Enzymatic degradation and partial biosynthetic reconstitution of microsomal and mitochondrial membranes

1979 ◽  
Vol 57 (10) ◽  
pp. 1237-1244 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Cytochrome C ◽  
Guinea Pig ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Mitochondrial Membranes ◽  
Original Activity ◽  
Pig Liver ◽  
Cytochrome C Reductase ◽  
Guinea Pig Liver ◽  
Nadph Cytochrome C Reductase

Guinea pig liver microsomal and mitochondrial membranes were degraded with phospholipase C and D followed by partial biosynthetic reconstitution. Activities of phosphatidylinositol synthetase in microsomal membranes and NADPH – cytochrome c reductase were almost completely lost after phospholipase C and D treatment; almost complete restoration of the original activity was achieved after biosynthesis of phosphatidylcholine in degraded microsomes, but was not reparable after biosynthesis of cytidinediphosphodiglycerides (CDP-diglycerides). The mitochondrial biosynthesis of polyglycerophosphatides was completely retained after degradation of these membranes with phospholipase C, but after similar treatment with phospholipase D, only about one-quarter of the original activity remained, the relative composition of polyglycerophosphatides being significantly different. The activity of NADPH – cytochrome c reductase of microsomes represented about 76% of the originalactivity after phospholipase C treatment, but only ~1% after treatment with phospholipase D. Although this activity could not be restored with CDP-diglyceride synthesis, it was restored to about 75% of the original activity after the biosynthesis of phosphatidylcholine in these fragments. These and additional experimental findings are discussed in terms of the relation between structural organization of lipids and proteins and enzymatic activities of membrane-bound phospholipid-synthesizing enzymes in microsomal and mitochondrial membranes isolated from guinea pig liver.

Biosynthesis, utilization, and translocation of endogenous isomeric spin-labelled radioactive cytidinediphosphodiglycerides from isolated guinea pig liver microsomal to mitochondrial membranes

1979 ◽  
Vol 57 (7) ◽  
pp. 1019-1025 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Cytochrome C ◽  
Guinea Pig ◽  
Reductase Activity ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Cytochrome C Reductase ◽  
Lipid Species ◽  
Microsomal Membranes ◽  
Guinea Pig Liver ◽  
Nadph Cytochrome C Reductase

When isolated guinea pig liver microsomal membranes were incubated with isomeric (5-, 12-, and 16-doxyl stearoyl) spin-labelled sn-3-[2-3H]phospfaatidic acid in the presence of CTP and Mg2+, formation of corresponding CDP-[2-3H]diglycerides (in an amount representing 16.5–17.4% of the labelled lipids), which were acceptable substrates in the microsomal biosynthesis of sn-3-[2-3H]phosphatidyl-myo-[U-l4C]inositols, took place. When microsomal membranes containing known amounts of labelled CDP-diglycerides were incubated with unlabeled mitochondrial membranes, reisolated mitochondria contained labelled lipids in an amount which could not be accounted for by the microsomal contamination of reisolated mitochondria, determined by the assay of NADPH – cytochrome c reductase activity, establishing therefore the translocation of labelled CDP-diglycerides (and other labelled lipids) from microsomal to mitochondrial membranes in an amount of ~50% of microsomal content. The rate of loss of paramagnetic lipid species in microsomal and in reisolated mitochondrial membranes was found to be quite different. When reisolated mitochondria containing trans-located isomeric spin-labelled CDP-[2-3H]diglycerides were further incubated with sn-3-[U-14C]glycerophosphate, the formation of labelled phosphatidylglycerophosphate and phosphatidylglycerol was detected. These findings established that the translocation of endogenously formed CDP-[2-3H]diglycerides occurred from isolated microsomal membranes to both outer and inner mitochondrial membranes.

Biosynthesis of phosphatidylglycerol and phosphatidylglycerolphosphate in submitoehondrial membranes isolated from guinea pig liver is absolutely dependent on CDP-diglycerides imported from microsomal membranes

1990 ◽  
Vol 68 (1) ◽  
pp. 111-116 ◽  
Author(s):  
Lidija Stuhne-Sekalec ◽  
Nikola Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Microsomal Membranes ◽  
Guinea Pig Liver

The biosynthesis of radioactively labelled phosphatidylglycerol via phosphatidylglycerophosphate in outer and inner mitochondrial membranes isolated from guinea pig liver was found to depend absolutely on CDP-diglycerides, which could not be biosynthesized in these membranes. The requirement for CDP-diglycerides in the biosynthesis of labelled phosphatidylglycerol could be fulfilled by the transfer of biosynthesized [3H]CDP-diglycerides from the microsomal membranes to the outer and inner mitochondrial membranes.Key words: submitoehondrial membranes, transfer, CDP-diglycerides, phosphatidylglycerol, phosphatidylglycerophosphate.

