Sphingomyelin synthase in rat liver nuclear membrane and chromatin.

The presence of phospholipids in chromatin has been demonstrated, as well as the difference in composition and turnover compared to those present in the nuclear membrane. Recently, some enzymes were also evidenced in chromatin: the base exchange protein complex and neutral sphingomyelinase. The latter has a particular relevance, since sphingomyelin is one of the phospholipids more represented in chromatin. We therefore decided to study the synthesis of sphingomyelin in chromatin and in nuclear membrane isolated from liver nuclei. The evaluation of the enzyme was made (i) using [(3)H]phosphatidylcholine as donor of radioactive phosphorylcholine and (ii) by identifying the product isolated by thin layer chromatography. In both fractions the enzyme phosphatidylcholine:ceramide phosphocholine transferase or sphingomyelin synthase was present, although with higher activity in nuclear membrane. The enzyme present in the chromatin differs in pH optimum and K(m), showing a higher affinity for the substrates than that of nuclear membrane. The results presented show that sphingomyelin synthase is present not only in the cytoplasm at the level of the Golgi apparatus, but also in the nuclei, at the level of either the nuclear membrane or the chromatin.     

CHOLINE BASE EXCHANGE ACTIVITY IN RAT HEPATOCYTE NUCLEI AND NUCLEAR MEMBRANES

Previous investigations have demonstrated the presence of phospholipids as a component of chromatin; however the mechanism of their synthesis, namely if they are synthesized in the nuclei or in the cytoplasm (microsomal fraction), from where they may eventually be transported to the nucleus, has not yet been clarified. The phosphatidylcholine, for example, can be formed, albeit in a limited amount, by an interconversion reaction between bases. The aim of the present research was to ascertain the presence of the enzyme complex responsible for this reaction in hepatocyte nuclei and in isolated nuclear membrane. The incorporation of [¹⁴C]-choline in phosphatidylcholine was assayed in microsomes, hepatocyte nuclei, liver nuclei and nuclear membranes of rat liver. The reaction was Ca²⁺-dependent and the specific activity was higher in microsomes but was present, albeit at a low level, also in nuclei and in nuclear membranes. Possible contaminations were excluded by specific microsomal markers and by the reaction time course. In fact, the nuclear reaction reached the maximum level slowly with respect to microsomes. Since the phosphatidylcholine extracted from the nuclei show an enrichment in unsaturated fatty acids of monoenoic fraction, such as oleic acid, the difference in reaction kinetics has been tentatively explained as due to the phosphatidylcholine fatty acid content. The presence of this base exchange enzyme complex may allow a fast change in chromatin phospholipid composition.     

Phospholipase C digestion induces the removal of nuclear RNA: A cytochemical quantitative study

Summary It has been reported that the incubation of isolated rat liver nuclear matrices with phospholipase C causes the digestion of the matrix-bound phospholipids and the release of most matrix-linked RNAs (Coccoet al., 1980). In this paper, the presence of phospholipids in nuclear substructures and the effects of their removal by phospholipase C digestion have been investigated by means of enzyme—colloidal gold cytochemistry. The nuclear phospholipids appear to be localized in the interchromatin areas and in the nucleolus and are virtually absent in the heterochromatin, when labelled with phospholipase C—colloidal gold. The double labelling test with ribonuclease A and phospholipase C conjugated with gold particles of different diameters shows that the nuclear phospholipids are co-localized with RNA-containing structures. The enzymatic digestion of phospholipids on thin sections of either isolated nuclei or pancreas embedded in LR White resin results in the decrease of the RNase-A colloidal gold labelling of nuclear RNA-containing structures, but not of the rough endoplasmic reticulum. The data confirm the presence of phospholipids in the nucleus in the absence of possible translocation due to isolation procedures and strengthen the hypothesis that they are involved in interactions between nucleic acids and proteins of the nuclear matrix.     

