Glucose-6-phosphatase localization

FIGURE 23.8 Glu cose-6-phosphatase is localized in the endoplasmic reticulum membrane. Conversion of glucose-6-phosphate to glucose occurs during transport into the ER. 

Although the enzyme sediments with intact cells, alkaline phosphatase appears in the supernate when broken cells are centrifuged. Malamy and Horecker (5) discovered that alkaline phosphatase is quantitatively released from the cell when E. coli are converted to spheroplasts by lysozyme and ethylenediaminetetraacetic acid (EDTA) in a sucrose medium. This evidence, supported by the observation that substrates such as glucose 6-phosphate are rapidly hydrolyzed by intact cells with release of most of the phosphate into the medium, led Malamy and Horecker (6) to suggest that alkaline phosphatase is localized in the periplasmic space, a region described by Mitchell (7) as lying between the protoplasmic membrane and the wall layer, and that it is not in association with the wall (8). 

Describe a possible means for the cytochemical detection and localization of the enzyme glucose 6-phosphatase it exists in liver and catalyzes the following reaction ... 

GC mode SECM experiments have been used to detect localized activity of a variety of enzymes (see Table 2). Since microelectrodes are used in the life sciences to detect neurotransmitters (36), metal cations (37), and free radicals (38), a wide variety of probes may be combined with SECM positioning technology to obtain spatially resolved information. The GC mode experiments may be carried out with potentiometric or amperometric tips depending on the species to be sensed. The tip must be chosen to determine a product or reactant consumed by the enzyme reaction, e.g., H+ for immobilized urease, H202 for immobilized glucose oxidase, or 4-aminophen-olate for alkaline phosphatase. A list of enzymes and the tips employed is given in Table 2. 

Among the most used labels are enzymes, such as peroxidise (HRP), glucose oxidase (Gox), and alkaline phosphatase (AP), that generate an electroactive product close to the transducer surface the formation of a relatively high local concentration of the enzyme product leads to a significant signal amplification. 

Studies of the distribution of glucose-6-phosphatase in hepatocytes after birth support the second view. During hepatocyte development in the fetus and the newborn rat, the rough endoplasmic reticulum appears first. The smooth endoplasmic reticulum expands a few days before birth, and glucose-6-phosphatase appears after birth. Electron microscopic localization of the glucose-6-phosphatase shows that the enzyme appears simultaneously in most hepatocytes in all parts of the endoplasmic reticulum. Therefore, there are no growing points for glucose-6-phosphatase, and the new enzyme molecules appear to be continuously inserted within the old framework. 

It has been suggested by Senior to classify such oases as type Ib with the assumption that although glucose-6-phosphatase is present in tissue it is not available for metabolism because of local factors, (2)... 

It was also beheved that the gluconeogenic machinery, including four critical enzymes pyruvate carboxylase, PEP carboxykinase, fructose-1,6-diphosphatase, and glucose-6-phosphatase could be fuUy expressed exclusively in the liver and in the kidney. Specifically, with the exception of hepatocytes and renal cortical cells, it was considered impossible that any glucose synthesized de novo or released Irom local glycogen stores could be released in the systemic circulation, because of the absence of gJucose-6-phosphatase. 

There is a recent, detailed account of methods for preparing and characterizing the components of the endoplasmic reticulum (DePierre and Dallner, 1976). Table 2 lists a number of enzymes associated with the endoplasmic reticulum. Several of these enzymes have been localized to the cytoplasmic surface of the reticular membranes, including cytochrome NADH-cytochrome bs-reductase, Mg " -ATPase, 5 -nucleotidase, nucleoside pyrophosphorylase, and GDP-mannosyl transferase. A number of others, including nucleoside diphosphatase, glucose-6-phosphatase, acetanilide-hydrolyzing esterase, and -glucuronidase have been localized to the luminal surface (DePierre and Ems-ter, 1977). Cytochrome P-450 appears to be present at both the cytoplasmic and luminal surfaces. 

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