Laboratory Guide to Biochemistry, Enzymology, and Protein Physical Chemistry

A Study of Aspartate Transcarbamylase
Softcover Nachdruck of the original 1. Auflage 1991
ISBN: 978-1-4613-6704-8
Verlag: Springer US

Buch, Englisch, 169 Seiten, Paperback, Format (B × H): 152 mm x 229 mm, Gewicht: 276 g

The study of a single well-chosen substance, here aspartate transcarb­ amylase, can provide an excellent basis for a laboratory course. The student is introduced to a variety of scientific ideas and to many experi­ mental and interpretive techniques. This enzyme is readily available, is relatively stable, has an extensive literature, and its behavior has many facets: substrate inhibition, a large change in structure upon homo­ tropic activation by substrates, allosteric stimulation by ATP, allosteric inhibition by CTP synergistic with VTP, positive cooperativity for sub­ strates, negative cooperativity for CTP binding, and dissociation and reassembly of subunits Cand R2 from the holoenzyme CI\5. In addition 3 6 to the known biochemical aspects of these properties, the results ob­ tained here can be interpreted in the light of the high-resolution X-ray diffraction structures of the T and R forms, the low-angle X-ray scattering results, and the large number of mutants now available by recombinant DNA methods. Future development of this course could also involve part of these methods, as well as the carefully chosen experiments described here. This approach resembles research more than the approaches one usually finds in biochemical laboratory courses. A consistent develop­ ment of ideas about a single enzyme, which shows so many facets in its behavior, is sure to hold the interest of the student. Moreover, one explores a depth, and reasons to move forward, that are an essential part of research.

Zielgruppe

Research

Autoren/Hrsg.

Maire, Marc Le

Hervé, Guy

Chabaud, Raymond

Fachgebiete

• Interdisziplinäres Wissenschaften Wissenschaften Interdisziplinär
• Naturwissenschaften Biowissenschaften Chemische Biologie, Synthetische Biologie
• Naturwissenschaften Chemie Physikalische Chemie

Inhaltsverzeichnis

1 Aspartate Transcarbamylase.- 2 Molecular Genetics: Regulation of Aspartate Transcarbamylase Biosynthesis.- 3 Purification of Aspartate Transcarbamylase and Its Subunits.- 4 Structural and Physicochemical Study of Aspartate Transcarbamylase.- 5 Enzymatic Catalysis and Regulation.- 6 Complementary Experiments.- 1. Equations and Units.- 2. Molar Mass and Molecular Mass.- 3. Units of Catalytic Activity.- 4. Units of Radioactivity.- 5. Units of Quantity.- 7. Calculation of Acceleration.- 8. Bacterial Strains.- 9. Solutions and Reagents.- 9.1. 0.8-M Tris-Acetate Buffer, pH 8.- 9.2. 0.2-M Acetic Acid.- 9.6. Elution Buffer for Chromatography: 10 × Stock Solution.- 9.7. 100-mM Aspartate, pH 8.- 9.8. 10-mM Carbamylaspartate, pH 8.- 9.9. 1-M Phosphate Buffer, pH 7.2.- 9.10. Buffer for Dilution of E.- 9.11. Buffer for Dilution of C.- 9.12. Buffer for Dilution of R and Recombination.- 9.13. 200-mM Cacodylate Buffer, pH 6, 7, and 7.5.- 9.14. 200-mM Tris-Acetate Buffer, pH 8 and 9.- 9.15. 200-mM Glycine Buffer, pH 10.- 9.16. 400-mM Succinate, pH 7.- 9.17. Reaction Medium: 20-mM CAP-200-mM Tris-Acetate, pH 8.- 10. Preparation of Standard Protein Mixtures for Column Calibration.- Answers To Questions.- References.