Temperature-induced changes of spin-labelled radioactive lipids in isolated guinea pig liver microsomal membranes before and in mitochondrial membranes after their translocation.

Lipids of isolated guinea pig liver microsomal membranes were labelled biosynthetically with isomeric doxyl stearic acid and temperature-induced changes of these membranes were studied by electron spin resonance. A noticeable discontinuity was detected at 10--12 degree C with 12- or 16-doxyl stearic acid containing membrane lipids which was attributed to the spin-labelled lipid--microsomal membrane protein interactions since no such discontinuity was detected in liposomes prepared from total lipid extracts of microsomal membranes. When microsomal membranes containing radioactive isomeric spin-labelled lipids were incubated with unlabelled mitochondria, reisolated mitochondrial membranes contained translocated radioactive isomeric spin-labelled lipids. Temperature-induced changes in these membranes showed no discontinuity with either isomeric doxyl stearic acid derivative, establishing a difference in the environment of translocated lipids in the membrane donor compared with that in the membrane acceptor. Microsomal membranes recovered from translocation experiments showed the same behaviour as the original membranes and exhibited the same discontinuity at 10--12 degree C, establishing that the translocation incubation itself did not alter the spin-labelled lipid interaction within these membranes. Studies of the loss of paramagnetism of spin-labelled lipids in microsomal membranes before and in mitochondrial membranes after their translocation showed a significant difference and suggested that both the outer and the inner mitochondrial membranes might have been involved.     

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

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 degrees C was attributed to the microsomal membrane phosphatidylcholine intrinsic microsomal membrane protein interaction(s), while discontinuities detected at 19-21 degrees C approximately and at 20-30 degrees 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.     

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

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.     

Triton solubilization of proteins from pig liver mitochondrial membranes

Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.     

Preparation and characterization of isolated parenchymal cells from guinea pig liver

1. A method is described for the preparation of isolated cells from guinea pig liver. This involved perfusion in situ, in the non-physiological direction, with collagenase. 2. The cell yield was 20--30%, comparable with those from the livers of other species. 3. The ratio of lactate dehydrogenase to glutamate dehydrogenase in the cells was similar to that in vivo, indicating that there was negligible leakage of cytoplasmic enzymes. 4. The concentrations of K+ and adenine nucleotides were initially lower than in the perfused liver; normal values were obtained on incubation, particularly in the presence of substrate. 5. The L-lactate: pyruvate ratio is 16:1, close to established values. The total beta-hydroxybutyrate: acetoacetate ratio indicates that the mitochondrial redox state is more oxidised than in the perfused liver, but the intracellular ratio is similar to that of the intact liver. 6. Rates of gluconeogenesis and ureogenesis, are within the physiological range. Maximal gluconeogeneis from L-lactate was preceded by a lag period. L-lysine stimulated glucose production from L-lactate but did not abolish the lag phase. 7. The effects of aminooxyacetate and octanoate on L-lactate gluconeogenesis were similar to those in the perfused liver.     

Ontogeny of microsomal activities of triacylglycerol synthesis in guinea pig liver

Because the onset of triacylglycerol-rich lipoprotein synthesis occurs in guinea pig liver during fetal life, we investigated the microsomal enzyme activities of triacylglycerol synthesis in fetal and postnatal guinea pig liver. Hepatic monoacylglycerol acyltransferase specific and total microsomal activities peaked by the 50th day of gestation and declined rapidly after birth to levels that were virtually unmeasurable in the adult. Peak fetal specific activity was more than 75-fold higher than observed in the adult. The specific activities of fatty acid CoA ligase and lysophosphatidic acid acyltransferase increased 2- to 3-fold before birth; lysophosphatidic acid acyltransferase increased a further 2.6-fold during the first week of life. Specific activities of phosphatidic acid phosphatase, microsomal glycerophosphate acyltransferase, and diacylglycerol acyltransferase varied minimally over the time course investigated. These data demonstrate that selective changes occur in guinea pig hepatic microsomal activities of triacylglycerol synthesis before birth. Because of an approximate 11-fold increase in hepatic microsomal protein between birth and the adult, however, major increases in total microsomal activity of all the triacylglycerol synthetic activities occurred after birth. The pattern of monoacylglycerol acyltransferase specific and total microsomal activities differs from that of the rat in occurring primarily during the last third of gestation instead of during the suckling period. This pattern provides evidence that hepatic monoacylglycerol acyltransferase activity probably does not function to acylate 2-monoacylglycerols derived from partial hydrolysis of diet-derived triacylglycerol.     

