A new cerium-based method for cytochemical localization of thiamine pyrophosphatase in the Golgi complex of rat hepatocytes

1984 ◽  
Vol 80 (2) ◽  
pp. 107-111 ◽  
Author(s):  
S. Angermüller ◽  
H. Fahimi
Keyword(s):  
Golgi Complex ◽  
Rat Hepatocytes ◽  
Cytochemical Localization ◽  
Thiamine Pyrophosphatase

scholarly journals Cytochemical localization of beta-NADPase in rat hepatocytes and Kupffer cells. Comparison with thiamine pyrophosphatase (TPPase).

1984 ◽  
Vol 32 (5) ◽  
pp. 541-546 ◽  
Author(s):  
S Angermüller ◽  
H D Fahimi
Keyword(s):  
Golgi Apparatus ◽  
Kupffer Cells ◽  
Intracellular Localization ◽  
Rat Hepatocytes ◽  
The Other ◽  
Cytochemical Localization ◽  
Liver Parenchymal ◽  
Thiamine Pyrophosphatase ◽  
The Difference ◽  
Parenchymal Cells

The intracellular localization of beta-NADPase in rat hepatocytes and Kupffer cells has been studied and compared with the pattern of TPPase in these cells. The reaction product for beta-NADPase is present in some but not all hepatocytes in two cisternae on the trans aspect of the Golgi apparatus. It is absent from the trans-most lamella and the GERL of hepatocytes. TPPase, on the other hand, is limited to the first Golgi cisterna on the trans aspect with sprinkles of reaction product in the second lamella. Considering that TPPase is a marker of the trans Golgi lamella and hepatocyte Golgi stacks contain usually 2-4 lamellae, our observations suggest that beta-NADPase is localized in the trans as well as in the intermediate Golgi lamellae of liver parenchymal cells. In Kupffer cells, the reaction product for both beta-NADPase and TPPase was found in some but not in all cells. The enzyme beta-NADPase was localized in the rigid lamella and the tubulovacuolar system of GERL. This pattern differed significantly from that for TPPase, which was found in 2-3 cisternae at the trans aspect of the Golgi complex in Kupffer cells. These observations demonstrate the difference in the localization of beta-NADPase in hepatocytes and Kupffer cells. Such differences should be taken into consideration in studies of Golgi fractions, when phosphatase reactions are used as specific markers of Golgi components.

scholarly journals The effects of low temperatures on intracellular transport of newly synthesized albumin and haptoglobin in rat hepatocytes

1986 ◽  
Vol 237 (1) ◽  
pp. 33-39 ◽  
Author(s):  
E Fries ◽  
I Lindström
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Low Temperatures ◽  
Intracellular Transport ◽  
Rat Hepatocytes ◽  
Subcellular Fractionation ◽  
Isolated Rat Hepatocytes ◽  
Different Temperatures ◽  
Lag Period ◽  
Time Courses

Isolated rat hepatocytes were pulse-labelled with [35S]methionine at 37 degrees C and subsequently incubated (chased) for different periods of time at different temperatures (37-16 degrees C). The time courses for the secretion of [35S]methionine-labelled albumin and haptoglobin were determined by quantitative immunoprecipitation of the detergent-solubilized cells and of the chase media. Both proteins appeared in the chase medium only after a lag period, the length of which increased markedly with decreasing chase temperature: from about 10 and 20 min at 37 degrees C to about 60 and 120 min at 20 degrees C for albumin and haptoglobin respectively. The rates at which the proteins were externalized after the lag period were also strongly affected by temperature, the half-time for secretion being 20 min at 37 degrees C and 200 min at 20 degrees C for albumin; at 16 degrees C no secretion could be detected after incubation for 270 min. Analysis by subcellular fractionation showed that part of the lag occurred in the endoplasmic reticulum and that the rate of transfer to the Golgi complex was very temperature-dependent. The maximum amount of the two pulse-labelled proteins in Golgi fractions prepared from cells after different times of chase decreased with decreasing incubation temperatures, indicating that the transport from the Golgi complex to the cell surface was less affected by low temperatures than was the transport from the endoplasmic reticulum to the Golgi complex.

