INDUCTION, PURIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS OF ISOMALTASE FROM SACCHAROMYCES CEREVISIAE.
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Title
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INDUCTION, PURIFICATION AND CHARACTERIZATION OF MULTIPLE FORMS OF ISOMALTASE FROM SACCHAROMYCES CEREVISIAE.
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Identifier
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AAI8501189
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identifier
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8501189
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Creator
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YOUNGLEIB, GARY LEWIS.
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Contributor
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Prof. Morton D. Glantz
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Date
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1981
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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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Four forms of isomaltase, (E.C.3.2.1.10), an enzyme possessing substantial hydrolytic activity at pH 7.0 towards isomaltose, (alpha)-methyl-D-glucoside, palatinose, sucrose and p-nitrophenyl-(alpha)-D-glucopyranoside (PNPG) have been isolated from a strain of Saccharomyces cerevisiae. Yeast cells grown on media containing maltose as the only added sugar produce predominantly the more acidic form (M), pI 4.86, while growth on (alpha)-methyl-D-glucoside induces predominantly the more basic one ((alpha)), pI 5.00. Growth on media containing both (alpha)-methyl-D-glucoside and maltose induces substantial amounts of both forms of the enzyme. The third form (B) is present at very low concentration with a pI intermediate between the other two (4.93), and can be concentrated into a protein peak upon hydroxylapatite chromatography. The fourth form (A) is present at low concentration with a more acidic pI than the M form of isomaltase. Each of the two major forms when added to a crude extract of the other shows no interconversion. Extraction in the presence of phenylmethanesulfonylfluoride, a serine protease inhibitor, produces the same distribution pattern as in the absence of inhibitor.;A near homogeneous preparation of the (alpha) and M form was achieved after 91 fold purification (31% yield). Homogeneity of the M form was established by isoelectric focusing in a pH 4-6 gradient, polyacrylamide disc gel electrophoresis and sodium dodecyl sulfate polyacrylamide disc electrophoresis. Homogeneity of the (alpha) form was established by isoelectric focusing in a pH 4-6 gradient. All four forms were subject to amino acid analysis and showed significant differences in amino acid composition with the M form containing half the glycine and three times the proline of the (alpha) form. A comparison of Km values at pH 7.0 and 25(DEGREES)C with PNPG for the maltose induced form (M) and the (alpha)-methyl-D-glucoside induced form ((alpha)) gave values of 8.54 x 10('-4)M and 3.2 x 10('-4)M respectively and Vmax values 3.18 x 10('4)nmolmin('-1)mg('-1) and 3.03 x 10('4)nmolmin('-1)mg('-1) respectively. Km values of the M form at pH 7.0 and 25(DEGREES)C for (alpha)-methyl-D-glucoside, isomaltose, palatinose, and sucrose were 2.02 x 10('-2)M, 2.22 x 10('-2)M, 2.25 x 10('-2)M, 5.68 x 10('-2)M respectively, and Vmax values of 8.42 (mu)molmin('-1)mg('-1), 12.9 (mu)molmin('-1)mg('-1), 9.16 (mu)molmin('-1)mg('-1) and 2.30 (mu)molmin('-1)mg('-1) respectively.;Thermolability studies performed on the M and (alpha) form of isomaltase gave first order decay constants k = -0.090 (+OR-) 0.021min-('1) and k = -0.100 (+OR-) 0.19 min-('1) respectively. Both the M and (alpha) form of isomaltase exhibited a pH optimum of 7.0-7.2, the (alpha) form demonstrating detectable activity from pH 4.6-7.7 and the M form from pH 4.6-8.5.
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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
