Robert C. Nordlie

[111] Glucose-6-phosphatase

Publisher Summary This chapter describes the assays for the determination of glucose-5-phosphatase and a modified method for measuring glucose-6-P hydrolysis. The methods discussed are glucose-6-P hydrolysis, PPi hydrolysis, and phosphotransferase activities. Sodium cacodylate buffers are employed in all assay systems because this compound buffers effectively over the range pH 5–7.4 and exerts no specific effect on the reactions. Sodium acetate buffer also may be employed in the phosphotransferase and inorganic pyrophosphatase assay systems; however it is not suitable for the glucose-6-P hydrolysis assay. Citrate recently has been found to inhibit competitively with respect to phosphate substrates, below pH 6. Glucose-6-phosphatase is a microsomal enzyme which has resisted attempts to obtain a satisfactory purification. A number of significant efforts to solubilize and purify this enzyme recently have been carried out. Moderate enrichments have been obtained by differential centrifugation of microsomal preparations after treatment with detergents or proteolytic enzymes, and by fractional precipitation of detergent-dispersed microsomal preparations with acetone in the presence of added magnesium++ (Mg++).

Metabolic Regulation by Multifunctional Glucose-6-phosphatase

Publisher Summary This chapter discusses metabolic regulation by multifunctional glucose-6-phosphatase. Glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9), once believed to be a rather specific hepatic phosphatase, is presently known to exhibit quite broad specificity, distribution, and multiplicity of functions. It is intrinsically the most potent hepatic glucose phosphorylating enzymatic activity yet discovered. The chapter describes a variety of new mechanisms for control of activities of the enzyme that are inherent in its multifunctional nature. Glucose-6-phosphatase is known to catalyze the hydrolysis of a variety of phosphate esters and phosphoanhydrides. In addition, the enzyme also effectively catalyzes the transfer of a phosphoryl group from such compounds to D-glucose, or with a lesser degree of efficiency, to various other sugars and polyols as well. Once believed to be confined exclusively to endoplasmic reticulum of the cell, glucose-6-phosphatase-phosphotransferase has presently been unequivocally established as present in the outer nuclear membrane also, where roughly 10%–15% of the total hepatic activity resides.

Glucose-6-Phosphatase Structure, Regulation, and Function: An Update

Abstract Work on the glucose-6-phosphatase system has intensified and diversified extensively in the past 3 years. The gene for the catalytic unit of the liver enzyme has been cloned from three species, and regulation at the level of gene expression is being studied in several laboratories worldwide. More than 20 sites of mutation in the catalytic unit protein have been demonstrated to underlie glycogenesis type 1a. Inhibition of glucose-6-P hydrolysis by several newly identified competitive and time-dependent, irreversible inhibitors has been demonstrated and in several instances the predicted effects on liver glycogen formation and/or breakdown and on blood glucose production have been shown. Refinements in and additions to the presently dominant “substrate transport-catalytic unit” topological model for the glucose-6-phosphatase system have been made. A new model alternative to this, based on the “combined conformational flexibility-substrate transport” concept, has emerged. Experimental evidence for the phosphorylation of glucose in liver by high-K m, glucose enzyme(s) in addition to glucokinase has continued to emerge, and new in vitro evidence supportive of biosynthetic functions of the glucose-6-phosphatase system in this role has appeared. High levels of multifunctional glucose-6-phosphatase have been shown present in pancreatic islet β cells. Glucose-6-P has been established as the likely insulin secretagog in β cells. Interesting differences in the temporal responses of glucose-6-phosphatase in kidney and liver have been demonstrated. An initial attempt is made here to meld the hepatic and pancreatic islet β-cell glucose-6-phosphatase systems, and to a lesser extent the kidney tubular and small intestinal mucosal glucose-6-phosphatase systems into an integrated, coordinated mechanism involved in whole-body glucose homeostasis in health and disease.

Glucose-6-phosphatase

Glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EG 3.1.3.9) is unique among gluconeogenic enzymes in a variety of respects. Not only is the enzyme either a part of, or extremely tightly bound to, membranes of the endoplasmic reticulum (Ernster et al., 1962) and nucleus (Gunderson and Nordlie, 1973, 1975), but it also manifests a multiplicity of functions including the synthesis of glucose-6-P* at rates which may equal or actually exceed that of glucose-6-P hydrolysis (Lueck et al., 1972). And intimate, activity-discriminant interrelationships exist between catalytic characteristics of the enzyme and the origin, nature, and physical state of the biomembranes with which it is associated.

Fine tuning of blood glucose concentrations

Abstract Blood glucose homeostasis is maintained by the correct balance between phosphorylation of glucose and hydrolysis of glucose 6-phosphate in the liver. The enzyme glucose-6-phosphatase can synthesize glucose 6-phosphate as well as degrade it, and its synthetic activity therefore acts as an adjunct to glucokinase activity. The ratio of this phosphotransferase activity of glucose-6-phosphatase to the activity of glucokinase may adjust to maintain the steady-state blood glucose concentration as the physiological circumstances demand.

Multifunctional Glucose-6-Phosphatase: A Critical Review

Glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase; EC 3.1.3.9) was reviewed in some detail in the first edition of this work (Nordlie and Jorgenson, 1976). Our intention here is to focus on recent developments since the literature search for that earlier chapter was completed. Accordingly, we attempt to consider here the literature concerning glucose-6-phosphatase which has appeared since 1975. The authors were pleased to note a renewed interest in this complex enzyme, worldwide, more than 150 papers on the subject having appeared in the past seven years. A detailed consideration of the contents of all these papers is impossible in this limited space; we have therefore chosen to allude to many of them through the use of tables. Most of these are straightforward, descriptive studies, the essence of which is included in tables along with the literature references.

