Date of Award

8-1-1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Abstract

The enzyme glucose-6-phosphatase is proposed to be a system consisting of at least six components--a catalytic unit (scD-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9), a stabilizing protein, and four or more specific substrate translocases, termed $\rm T\sb1$ (glucose-6-phosphate transport), $\rm T\sb2\beta$ (carbamyl phosphate, inorganic phosphate, and inorganic pyrophosphate transport), $\rm T\sb2\alpha$ (inorganic phosphate transport), and $\rm T\sb3$ (glucose transport). Several of these components have been identified and characterized, but $\rm T\sb1,$ the putative glucose-6-phosphate translocase, has not. Identification and characterization of $\rm T\sb1$ would support the current multicomponent model of the system, and substantiate the existence of a critical component of the system at which discriminant regulation of glucose-6-phosphatase activity may occur.Putative $\rm T\sb1,$ as a focus of regulation, has been demonstrated in studies indicating hysteretic behavior with activation by critical intramicrosomal concentrations of glucose-6-phosphate, by kinetic studies showing immediate and time-dependent inhibition by 3-mercaptopicolinate at $\rm T\sb1,$ and by liver perfusion studies indicating enhanced glycogenesis from glucose and inhibited glycogenolysis by 3-mercaptopicolinate.The utility of the compound, N-bromoacetylethanolamine phosphate (BAEP), as a probe of functional components of the glucose-6-phosphatase system is demonstrated. This compound is shown kinetically to have 2 or 3 sites of inhibitory action with the glucose-6-phosphatase system. Immediate and time-dependent inhibition studies demonstrate that BAEP binds irreversibly with putative translocase $\rm T\sb1$ and reversibly at the catalytic unit of the glucose-6-phosphatase system. A third site of potential reversible inhibition at $\rm T\sb2$ is suggested from these studies.Finally, the synthesis of $\lbrack\rm\sp{32}P\rbrack$-BAEP has been accomplished, and this compound has been utilized to label and then identify several potentially critical components of the glucose-6-phosphatase system. The irreversible labeling of 74.1, 56.6, 50.7, 15.9, and 14.1-kDa polypeptides from microsomes has been demonstrated. One or more of these polypeptides appear(s) to be the putative translocase, $\rm T\sb1,$ or at the very least an auxiliary polypeptide of the glucose-6-phosphatase system bestowing sensitivity to inhibition of the system by BAEP. The 103.7 and 96.6-kDa polypeptide doublet reversibly labeled by $\lbrack\rm\sp{32}P\rbrack$-BAEP is suggested from observations here to be the catalytic unit of the glucose-6-phosphatase system, and data further suggest that the 40.7 and 37.6 kDa polypeptide doublet may be a time-dependent degradation product of this peptide complex.

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