Phosphatidylcholine/sphingomyelin metabolism crosstalk inside the nucleus

It is known that phospholipids represent a minor component of chromatin. It has been highlighted recently that these lipids are metabolized directly inside the nucleus, thanks to the presence of enzymes related to their metabolism, such as neutral sphingomyelinase, sphingomyelin synthase, reverse sphingomyelin synthase and phosphatidylcholine-specific phospholipase C. The chromatin enzymatic activities change during cell proliferation, differentiation and/or apoptosis, independently from the enzyme activities present in nuclear membrane, microsomes or cell membranes. This present study aimed to investigate crosstalk in lipid metabolism in nuclear membrane and chromatin isolated from rat liver in vitro and in vivo. The effect of neutral sphingomyelinase activity on phosphatidylcholine-specific phospholipase C and sphingomyelin synthase, which enrich the intranuclear diacylglycerol pool, and the effect of phosphatidylcholine-specific phospholipase C activity on neutral sphingomyelinase and reverse sphingomyelin synthase, which enrich the intranuclear ceramide pool, was investigated. The results show that in chromatin, there exists a phosphatidylcholine/sphingomyelin metabolism crosstalk which regulates the intranuclear ceramide/diacylglycerol pool. The enzyme activities were inhibited by D609, which demonstrated the specificity of this crosstalk. Chromatin lipid metabolism is activated in vivo during cell proliferation, indicating that it could play a role in cell function. The possible mechanism of crosstalk is discussed here, with consideration to recent advances in the field.     

Reverse sphingomyelin-synthase in rat liver chromatin

The chromatin phospholipid fraction is enriched in sphingomyelin content which changes during cell maturation and proliferation. Recently, we have demonstrated that the sphingomyelin variations can be due to chromatin neutral sphingomyelinase and sphingomyelin-synthase activities which differ in pH and K(m) optima from those present in nuclear membranes. The sphingomyelin can be used also as a source of phosphorylcholine for phosphatidylcholine synthesis by reverse sphingomyelin-synthase. In the present work we have studied the possible existence of reverse sphingomyelin-synthase activity in nuclear membrane and chromatin. A very low activity was detected in the homogenate, cytosol and nuclear membrane (0.93+/-0.14, 2.61+/-0.33 and 0.87+/-0.13 pmol/mg protein/min, respectively), whereas the activity present in chromatin was 37.09+/-2.05 pmol/mg protein/min. The reverse sphingomyelin-synthase decreases the intranuclear diacylglycerol pool and increases the intranuclear ceramide pool, whereas sphingomyelin-synthase has an opposite effect. The possible correlation between these enzymes is discussed.     

Phospholipids in plant and animal chromatin

Isolated hepatic nuclei and hepatic chromatin have been analysed for their DNA, RNA, protein and phospholipid content. The protein/DNA ratio is 3 for nuclei and 1.95 for chromatin extracted from Triton X-100 treated nuclei. The phospholipids, (2.36 +/- 0.91 (S.D.) per cent of the total nuclear material), are lost during the chromatin preparation mainly during the Triton X-100 washings of the nuclei. Nevertheless, 10 per cent of the total nuclear phospholipids remain bound to the chromatin. The comparative analysis of both nuclei and chromatin shows a difference in phospholipids and fatty acid composition. Thus, the chromatin-associated phospholipid cannot be attributed simply to contaminating nuclear membrane. This is supported by the autoradiographic study of semi-thin sections of interphase nuclei from root apices of Vicia faba in which [3H] ethanolamine is clearly localized in the chromatin and nucleolar regions of the nuclei.     

Incorporation of 1-(14)C linoleic acid in rat liver nuclei and chromatin fractions

The incorporation of 1-(14)C linoleic acid in several chromatin fractions of rat liver nuclei was investigated using two different procedures: (1) rat liver nuclei were incubated with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid. After 40 min at 37 degrees C the chromatin obtained by sonication of nuclei suspended in 0.25 M sucrose was fractionated by differential sedimentation; (2) chromatin fractions obtained by differential sedimentation were incubated separately with ATP, CoASH, Mg(++) and 1-(14)C linoleic acid 40 min at 37 degrees C in order to characterize the fatty acid incorporation in isolated chromatin. A comparative study of the incorporation of 1-(14)C linoleic acid in microsomes and nuclei isolated from rat liver is also presented for the purpose of comparison. Linoleic acid was incorporated into nuclear lipids as well as in chromatin fractions. The fatty acid incorporation was stimulated considerably in the acylation system when compared to control, it appears to be highly dependent on the state of condensation of chromatin, being barely detectable in the lowest density fraction. The major proportion of 1-(14)C linoleic acid was found in phospholipids and in a lesser proportion it remained esterified to triglycerides and cholesteryl esters. The distribution of radioactivity in different classes of phospholipids present in microsomes and nuclei isolated from rat liver, showed a similar profile of distribution. The major proportion of radioactivity, approximately 50% was found in phosphatidylcholine and in a lesser proportion in sphingomyelin, phosphatidylserine and phosphatidylethanolamine. When chromatin fractions were incubated separately, it was observed that the major proportion of 1-(14)C linoleic acid in phospholipids was found in heavy chromatin fractions whereas low density chromatin fraction only incorporated in a lesser proportion.     

