Enzyme-Structure Relationships in the Endoplasmic Reticulum of Rat Liver

Ernster, Lars; Siekevitz, Philip; Palade, George E.

doi:10.1083/jcb.15.3.541

Cited by 718 publications

(156 citation statements)

References 47 publications

(63 reference statements)

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“…Microsomes were subfractionated into ribosomes, a membrane fraction, and a microsomal content fraction following the method of Ernster et al . (18) as previously modified (8) . It is important in this procedure that the microsomes be washed thoroughly in 0 .25 M sucrose after incubation and that microsomes from at least 5 g of liver be used for each 10 ml of suspension before the addition of the 0 .26% sodium deoxycholate.…”

Section: Methodsmentioning

confidence: 99%

“…Methods have been developed, using limiting amounts of deoxycholate, which can separate the microsomes into a ribosomal fraction, a membrane fraction, and the contents of the cisternae of the microsomal vesicle (18) . In order to determine which of these microsomal subfractions contained the radio-23 6 THE JOURNAL OF CELL BIOLOGY • VOLUME 52,1972 active proteins, microsomes were incubated with either leucine-14C or the appropriate radioactive nucleotide-sugars, and then the washed microsomes were subfractionated into the three microsomal subfractions .…”

Section: Localization Of the Radioactive Proteins On Microsomal Subfrmentioning

confidence: 99%

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The Secretory Pathways of Rat Serum Glycoproteins and Albumin

Redman

Cherian

1972

The Journal of Cell Biology

147

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These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein . N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membraneattached polysomes and not into proteins from free polysomes . Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes .Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae . Albumin was mostly recovered (98%0 ) in the cisternae, with negligible amounts in the membranes . To determine whether the radioactive sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the "C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins . The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae .

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Section: Methodsmentioning

confidence: 99%

Section: Localization Of the Radioactive Proteins On Microsomal Subfrmentioning

confidence: 99%

The Secretory Pathways of Rat Serum Glycoproteins and Albumin

Redman

Cherian

1972

The Journal of Cell Biology

147

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“…Total microsomes from rat liver were prepared according to Ernster et al (38). The microsomal pellets were suspended in 0.5 M sucrose when routine experiments were made for determination of water compartments.…”

Section: F Actionationsmentioning

confidence: 99%

Permeability of Microsomal Membranes Isolated From Rat Liver

Nilsson

Peterson

Dallner

1973

The Journal of Cell Biology

117

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Water compartments, permeability, and the possible active translocation of various substances in rat liver microsomes were studied by using radioactive compounds and ultracentrifugation. The total water of the microsomal pellet, 3.4 #l/rag dry weight, is the sum of water in the extramicrosomal and intramicrosomal spaces, or 56 and 44%, respectively. Sucrose space accounts for 77% of the intramicrosomal water and the hydration water ~ 14 %, leaving almost no sucrose-impermeable space when using the ultracentrifugation approach. With increasing sucrose concentration, microsomes do not show an osmotic response. The intramicrosomal water decreases greatly-in the presence of Cs + and Mg ++ in rough but not in smooth microsomes. Uncharged substances of molecular weight of up to at least 600 freely penetrate microsomal membranes, which already become impermeable to charged substances at a molecular weight of 90. These substances also induce an osmotic response. The vesicles can be made permeable to charged substances after water treatment and cooling, which, however, does not increase glucose-6-phosphatase and inosine diphosphatase (IDPase) activities, and these enzymes can still be activated by deoxycholate. IDPase, reduced nicotinamide adenine dinucleotide-cytochrome c reductase, and reduced nicotinamide adenine dinucleotide phosphate-dependent hydroxylation reactions, performed in vitro, also disproved the hypothesis of an accumulation of charged substances inside of vesicles of being a major pathway. The products of the enzymic reactions as well as the glucuronidated form of a hydroxylated product can be recovered on the cytoplasmic side of membranes, and little accumulation occurs in the intravesicular compartment.

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“…The rats were sacrificed at various times after the initial injection and a portion of the liver was used to make microsomes according to Ernster et al [7] . Microsomal phospholipid was extracted with chloroform-methanol [8] and lipid phosphorus was determined according 240 to Bartlett [9].…”

Section: Methodsmentioning

confidence: 99%