Evidence that the enzymes involved in the methylation of phosphatidylethanolamine are on the external side of the microsomal vesicles

Audubert, François; Vance, Dennis E.

doi:10.1016/0005-2760(84)90204-2

Cited by 17 publications

(4 citation statements)

References 30 publications

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“…Although early trypsin-proteolysis studies suggested that certain domains of PEMT were localized external to the microsomal membranes, the specific membrane topography of PEMT had, until now, remained elusive (34). Here, we present data that are consistent with the tetra-span membrane topography model of PEMT shown in Fig.…”

Section: Discussionsupporting

confidence: 82%

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Shields¹,

Lehner²,

Agellon³

et al. 2003

Journal of Biological Chemistry

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In liver, phosphatidylethanolamine is converted to phosphatidylcholine through a series of three sequential methylation reactions. Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes each transmethylation reaction, and S-adenosylmethionine is the methyl group donor. Biochemical analysis of human liver revealed that the methyltransferase activity is primarily localized to the endoplasmic reticulum and mitochondria-associated membranes. Bioinformatic analysis of the predicted amino acid sequence suggested that the enzyme adopts a polytopic conformation in those membranes. To elucidate the precise membrane topography of PEMT and thereby provide the basis for in-depth functional characterization of the enzyme, we performed endoproteinase-protection analysis of epitopetagged, recombinant protein. Our data suggest a topographical model of PEMT in which four transmembrane regions span the membrane such that both the N and C termini of the enzyme are localized external to the ER. Two hydrophilic connecting loops protrude into the luminal space of the microsomes whereas a corresponding loop on the cytosolic side remains proximate to the membrane. Further support for this model was obtained following endoproteinase-protection analysis of mutant recombinant PEMT derivatives in which specific protease cleavage sites had been genetically engineered or ablated.

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Section: Discussionsupporting

confidence: 82%

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Shields¹,

Lehner²,

Agellon³

et al. 2003

Journal of Biological Chemistry

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“…This possibility seems unlikely since there is no clear example of proteolytic inactivation of a protein with the active site on the internal side of microsomal vesicles in the absence of a membrane-disrupting agent (Vance, Choy, Farren, Lim and Schneider 1977). Moreover, it has been demonstrated that microsomal vesicles remain impermeable to trypsin even after a 30 min-incubation with 3 mg trypsin/ml at 30°C (Audubert and Vance 1984). When the receptor degradation was studied by using a high trypsin concentration during short periods of time, no differences in the degradation kinetics of both exposed and masked receptors were observed (Fig.…”

Section: Discussionmentioning

confidence: 89%

Effect of Proteolytic Enzymes on Masked Insulin Receptors in Rat Submaxillary Gland Microsomes

Scacchi

Wald²,

Turyn³

et al. 1988

Horm Metab Res

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Trypsin and alpha-chymotrypsin effects on masked insulin receptors were studied. Phospholipase C treatment, incubation in a high ionic strength buffer or solubilization were used as alternative procedures for the unmasking of insulin receptors. These three methods expose receptor structures which are inaccessible to insulin in the current experimental conditions of binding assays without any significant change in binding affinity. Both exposed and masked receptors proved to be equally sensitive to trypsin and alpha-chymotrypsin degradation. At 25 degrees C, about 5 micrograms trypsin/ml for 50 min or 80 micrograms alpha-chymotrypsin/ml for 200 min were necessary in each case to cause a 50% inhibition of the binding of 125I-iodo insulin to microsomes. The results suggest that masked receptors are only nonfunctional to bind insulin but they are not located in compartments inaccessible to molecules present in the medium.

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“…PLMT could also be localized in endoplasmic reticulum (Audubert & Vance, 1984) or Golgi bodies (Higgins & Fieldsend, 1987) as previ¬ ously described for liver tissues. Although this enzyme has been mainly considered to be a microsomal enzyme (Van Golde et al 1974), its presence at the level of the Leydig cell plasma membrane playing a regulatory role in hormonal signal transduction, should also be considered (Crews et al 1980;Kelly et al 1984;Dudeja et al 1986).…”

Section: Discussionmentioning

confidence: 91%

Phospholipid methylation decreases in human chorionic gonadotrophin-induced desensitized rat Leydig cells

Ronco

Valladares²

1993

Journal of Endocrinology

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Phospholipid methylation in Leydig cells from desensitized rats was studied. The incorporation of L-[methyl-3H]methionine into phospholipids in intact Leydig cells decreased when animals were injected with a single high dose of human chorionic gonadotrophin (hCG). This effect was detected on the first day after hCG injection and remained up to 12 days after treatment. The inhibition was not due to a reduced uptake of L-[methyl-3H]methionine. A decreased phospholipid methylation with unaltered phospholipid methyltransferase activity was observed on days 1, 6 and 12 after the hCG. On day 3 after hCG injection, phospholipid methyltransferase activity and phospholipid methylation in intact Leydig cells were both inhibited by 40%. Also, a minimal amount of LH free receptors and the lowest number of total receptors was observed at this time. Thus, a relationship between the reduced enzymatic activity and the maximal decrease in LH surface receptors is suggested. In addition, the decreased incorporation of L-[methyl-3H]methionine into phospholipids on days 1, 6 and 12 after hCG injection, could be associated with other cellular changes related to the desensitization process.

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Evidence that the enzymes involved in the methylation of phosphatidylethanolamine are on the external side of the microsomal vesicles

Cited by 17 publications

References 30 publications

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Membrane Topography of Human Phosphatidylethanolamine N-Methyltransferase

Effect of Proteolytic Enzymes on Masked Insulin Receptors in Rat Submaxillary Gland Microsomes

Phospholipid methylation decreases in human chorionic gonadotrophin-induced desensitized rat Leydig cells

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