Two chromatin fractions with different metabolic properties of non-histone proteins and of newly synthesized RNA.

Digestion of chromatin with micrococcal nuclease under mild conditions results in the release of a minor chromatin fraction showing an increased RNA and non-histone protein content, a fast turnover of the non-histone proteins and the presence of rapidly labelled heterogeneous nuclear RNA (hnRNA) with half-life of about 20 min. Further digestion of the chromatin leads to the elimination of about 19% of the initial chromosomal DNA, thus leaving a second chromatin fraction relatively resistant to nuclease attack. This fraction has a low protein and RNA content and contains only metabolically stable non-histone proteins. No differences in the histone complement of the two fractions was found except for a 40% deficiency of H1 in the minor fraction.     

Co-ordination between membrane phospholipid synthesis and accelerated biosynthesis of cytoplasmic ribonucleic acid and protein

1. The rate of synthesis of membrane phospholipid was studied in rat liver and seminal vesicles by following the incorporation of [(32)P]orthophosphate, [(14)C]choline and [(14)C]glycerol. Particular emphasis was laid on the endoplasmic reticulum, which was fractionated into smooth microsomal membranes, heavy rough membranes, light rough membranes and free polyribosomes. 2. Phospholipid labelling patterns suggested a heterogeneity in the synthesis and turnover of the different lipid moieties of smooth and rough endoplasmic membranes. The major phospholipids, phosphatidylcholine and phosphatidylethanolamine, were labelled relatively rapidly with (32)P over a short period of time whereas incorporation of radioisotope into the minor phospholipids, sphingomyelin, lysolecithin and phosphatidylinositol proceeded slowly but over a longer period of time. 3. The incorporation of orotic acid into RNA and labelled amino acids into protein of the four submicrosomal fractions was also studied. 4. Rapid growth of the liver was induced by the administration of growth hormone and tri-iodothyronine to hypophysectomized and thyroidectomized rats and by partial hepatectomy. Growth of seminal vesicles of castrated rats was stimulated with testosterone propionate. 5. The rate of labelling of membrane phospholipids was enhanced in all major subcellular particulate fractions (nuclear, mitochondrial and microsomal) during induced growth. However, it was in the rough endoplasmic reticulum that the accumulation of phospholipids, RNA and protein was most marked. The effect of hormone administration was also to accelerate preferentially the labelling with (32)P of sphingomyelin relative to that of phosphatidylcholine or phosphatidylethanolamine. 6. Time-course analyses showed that, in all four growth systems studied, the enhancement of the rate of membrane phospholipid synthesis coincided with the rather abrupt increase in the synthesis of RNA and protein of the rough endoplasmic reticulum. Growth hormone and tri-iodothyronine administered to hypophysectomized rats had additive effects in all the biosynthetic processes. The latent period of action of each hormone was maintained so that two waves of proliferation of endoplasmic reticulum occurred if the hormones were administered simultaneously. 7. It is concluded that there is some mechanism in the cell that tightly co-ordinates the formation of membranes, especially those of the endoplasmic reticulum, when an increased demand is made for protein synthesis.     

Effect of insulin on the composition of chromatin phospholipid in rat liver cells

In vivo the action of insulin on rat liver chromatin phospholipid composition was investigated. It was shown that the hormone led to reliable increase (nearly 20%) of total phospholipid content. The same phenomenon was shown also in the fraction of active chromatin while the phospholipids content of non-active chromatin didn't changed. It was also shown that the content of three from six phospholipid fractions altered under the insulin action. The content of sphingomyelin and phosphatidilethanolamine increased and the quantity of phosphatidylinositol decreased under the hormone action. The decrease of monophosphoinositides content was accompanied by the reliable increase of triphosphoinositides amount. It was suggested that the fractions of chromatin polyphosphoinositides were redistributed under the action of insulin.     

The Phospholipid Composition of Nuclear Subfractions from Germinating Wheat Embryos

The content of phospholipids in chromatin, nuclear matrix, and nuclear membrane from wheat (Triticum aestivum L.) embryos was studied. Subfractions of intact nuclei from dry embryos were shown to differ in the content and composition of particular phospholipids. Embryo germination resulted in the redistribution of phospholipid between nuclear subfractions. A functional role of structural changes in the nuclear membrane due to this phospholipid redistribution is discussed. It is supposed that these rearrangements change nuclear membrane permeability and its surface charge.     

