Date of Award


Document Type

Dissertation - Restricted

Degree Name

Doctor of Philosophy (PhD)



First Advisor

Deane N. Calvert

Second Advisor

John J. Lech

Third Advisor

James M. Fujimoto

Fourth Advisor

Harris L. Friedman

Fifth Advisor

John P. Kampine


Present concepts of the sequence of reactions in triglyceride breakdown indicates a role for monoglyceride lipases in the complete hydrolysis of triglyceride by various tissues. The present investigation has revealed the presence of two monoglyceride hydrolyzing enzymes in rat adipose tissue which have been tentatively designated as monolaurin lipase and monoolein lipase. These two enzymes c n be differentiated on the basis of column chromatographic behavior, pH optimum, substrate preference, inhibition character­istics and heat sensitivity. The mobility of monolaurin lipase on Sephadex G-200 and disc gel electrophoresis is similar to that of a previously reported fluoride-sensitive tributyrinase enzyme but can be differentiated from it on the basis of its inability to hydrolyze tributyrin and its relative insensitivity to fluoride inhibition. The physiological role of monolaurin lipase is uncertain at this time, but since adipose tissue triglyceride contains relatively small quantities of lauric acid, this enzyme appears to be implicated in lipid metabolism.

The utilization of discontinuous sucrose gradient centrifugation has resulted in a greatly increased specific activity of monoolein lipase and has allowed separation of this enzyme from the monolaurin lipase and the fluoride-sensitive tributyrinase. Monoolein lipase shows a high specificity for monoglyceride substrates with no hydrolytic activity against the corresponding triglycerides or methyl esters. The synthesis and assay of various monoglyceride substrates have indicated that those substrates containing unsaturation in the fatty acid moiety are more readily hydrolyzed than the corresponding monoglycerides containing saturated fatty acids. The results of kinetic studies have suggested that both unsaturation in the fatty acid component and initial chain structure from the ester group as influenced by the position and geometry of the double bond may be important properties for monoglyceride substrates to be hydrolyzed by monoolein lipase. Monoolein lipase is highly sensitive to inactivation by p-chloromercuribenzoate, and dithiothreitol is capable of reversing and preventing this inhibition. Since mono­olein lipase is capable of hydrolyzing monoglycerides containing those fatty acids (i. e., myristic, palmitic, palmitoleic, stearic, oleic, linoleic) known to be present in adipose tissue triglycerides, it is rather easy to envision an important physiological role for the enzyme in lipid mobilization.

This investigation has also presented evidence for the presence of two enzymes in rat ventricular tissue which are capable of hydrolyzing monoglyceride substrates. These enzyme activities, which are associated with particulate cellular fractions, can be released from the bound form with Triton X-100. On the basis of substrate speci­ficity, pH optimum, inhibition characteristics and heat sensitivity, one of these enzymes, which has been substantially purified by discontinuous sucrose gradient centrifugation, appears to be identical to the monoolein lipase from rat adipose tissue. On the basis of high specificity for monolaurin, slight inhibition by p-chloro­mercuribenzoute and sodium fluoride, and heat stability, the second enzyme activity, although not highly purified, appears to be similar to the monolaurin lipase of rat adipose tissue.



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