The presence of acetone in diabetic urine was reported more than a century ago. Since that time considerable effort has been devoted to studies on ketosis and ketogenesis. Recent studies in several laboratories have revealed the precise enzymatic mechanism for the formation of acetoacetate, the primary ketone body. For these studies, investigators have used liver extracts obtained from various species such as beef, dog, rabbit, pigeon and chicken, as well as omasum and rumen epithelium obtained from sheep.
A study of acetoacetate formation in extracts of chicken liver is complicated by the presence of a factor which inhibits: in vitro acetoacetate formation in the assay used. This "inhibitor" also depresses acetoacetate formation by beef liver extracts. A study of the nature of this inhibitor action was begun in this laboratory in the hope that an understanding of its action might shed some light on the mechanism of acetoacetate formation. The mechanism of acetoacetate formation was subsequently worked out by independent means. It was still of interest, however, to clarify the nature of the inhibitor factor. This thesis constitutes such a study.
The "inhibitor" protein has been purified 300-fold from extracts of fresh chicken liver. The various possible sites of action on the in vitro acetoacetate-forming system have been examined. Preliminary studies revealed that the factor does not act on any of the enzymatic components of acetoacetate formation. Evidence is presented that it inhibits acetoacetate formation through the inactivation of both the free and esterified forms of coenzyme A. The product of its action on coenzyme A has been isolated and characterized by paper and ion-exchange chromatography and chemical analysis as 3'-dephosphocoenzyme A. The inhibitor protein can thus be more properly designated as a "coenzyme A 3'-phosphohydrolase." In retrospect, it is now clear that this enzyme could have afforded no knowledge concerning the enzymatic mechanism of acetoacetate formation.
The enzyme exhibits maximal activity at pH 3.6, with half-maximal activity at a "plateau" between pH 5.0 and pH 6.0, and at some point below pH 2.5. The rate of inactivation of coenzyme A by the phosphohydrolase is neither enhanced nor depressed by divalent cation or metal-binding agents. Although the most highly purified enzyme fraction obtained exhibits phosphohydrolase activity against a wide variety of phosphate esters, evidence is presented to indicate that the coenzyme A 3'-phosphohydrolase activity may well be a specific and unique enzyme. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate
| Identifer | oai:union.ndltd.org:UBC/oai:circle.library.ubc.ca:2429/37884 |
| Date | January 1964 |
| Creators | Caldwell, Ian Carl |
| Publisher | University of British Columbia |
| Source Sets | University of British Columbia |
| Language | English |
| Detected Language | English |
| Type | Text, Thesis/Dissertation |
| Rights | For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use. |
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