scholarly journals Both the outer mitochondrial membrane and the microsomal forms of cytochrome b5 reductase contain covalently bound myristic acid. Quantitative analysis on the polyvinylidene difluoride-immobilized proteins

1990 ◽  
Vol 266 (2) ◽  
pp. 341-347 ◽  
Author(s):  
N Borgese ◽  
R Longhi
Keyword(s):  
Fatty Acids ◽  
Endoplasmic Reticulum ◽  
Myristic Acid ◽  
Cytochrome B5 ◽  
Molar Ratio ◽  
Mitochondrial Membranes ◽  
Polyvinylidene Difluoride ◽  
Cytochrome B5 Reductase ◽  
Fatty Acylation ◽  
Mitochondrial Fractions

NADH-cytochrome b5 reductase is known to be located on two distinct membranes, i.e. endoplasmic reticulum and outer mitochondrial membranes. The endoplasmic-reticulum-associated form of the enzyme contains myristic acid in an amide linkage to its N-terminal glycine [Ozols, Carr & Strittmatter (1984) J. Biol. Chem. 259, 13349-13354]. To investigate whether the dual subcellular localization of the reductase corresponds to a difference in fatty acylation, the enzyme was purified from well-characterized rat liver microsomal and mitochondrial fractions and analysed by a new quantitative analytical procedure. The purified reductases were run on SDS/polyacrylamide gels and blotted on to polyvinylidene difluoride membranes. The reductase-containing bands were treated with hydroxylamine, and amide-linked fatty acids were then detached by acid hydrolysis. The detached fatty acids were extracted, derivatized and analysed as phenylacyl esters by reverse-phase h.p.l.c., and the protein content of the samples was determined by amino acid analysis of the acid hydrolysates. Myristic acid was found in both the microsomal and mitochondrial reductases in a molar ratio of 1:1 with protein. These results demonstrate for the first time the presence of a myristylated protein on outer mitochondrial membranes, and show that the microsomal and mitochondrial reductases are also identical in their fatty acylation.

scholarly journals A role for N-myristoylation in protein targeting: NADH-cytochrome b5 reductase requires myristic acid for association with outer mitochondrial but not ER membranes.

1996 ◽  
Vol 135 (6) ◽  
pp. 1501-1513 ◽  
Author(s):  
N Borgese ◽  
D Aggujaro ◽  
P Carrera ◽  
G Pietrini ◽  
M Bassetti
Keyword(s):  
Protein Interactions ◽  
Myristic Acid ◽  
Protein Targeting ◽  
Mdck Cells ◽  
Cytochrome B5 ◽  
Cell Fractionation ◽  
Protein Protein Interactions ◽  
Cytochrome B5 Reductase ◽  
Nadh Cytochrome ◽  
Triton X

N-myristoylation is a cotranslational modification involved in protein-protein interactions as well as in anchoring polypeptides to phospholipid bilayers; however, its role in targeting proteins to specific subcellular compartments has not been clearly defined. The mammalian myristoylated flavoenzyme NADH-cytochrome b5 reductase is integrated into ER and mitochondrial outer membranes via an anchor containing a stretch of 14 uncharged amino acids downstream to the NH2-terminal myristoylate glycine. Since previous studies suggested that the anchoring function could be adequately carried out by the 14 uncharged residues, we investigated a possible role for myristic acid in reductase targeting. The wild type (wt) and a nonmyristoylatable reductase mutant (gly2-->ala) were stably expressed in MDCK cells, and their localization was investigated by immunofluorescence, immuno-EM, and cell fractionation. By all three techniques, the wt protein localized to ER and mitochondria, while the nonmyristoylated mutant was found only on ER membranes. Pulse-chase experiments indicated that this altered steady state distribution was due to the mutant's inability to target to mitochondria, and not to its enhanced instability in that location. Both wt and mutant reductase were resistant to Na2CO3 extraction and partitioned into the detergent phase after treatment of a membrane fraction with Triton X-114, demonstrating that myristic acid is not required for tight anchoring of reductase to membranes. Our results indicate that myristoylated reductase localizes to ER and mitochondria by different mechanisms, and reveal a novel role for myristic acid in protein targeting.

scholarly journals The effect of linseed oil supplementation of the diet on the content of fatty acids in the egg yolk

2012 ◽  
Vol 81 (2) ◽  
pp. 159-162 ◽  
Author(s):  
Petra Hudečková ◽  
Lucie Rusníková ◽  
Eva Straková ◽  
Pavel Suchý ◽  
Petr Marada ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Soybean Oil ◽  
Fatty Acid Profile ◽  
Myristic Acid ◽  
Linseed Oil ◽  
Control Group ◽  
Heptadecanoic Acid ◽  
Egg Yolks ◽  
Experimental Group

The aim of this study was to compare the effect of two different types of oils in diet on the fatty acid profile in the eggs of layers and to include a particular type of oil as a supplement of feeding mixtures for layers in order to support the development of functional foodstuffs. Thirty layers fed a diet containing soybean oil constituted the control group (soybean oil is the most frequently used oil added to feeding mixtures). In the experimental group (thirty layers), soybean oil was replaced with linseed oil at the same amount (3 kg of oil per 100 kg of feeding mixture). Feeding was provided ad libitum for all days of the month. After one month, egg yolks were analysed and the fatty acid profile was compared. Significant differences (P ≤ 0.05) were found in the concentration of myristic acid that belongs to the group of saturated fatty acids. Eggs in the experimental group showed higher concentrations of myristic acid compared to the control group (0.20 g/100 g of fat and 0.18 g/100 g of fat, respectively). Highly significant differences (P ≤ 0.01) were found for heptadecanoic acid but the trend was opposite to that of myristic acid; concentrations of heptadecanoic acid in the experimental group were lower than those in the control group. Highly significant differences (P ≤ 0.01) were found for n-9 monounsaturated fatty acids where egg yolks in eggs from layers fed linseed oil contained higher concentrations of oleic acid, myristoleic acid, and palmitoleic acid. Lower concentrations of n-6 fatty acids (P ≤ 0.01) were found after the addition of linseed oil in eggs. Linseed oil showed a positive effect on n-3 fatty acids (α-linolenic acid), its concentration in the control and experimental group was 0.82 g/100 g of fat and 5.63 g/100 g of fat, respectively. The possibility of influencing the fatty acid profile in eggs is very important for the development of functional foods.

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