Intracellular Distribution of Some Enzymes Catalyzing Reactions in the Biosynthesis of Complex Lipids

619

483

Abstract. The interrelationships of the endoplasmic reticulum (ER), microtubules, and intermediate illaments were studied in the peripheral regions of thin, spread fibroblasts, epithelial, and vascular endothelial cells in culture. We combined a fluorescent dye staining technique to localize the ER with immunofluorescence to localize microtubules or intermediate filaments in the same cell.Microtubules and the ER are sparse in the lamellipodia, but intermediate filaments are usually completely absent. These relationships indicate that microtubules and the ER advance into the lamellipodia before intermediate filaments.We observed that microtubules and tubules of the ER have nearly identical distributions in lamellipodia, where new extensions of both are taking place. We perturbed microtubules by nocodazole, cold temperature, or hypotonic shock, and observed the effects on the ER distribution. On the basis of our observations in untreated cells and our experiments with microtubule perturbation, we conclude that microtubules and the ER are highly interdependent in two ways: (a) polymerization of individual microtubules and extension of individual ER tubules occur together at the level of resolution of the fluorescence microscope, and (b) depolymerization of microtubules does not disrupt the ER network in the short term (15 min), but prolonged absence of microtubules (2 h) leads to a slow retraction of the ER network towards the cell center, indicating that over longer periods of time, the extended state of the entire ER network requires the microtubule system.

Section: Discussionmentioning

confidence: 99%

Microtubules and the endoplasmic reticulum are highly interdependent structures.

Terasaki¹,

Chen²,

Fujiwara³

1986

619

483

“…The present demonstration that phosphorylcholine-glyceride transferase is specifically associated with the membranes of the endoplasmic reticulum refines and confirms earlier conclusions based on the fractionation of cell extracts by differential centrifugation (25) . Although those fractionation techniques do not achieve a clean separation of organelle fractions, phosphorylcholine-glyceride transferase was predominantly recovered in microsomal pellets in studies involving both animal (28,44) and plant (8, 20, 31) tissues. Such crude microsomal pellets from plants were previously shown to contain NADHand NADPH-cytochrome c reductases and NADH diaphorase (16,26,27,35) as well as cytochromes b5 and P 450 (5,16,32,37) .…”

Section: Effect Of Mg++ On Sedimentation Behaviormentioning

confidence: 99%

Endoplasmic Reticulum as the Site of Lecithin Formation in Castor Bean Endosperm

Lord

Kagawa

Moore

et al. 1973

320

201

The properties of a discrete membranous fraction isolated on sucrose gradients from castor bean endosperm have been examined . This fraction was previously shown to be the exclusive site of phosphorylcholine-glyceride transferase . The distribution of NADPH-cytochrome c reductase and antimycin insensitive NADH-cytochrome c reductase across the gradient followed closely that of the phosphorylcholine-glyceride transferase . This fraction also had NADH diaphorase activity and contained cytochromes b5 and P 450. On sucrose gradients containing 1 mM EDTA this fraction had a mean isopycnic density of 1 .12 g/cma and sedimented separately from the ribosomes ; electron micrographs showed that it was comprised of smooth membranes . When magnesium was included in the gradients to prevent the dissociation of membrane-bound ribosomes, the isopycnic density of the membrane fraction with its associated enzymes was increased to 1 .16 g/cma and under these conditions the electron micrographs showed that the membranes had the typical appearance of rough endoplasmic reticulum . Together these data show that the endoplasmic reticulum is the exclusive site of lecithin formation in the castor bean endosperm and establish a central role for this cytoplasmic component in the biogenesis of cell membranes .

“…The liver endoplasmic reticulum (ER) plays a central role in the synthesis of plasma proteins (14,15), various types of lipoproteins (16)(17)(18), cholesterol (19,20), and glycoproteins (21,22), and in the metabolism of small molecules of physiological as well as nonphysiological origin (cf. reference 23).…”

Section: Introductionmentioning

confidence: 99%

Permeability of Microsomal Membranes Isolated From Rat Liver

Nilsson

Peterson

Dallner

1973

117

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.