Expression of human prolidase in E. coli: Kinetic and metal binding properties. Massive iminodipeptiduria, severe skin ulcers, and mild mental retardation mark Prolidase Deficiency, a rare autosomal recessive disorder. In vitro, Prolidase requires incubation with Mn 2+ for maximal activity, however, the in vivo role of metal in prolidase catalysis has not been clarified. / Human Prolidase cDNA was cloned into the bacterial expression vector pRSET C. The plasmid expresses a chimeric protein consisting of a hexahistidine tag and enterokinase site at the N-terminus of prolidase. This protein was purified to homogeneity. Several properties of the purified prolidase were examined. Prolidase preferentially bound Mn in vivo. In vitro incubation and atomic absorption studies revealed a positive correlation between activity and Mn2+ content, and a negative correlation between Zn content and activity. Prolidase has two distinct metal binding sites each binding two metals with different metal binding affinities towards Mn2+ and Zn2+. Prolidase thermostability was directly proportional to percent Mn2+ content. In addition, chelators inhibited prolidase activity.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.21521 |
| Date | January 1998 |
| Creators | Carriles, Claudia. |
| Contributors | Hechtman, Peter (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Master of Science (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001659004, proquestno: MQ50732, Theses scanned by UMI/ProQuest. |
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