Title page for ETD etd-04272000-10510025


Type of Document Master's Thesis
Author Blanchard, David Joseph
Author's Email Address dablanch@ll.mit.edu, dablanch@vt.edu
URN etd-04272000-10510025
Title Characterization of a Beta-glucosidase Aggregating Factor Responsible for the Null Beta-glucosidase Phenotype in Maize (Zea mays L.)
Degree Master of Science
Department Biology
Advisory Committee
Advisor Name Title
Esen, Asim Committee Chair
Newton, William E. Committee Member
Rutherford, Charles L. Committee Member
Keywords
  • Beta-glucosidase
  • antigen-antibody interactions
  • Beta-glucosidase aggregating factor (BGAF)
  • small heat-shock protein (sHsp)
Date of Defense 2000-04-25
Availability unrestricted
Abstract
Beta-Glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) catalyzes the hydrolysis of aryl and alkyl beta-D-glucosides as well as glucosides with a carbohydrate moiety such as cellobiose and other beta-linked oligosaccharides. In maize (Zea mays L.), beta-glucosidase exists as 120 kD homodimers, but also forms high-molecular-weight (HMW) aggregates in certain maize inbreds (nulls). In this study we show that the null beta-glucosidase phenotype is caused by the formation of HMW enzyme aggregates (>1.5 X 106 Daltons), caused by a beta-glucosidase aggregating factor (BGAF). BGAF is a 32 kD protein that binds specifically to beta-glucosidase and renders it insoluble during extraction. The data unequivocally demonstrate that BGAF is solely responsible for beta-glucosidase aggregation and insolubility, and thus, the apparent null phenotype. Additionally, I have isolated the cDNA encoding BGAF and have identified BGAF as a member of the small heat-shock protein (sHsp) family.

Interestingly, BGAF binds to both maize beta-glucosidase isozymes (Glu1 and Glu2), but does not bind to their sorghum homolog Dhurrinase-1 (Dhr1; Sorghum beta-glucosidase), that shares 70% sequence identity with Glu1 and Glu2. Therefore, these proteins provide an excellent system to study functional differences at nonconserved residues and elucidate the mechanism of enzyme aggregation and insolubility. By examining the behavior of beta-glucosidase chimeras in binding assays, I demonstrate that BGAF binding is conformation dependent, highly specific, and reminiscent of antigen-antibody interactions. Additionally, I have identified two disparate polypeptide segments in the primary structure of the maize beta-glucosidase isozyme Glu1 that form a BGAF binding site in the tertiary structure of the enzyme.

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  Chapter1.pdf 171.68 Kb 00:00:47 00:00:24 00:00:21 00:00:10 < 00:00:01
  Chapter2.pdf 17.60 Kb 00:00:04 00:00:02 00:00:02 00:00:01 < 00:00:01
  Chapter3.pdf 195.91 Kb 00:00:54 00:00:27 00:00:24 00:00:12 00:00:01
  cover.pdf 18.20 Kb 00:00:05 00:00:02 00:00:02 00:00:01 < 00:00:01
  cover.pdf 18.20 Kb 00:00:05 00:00:02 00:00:02 00:00:01 < 00:00:01
  vitae.pdf 11.39 Kb 00:00:03 00:00:01 00:00:01 < 00:00:01 < 00:00:01

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