Cytochemical localization of glutaraldehyde-resistant NAD(P)H-oxidase in rat hepatocytes

1983 ◽  
Vol 79 (2) ◽  
pp. 259-267 ◽  
Author(s):  
Y. Mizukami ◽  
F. Matsubara ◽  
S. Matsukawa ◽  
R. Izumi
Keyword(s):  
Rat Hepatocytes ◽  
Cytochemical Localization

scholarly journals The cytochemical demonstration of GERL in rat hepatocytes during lipoprotein mobilization.

1978 ◽  
Vol 26 (1) ◽  
pp. 1-13 ◽  
Author(s):  
P M Novikoff ◽  
A Yam
Keyword(s):  
Acid Phosphatase ◽  
Orotic Acid ◽  
Rat Hepatocytes ◽  
Semisynthetic Diet ◽  
Hepatocyte Ultrastructure ◽  
Cytochemical Demonstration ◽  
Normal Hepatocyte ◽  
Golgi Element ◽  
Thiamine Pyrophosphatase ◽  
Normal Rat

When a semisynthetic diet containing 1% orotic acid (OA) is fed to rats, the endoplasmic reticulum (ER) of hepatocytes vesiculates and lipoprotein (LP) droplets accumulate within the vesicles. When clofibrate (ethyl chlorophenoxyisobutyrate, CPIB) is added to the orotic acid-rich diet, the ER cisternae reform and the LP is mobilized through the reconstituted ER. A remarkable restoration of normal hepatocyte ultrastructure occurs except for a few organelles. From their morphological appearance it was suggested that cisternae which became dilated with small LP particles were part of GERL, abnormally enlarged. The present communication validates this interpretation through ultrastructural cytochemistry which can distinguish GERL from the adjacent Colgi apparatus. GERL shows acid phosphatase (AcPase) but not thiamine pyrophosphatase (TPPase) activity. In contrast, the adjacent Golgi element shows thiamine pyrophosphatase but not acid phosphatase activity. From such cytochemical studies we have recently proposed that GERL in normal rat hepatocytes may be involved in transforming LP particles, by enzymes like lipases that were presumed to be present in this hydrolase-rich portion of smooth ER. In the situation studied in this communication, the addition of ethyl chlorophenoxyisobutyrate to the diet causes the release from the ER of large amounts of LP to the Golgi apparatus and to GERL. Apparently the capacity of GERL to metabolize LP is exceeded and lipid accumulates in the residual bodies.

scholarly journals Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes.

1983 ◽  
Vol 97 (5) ◽  
pp. 1582-1591 ◽  
Author(s):  
E S Sztul ◽  
K E Howell ◽  
G E Palade
Keyword(s):  
Membrane Proteins ◽  
Golgi Complex ◽  
Rat Hepatocytes ◽  
Secretory Component ◽  
Bile Canaliculus ◽  
Sds Page ◽  
Iga Antibody ◽  
Co Precipitation ◽  
Kinetics Of

The intra- and transcellular transports of hepatic secretory and membrane proteins were studied in rats in vivo using [3H]fucose and [35S]cysteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile, and plasma were separated by SDS PAGE and identified by fluorography. 3H-radioactivity in Golgi fractions peaked at 10 min postinjection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from Golgi occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 30 min later than the bulk of content proteins. A major 80,000-dalton form of secretory component (SC) was identified in the bile by co-precipitation with (IgA)2 by an anti-IgA antibody. An antibody (raised in rabbit) against the biliary 80,000-dalton peptide recognized two larger forms (116,000 and 94,000 dalton), presumably precursors, in Golgi membranes. A comparative study of kinetics of transport of 35S-SC and 35S-albumin showed that albumin peaked in bile at approximately 45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins that are delivered to the bile canaliculus.

