An affinity column for cathepsin D was prepared making use of the strong affinity of pepstatin for cathepsin D. Pepstatin is an N-acylated pentapeptide from Actinomycetes with the following structure: isovaleryl-L-valyl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L-alanyl-4-amino-3-hydroxy- 6-methyl heptanoic acid. A relatively rapid and efficient method for cathepsin D purification has been developed; Steps include homogenization, ammonium sulfate fractionation, and chromatography on pepstatin-Sepharose column. The final preparation has a specific activity of 38 units/mg. and shows a single protein band on polyacrylamide gel electrophoresis in sodium dodecyl sulfate corresponding to a subunit molecular weight of 42,000. Polyacrylamide gel electrophoresis studies did not reveal any impurities. The proteolytic activity of isolated cathepsin D on bovine myofibrils and myosin was examined at pH 3.80, 37 °C. The heavy chains of myosin, as well as other smaller regulatory proteins of the myofibrils were degraded. Actin was degraded less rapidly than myosin heavy chain. Degradation became more extensive when the substrate-enzyme incubation time was increased.
| Identifer | oai:union.ndltd.org:UTAHS/oai:digitalcommons.usu.edu:etd-6335 |
| Date | 01 May 1981 |
| Creators | Fan, Paul Hwaleun |
| Publisher | DigitalCommons@USU |
| Source Sets | Utah State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | All Graduate Theses and Dissertations |
| Rights | Copyright for this work is held by the author. Transmission or reproduction of materials protected by copyright beyond that allowed by fair use requires the written permission of the copyright owners. Works not in the public domain cannot be commercially exploited without permission of the copyright owner. Responsibility for any use rests exclusively with the user. For more information contact digitalcommons@usu.edu. |
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