Date of Award

9-18-1998

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

First Advisor

Robert C. Nordlie

Abstract

The enzyme glucose-6-phosphatase (Glc-6-Pase) (E.C. 3.1.9.3) plays a major role in the maintenance of blood glucose levels. Besides its classical role of hydrolyzing Glc-6-P, Glc-6-Pase also hydrolyzes mannose-6-phosphate (Man-6-P) and phosphorylates glucose via its biosynthetic activity. The enzyme is routinely studied in rat liver microsomes in the presence and absence of an activating/disrupting agent. The activator histone II-A reportedly increases Glc-6-Pase activity without destroying the microsomal membrane (St.-Denis et al. (1995) Biochem. J. 310, 221-224). We examined whether histone II-A disrupts microsomes by using the inhibitor N-bromoacetylethanolamine phosphate (BAEP). This compound inhibits Glc-6-Pase only in native microsomes. We found that BAEP was able to inhibit Glc-6-Pase after treatment with histone II-A, suggesting that histone II-A activates without destroying microsomal membranes.The low-$\rm K\sb{m}$ activity of Man-6-Pase is routinely used to measure the structural integrity of microsomes. The report that histone II-A activates Man-6-Pase without disrupting microsomal vesicles (St.-Denis et al., op. cit.) suggests that some low-$\rm K\sb{m}$ Man-6-Pase activity may be an intrinsic characteristic even of "non-leaky" microsomes. We further studied the relationship of Man-6-Pase with microsomal integrity and found that (1) increases in the hydrolysis of Glc-6-P and Man-6-P were not parallel when microsomes were treated with low but increasing concentrations of detergent and (2) mannose-6-phosphate is hydrolyzed by untreated microsomes by more than a single mechanism. We propose that Man-6-Pase is not a reliable index of the integrity of microsomes.The stimulation by amino acids of glycogenesis from glucose has been explained by the regulatory volume decrease mechanism (Meijer et al. (1992) J. Biol. Chem. 267, 5823-5828). It involves amino acid-induced swelling of hepatocytes resulting in loss of chloride ions which leads to deinhibition of glycogen synthase. We studied the effects of amino acids and chloride ion on Glc-6-Pase and correlated our results with those reported by others with glycogen synthase. We found that (1) chloride ion inhibits biosynthetic more extensively than hydrolytic activity of Glc-6-Pase and (2) inhibition by chloride ion and activation due to ionic strength may be important considerations when assessing in vitro Glc-6-Pase activities. We propose that amino acids may play a role in increasing biosynthetic activity of Glc-6-Pase, as well as previously characterized glycogen synthase via the regulatory volume decrease mechanism through diminished chloride ion inhibition.

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