Files in this item



application/pdf9411734.pdf (6MB)Restricted to U of Illinois
(no description provided)PDF


Title:Overexpression, Purification and Characterization of Recombinant Human Phosphoribosylpyrophosphate Synthetase Isozymes I and II
Author(s):Nosal, Jane Marie
Doctoral Committee Chair(s):Switzer, Robert L.
Department / Program:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Molecular
Chemistry, Biochemistry
Abstract:Phosphoribosylpyrophosphate (PRPP) synthetase catalyzes the formation of PRPP, an essential substrate in the biosynthesis of purine, pyrimidine and pyridine nucleotides. At least three isozymes of mammalian PRPP synthetase exist, and the corresponding genes are differentially expressed in tissues, suggesting the existence of functional differences in catalytic and/or regulatory properties between these isozymes. Previous studies of human PRPP synthetase were carried out prior to knowledge of the existence of multiple isozymes.
Using the polymerase chain reaction, a system was developed for the expression of plasmid-encoded human PRPP synthetase that allowed for the independent isolation of individual isozymes. Expression of two of three known isozymes was obtained at levels as high as 2,000-fold those published for crude erythrocyte lysates, the previous source of the enzyme. Purification of recombinant human PRPP synthetase isozymes I and II (PRSI and PRSII) from E. coli HO700, a strain containing a deletion for the bacterial PRPP synthetase enzyme, proceeded to greater than 95% in simple 24-hour procedures. This was a significant improvement over purification from erythrocytes, which typically requires three to four days. The rapid and simple purification procedure presented here resulted in specific activities for human PRPP synthetase isozymes that were five to ten-fold higher than any previously reported.
Human PRSI and PRSII were characterized in pure form. Despite 95% amino acid sequence identity, these isozymes differed significantly in several physical and kinetic properties, including thermal stability, subunit aggregation, K$\sb{\rm m}$ values for substrates, pH-activity profiles, and response to enzyme activators and inhibitors. Differences between the two isozymes are consistent with the idea that PRSII is preferentially expressed in metabolically active, rapidly dividing cells.
Superactive mutants in human PRPP synthetase have been associated with inherited disease including gout and sensorineural deafness. Mutations in the structural gene have previously been identified for three of these superactive forms. Attempts to express one of these mutants in the same system that was used to express wild type human PRSI and PRSII were unsuccessful. Thus, this mutant may be toxic to E. coli when overexpressed, perhaps due to resistance to normal feedback regulation by purine nucleotides.
This study provides potential means for differentiating between human PRSI and PRSII in tissue extracts. It introduces a system for expression and characterization of other normal PRPP synthetase isozymes as well as mutant forms responsible for human disease. Information obtained in this study provides a better understanding of the potential physiological roles played by multiple PRPP synthetase isozymes. Differences in properties between human PRSI and PRSII allow for speculations regarding possible structure-function relationships for this enzyme, for which the crystal structure is unknown. Possible clinical implications of these findings are discussed.
Issue Date:1993
Description:156 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1993.
Other Identifier(s):(UMI)AAI9411734
Date Available in IDEALS:2014-12-17
Date Deposited:1993

This item appears in the following Collection(s)

Item Statistics