Regulation of liver microsomal inorganic pyrophosphate-glucose phosphotransferase, glucose-6-phosphatase and inorganic pyrophosphatase

Abstract Levels of liver inorganic pyrophosphate-glucose phosphotransferase, acid inorganic pyrophosphatase, and glucose-6-phosphate phosphohydrolase (EC 3.1.3.9) have been assayed in normal, alloxan-diabetic, adrenalectomized and cortisone-treated adrenalectomized rats. Assays were carried out in the absence of added detergent, and with homogenates which had been supplemented with sodium deoxycholate or Triton X-100 to final concentrations of 0.004%, 0.012%, 0.04%, 0.20%, and 0.40%, w/v (deoxycholate) or v/v (Triton). Increases in levels of all activities were observed in the absence of detergent in cortisone-treated adrenalectomized rats compared with untreated adrenalectomized animals, and in diabetic compared with normal rats. Both detergents activated all three activities in all groups of animals. However, basic differences in the modes of response of these activities to cortisone administration as contrasted with insulin deprivation were apparent when assays were conducted with detergent-supplemented homogenates. As detergent concentration was increased, a progressive diminution was noted in the ratios of activity in cortisone-treated compared with untreated adrenalectomized animals. Statistically significant differences in activity level values for the two groups of animals disappeared when deoxycholate concentrations were 0.20% or 0.40%; Triton X-100 also effected a marked diminution in activity level ratios under these conditions. In contrast, differences between comparable activity level values for diabetic compared with normal animals were progressively magnified with increasing concentration of both detergents. These observations indicate that the detergent effects involved are general in nature, and point up the complexity of mechanisms involved in responses of activities of this enzyme to hormonal manipulations. They also raise a question as to the conditions which properly should be employed to assess also raise a question as to the conditions which properly should be employed to assess alterations in functional levels, in vivo, of these activities in future horm and nutritional studies.

The biochemistry and molecular biology of the glucose-6-phosphatase system.

Progress has continued to be made over the past 4 years in our understanding of the glucose-6-phosphatase (G6Pase) system. The gene for a second component of the system, the putative glucose-6-P transporter (G6PT), was cloned, and mutations in this gene were found in patients diagnosed with glycogen storage disease type 1b. The functional characterization of this putative G6PT has been initiated, and the relationship between substrate transport via the G6PT and catalysis by the systems catalytic subunit continues to be explored. A lively debate over the feasibility of various aspects of the two proposed models of the G6Pase system persists, and the functional/structural relationships of the individual components of the system remain a hot topic of interest in G6Pase research. New evidence supportive of physiologic roles for the biosynthetlc functions of the G6Pase system in vivo also has emerged over the past 4 years.

Carbamyl phosphate: Glucose phosphotransferase activity of hepatic microsomal glucose 6-phosphatase at physiological pH☆

Abstract Evidence is presented indicating that classical hepatic microsomal D-glucose 6-phosphate phosphohydrolase (EC 3.1.3.9) possesses potent carbamyl phosphate: glucose phosphotransferase activity. Unlike phosphotransferase activity of this enzyme observed with nucleotides, phosphoenolpyruvate, or to a less pronounced extent inorganic pyrophosphate as phosphoryl donors, activity with carbamyl phosphate remains high even at pH 7.0 and 7.5. For example, at the latter pH in the presence of deoxycholate glucose 6-phosphate production with 10 mM carbamyl phosphate and 180 mM D-glucose is approximately double the rate of hydrolysis of 10 mM glucose 6-phosphate. A physiologically significant synthetic role for this phosphotransferase is suggested.

Multifunctional glucose-6-phosphatase: cellular biology.

Abstract Glucose-6-phosphatase is a multifunctional enzyme, displaying potent ability to synthesize as well as hydrolyze Glc-6-P. These multifunctional characteristics have been exploited in studies of the extended distribution of the enzyme, and their physiological significance has been examined. The enzyme is considerably more widely distributed than previously suspected. It has been found in pancreas, adrenals, lung, testes, spleen, and brain as well as in liver, kidney, and mucosa of small intestine. Approximately 15–20% of total hepatic glucose-6-phosphatase-phosphotransferase is present in nuclear membrane, 75–80% is found in endoplasmic reticulum, and small amounts have been detected also in plasma membrane and repeatedly-washed mitochondria. Both hydrolytic and synthetic functions, in constant proportions, have been found in livers of 21 species of birds, amphibia, reptiles, crustacea, fishes, and mammals (including man) studied. With 5 mM phosphoryl donor and 100 mM D-glucose as substrates, carbamyl-P:glucose phosphotransferase activity of glucose-6-phosphatase exceeded that of glucokinase by 5–50 fold. While latencies of activities of isolated microsomal preparations are extensive, those of nuclear membranes are not. Latencies of activities of intact endoplasmic reticulum of permeable hepatocytes are 28% for Glc-6-P phosphohydrolase and 56% for carbamyl-P:glucose phosphotransferase. Studies with isolated perfused livers from fasted rats suggest rather convincingly that such phosphotransferase activities may function as an hepatic glucose-phosphorylating system supplemental to glucokinase and hexokinase. This conclusion is based both on comparisons of rates of glucose uptake with hepatic enzyme levels (glucokinase, hexokinase, phosphotransferase), and on observed inhibitibility of glucose uptake by ornithine and 3-0-methyl-D-glucose. The question of availability of adequate concentrations of suitable phosphoryl donor(s) in cytosol of the liver cell constitutes a principal focus for continuing studies regarding physiological functions of this enzyme.