Differentiation of Endoplasmic Reticulum in Hepatocytes

Leskes, A.; Siekevitz, Philip; Palade, George E.

doi:10.1083/jcb.49.2.288

Cited by 123 publications

(38 citation statements)

References 22 publications

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“…In contrast, the density alteration described in this and other (8,19,25) papers is performed in vitro on subcellular organelles whose equilibrium density is initially unaffected.…”

Section: Analytical and Preparative Applications To Subcellular Organmentioning

confidence: 93%

Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles.

Courtoy¹,

Quintart²,

Baudhuin³

1984

The Journal of Cell Biology

133

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Galactosylated BSA (gaIBSA) and its conjugate to horseradish peroxidase (gaIBSA-HRP) enter the galactose-specific pathway of hepatocytes . 10 min after intravenous injection, structures containing either ligand sediment mostly between 33,000 and 3 x 106 g~min (LP fraction) and have an equilibrium density of 1 .11-1 .13 g/ml in sucrose gradients (Quintart, J., P. J . Courtoy, J . N . Limet, and P. Baudhuin, 1983, Eur . J. Biochem ., 131 :105-112) .Such low density fractions, prepared from rats given gaIBSA-HRP, were incubated for 30 min at 25°C in 5.5 mM 3,3'-diaminobenzidine (DAB) and 11 mM H202 in buffered sucrose . Upon equilibration in a second sucrose gradient, the gaIBSA-HRP distribution shifted towards higher 01 .19 g/ml) density, but the bulk of protein remained at low density . In the absence of H202, gaIBSA-HRP distribution was also found at low density . As observed by electron microscopy, particles equilibrating at higher density after DAB cytochemistry were largely made of vesicles or tubules filled with DAB reaction product. The density shift of gaIBSA-HRP-containing organelles after incubation with DAB and H zOz is attributed to the trapping of HRP-oxidized DAB inside the host organelles .If the low density fractions isolated from a rat injected with [3 H]gaIBSA-HRP were mixed in vitro with similar fractions from another rat given [' 4C]gaIBSA, the 3 H distribution shifted after DAB cytochemistry, but the '4C distribution was essentially unaffected . By contrast, if both derivatives were injected simultaneously, a concomitant density shift was observed .In conclusion, the DAB-induced density shift was specific to ligand-HRP-containing organelles . The potentials of the method include the purification of HRP-containing particles and the study of their association to ligands, fluid-phase tracers, or marker enzymes .After receptor-mediated internalization (3, 28), ligands are rapidly transferred to an "intermediate compartment" (34,37), often referred to as "endosomes" (14) or "receptosomes" (37). To assess the purity of subcellular fractions enriched in ligand-containing organelles, we recently used ligands conjugated to horseradish peroxidase (HRP),' an approach that proved successful in intact liver (29,33). Glutaraldehydefixed fractions were incubated in 3,3'-diaminobenzidine ' Abbreviations used in this paper: DAB, 3,3'-diaminobenzidine; gaIBSA, galactosylated BSA; gaIBSA-HRP, gaIBSA conjugated to horseradish peroxidase (HRP) .(DAB) and HZOz (12) and processed for electron microscopy . The organelles containing ligand-HRP were identified by the DAB reaction product (23). We report here that the DAB procedure can also be applied to unfixed subcellular fractions. Moreover, the accumulation of polymerized DAB inside HRP-containing organelles induces a major increase in their equilibrium density, while other organelles of the same fraction are essentially unaftècted.It has already been reported that cytochemical procedures may be applied to the isolation of specific organelles with endogeno...

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“…In contrast, the density alteration described in this and other (8,19,25) papers is performed in vitro on subcellular organelles whose equilibrium density is initially unaffected.…”

Section: Analytical and Preparative Applications To Subcellular Organmentioning

confidence: 93%

Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles.

Courtoy¹,

Quintart²,

Baudhuin³

1984

The Journal of Cell Biology

133

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“…It should, however, be borne in mind that the absence of visible deposits in many vesicles does not mean that these vesicles are devoid of either deposit or glucose 6-phosphatase activity, since deposits may have been missed in the particular plane in which the vesicles are sectioned. Leskes et al (1971b), using a much higher Pb(N03)2 concentration in the incubation medium, also found vesicles that contained no deposit. The specific activities of glucose 6-phosphatase in the bands obtained by the technique described in this paper are given in Tables 2 and 3.…”

Section: Explanation Of Platementioning

confidence: 99%

“…During the development of this technique, Leskes et al (1971b) published their, rather different, procedure for separating microsomal vesicles containing glucose 6-phosphatase from those devoid of such activity. Our method is also of general applicability and can be used, for example, to separate vesicles derived from the endoplasmic reticulum from plasma membrane, Golgi elements and other components of the microsomal fraction as well as separating vesicles containing different amounts of glucose 6-phosphatase activity.…”