Lipid asymmetry in rabbit small intestinal brush border membrane as probed by an intrinsic phospholipid exchange protein.

All classes of phospholipids present in brush border membrane are exchanged in a 1:1 ratio for egg phosphatidylcholine when brush border membrane vesicles from rabbit small intestine are incubated with small unilamellar vesicles of egg phosphatidylcholine. The exchange reaction exhibits biphasic kinetics similar to those of the hydrolysis of brush border membrane phospholipids by phospholipase A2 and sphingomyelinase C. In both reactions there is an initial fast phase followed by a markedly slower one. The phospholipid exchange appears to be catalyzed by intrinsic brush border membrane protein(s), while the digestion by phospholipases is mediated by externally added enzymes. From a comparison of the kinetics of phospholipid exchange and phospholipid hydrolysis, the following conclusions can be drawn: Both sets of experiments indicate the presence of two phospholipid pools differing in the rate of phospholipid exchange and hydrolysis. Except for sphingomyelin, the size of the two phospholipid pools derived from phospholipid exchange is in good agreement with that derived from phospholipid hydrolysis. This is the main finding of this work, and on the basis of this result the two lipid pools are tentatively assigned to phospholipid molecules located on the outer and inner layer of the brush border membrane. The slow rate of phospholipid exchange reflects the rate of transverse or flip-flop movement of phospholipids. The half-time of this motion is approximately 8 h for isoelectric (neutral) phospholipids such as phosphatidylethanolamine and approximately 80 h for negatively charged phosphatidylserine and phosphatidylinositol. Isoelectric phospholipids (phosphatidylcholine, phosphatidylethanolamine) are preferentially located on the inner (cytoplasmic) side (to about 70%) while the negatively charged phospholipids are more evenly distributed: 55-60% are located on the inner side.     

Evidence for Two Metabolically Distinct Types of Ribonucleic Acid in Chromatin and Nucleoli

Patterns of radioisotope incorporation are useful characteristics in describing cellular RNA fractions, and have indicated a distinctive "nuclear" RNA. In order to characterize the RNA fractions of the two nuclear components, nucleoli and chromatin, and to determine thereby the precise localization of the RNA typical of isolated nuclei, time-courses of P³² incorporation into nucleolar, chromosomal, and cytoplasmic RNA of Drosophila salivary glands have been determined from autoradiograms. Two experiments are reported which cover 12 and 18 hour periods, including an initial 2 hour feeding on P³². Concentrations of RNA-P³² (identified by ribonuclease digestion) were determined by grain counts. After 1 hour only the nucleolar RNA is labelled. Activity is detectible in chromosomal and cytoplasmic RNA after the 2nd hour. The nucleolar fraction reaches its maximum activity shortly after transfer of the larvae to non-radioactive food, the other fractions several hours later. Maximum activities persist in the chromosomal and cytoplasmic fractions; nucleolar activity decreases after the 9th hour. The observed differences in times at which incorporation begins and maximum activities are reached, and in maintenance of maximum activities indicate that chromosomal and nucleolar RNA are distinct fractions. The metabolic characteristics which have been ascribed to "nuclear" RNA apply only to the nucleolar fraction.     

Action of phospholipase C (Bacillus cereus) on isolated myelin sheath preparations

The action of phospholipase C (Bacillus cereus) on the phospholipids of myelin sheath preparations has been investigated. With freshly isolated bovine brain myelin about 40% of the total phospholipid could be hydrolyzed by this enzyme. With bovine spinal cord myelin the phospholipid seemed more resistant to attack whereas the opposite was the case with myelin from guinea-pig brain or rat brain. With fresh bovine brain myelin, phosphatidylcholine and the ethanolamine-containing phospholipids were the main targets for the enzyme with lesser extents of hydrolysis occurring with phosphatidylserine and sphingomyelin. The effect of exposing bovine brain myelin to structural perturbants prior to enzyme digestion indicated that trypsin pretreatment had no significant effect, whereas marked enhancement of the extent of phospholipid hydrolysis occurred following lyophilization + rehydration, or pretreatment of myelin with HCl, Triton TX-100/ammonium acetate or deoxycholate. The effect of myelin pretreatment on the degradation of the individual phospholipid classes was also studied.     

