PURIFICATION AND CHARACTERIZATION OF PHOSPHOPROTEIN PHOSPHATASE-1 AND ITS PROTEIN ACTIVATOR, FA, FROM BOVINE HEART (FC, DEPHOSPHORYLATION, PHOSPHORYLATION).
Item
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Title
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PURIFICATION AND CHARACTERIZATION OF PHOSPHOPROTEIN PHOSPHATASE-1 AND ITS PROTEIN ACTIVATOR, FA, FROM BOVINE HEART (FC, DEPHOSPHORYLATION, PHOSPHORYLATION).
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Identifier
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AAI8501178
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identifier
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8501178
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Creator
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TABARINI, DIANE.
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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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Biophysics, General
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Abstract
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Phosphoprotein phosphatase activity in bovine heart was studied by using Ser(P)-phosphorylase a, Ser(P)-casein, and Thr(P)-inhibitor-1 as substrates. Four distinct isozyme species can be separated from cardiac muscle extracts by ammonium sulfate fractionation and ion exchange chromatography. These are termed phosphatases-1, -2, -3, and -4. Phosphatase-1 requires preincubation with Mg('2+), ATP, and a protein activator termed Fa, for expressing its activity. Phosphatase-2 is dependent on Ca('2+) and calmodulin. Phosphatase-3 is spontaneously active but can be stimulated several-fold by transition metal ions. Phosphatase-4 is divalent cation (Mg('2+) or Mn('2+))-dependent. Phosphatase-1, -3, and Fa have been extensively purified and characterized.;Phosphatase-1 has been purified over 3000-fold. The purified enzyme has an apparent M(,r)=58K. Evidence indicates that it is composed of a catalytic (C) subunit of M(,r) = 38K and a regulatory (R) subunit of M(,r) = 31K. The C-subunit is trypsin resistent but heat labile. On the other hand, the R-subunit is heat-stable but is very sensitive to trypsin digestion. Activation of phosphatase-1 by preincubation with Mg('2+), ATP and Fa is accompanied by phosphorylation of the R-subunit. Limited trypsinization of the enzyme results in selective digestion of the R-subunit with a concomitant decrease in the Fa-activated and an increase in the Mn('2+)-activated phosphatase activity. These observations collectively suggest that activation of phosphatase-1 by Fa occurs as follows: (a) phosphorylation of the R-subunit by Fa releases its inhibitory effects on the C-subunit and transforms the C-subunit into a relaxed conformation and (b) in the presence of Mg('2+), an essential cofactor for the phosphatase reaction, the enzyme becomes catalyticly active.;Fa has been purified over 1000-fold. The major active species has a M(,r) = 48-50K and appears to be composed of a single polypeptide chain. An additional minor active species of M(,r) = 24K was also found. In addition to acting as an activator of phosphatase-1, Fa also possesses glycogen synthase kinase activity and is capable of catalyzing autophosphorylation. Studies on the Fa activation of phosphatase-1 demonstrate the following: (a) activation occurs in a time and concentration (Fa) dependent manner, (b) activation requires the presence of a nucleotide triphosphate as well a divalent cation and (c) both the activation reaction of phosphatase-1 as well as the activity was maximum between pH 7.0-8.0.
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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