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confidence: 99%

Heterogeneous distribution of glucose 6-phosphatase in rat liver microsomal fractions as shown by adaptation of a cytochemical technique

Lewis¹,

Tata²

1973

Biochemical Journal

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1. A novel technique for the subfractionation of rat liver smooth and rough microsomal fractions according to their content of glucose 6-phosphatase is described. This technique, based on the Gomori lead histochemical procedure, involves incubation of smooth and rough microsomal fractions with low concentrations of Pb(NO3)2 and glucose 6-phosphate. Control experiments, in which enzyme was assayed in the presence of various amounts of Pb(NO3)2 or in which microsomal fractions were reisolated after incubation with low concentrations of Pb(NO3)2 and glucose 6-phosphate, showed that lead does not interfere with glucose 6-phosphatase activity. 2. Discontinuous sucrose-densitygradient centrifugation of microsomal fractions which had previously been incubated with various amounts of Pb(N03)2 and glucose 6-phosphate showed that it is possible to subfractionate both smooth-and rough-microsomal fractions into several bands, owing to a differential modification of the density of the microsomal vesicles by the trapping of lead phosphate within them. 3. When the material in the bands obtained by density-gradient centrifugation ofincubated microsomal fractions was assayed for glucose 6-phosphatase activity, it was found that the modification of the density ofthe microsomal fractions was directly related to their relative enrichment in glucose 6-phosphatase activity. Control experiments, in which microsomal fractions were incubated with Pb(NO3)2 and glucose 6-phosphate and then treated with EDTA, showed that the subfractionation was not due to aggregation of microsomal vesicles, lead and glucose 6-phosphate. Thus the resolution of microsomal preparations into subfractions with different glucose 6-phosphatase activities is interpreted as indicating heterogeneity of glucose 6-phosphatase distribution in the microsomal vesicles. 4. Electron micrographs of both smooth-and rough-microsomal subfractions show deposits of lead phosphate within the microsomal vesicles. The frequency and extent of these deposits correlate with the different amounts of glucose 6-phosphatase activity measured biochemically. 5. The nature of the heterogeneous distribution of glucose 6-phosphatase is discussed and the more general applicability of the technique for studying membrane fractions containing a heterogeneous distribution of phosphatases is indicated.

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“…The lack of glucose accumulation indicates that G1P is transported into vesicles lacking G6Pase enzyme activity, since G1P is a good substrate for the G6Pase enzyme [26]. Classic reports on the localization of the G6Pase enzyme activity in liver ER in situ [29], and in isolated liver microsomal vesicles [30], indicated that the enzyme is not present in all vesicles, even though it is evenly distributed throughout the entire ER network. In liver, GPT activity is therefore probably not involved in G6P hydrolysis by the G-6-Pase enzyme.…”

Section: Discussionmentioning

confidence: 99%

Glucose 6-phosphate transport in fibroblast microsomes from glycogen storage disease type 1b patients: evidence for multiple glucose 6-phosphate transport systems

Leuzzi

Fulceri

Marcolongo

et al. 2001

Biochemical Journal

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In liver endoplasmic reticulum the intralumenal glucose-6-phosphatase activity requires the operation of a glucose 6-phosphate transporter (G6PT1). Mutations in the gene encoding G6PT1 cause glycogen storage disease type 1b, which is characterized by a loss of glucose-6-phosphatase activity and impaired glucose homoeostasis. We describe a novel glucose 6-phosphate (G6P) transport activity in microsomes from human fibroblasts and HeLa cells. This transport activity is unrelated to G6PT1 since: (i) it was similar in microsomes of skin fibroblasts from glycogen storage disease type 1b patients homozygous for mutations of the G6PT1 gene, and in microsomes from human control subjects; (ii) it was insensitive to the G6PT1 inhibitor chlorogenic acid; and (iii) it was equally active towards G6P and glucose 1-phosphate, whereas G6PT1 is highly selective for G6P. Taken together, our results provide evidence for the presence of multiple transporters for G6P (and other hexose phosphoesters) in the endoplasmic reticulum.

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Differentiation of Endoplasmic Reticulum in Hepatocytes

Cited by 123 publications

References 22 publications

Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles.

Shift of equilibrium density induced by 3,3'-diaminobenzidine cytochemistry: a new procedure for the analysis and purification of peroxidase-containing organelles.

Heterogeneous distribution of glucose 6-phosphatase in rat liver microsomal fractions as shown by adaptation of a cytochemical technique

Glucose 6-phosphate transport in fibroblast microsomes from glycogen storage disease type 1b patients: evidence for multiple glucose 6-phosphate transport systems

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