Synthesis of chromatin phospholipids

The presence of a phospholipid fraction associated with chromatin has been demonstrated by biochemical technique in rat hepatocytes. The composition of this fraction determined by chromatography with respect to that of the nuclei is characterized by low content of phosphatidylserine and high content in phosphatidylethanolamine. Also the synthesis and turnover studied after injection of [32P]O4(2-) show a different behaviour: the peak of activity is after 6 hrs in nuclei and microsomes, whereas in chromatin it occurs after 9 hrs. A second peak is evident after 24 hrs in chromatin and microsome phospholipids. Differences have been also shown by analyzing the single phospholipid radioactivity in time. The behaviour of chromatin phospholipids has also been studied during DNA premitotic synthesis in regenerating liver. It has been shown that there is no difference in synthesis in relation to that of DNA in nuclear phospholipids, whereas the specific activity of chromatin phospholipids begins to increase twelve hours after hepatectomy and continues throughout the period of the first mitotic wave, thus bringing to a summation with the beginning of the second wave. The role of this phospholipid fraction in relation to DNA synthesis and gene expression is discussed.     

Structural and functional aspects of nuclear lipids in normal and tumor cells

This review summarizes available data on the structural and functional role of neutral lipids and phospholipids in normal and tumor eukaryotic cells. The role of acidic phospholipids (cardiolipin, phosphatidylinositol, and phosphatidylglycerol) in regulation of activities of DNA- and RNA-polymerases, DNA-topoisomerases I and II, DNA-methylases, and replication initiation proteins (dnaA and T-antigen) is discussed. The role of sphingolipids is emphasized considering, on one hand, the involvement of sphingosines in signal transduction, chromatin association-dissociation, and regulation of DNA and RNA synthesis and protein kinase C and, on the other hand, participation of ceramides and dihydroceramides in apoptosis. The possible role of sphingomyelin, sphingosine, cardiolipin, and diglycerides in the contacts of DNA loops with nuclear matrix is analyzed. Lipid hormones indirectly influence supercoiled DNA conformation; the effect of hormones on metabolism of phospholipids and neutral lipids in chromatin and nuclear matrix is reviewed. Characteristics of lipid composition in chromatin and nuclear matrix of the tumor cells are discussed.     

PHOSPHOLIPID METABOLISM IN LIGHT AND DARK ADAPTED EXCISED RETINA

Abstract— The phospholipid composition of, and the incorporation of labelled phosphorus into the different phospholipids of rat and calf retina have been studied. The influence of various conditions, such as dark and light adaptation, during the preparation of retina, lipid extraction and incubation of retina with radioactive phosphorus was investigated.
The phospholipid composition of rat retina did not differ significantly from that of calf retina and the different conditions of preparation and incubation did not modify the distributions.
The specific radioactivities of the different phospholipids of calf and rat retina, incubated in the presence of ³²P, distinguished in both species two groups of components characterized by the rate of labelling. Phosphatidic acid (PA) and inositol glycerophospholipids (PI) belonged to the first group and showed the highest uptake of labelled phosphorus; the second group, comprising choline glycerophospholipids (PC), serine glycerophospholipids (PS), sphingomyelin (SP), ethanolamine glycerophospholipids (PE) and cardiolipin (CL) showed low incorporation activities. Only SP was labelled differently in rat and calf retina. With the exception of PS, there was no evidence for the influence of light on the turnover of individual phospholipids. The finding that PS showed higher specific radioactivities when adaptation and incubation proceeded in the dark, seems to be of interest and needs further study.     

Control of salivary phospholipid content and composition.

The mediation of phospholipid secretion in rat sublingual salivary gland cells maintained in the presence of [3H]choline was investigated. The secretion of [3H]choline-containing phospholipids was enhanced by beta-adrenergic agonist, isoproterenol, to a greater extent than the cholinergic agonist carbachol. A 2.9-fold increase in phospholipid secretion occurred with isoproterenol, while carbachol evoked only about 1.3-fold increase. In contrast to carbachol, the enhanced phospholipid secretion due to isoproterenol was accompanied by an increase in cAMP concentration. The secretion of phospholipids was also stimulated by dibutyryl-cAMP and the protein kinase C activator, phorbol myristate acetate, but not by 4 alpha-phorbol 12, 13-didecanoate which does not activate protein kinase C. Furthermore, the effects of dibutyryl-cAMP and phorbol myristate acetate were additive. The phospholipids secreted in response to isoproterenol exhibited a 52% decrease in lysophosphatidylcholine, while those secreted in response to carbachol showed a 23% lower content of phosphatidylcholine, and were enriched in lysophosphatidylcholine (2.8-fold) and sphingomyelin (1.4-fold). The results suggest that salivary phospholipid secretion remains mainly under beta-adrenergic control, while the phospholipid makeup is under cholinergic regulation.