Date of Award

1989

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Thomas S. Moore, Jr

Abstract

An ethanolamine base exchange enzyme has been assayed in the endoplasmic reticulum (ER) of castor bean (Ricinus communis L. var. Hale) endosperm. The activity was maximal at ethanolamine concentrations of 30 uM in the presence of 2 mM CaCl$\sb2$. The optimum pH was 7.8 using HEPES buffer. Under these conditions, the enzyme had a maximum enzyme velocity of approximately 0.3 nmol h$\sp{-1}$ mg$\sp{-1}$ protein and an apparent Km of 5.0 uM for ethanolamine. A maximum velocity of approximately 1.7 nmol h$\sp{-1}$ mg$\sp{-1}$ protein was obtained under similar conditions with L-serine at about 400 uM in a serine base exchange reaction. The divalent cation requirement and optimal pH for the serine base exchange reaction were the same as those for the ethanolamine base exchange reaction. Ethanolamine and serine were noncompetitive inhibitors of the serine and ethanolamine base exchange enzymes, respectively. The ethanolamine base exchange enzyme was solubilized from ER by using the zwitterionic detergent, 3-(3-cholamidopropyl)dimethyl-ammonio-2-hydroxyl-1-propanesulfonate (CHAPSO). At 2 mM, CHAPSO was the most effective detergent for solubilizing the enzyme activity from the membrane. The CHAPSO solubilized enzyme was partially purified by gel filtration chromatography. Attempts at further purification of the enzyme were unsuccessful due to an irretrievable loss of activity. Reconstitution of gel filtration purified enzyme activity was performed by the addition of exogenous phospholipids. A mixture of soya phospholipids, asolectin, was most effective for the recovery of activity. The ethanolamine and serine base exchange enzymes were found to be localized on the lumenal side of the ER membrane, while CDP-ethanolamine: 1,2 diacylglycerol ethanolaminephosphotransferase occurred on the cytoplasmic side. Most of phosphatidylethanolamine formed by the CDP-ethanolamine pathway (70%) and base exchange reactions (80%) was found to be located in the outer surface of the membrane.

Pages

114

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