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Title
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ISOLATION AND CHARACTERIZATION OF BOVINE CEREBELLAR PROTEIN PHOSPHATASES (PHOSPHORYLATION, G-SUBSTRATE).
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Identifier
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AAI8501175
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identifier
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8501175
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Creator
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SIMONELLI, PAUL FLAUIAN.
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Contributor
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Heng-Chun Li
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Date
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1984
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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, General
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Abstract
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A systematic study of cerebellar protein phosphatases (phosphoprotein phosphohydrolase, EC 3.1.3.16) was conducted using as substrates ('32)P-labeled phosphorylase a and G-substrate, a neuron- and cerebellum-specific substrate for cyclic GMP-dependent protein kinase (Aswad, D., & Greengard, P., J. Biol. Chem. 256, 3487-3493, 1981). Four major phosphatase activities termed I, -2, II, and III were eluted by DEAE-cellulose chromatography of fresh cerebellar homogenates at 0.12, 0.15, 0.20, & 0.35 M KCl, respectively. Phosphatase I (M(,r) 62,800, SR 3.8 nm, s(,20,w) 4.0) was preferentially active towards phosphorylase a, could be activated by Mn('2+) or by preincubation with Mg(.)ATP + F(,a), and was the only phosphatase sensitive to heat-stable inhibitor-1 (K(,i) 0.7 nM) and inhibitor-2. Phosphatase-2 (M(,r) 80,000) was activated by Mn('2+) or Ca('2+)-calmodulin, and preferentially dephosphorylated G-substrate. The ratio of maximal velocities indicated that phosphatase II (M(,r) 187,300, SR 5.4 nm, s(,20,w) 8.4) preferentially dephosphorylated G-substrate (G-substrate : phosphorylase a 3:1), whereas phosphatase III (M(,r) 101,700, SR 4.4 nm, s(,20,w) 5.6) was preferentially active towards phosphorylase a (V(,max) ratio 1:5). The K(,m) parameters displayed by the two phosphatases were similar (0.2 uM G-substrate, 5-17 uM phosphorylase a). Like phosphatase I, G-substrate dephosphorylation catalyzed by phosphatases II and III required the simultaneous presence of Mn('2+) (K(,a) 0.2 nM); other metals were less effective. Dephosphorylation of phosphorylase a by phosphatase II was also Mn('2+)-dependent (K(,a) 0.2 mM), but phosphatase III displayed near-maximal activity towards this substrate in the presence of 2 mM EDTA. Under these conditions, G-substrate inhibited phosphatase III activity towards phosphorylase a in a concentration-dependent, non-competitive manner (K(,i) 0.15 uM), an effect that was reversible by dilution. In its dephosphorylated form (catalyzed by phosphatase II), G-substrate was ineffective as an inhibitor, but its potency could be restored by subsequent phosphorylation catalyzed by cyclic GMP-dependent protein kinase. SDS-gel electrophoresis indicated that no changes other than an altered ('32)P-content were apparent. Together, these findings indicate that G-substrate could function as a specific inhibitor of phosphatase III, and that its potency can be regulated by a cycle of phosphorylation/dephosphorylation.
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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.
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Program
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Biomedical Sciences
