Guinea Pig Liver Microsomal Membranes Research Articles

Glucose-6-phosphate phosphohydrolase activity in guinea pig liver microsomes is influenced by phosphatidylcholine. Interaction with cholesterol-enriched membranes

Guinea pig liver microsomal membranes were cholesterol-enriched by feeding guinea pigs a high-cholesterol diet. Cholesterol enrichment as well as partial lipid removal of normal native microsomes by acetone-butanol extraction resulted in 40-50% loss in activity of the glucose-6-phosphate phosphohydrolase (G-6-Pase) (EC 3.1.3.9) enzyme system. The activity was restored by supplementation of microsomal total phospholipid (PL) and its phosphatidylcholine (PC) species but not with microsomal neutral lipids, cholesterol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin or diphosphatidylglycerol (cardiolipin). The activity was decreased by sodium deoxycholate but enhanced by dimethylsulfoxide. Egg-yolk PC and asolectin influenced the activity of the enzyme to the same extent as microsomal PC did. Lipid depletion and cholesterol produced an increase in Km while the Vmax was lowered. The non-linearity in the Arrhenius plot of the native microsomes was lost on lipid removal and cholesterol enrichment. The energy of activation (Ea) calculated from the continuous line was found to be lowered to the level that was observed above the break points in intact microsomes. Addition of microsomal PC to the assay system decreased the Km of the enzymatic reaction in native membranes, in partially lipid-depleted and cholesterol-enriched membranes, but did not alter the Vmax values and only marginally influenced the non-linear relationship of the Arrhenius expression of temperature dependence. The ability of immature rat liver phospholipid exchange protein to introduce alien PL into microsomal membrane was used to study the lipid dependence of G-6-Pase. Protein-catalyzed and detergent (cholate)-mediated membrane PL exchange for egg-yolk PC from the PC/cholesterol unilamellar liposomes resulted in substantial loss of enzyme activity. The discrepancies in the influence of PC on G-6-Pase were interpreted by assuming that the enzyme was a two-component system, a surface-located substrate transporter unit and a membrane integral catalytic phosphohydrolase unit. The lipid microenvironment and PL requirement in particular, could be different for the two components, although they represented a single functional unit at the time of enzymatic reaction.

Biosynthetic incorporation of cis-parinaric acid into radioactive sn-3-phosphatidic acid.

Isolated guinea pig liver microsomal membranes catalyzed the incorporation of naturally occurring cis-parinaric acid into sn-3-[U-14C]glycerophosphate. This resulted in the formation of sn-3-[14C](parinaroyl)phosphatidic acid, which was isolated by Chelex-100 and DEAE-cellulose column chromatography and further purified by Sephadex-G 25. The sn-3-[14C](parinaroyl)phosphatidic acid thus obtained exhibited absorption and fluorescence spectra substantially different from the cis-parinaric acid. Distribution of the incorporated cis-parinaric acid between the hydroxyl groups of biosynthesized sn-3-[14C]phosphatidic acid was determined by degradation with Crotalus adamanteus venom. This established that the major portion of the incorporated cis-parinaric acid esterified the secondary hydroxyl group in the sn-3-[14C]phosphatidic acid, while the primary hydroxyl group was esterified to a significantly lesser degree. The similarity between the biochemical incorporation of isomeric doxyl stearic acids into lipids of biological membranes and that of cis-parinaric acid into sn-3-phosphatidic acid described in this communication are discussed in relation to the possible use of these probes in studies of intact biological membranes.

The phospholipid dependence of UDP-glucuronyltransferase: Conformation/reactivity studies with purified enzyme

Highly-active purified UDP-glucuronyltransferase from guinea-pig liver microsomal membranes is associated with phospholipids. Removal of these phospholipids inactivated the transferase and caused profound changes in the enzyme's circular dichroism spectrum indicating that its secondary structure was drastically altered. Treatment of the delipidated fraction with phosphatidylcholine restored the enzyme to a much more helical, high reactivity conformation. These results show clearly that an intact phospholipid environment is required to maintain the transferase in a reactive conformation.

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

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.

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

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.

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.

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.

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

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 X 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 acic 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.

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Phospholipid depletion and re-activation of guinea-pig liver microsomal enzyme

More than 80% of the phospholipid component of guinea-pig liver microsomal membranes (prepared with 154mM-KCl) was removed by treatment with phospholipase A followed by extraction of the lysophosphatides and fatty acids produced with albumin. Delipidation strongly inactivated the highly active UDP-glucuronyltransferase of these preparations and activity was restored by mixtures of phosphatidylcholine and lysophosphatidylchlone. However, small quantities of lysophosphatides were still associated with the delipidated fractions after extraction with albumin and might have influenced the inactivation and re-activation observed. To eliminate these uncertainties, microsomal proteins and phospholipids were separated by gel filtration on Sephadex G-150 in the presence of cholate. This technique also strongly inactivated the enzyme but did not generate membrane-active phospholipid degradation products. High transferase activity was again restored to the delipidated protein by choline glycerophosphatides. These results confirm the view that the fully active form of microsomal UDP-glucuronyltransferase is phospholipid-dependent.