX-sensitive PLC pathway.;PLC-{dollar}X\beta{dollar} has been functionally expressed in Sf9 cells by using the baculovirus expression system. In the in vitro reconstitution assay, the recombinant PLC-{dollar}X\beta{dollar} is stimulated by {dollar}\rm\alpha\sb{lcub}q{rcub},{dollar} similar to all of the other cloned mammalian PLC-{dollar}\beta{dollar}s. However, {dollar}\rm\alpha\sb{lcub}o{rcub}\ as\ well\ as\ \beta\gamma{dollar} subunits are unable to stimulate PLC-{dollar}X\beta.{dollar} The regulation of PLC-{dollar}X\beta{dollar} by {dollar}\rm G\sb{lcub}o{rcub}{dollar} in the intact oocyte but not in vitro indicates that the stimulatory effect is not direct. Additional as yet unknown component(s) may be required to communicate signals from {dollar}\rm G\sb{lcub}o{rcub}{dollar} to PLC-{dollar}X\beta.{dollar}.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
