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Role of Enzyme-Enzyme Interactions in the Regulation of Gluconeogenesis

PROPERTIES AND SUBUNIT STRUCTURE OF FRUCTOSE 1,6-DIPHOSPHATASE FROM SWINE KIDNEY
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      Fructose 1,6-diphosphatase has been isolated from swine kidney extracts by a procedure which yields large quantities of homogeneous enzyme. The purified preparation has an extinction coefficient (E280nm1%) of 8.9 in 0.05 m Tris-HCl, pH 8.0 and a specific activity of 25 to 30 µmoles per min per mg. The molecular weight of the native protein, as determined by sedimentation velocity, sedimentation equilibrium analysis, sucrose gradient centrifugation, and gel filtration on Sephadex G-200 is 130,000 ± 2,500. Sedimentation equilibrium analysis of the enzyme denatured in 4 m guanidine hydrochloride indicated that four polypeptide chains might be present. These results were confirmed by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate which showed that the native enzyme contained 4 identical subunits with molecular weights of 34,000 ± 1,500. Further evidence for the presence of 4 subunits was obtained from a determination of the number of peptides formed after digestion of the enzyme with trypsin. The COOH-terminal amino acid residues of all 4 subunits were identified as alanine by hydrozinolysis and by digestion with carboxypeptidases. The NH2-terminal amino acid was serine. Quantitative Edman degradations on oxidized denatured samples showed that the native enzyme contained 4 serine NH2-terminal residues per mole. All of the determinations gave nearly the same values for the subunit molecular weight of about 34,000, which is in good agreement with a tetrameric structure for the native enzyme with a molecular weight of 130,000. Examination of fructose 1,6-diphosphatase in the electron microscope by the negative staining technique revealed the presence of a flattened ellipsoidal particle with cross-section diameters of 60 and 120 A and a width of about 30 A.

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