Graduate Thesis Or Dissertation
 

Studies on sterol : UDP glucose glucosyltransferase in germinating pea seedling

Public Deposited

Downloadable Content

Download PDF
https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/q524js434

Descriptions

Attribute NameValues
Creator
Abstract
  • Steryl glycoside is synthesized from mevalonate [2- ¹⁴C] in both maturing pea seeds and etiolated pea seedlings. The maturing seed appeared more active than the seedlings for this synthesis. The sugar moiety was identified predominantly as glucose by acid hydrolysis and paper chromatography of the aqueous extract of the hydrolysate. The lipid moiety extracted from the hydrolysate was found to be mainly sterol by thin-layer chromatography and radiochromatographic scanning. Etiolated shoot-root axes were used for the isolation of the enzyme preparation because of the convenience of obtaining large amounts of material. UDP-glucose labelled with ¹⁴C on the glucose moiety and exogenous sitosterol were used as substrates for the study of sterol:UDP-glucose glucosyltransferase activity. The highest specific activity for this enzyme was found in the shoot, root and shoot-root axial tissues of seven-day old seedlings. Fractionation of the seedling homogenate by centrifugation showed that the 13,000-25,000 g fraction has the highest specific activity in tissue homogenate. The fraction appeared to be rich in Golgi membranes by the presence of the marker enzyme, IDPase. The glucosyltransferase in this membrane-rich fraction was stimulated best by Ca⁺⁺ and by Mg⁺⁺. Marked inhibition was observed in the presence of Zn⁺⁺. The presence of ATP has different effects on the enzyme activity and depends on the kind and quantity of metal ion used in the reaction mixture. In addition, the enzyme activity was enhanced further in the presence of a small amount of methanol and low buffer concentration. Under the assay conditions employed, the enzyme has an apparent Km of 3.33 mM and a V[subscript max] of 480 nmoles/ hr for UDP-glucose. It is inhibited by concentrations of UDP-glucose greater than 4 mM. The enzymatic reaction proceeds preferentially with endogenous sterol as the glucose acceptor and UDP-glucose as the glucose donor. Glucosyltransferase activity suffers a sharp decline during the course of the assay. The activity could be partially maintained by repeated addition of ATP during the assay. The role of ATP and the possible relationship between ATPase, protein kinase and the glucosyltransferase still needs to be explored. Attempts to purify this enzyme from the 13,000-25,000 g fraction have been made, but failed. Transferase activity was inhibited by treatments with phospholipases A, C, and D. The lost activity can be fully recovered by adding phosphatidyl serine, or phosphatidyl choline and even further enhanced by adding phosphatidyl ethanolamine. A transferase with high specific activity of the membrane-rich fraction was separated by sucrose density gradient centrifugation. This method may provide an avenue for further characterization of sterol:UDP-glucose glucosyltransferases in plant tissues.
Resource Type
Date Available
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Committee Member
Academic Affiliation
Non-Academic Affiliation
Subject
Rights Statement
Publisher
Peer Reviewed
Language
Digitization Specifications
  • File scanned at 300 ppi (Monochrome) using ScandAll PRO 1.8.1 on a Fi-6770A in PDF format. CVista PdfCompressor 5.0 was used for pdf compression and textual OCR.
Replaces

Relationships

Parents:

This work has no parents.

In Collection:

Items

Thumbnail Title Date Uploaded Visibility Actions
file details FangTa-Yun1976.pdf 2017-08-23 Public Download