Purification and properties of cytosolic malic enzyme from human skeletal muscle
| ISSN: |
0020-711X
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|---|---|
| Source: |
Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
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| Topics: |
Biology
Chemistry and Pharmacology
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| Type of Medium: |
Electronic Resource
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| URL: |
| _version_ | 1798290881190559745 |
|---|---|
| autor | Taroni, F. Donato, S.D. |
| autorsonst | Taroni, F. Donato, S.D. |
| book_url | http://linkinghub.elsevier.com/retrieve/pii/0020-711X(88)90075-4 |
| datenlieferant | nat_lic_papers |
| fussnote | 1.An NADP*-dependent malic enzyme was purified 7940-fold from the cytosolic fraction of human skeletal muscle with a final yield of 55.8% and a specific activity of 38.91 units/mg of protein.2. The purification to homogeneity was achieved by ammonium sulfate fractionation, DEAE-Sepharose chromatography, affinity chromatography on NADP^+-Agarose, gel filtration on Sephacryl S-300 and rechromatography on the affinity column.3. Either Mn^2^+ or Mg^2^+ was required for activity: the pH optima with Mn^2^+ and Mg^2^+ were 8.1 and 7.5, respectively. The enzyme showed Michaelis-Menten kinetics. At pH 7.5 the apparent K"m values with Mn^2^+ and Mg^2^+ for l-malate and NADP^+ were 0.246 mM and 5.8 μM, and 0.304 mM and 5.8 μM, respectively. The K"m values with Mn^2^+ for pyruvate, NADPH and bicarbonate were 8.6 mM, 6.1 μM and 22.2 mM, respectively.4. The enzyme was also able to decarboxylate malate in the presence of NAD^+. At pH 7.5 the reaction rate was approximately 10% of the rate in the presence of NADP^+, with a K"m value for NAD^+ of 13.9 mM.5. The following physical parameters were established: s"2"0","w^0=10.48, Stokes' radius = 5.61 nm, pI = 5.72, M"r of the dissociated enzyme = 61,800. The estimates of the native apparent M"r yielded a value of 313,000 upon gel filtration, and 255,400 with /"0 = 1.33 by combining the Chromatographie data with the sedimentation measurements.6. The electron microscopy analysis of the uranyl acetate-stained enzyme revealed a tetrameric structure.7. Investigations to detect sugar moieties indicated that the enzyme contains carbohydrate side chains, a property not previously reported for any other malic enzyme. |
| hauptsatz | hsatz_simple |
| identnr | NLZ186258658 |
| issn | 0020-711X |
| journal_name | International Journal of Biochemistry |
| materialart | 1 |
| package_name | Elsevier |
| publikationsort | Amsterdam |
| publisher | Elsevier |
| reference | 20 (1988), S. 857-866 |
| search_space | articles |
| shingle_author_1 | Taroni, F. Donato, S.D. |
| shingle_author_2 | Taroni, F. Donato, S.D. |
| shingle_author_3 | Taroni, F. Donato, S.D. |
| shingle_author_4 | Taroni, F. Donato, S.D. |
| shingle_catch_all_1 | Taroni, F. Donato, S.D. Purification and properties of cytosolic malic enzyme from human skeletal muscle 0020-711X 0020711X Elsevier |
| shingle_catch_all_2 | Taroni, F. Donato, S.D. Purification and properties of cytosolic malic enzyme from human skeletal muscle 0020-711X 0020711X Elsevier |
| shingle_catch_all_3 | Taroni, F. Donato, S.D. Purification and properties of cytosolic malic enzyme from human skeletal muscle 0020-711X 0020711X Elsevier |
| shingle_catch_all_4 | Taroni, F. Donato, S.D. Purification and properties of cytosolic malic enzyme from human skeletal muscle 0020-711X 0020711X Elsevier |
| shingle_title_1 | Purification and properties of cytosolic malic enzyme from human skeletal muscle |
| shingle_title_2 | Purification and properties of cytosolic malic enzyme from human skeletal muscle |
| shingle_title_3 | Purification and properties of cytosolic malic enzyme from human skeletal muscle |
| shingle_title_4 | Purification and properties of cytosolic malic enzyme from human skeletal muscle |
| sigel_instance_filter | dkfz geomar wilbert ipn albert fhp |
| source_archive | Elsevier Journal Backfiles on ScienceDirect 1907 - 2002 |
| timestamp | 2024-05-06T08:23:46.523Z |
| titel | Purification and properties of cytosolic malic enzyme from human skeletal muscle |
| titel_suche | Purification and properties of cytosolic malic enzyme from human skeletal muscle 1.An NADP*-dependent malic enzyme was purified 7940-fold from the cytosolic fraction of human skeletal muscle with a final yield of 55.8% and a specific activity of 38.91 units/mg of protein.2. The purification to homogeneity was achieved by ammonium sulfate fractionation, DEAE-Sepharose chromatography, affinity chromatography on NADP^+-Agarose, gel filtration on Sephacryl S-300 and rechromatography on the affinity column.3. Either Mn^2^+ or Mg^2^+ was required for activity: the pH optima with Mn^2^+ and Mg^2^+ were 8.1 and 7.5, respectively. The enzyme showed Michaelis-Menten kinetics. At pH 7.5 the apparent K"m values with Mn^2^+ and Mg^2^+ for l-malate and NADP^+ were 0.246 mM and 5.8 μM, and 0.304 mM and 5.8 μM, respectively. The K"m values with Mn^2^+ for pyruvate, NADPH and bicarbonate were 8.6 mM, 6.1 μM and 22.2 mM, respectively.4. The enzyme was also able to decarboxylate malate in the presence of NAD^+. At pH 7.5 the reaction rate was approximately 10% of the rate in the presence of NADP^+, with a K"m value for NAD^+ of 13.9 mM.5. The following physical parameters were established: s"2"0","w^0=10.48, Stokes' radius = 5.61 nm, pI = 5.72, M"r of the dissociated enzyme = 61,800. The estimates of the native apparent M"r yielded a value of 313,000 upon gel filtration, and 255,400 with /"0 = 1.33 by combining the Chromatographie data with the sedimentation measurements.6. The electron microscopy analysis of the uranyl acetate-stained enzyme revealed a tetrameric structure.7. Investigations to detect sugar moieties indicated that the enzyme contains carbohydrate side chains, a property not previously reported for any other malic enzyme. |
| topic | W V |
| uid | nat_lic_papers_NLZ186258658 |