ISOLATION AND CHARACTERIZATION OF HUMAN LIVER GUANINE DEAMINASE.
Item
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Title
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ISOLATION AND CHARACTERIZATION OF HUMAN LIVER GUANINE DEAMINASE.
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Identifier
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AAI8212194
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identifier
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8212194
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Creator
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GUPTA, NARENDRA K.
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Contributor
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Morton D. Glantz
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Date
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1982
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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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Chemistry, Biochemistry
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Abstract
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Guanine deaminase (E.C.3.5.4.3, guanine aminohydrolase {lcub}GAH{rcub}) was purified 3248 fold from human liver by a combination of ammonium sulfate fractionation, DEAE-cellulose, hydroxylapatite and affinity chromatography to the homogeneity. The enzyme is a dimer protein of the molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and SDS-gel electrophoresis. GAH is an acidic protein as evidenced by the amino acid analysis and isoelectric focusing with a pI of 4.76, and is enriched with glutamic acid, aspartate, alanine and glycine. It shows a sharp pH-optimum of 8.0 and was very sensitive to p-hydroxymercuribenzoate inhibition. A Ki of 5 x 10('-5)M and 1.53 x 10('-5)M was obtained for 5-aminoimidazole-4-carboxamide and p-hydroxymercuribenzoate respectively demonstrating strong inhibition. The inhibition with idoacetic acid showed only a 7% loss in the activity at 1 x 10('-4)M and 24% loss at 1 x 10('-3)M concentration after 30 minutes of incubation. Guanine was the substrate for GAH in all the inhibition studies. p-Hydroxymercuribenzoate incubation for 30 minutes, however, resulted in a loss of 91% of the activity at a concentration of 1 x 10('-4)M. Enzyme was found to be stable up to 40(DEGREES)C but lost almost all of its activity at 65(DEGREES)C at 30 minutes incubation. Km value of 2 x 10('-4)M for 8-azaguanine was obtained at pH 6.0, while a Km of 1.538 x 10('-5)M was obtained for guanine as substrate at pH 7.0. The two pKa values obtained in the pH studies were 5.85 and 8.0. The plots of log Vmax/Km vs. pH, Vmax vs. pH and pKm vs. pH were made. The N-terminal amino acid was found to be valine while the C-terminal residue was alanine.
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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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Biochemistry
