Date of Award

12-1979

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biochemistry

Research Advisor

Edsel Bucovaz

Abstract

A multienzyme complex contained in Bakers' yeast (Saccharomyces cerevisiae) which synthesizes CoA has been named the coenzyme A-synthesizing protein complex (CoA-SPC). The CoA-SPC has been shown to be insoluble in the crude Bakers' yeast cell lysate formed by exposing the yeast cell to ether and dry ice. Only after solubilization has this multienzyme complex been shown to catalyze the formation of bound dephospho-CoA utilizing the substrates adenosine triphosphate, D-pantothenic acid and L-cysteine. A low molecular weight component or components of the soluble fraction of the yeast cell and chloride ion appears to be responsible for the solubilization of CoA-SPC. This endogenous component of the yeast cell is referred to as t-Factor.

Purification of t-Factor has been accomplished by techniques which have included dialysis, ultrafiltration and paper, permeation, adsorption and ion-exchange chromatography. Although the t-Factor has not been identified, several properties have been demonstrated: (1) the molecular weight is between 400 to 1,000; (2) it is stable to heat at 80°C for 24 hours; (3) it is resistant to hydrolysis by trypsin and protease; (4) it is inactivated by ashing; and (5) has no ultraviolet absorption at 260 mu and 280 my.

Replacement of t-Factor by commercially available compounds or solubilizing agents has failed to reveal its identity. A "one-step" purification of CoA-SPC may be obtained by combining partially purified t-Factor with insoluble yeast cell residue.

An alternate procedure was developed for the preparation of CoA-SPC. This procedure involves the slow drying and rehydration of the yeast cells, but the need for t-Factor still remains. Sonic oscillation and passage through a french pressure cell, two of the more classical cell breakage techniques, fail to produce active CoA-SPC preparations.

DOI

10.21007/ptd.cghs.1979.0545

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