Cytochemical Studies of Protein Uptake by Adrenal Cortical Cells of the Golden Hamster

1973 ◽  
Vol 31 ◽  
pp. 684-685
Author(s):  
J. R. Hillman ◽  
W. G. Seliger ◽  
P. E. Burk
Keyword(s):  
Acid Phosphatase ◽  
Golden Hamster ◽  
Adult Animal ◽  
Zona Glomerulosa ◽  
Cortical Cells ◽  
Cytochemical Localization ◽  
Protein Uptake ◽  
Dense Bodies ◽  
Thiamine Pyrophosphatase ◽  
Adrenal Cortical Cells

In a previous histochemical study, high levels of acid phosphatase were found in the developing adrenal cortex of the golden hamster. The present study describes the cytochemical localization of acid phosphatase and thiamine pyrophosphatase in the adult animal. These localizations are then related to protein uptake by adrenal cortical cells using the exogenous horseradish peroxidase as a tracer protein. Animals were sacrificed at times ranging from 15 minutes to 24 hours after injection of peroxidase. The techniques described by Novikoff et al. were utilized for localization of acid phosphatase and thiamine pyrophosphatase.In acid phosphatase preparations (Fig. 1), dense bodies containing reaction product are found in the cytoplasm of most cortical cells and are often associated with small coated vesicles near the Golgi area. Acid phosphatase activity is present in cells of the zona glomerulosa as well as those of the other two cortical zones.

Effects of Colchicine on the Golgi Complex and Gerl of Cultured Rat Peritoneal Macrophages and Epiphyseal Chondrocytes

1980 ◽  
Vol 45 (1) ◽  
pp. 41-58
Author(s):  
JOHAN THYBERG ◽  
ANDRZEJ PIASEK ◽  
STANISLAW MOSKALEWSKI
Keyword(s):  
Golgi Complex ◽  
Structural Integrity ◽  
Cell Types ◽  
Peritoneal Macrophages ◽  
Electron Microscopic ◽  
Thiamine Pyrophosphatase ◽  
Cytoplasmic Microtubules ◽  
The Individual ◽  
Functionally Diverse

Thioglycollate-elicited rat peritoneal macrophages and epiphyseal chondrocytes were cultured in vitro, treated with colchicine, and then studied by electron-microscopic and cytochemical techniques. Colchicine, but not lumicolchicine, caused disappearance of cytoplasmic microtubules and breakup of the Golgi complex with spreading of its dictyosomes from a well defined juxta nuclear area throughout the cytoplasm. There was also an altered distribution of lysosomes, which oriented themselves close to the dictyosomes both in controland colchicine-treated cells. Further, the structure of the individual dictyosomes was changed, especially in the chondrocytes. GERL equivalents were observed in control cells but were difficult to detect after exposure to colchicine. Reaction product for thiamine pyrophosphatase was found in narrow cisternae on the inner side of the dictyosomes in control cells but invacuole-like structures in colchicine-treated cells. Reaction product for acid phosphatase was present in GERL equivalents and lysosomes in control cells but mainly in lysosomes incolchicine-treated cells. Nevertheless, the total specific activities of these enzymes as well as of 5′-nucleotidase, a plasma membrane marker, remained unaffected by the drug treatment. These observations show that cytoplasmic microtubules play an important and, in many respects similar, cytoskeletal role in two so functionally diverse cell types as macrophages and chondrocytes. They are particularly important for the structural integrity of the Golgi complex, which in both cells is normally organized in the area around the centrioles, from which numerous microtubules radiate into the cytoplasm. The observations further suggest that GERL is an integrated part of the Golgi complex in these cells.

scholarly journals Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

1997 ◽  
Vol 8 (10) ◽  
pp. 1911-1931 ◽  
Author(s):  
Randall S. Taylor ◽  
Steven M. Jones ◽  
Rolf H. Dahl ◽  
Mark H. Nordeen ◽  
Kathryn E. Howell
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Secretory Pathway ◽  
Calcium Uptake ◽  
Rat Hepatocytes ◽  
Calcium Atpase ◽  
Endoplasmic Reticulum Calcium ◽  
Calcium Atpases ◽  
In Transit

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.

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