Selective degradation with phospholipases D and C of radioactive isomeric spin-labelled lipids bound to guinea pig liver microsomal membranes

1978 ◽  
Vol 56 (10) ◽  
pp. 943-951 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Microsomal Membrane ◽  
Spectral Parameters ◽  
Pig Liver ◽  
Selective Degradation ◽  
Bound Lipids ◽  
Before And After ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Guinea Pig Liver

Membrane-bound lipids of isolated guinea pig liver microsomal membranes were selectively enzymatically labelled with isomeric (5-, 12-, and 16-)doxyl stearic acid. After reisolation, the membranes were degraded with phospholipases D and C under conditions not requiring detergents or organic solvent activators. The degradation of membrane-bound lipids occurred according to the recognized specificity of phospholipases D and C. Temperature-induced changes of degraded membranes containing radioactive spin-labelled isomeric lipids were followed by the electron spin resonance and spectral changes correlated with the lipid composition of membranes. Discontinuities in plots of experimental spectral parameters versus temperature detected in the case of microsomal membranes before and after degradation with phospholipases D and C were attributed to lipid–protein and lipid–lipid interaction(s). On the basis of these and control experiments, discontinuity at around 10–12 °C was attributed to the microsomal membrane phosphatidylcholine intrinsic microsomal membrane protein interaction(s), while discontinuities detected at 19–21 °C approximately and at 20–30 °C approximately were attributed to the phase separation of Ca or Zn salts of membranous phosphatidic acid and to the similar phenomenon involving membrane-bound diglycerides respectively.

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.

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

1979 ◽  
Vol 57 (6) ◽  
pp. 618-624 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Liver Mitochondria ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Guinea Pig Liver

Translocation of membrane-bound isomeric (5-, 12-, and 16-doxyl) spin-labelled radioactive cytidinediphosphodiglycerides (CDP-diglycerides) from guinea pig liver microsomal membranes to mitochondrial membranes was studied. When microsomal membranes containing known amounts of isomeric spin-labelled radioactive CDP-diglycerides were incubated with unlabelled mitochondrial membranes, reisolated mitochondria contained labelled lipids in an amount which could not be accounted for by microsomal contamination, indicating that translocation of labelled CDP-diglycerides from microsomal to mitochondrial membranes had occurred. The rate of loss of paramagnetism in microsomal and in reisolated mitochondrial membranes was found to be different, supporting the conclusion that the translocation of labelled lipids between membranes took place. When reisolated mitochondria containing translocated isomeric spin-labelled radioactive CDP-diglycerides were further incubated with sn-3-glycerophosphate, the formation of labelled phosphatidylglycerol was detected. Data from these studies established that the translocation of labelled CDP-diglycerides from microsomal membranes to both outer and inner mitochondrial membranes had occurred.This study established that the isolated guinea pig liver mitochondria are capable of biosynthesis of polyglycerophosphatides (phosphatidylglycerolphosphate, phosphatidylglycerol, and diphosphatidylglycerol or cardiolipin) but depend on the microsomal supply of CDP-diglyceride, an obligatory precursor in the formation of polyglycerophosphatides. This liponucleotide can be translocated, as shown here, to outer and inner mitochondrial membranes for further biosynthetic utilization.

On the mechanism of spontaneous transfer of lipids from isolated microsomal to mitochondrial membranes

1982 ◽  
Vol 60 (2) ◽  
pp. 137-143 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Phosphatidic Acid ◽  
Close Contact ◽  
Lipid Transfer ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Spontaneous Transfer ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Guinea Pig Liver

Spontaneous (protein independent) transfer of endogenously biosynthesized radioactive lipids (phosphatidic acid, phosphatidylcholine, and diglycerides) from isolated guinea pig liver microsomal to unlabeîled mitochondrial membranes was studied as a function of addition of ATP, duration of incubation, and protein concentration of microsomal and mitochondrial membranes. It was found that transfer of phosphatidic acid, phosphatidylcholine, and diglycerides was not absolutely dependent on addition of ATP, although its presence enhanced the transfer of phosphatidylcholine. The highest amount of transferred biosynthesized phosphatidic acid, phosphatidylcholine, and diglycerides was observed after a relatively short incubation and amounted to around one-third of all biosynthesized microsomal lipids, but decreased with increasing incubation time. With increasing concentrations of proteins of mitochondria, a significant decrease of transfer of radioactive lipids from microsomal membranes was established. The same tendency, although to a lesser degree, was established with increasing concentrations of microsomal proteins.These results, together with previously published experiments from this laboratory, were taken as the evidence for the transfer of membrane-bound biosynthesized phosphatidic acid, phosphatidylcholine, and diglycerides from isolated guinea pig liver microsomal to mitochondrial membranes, by a mechanism which involves a close contact between the membrane–donor and the membrane–acceptor. Mechanisms for lipid transfer in biological membranes and in model membranes (liposomes) were compared and discussed.

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.

