THE STRUCTURE OF THE MULTIENZYME COMPLEX OF FATTY ACID OXIDATION FROM ESCHERICHIA COLI.
Item
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Title
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THE STRUCTURE OF THE MULTIENZYME COMPLEX OF FATTY ACID OXIDATION FROM ESCHERICHIA COLI.
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Identifier
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AAI8203314
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identifier
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8203314
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Creator
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PAWAR, SHASHI.
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Contributor
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Horst Schulz
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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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The purified multienzyme complex of fatty acid oxidation from E. coli B was found to possess 3-hydroxyacyl-CoA epimerase (EC 5.1.2.3) and cis-(DELTA)('3)-trans-(DELTA)('2)-enoyl-CoA isomerase (EC 5.3.3.3) activities in addition to the previously identified enoyl-CoA hydratase (EC 4.2.1.17), L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-ketoacyl-CoA thiolase (EC 2.3.1.16). Co-chromatography of these enzymes on phosphocellulose, their co-migration on polyacrylamide gel electrophoresis and their parallel inactivation by Tris-hydrochloride represent sufficient proof for the association of the five enzymes in a multienzyme complex. The structure of the purified multienzyme complex of fatty acid oxidation from E. coli B has been studied. The molecular weight of the native complex was estimated by two methods to be close to 260,000. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate led to the identification of two types of subunits whose molecular weights were estimated to be 78,000 and 42,000 respectively. The two subunits are present in the complex in equimolar amounts. When the complex was rapidly isolated by immunoprecipitation in the presence of several protease inhibitors, its subunit structure was found to be identical to that of a preparation purified by the standard procedure. It is therefore concluded that the complex is composed of two copies each of the 78,000 dalton and 42,000 dalton subunit. The complex contains additionally phospholipids (63 nmoles of lipid phosphate per mg of protein) which were identified as phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Immunotitration of a soluble E. coli extract with antibodies raised against the pure complex provided evidence for the association of all 3-ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase and crotonase activities with the complex. However, a long chain enoyl-CoA hydratase exists as a separate protein. By specifically labeling 3-ketoacyl-CoA thiolase and cis-(DELTA)('3)-trans-(DELTA)('2)-enoyl-CoA hydratase with N-{lcub}2-('14)C{rcub}ethylmaleimide, it was shown that the former component enzyme resides on the 42,000 dalton subunit, whereas the latter one is associated with the 78,000 polypeptide. Various attempts to prove the location of 3-hydroxyacyl-CoA dehydrogenase and enoyl-CoA hydratase on the 78,000 dalton subunit have failed so far.
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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
