Multifunctional glucose-6-phosphatase studied in permeable isolated hepatocytes.

Jorgenson, Roger A.; Nordlie, Robert C.

doi:10.1016/s0021-9258(19)70717-0

Cited by 71 publications

(3 citation statements)

References 35 publications

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“…Latency is calculated as 100 (detergent-treated activity -untreated activity)/detergent-treated activity. * From the procedure of Jorgenson and Nordlie (6). t From the procedure of Gankema et al (7).…”

Section: Assays Of Glucose-6-phosphatase Activity In A-toxin-treated Cellsmentioning

confidence: 99%

“…In spite of this selective interaction, the use of filipin or digitonin can cause extensive changes in cell morphology (12,16,17). 2 The rough ER is especially vulnerable, and other studies show that these agents alter the kinetics of ER enzymes (Lange, A. J., B. F. McEwen, and W. J. Arion, unpublished observations; and references [18][19][20], which suggests the agents interact with the ER membrane or ER proteins. Because of the structural similarity of saponin to digitonin (21), saponin interaction with the ER membrane is also expected.…”

mentioning

confidence: 97%

“…Filipin (3)(4)(5)(6)(7), digitonin (8)(9)(10)(11)(12), and saponin (13)(14)(15), all of which complex with membrane-bound cholesterol, have been used to create permeability in cells to a variety of substances. Since the plasma membrane has a higher cholesterol content than most internal membranes (e.g., mitochondria and the 1922 endoplasmic reticulum [ER]'), it has been postulated that these agents would react preferentially with the plasma membrane (6, 7, 1 l, 13).…”

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Permeabilization of rat hepatocytes with Staphylococcus aureus alpha-toxin.

McEwen¹,

Arion²

1985

The Journal of Cell Biology

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Pathogenic staphylococci secrete a number of exotoxins, including a-toxin, aToxin induces lysis of erythrocytes and liposomes when its 3S protein monomers associate with the lipid bilayer and form a hexomeric transmembrane channel 3 nm in diameter. We have used a-toxin to render rat hepatocytes 93-100% permeable to trypan blue with a lactate dehydrogenase leakage <_22%. Treatment conditions included incubation for 5-10 min at 37°C and pH 7.0 with an a-toxin concentration of 4-35 human hemolytic U/ml and a cell concentration of 13-21 mg dry wt/ml. Scanning electron microscopy revealed signs of swelling in the treated hepatocytes, but there were no large lesions or gross damage to the cell surface. Transmission electron microscopy indicated that the nucleus, mitochondria, and cytoplasm were similar in control and treated cells and both had large regions of well-defined lamellar rough endoplasmic reticulum. Comparisons of the mannose-6-phosphatase and glucose-6-phosphatase activities demonstrated that 5-10 U/ml a-toxin rendered cells freely permeable to glucose-6-phosphate, while substantially preserving the selective permeability of the membranes of the endoplasmic reticulum and the functionality of the glucose-6-phosphatase system. Thus, a-toxin appears to have significant potential as a means to induce selective permeability to small ions. It should make possible the study of a variety of cellular functions in situ.The study of the relationship of enzymic activities to internal cell structure requires the development of assay systems that can determine these activities in situ. For most reactions, the development of such assays requires circumventing the permeability barrier presented by the plasma membrane. The use of toluene (1, 2) or detergents to render the plasma membrane permeable to ionic substrates is unsatisfactory since such agents induce permeability changes in intracellular membranes. Toluene treatment also results in large morphological changes in the internal cell structure (2). The ideal reagent would be one that selectively reacts with the plasma membrane, does not induce any alterations of cellular morphology, and does not cause leakage of cytosolic enzymes.Filipin (3-7), digitonin (8-12), and saponin (13-15), all of which complex with membrane-bound cholesterol, have been used to create permeability in cells to a variety of substances. Since the plasma membrane has a higher cholesterol content than most internal membranes (e.g., mitochondria and the 1922 endoplasmic reticulum [ER]'), it has been postulated that these agents would react preferentially with the plasma membrane (6, 7, 1 l, 13). In spite of this selective interaction, the use of filipin or digitonin can cause extensive changes in cell morphology (12,16,17). 2 The rough ER is especially vulnerable, and other studies show that these agents alter the kinetics of ER enzymes (Lange, A. J., B. F. McEwen, and W. J. Arion, unpublished observations; and references 18-20), which suggests the agents interact with the ER membrane or E...

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Section: Assays Of Glucose-6-phosphatase Activity In A-toxin-treated Cellsmentioning

confidence: 99%

mentioning

confidence: 97%

mentioning

confidence: 99%

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Permeabilization of rat hepatocytes with Staphylococcus aureus alpha-toxin.

McEwen¹,

Arion²

1985

The Journal of Cell Biology

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Endoplasmic Reticulum

B��nhegyi¹,

Benedetti

Csala

2008

Wiley Encyclopedia of Chemical Biology

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Several pathways are compartmentalized in the endoplasmic reticulum (ER). These intraluminal activities require the passage of substrates, cofactors, and products through the ER membrane. The arguments for a general permeability of the ER membrane are contradicted by strong biochemical, pharmacological, clinical, and genetic evidence, which indicates that the lipid bilayer has a barrier function and that specific transport activities are needed in the membrane. Consequently, the ER lumen can be regarded as a separate metabolic compartment. This article overviews the best characterized intraluminal processes in which the compartmentation is important either by defining an intraluminal milieu, by limiting the rate of the reaction, by determining the specificity, or by creating a common substrate pool because of the colocalization.

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Glucose‐6‐Phosphatase: Two Concepts of Membrane‐Function Relationship

Sukalski

Nordlie

1989

Advances in Enzymology - And Related Areas of Molecular Biology

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Multifunctional glucose-6-phosphatase studied in permeable isolated hepatocytes.

Cited by 71 publications

References 35 publications

Permeabilization of rat hepatocytes with Staphylococcus aureus alpha-toxin.

Permeabilization of rat hepatocytes with Staphylococcus aureus alpha-toxin.

Endoplasmic Reticulum

Glucose‐6‐Phosphatase: Two Concepts of Membrane‐Function Relationship

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