Phosphatidylinositol movement between isolated microsomal and mitochondrial membranes

1983 ◽  
Vol 61 (12) ◽  
pp. 1282-1291 ◽  
Author(s):  
J. Chudzik ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Direct Contact ◽  
Reaction Medium ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Liver Cytosol ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Phosphatidylinositol Transfer ◽  
Guinea Pig Liver

Transfer of membrane-bound phosphatidyl-[2′-3H]inositol from microsomal to unlabelled mitochondrial and from mitochondrial to unlabelled microsomal membranes was studied using partially purified cytosol proteins isolated from guinea pig liver cytosol. In the absence and presence of these proteins the amounts of phosphatidylinositol transfer from microsomal to mitochondrial membranes were approximately 21 and 33%, respectively, and the amounts from mitochondrial to microsomal membranes were approximately 31 and 39%, respectively. The release of phosphatidyl-[2′-3H]inositol from microsomal membranes in the absence of mitochondria was dependent on concentration of cytosol proteins. Two mechanisms for movement between membranes are proposed. In cytosol-protein-independent movement of phosphatidyl-[2′-3H]inositol from microsomal to mitochondrial membranes, a direct contact between membranes is required, since phosphatidyl-[2′-3H]inositol was not detected in the reaction medium. In the cytosol-protein-catalyzed transfer, formation of phosphatidyl-[2′-3H]inositol – cytosol protein complex is postulated, since phosphatidyl-[2′-3H]inositol was released into the reaction medium and its movement proceeded from mitochondrial to microsomal membranes in the presence of partially purified cytosol proteins. Thus, contact between the two membranes is probably not necessary for this transfer. Implications for the movement of phospholipids between biological membranes are discussed.

Translocation of spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes

1977 ◽  
Vol 55 (11) ◽  
pp. 1159-1165 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Experimental Data ◽  
Guinea Pig ◽  
Biological Membranes ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Guinea Pig Liver ◽  
Lipid Translocation

Translocation of membrane-bound labelled lipids from guinea pig liver microsomal to mitochondrial membranes was studied. When microsomal membranes containing known amounts and composition of spin-labelled radioactive lipids were incubated with unlabelled mitochondrial membranes, reisolated mitochondria contained spin- and radioactive-lipids in an amount which could not be accounted for by the microsomal contamination of reisolated mitochondria, establishing therefore the translocation of labelled lipids from microsomal to mitochondrial membranes. The effect of addition of crude 105 000 × g supernatant on the translocation was studied. The translocation of labelled lipids in aged membranes was also described. The rate of loss of paramagnetism in microsomal and reisolated mitochondrial membranes was measured and found to be different, supporting the conclusion that the translocation of labelled lipids between membranes took place. Data from these studies suggested that the translocation of labelled lipids of microsomal membranes to both outer and inner mitochondrial membranes probably occurred. Furthermore, our results suggest that the mechanism by which lipid translocation takes place does not necessarily involve soluble cytosol proteins. In view of experimental data, possible mechanisms for the translocation of lipids between biological membranes were discussed.

Cytosol protein-independent translocation of isomeric spin-labelled radioactive lipids from isolated guinea pig liver microsomal to mitochondrial membranes

1978 ◽  
Vol 56 (6) ◽  
pp. 407-413 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Phosphatidic Acid ◽  
Stearic Acid ◽  
Biological Membranes ◽  
Probable Mechanism ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Guinea Pig Liver

Intermembranous translocation of membrane-bound radioactive lipids covalently labelled with 5-, 12-, and 16-doxyl stearic acid was studied. Guinea pig liver microsomal membranes containing known amounts of isomeric spin-labelled radioactive phosphatidic acid, phosphatidylcholine, and diglycerides were incubated with unlabelled mitochondria; reisolated mitochondria contained around 28–31% of microsomal labelled lipids above the microsomal contamination. The effect of adding crude or'pH 5.1' 105 000 × g cytosol supernatant on the amount and composition of translocated labelled lipids was studied. While the translocation of labelled phosphatidylcholine was slightly stimulated by the addition of these cytosol supernatants, no significant increase of the amount of translocated labelled phosphatidic acid and diglycerides was observed by this addition. In view of these results, a probable mechanism for the cytosol protein-independent translocation of lipids between biological membranes is proposed.

Sign in / Sign up

Export Citation Format

Share Document