Age-related changes in the translocation of phosphatidate phosphohydrolase from the cytosol to microsomal membranes in rat liver

The effects of oleate, spermine and chlorpromazine were assayed in the presence or absence of 0.15 M KCl on the translocation of phosphatidate phosphohydrolase activity from cytosol to endoplasmic reticulum membranes in liver homogenates obtained from rats aged 1, 30, 60, 180 and 360 days. Marked age-associated decreases in phosphatidate phosphohydrolase distribution onto the membranes were demonstrated under nearly all conditions. In liver homogenates taken from 1-day-old rats and incubated with 0.15 M KCl, most of the enzyme was active (associated with the membranes). Physiological salt concentration (0.15 M KCl) produced a 2-fold increase of oleate-induced translocation of phosphatidate phosphohydrolase activity in liver homogenates from 1-day-old rats; it had no effect on those from 60-day-old rats, and produced a notable decline in liver homogenates obtained from 180- and 360-day-old rats. The promoting effect of spermine on oleate-induced translocation of this enzyme activity was higher in younger rats when incubated in the absence of 0.15 M KCl. Chlorpromazine did not show its usual antagonizing effect on oleate-induced translocation of phosphatidate phosphohydrolase when added to homogenates taken from 1-day-old rats. The antagonizing effect was slightly apparent in liver homogenates from 30-day-old rats and was more pronounced in those from 60-day-old rats in which the values diminished to one-half and to one-third either in the presence or absence of 0.15 M KCl.     

Kinetic properties of guinea pig liver microsomal NADPH-cytochrome P-450 reductase

NADPH-cytochrome P-450 reductase was purified to apparent homogeneity from detergent-solubilized guinea pig liver microsomes. The reductase had a mol. wt of 78,000 and contained one mole each of FAD and FMN. Electron transfer activity to cytochrome c was optimal at a pH of 8.0 and an ionic strength of 0.43. The results of kinetic experiments were consistent with a ternary-complex mechanism for the interaction of the reductase with cytochrome c and NADPH. Km values for NADPH and cytochrome c were 3.1 and 26.7 microM, respectively. Inhibition by NADP+ and 2'-AMP was competitive with respect to NADPH; Ki values were 12.1 microM for NADP+ and 46.7 microM for 2'-AMP.     

Localisation of a particulate luliberin hydrolysing activity in microsomal membranes of guinea pig brain

A particulate luliberin hydrolysing enzyme has been described for guinea pig brain. Examination of subcellular fractions generated under different conditions indicated that particulate luliberin hydrolysing activity was most closely associated with the microsomal marker, rotenone-insensitive NADH cytochrome C reductase. The results obtained indicate that luliberin hydrolysing activity is not associated with synaptosomal membrane preparations and that such luliberin hydrolysing activity as is observed in synaptosomal membranes is probably the result of contamination by microsomes. The enzyme could be released from microsomes by Triton X-100 treatment and the solubilised enzyme was found to be inhibited by puromycin and sulphydryl reagents but to be unaffected by phosphoramidon, captopril, phenylmethyl sulphonyl fluoride and by chelating agents except 1,10-phenanthroline.     

Oleate-induced translocation of protein kinase C to hepatic microsomal membranes

The incubation of rat liver homogenates in the presence of oleate induces the translocation of protein kinase C from the cytosol to the endoplasmic reticulum membranes. The half-maximal effect was obtained at 0.3 mM oleate. The redistribution of this enzyme induced by oleate was also obtained with purified protein kinase C and hepatic microsomal membranes. This effect seems to be mediated by long-chain fatty acids since translocation was not obtained with esterified derivatives.     

