Purification,mRNA expression and regulation of pyroglutamyl peptidase II.
Item
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Title
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Purification,mRNA expression and regulation of pyroglutamyl peptidase II.
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Identifier
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AAI9720110
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identifier
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9720110
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Creator
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Lin, Jun.
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Contributor
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Adviser: Sherwin Wilk
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Date
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1997
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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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Health Sciences, Pharmacology | Biology, Molecular | Biology, Neuroscience
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Abstract
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We have purified the specific thyrotropin releasing hormone (TRH) degrading enzyme pyroglutamyl peptidase II from rabbit brain to apparent homogeneity as judged by SDS-PAGE and nondissociating gel electrophoresis. Its subunit structure was characterized as a homodimer with a native molecular mass of 230 kDa and a subunit molecular mass of 115 kDa. The specific enzymatic activity was estimated as 135 units/mg. The amino acid sequences of two tryptic peptides were obtained with the purified enzyme and they were found within the later reported cDNA sequence. The biochemical properties are consistent with a glycosylated membrane protein.;The distribution and quantitation of pyroglutamyl peptidase II mRNA in rat major organs and brain regions were determined by a reliable and sensitive solution hybridization ribonuclease protection assay. The highest level of pyroglutamyl peptidase II mRNA was found in brain. Of the brain regions studied, the highest level was found in posterior cortex. Significant levels of the mRNA were also found in lung and pituitary, whereas lower but detectable amounts were found in other peripheral organs. In rats treated with a single dose of T{dollar}\sb3{dollar}, the level of pyroglutamyl peptidase II mRNA was markedly increased in the pituitary (p {dollar}<{dollar} 0.01), and in the liver (p {dollar}<{dollar} 0.05). A single dose of TRH had no significant effect on pyroglutamyl peptidase II mRNA levels. Elevation of pyroglutamyl peptidase II mRNA in liver by T{dollar}\sb3{dollar} suggests that this organ is the source of the enzyme in serum.;The regulation of pyroglutamyl peptidase II mRNA was investigated in GH3 cells. TRH decreased pyroglutamyl peptidase II mRNA with a maximal effect seen 16h after exposure to 10{dollar}\sp{lcub}-7{rcub}{dollar} M TRH. The protein kinase C (PKC) activators phorbol-12{dollar}\beta{dollar}-myristate-13-acetate (PMA), and phorbol-12,13-dibutyrate also decreased pyroglutamyl peptidase II mRNA levels, whereas the inactive phorbol ester analog 4-{dollar}\alpha{dollar}-TPA had no effect. The protein kinase C inhibitor H-7 inhibited the decrease in pyroglutamyl peptidase II mRNA produced by TRH and PMA. Exposure to T{dollar}\sb3{dollar} did not change pyroglutamyl peptidase II mRNA levels. EGF treatment (5 {dollar}\times{dollar} 10{dollar}\sp{lcub}-9{rcub}{dollar} M for 5 days) produced a small (30%) but significant increase in pyroglutamyl peptidase II mRNA in GH3 cells. The down-regulation of pyroglutamyl peptidase II mRNA by TRH may provide a positive feedback mechanism for the action of TRH on pituitary lactotrophs.
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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.