Mitochondrial importation of lipids and liponucleotides from microsomes independent of and facilitated by purified cytosol proteins

1980 ◽  
Vol 58 (10) ◽  
pp. 1082-1090 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
N. Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Neutral Lipids ◽  
Protein S ◽  
Mitochondrial Import ◽  
Pig Liver ◽  
Conventional Procedure ◽  
Independent Mechanism ◽  
Microsomal Membranes ◽  
Experimental Findings ◽  
Guinea Pig Liver

The mitochondrial importation of microsomal lipids and liponucleotides in the presence and in the absence of partially purified cytosol protein(s) isolated from guinea pig liver was studied by the aid of isomeric (5-, 12-, and 16-(N-oxyl-4′,4′-dimethyloxazolidine)stearoyl) spin-labelled radioactive phosphatidic acid, phosphatidylcholine, neutral lipids, and CDP-diglycerides. Using a conventional procedure for the protein purification, cytosol protein(s) was purified approximately 1000-fold in respect to its ability to catalyze the translocation of isomeric spin-labelled lipids and liponucleotides from the microsomal to mitochondrial membranes. The highest activity of this protein was exhibited with biosynthesized spin-labelled lipids and liponucleotides bound to the microsomal membranes as substrates and the lowest, with the synthetic liponucleotides and derived lipids bound to the microsomal membranes. The partially purified protein was active in catalyzing the mitochondrial import of phospholipids from microsomes after heat treatment up to 90 °C.In addition to the cytosol protein catalyzing mechanism of mitochondrial import of lipids and liponucleotides from microsomal membranes, another cytosol protein independent mechanism of the mitochondrial importation of the same lipids and liponucleotides was also demonstrated in an agreement with our previous reports on the existence of cytosol protein independent intermembranous translocation of phospholipids. These experimental findings are discussed in terms of possible physiological significance and reaction mechanisms involved in the mitochondrial import of lipids and liponucleotides from the microsomal membranes of 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.

Mechanism and localization of cardiolipin biosynthesis revisited: evidence for the identical mechanism and different localization in mitochondrial and submitochrondrial membranes isolated from guinea pig and rat liver

1990 ◽  
Vol 68 (6) ◽  
pp. 922-935 ◽  
Author(s):  
Lidija Stuhne-Sekalec ◽  
Nikola Z. Stanacev
Keyword(s):  
Guinea Pig ◽  
Rat Liver ◽  
Divalent Cations ◽  
Experimental Results ◽  
Mitochondrial Membranes ◽  
Pig Liver ◽  
Membrane Bound ◽  
Rat Livers ◽  
Guinea Pig Liver

The mechanism of cardiolipin (diphosphatidylglycerol) biosynthesis was examined in mitochondria and outer and inner mitochondrial membranes prepared from guinea pig and rat livers to determine whether this formation from phosphatidylglycerol was absolutely dependent on cytidinediphosphodiglyceride, as previously reported for intact mitochondria. Experimental results confirmed that the biosynthesis of cardiolipin, from the membrane-bound radioactive phosphatidylglycerol in intact mitochondria isolated from guinea pig and rat liver, was absolutely dependent on CDP-diglycerides and required the addition of divalent cations. Furthermore, the same mechanism for the biosynthesis of cardiolipin was operational in the outer and inner mitochondrial membranes. This biosynthesis was associated with both the outer and inner mitochondrial membranes prepared from guinea pig liver, but only with the inner mitochondrial membranes prepared from rat liver. The release of radioactive glycerol was also measured, but the amount obtained did not satisfy the stoichiometric requirement for CDP-diglyceride-independent biosynthesis of cardiolipin from 2 mol of phosphatidylglycerol with the liberation of 1 mol of glycerol. Therefore, it was concluded that this mechanism is not involved in the biosynthesis of cardiolipin in mitochondrial and submitochondrial membranes prepared from guinea pig and rat liver.Key words: mitochondria, outer mitochondrial membranes, inner mitochondrial membranes, cardiolipin, biosynthesis.

Effect of chlorpromazine on the synthesis, hydrolysis, and transfer of microsomal cytidine liponucleotides and mitochondrial polyglycerophosphatides

1987 ◽  
Vol 65 (3) ◽  
pp. 377-384 ◽  
Author(s):  
L. Stuhne-Sekalec ◽  
J. Chudzik ◽  
N. Z. Stanacev
Keyword(s):  
Phosphatidic Acid ◽  
Dependent Manner ◽  
Mitochondrial Membranes ◽  
Concentration Dependent Manner ◽  
Pig Liver ◽  
Phosphatidate Phosphohydrolase ◽  
Microsomal Membranes ◽  
Membrane Bound ◽  
Guinea Pig Liver ◽  
Stimulation Of

The effect of chlorpromazine on subcellular biosynthesis, hydrolysis, and transfer of lipids and liponucleotides participating in the biosynthesis of polyglycerophosphatides in guinea pig liver was studied. Chlorpromazine showed an apparent stimulation of accumulation of phosphatidic acid and CDP-diglycerides in microsomal membranes and phosphatidylglycerolphosphate in mitochondrial membranes in a concentration-dependent manner that was influenced by incubation time and the nature of fatty acids in CDP-diglycerides. Transfer of membrane-bound CDP-diglycerides from microsomal to mitochondrial membranes was established by the CDP-diglyceride-dependent biosynthesis of phosphatidylglycerolphosphate and phosphatidylglycerol and appeared to be inhibited by the addition of chlorpromazine by about 20%. Evidence was obtained for the formation of a molecular complex between phosphatidic acid and chlorpromazine; this was thought to be responsible for the protection from phosphatidate phosphohydrolase at the concentrations of chlorpromazine and Mg2+ examined.

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