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

Influence of dietary lipids on microsomal membranes from chick breast muscle

1. The effect of different dietary fat intake on the lipid composition and fluidity of microsomal membranes as well as in the enzymatic activity of the Ca2+-ATPase from chick breast muscle was investigated. 2. When a standard diet was supplemented with 10% sunflower seed oil, an increase in the relative amounts of unsaturated fatty acids and membrane fluidity and a decrease in the cholesterol content was observed. 3. The presence of 6% cholesterol in the diet does not modify the fatty acid composition and the fluidity of the membrane but increased, in a low extension, the cholesterol content. 4. The provision of the sunflower seed oil-rich diet supplemented with cholesterol just 48 hr before death promoted an increase in the relative amounts of unsaturated fatty acids and cholesterol content whereas the membrane fluidity decreased in a significant extent. 5. Despite that dietary lipids gave rise in some cases to changes in lipid composition and in the physical state of the microsomal membrane, neither the Ca2+ uptake capacity nor the ATPase activity were significantly affected.     

Comparison of cytidinediphospho-sn-1,2-diglyceride transfer from microsomal and liposomal to mitochondrial membranes

Transfer of [3H]CDP-diglycerides from isolated guinea pig liver microsomal and liposomal membranes to guinea pig mitochondrial membranes was studied by incubating microsomal or liposomal membranes carrying [3H]CDP-diglycerides with mitochondrial membranes and determining the CDP-diglyceride-dependent incorporation of sn-3-[14C]glycerolphosphate into mitochondrial [14C]polyglycerophosphatides. A significant difference in the amount of transferred [3H]CDP-diglycerides and the composition of mitochondrial [14C]polyglycerophosphatides was found depending on whether [3H]CDP-diglycerides were transferred from microsomal or liposomal membranes. This amount was around 12% when [3H]CDP-diglycerides were transferred from the microsomal membranes and around 4.6% when they were transferred from the liposomal membranes. Furthermore, about 60% of [14C]phosphatidylglycerol and 35% of [14C]phosphatidylglycerophosphate were found in the microsomes-mitochondria system and about 9% of [14C]phosphatidylglycerol and 79% of [14C]phosphatidylglycerophosphate were found in the liposomes-mitochondria system, establishing an important role for the membrane donor in the transfer of [3H]CDP-diglycerides to mitochondria. Furthermore, if the transfer of [3H]CDP-diglycerides from the microsomal to the mitochondrial membranes was assayed by the determination of [3H]CDP-diglycerides in reisolated mitochondrial membranes without further incorporation into mitochondrial polyglycerophosphates, it amounted to about 38%.     

Comparative in vitro effects of guinea pig VIP and common VIP on liver and lung membranes from guinea pig and rat and on human lymphoblastic SUP-T1 membranes

Guinea pig VIP differs from VIP of several mammals by its amino acids in positions 5, 9, 19 and 26. We tested a) its ability to occupy VIP receptors in liver and lung membranes of rat and guinea pig and in the human lymphoblastic SUP-T1 cell line and b) the ensuing adenylate cyclase stimulation. In liver and lung membranes from rat, guinea pig VIP was less potent than common VIP to occupy high and low affinity VIP receptors. In rat liver both VIP activated adenylate cyclase mostly through high affinity receptors. In rat lung, guinea pig VIP activated the enzyme mostly through high affinity receptors and was less efficient than common VIP acting through both classes of receptors. In guinea pig liver and lung membranes, binding inhibition curves were steeper than with rat preparations and adenylate cyclase appeared to be mostly activated through high affinity VIP receptors in liver and through both classes of receptors in lung. On human lymphoblastic SUP-T1 membranes both VIP were equally potent and efficient to inhibit tracer binding and activate adenylate cyclase.     

Ethanol sensitivity and fatty acid composition of chick liver mitochondrial and microsomal membranes

Fatty acid composition of hepatic mitochondrial and microsomal membranes was studied in 2-day-old chicks exposed to ethanol for 60 h (short treatment) or 18 days (chronic treatment). Short ethanol treatment induced in mitochondria an increase in the 18:1/18:0 ratio as a consequence of both an increase in the percentage of oleic and a decrease in that of stearic acid. Likewise, a clear decrease in the polyunsaturated fatty acids and in the 20:4/18:2 ratio was found in mitochondria after short ethanol administration. Microsomal membranes were practically unaffected by this treatment. However, chronic ethanol exposure produced a significant increase in the percentages of polyunsaturated fatty acids in both mitochondria and microsomes as well as a decrease in the 18:1/18:0 ratio. These results suggest that delta 9 desaturase modifies its activity in response to ethanol treatment with a different pattern to those showed by delta 6 and delta 5 desaturase activities.