Characterization of a Chloride-Selective Channel from Rough Endoplasmic Reticulum Membranes of Rat Hepatocytes: Evidence for a Block by Phosphate

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.

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Biogenesis of Smooth Endoplasmic Reticulum in Hepatocytes of Phenobarbital Treated Rats

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051554 ◽

1974 ◽

Vol 32 ◽

pp. 308-309

Author(s):

Joan A. Higgins

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Specific Activity ◽

Smooth Endoplasmic Reticulum ◽

Rat Hepatocytes ◽

Membrane Formation ◽

Drug Detoxification ◽

Marked Proliferation

In response to intraperitoneal injections of phenobarbital there is a marked proliferation of smooth endoplasmic reticulum membranes (s.e.r.) of rat hepatocytes, with little change in other membranous organelles. This increased membrane formation is accompanied by a rise in the specific activity of the enzymes involved in drug detoxification initially in the rough endoplasmic reticulum (r.e.r.) followed by a rise in the s.e.r. There is also an increased accumulation of glycerophospholipid in the newly formed s.e.r.

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Dilated rough endoplasmic reticulum, abnormal distribution of collagen fibril diameters and gene abnormalities as clues to the diagnosis and characterization of Ehlers-Danlos type IV

Journal of Dermatological Science ◽

10.1016/0923-1811(93)91399-f ◽

1993 ◽

Vol 6 (1) ◽

pp. 117

Author(s):

J.D. Marden ◽

H. Kuivaniemi ◽

J.C. Pierson ◽

J. McMahon ◽

T.N. Helm ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Collagen Fibril ◽

Type Iv ◽

Abnormal Distribution

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Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum

Journal of Cellular Biochemistry ◽

10.1002/(sici)1097-4644(20000201)76:2<244::aid-jcb8>3.0.co;2-k ◽

2000 ◽

Vol 76 (2) ◽

pp. 244-253 ◽

Cited By ~ 26

Author(s):

Woo-kyoung Lee ◽

Seungjin Shin ◽

Soo Seock Cho ◽

Jong-sang Park

Keyword(s):

Endoplasmic Reticulum ◽

Glutamate Dehydrogenase ◽

Rough Endoplasmic Reticulum ◽

Purification And Characterization

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Radioautographic characterization of successive compartments along the rough endoplasmic reticulum-Golgi pathway of collagen precursors in foot pad fibroblasts of [3H]proline-injected rats.

The Journal of Cell Biology ◽

10.1083/jcb.98.5.1705 ◽

1984 ◽

Vol 98 (5) ◽

pp. 1705-1709 ◽

Cited By ~ 23

Author(s):

F Marchi ◽

C P Leblond

Keyword(s):

Endoplasmic Reticulum ◽

Rough Endoplasmic Reticulum ◽

Extracellular Space ◽

Unit Volume ◽

Secretory Granules ◽

Migration Pathway ◽

Precursor Proteins ◽

Foot Pad ◽

Silver Grains

Young rats given an intravenous injection of [3H]proline were killed at successive times from 4 to 80 min later. Fibroblasts from the front foot pad were radioautographed ; silver grains were counted over several of the organelles and the results were expressed as percent radiolabel per unit volume. These percentages reached a peak over rough endoplasmic reticulum cisternae at 4 min, intermediate vesicles and tubules at 10 min, spherical distensions of cis-side Golgi saccules at 20 min, cylindrical distensions of trans-side saccules between 40 and 60 min, and secretory granules at 60 min. It is proposed that the succession of peaks corresponds to the migration pathway of collagen precursor proteins within fibroblasts; that is, the proteins synthesized in rough endoplasmic reticulum are delivered by intermediate vesicles and/or tubules to the spherical distensions of cis-side saccules, somehow pass from there to the cylindrical distensions of trans-side saccules and, finally, are carried by secretory granules to the extracellular space.

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Purification and characterization of autophagosomes from rat hepatocytes

Biochemical Journal ◽

10.1042/bj3350217 ◽

1998 ◽

Vol 335 (2) ◽

pp. 217-224 ◽

Cited By ~ 104

Author(s):

Per Eivind STRØMHAUG ◽

Trond Olav BERG ◽

Monica FENGSRUD ◽

Per O. SEGLEN

Keyword(s):

Endoplasmic Reticulum ◽

Density Gradient ◽

Rat Hepatocytes ◽

Isolated Hepatocytes ◽

Protein Markers ◽

Intact Cells ◽

Cytoskeletal Elements ◽

Intermediate Density ◽

Glucose Regulated Protein

To investigate the properties and intracellular origin of autophagosomes, a procedure for the purification and isolation of these organelles from rat liver has been developed. Isolated hepatocytes were incubated with vinblastine to induce autophagosome accumulation; the cells were then homogenized and treated with the cathepsin C substrate glycyl-l-phenylalanine 2-naphthylamide to cause osmotic disruption of the lysosomes. Nuclei were removed by differential centrifugation, and the postnuclear supernatant was fractionated on a discontinuous Nycodenz density gradient. The autophagosomes, recognized by their content of autophagocytosed lactate dehydrogenase (LDH), could be recovered in an intermediate-density fraction, free from cytosol and mitochondria. Finally, the autophagosomes were separated from the endoplasmic reticulum and other membranous elements by centrifugation in a Percoll colloidal density gradient, followed by flotation in iodixanol to remove the Percoll particles. The final autophagosome preparation represented a 24-fold purification of autophagocytosed LDH relative to intact cells, with a 12% recovery. The purified autophagosomes contained sequestered cytoplasm with a normal ultrastructure, including mitochondria, peroxisomes and endoplasmic reticulum in the same proportions as in intact cells. However, immunoblotting indicated a relative absence of cytoskeletal elements (tubulin, actin and cytokeratin), which may evade autophagic sequestration. The autophagosomes showed no enrichment in protein markers typical of lysosomes (acid phosphatase, cathepsin B, lysosomal glycoprotein of 120 kDa), endosomes (early-endosome-associated protein 1, cation-independent mannose 6-phosphate receptor, asialoglycoprotein receptor) or endoplasmic reticulum (esterase, glucose-regulated protein of 78 kDa, protein disulphide isomerase), suggesting that the sequestering membranes are not derived directly from any of these organelles, but rather represent unique organelles (phagophores).

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