Developmental patterns in rat brain of phosphatidylinositol synthetic enzymes and phosphatidylinositol transfer protein

Phosphatidylinositol synthetic and intermembrane transfer activities were studied in rat in the developing whole brain and isolated cerebellum. Specific activities of CTP: phosphatidate cytidylyltransferase and CDPdiacylglycerol: inositol phosphatidyltransferase were found to have similar developmental patterns. Levels of phosphatidyltransferase seen in fetal animals (whole brain only) and neonatal (whole brain and cerebellum) were maintained through approximately postnatal day 15, peaked at day 28, and then declined to somewhat higher than fetal levels at day 60. Cytidylyltransferase activity varied from the phosphatidylinositol synthesizing enzyme in that specific activity continued to increase up to day 60. Whole brain phosphatidylinositol transfer specific activity showed a sharp peak at postnatal day 9 after which activity was maintained at or above the fetal levels to day 60. Cerebellum phosphatidylinositol transfer specific activity had a similar peak which was delayed 7–10 days compared to the whole brain. Phosphatidylinositol transfer protein was also determined immunologically: whole brain levels increased dramatically from fetal day 16 to 18 and then remained relatively constant, while cerebellum levels (measured from postnatal day 7) displayed a variable profile between days 7 and 28. The developmental pattern of CTP: phosphatidate cytidylyltransferase in rat brain is reported here for the first time.     

Regional distribution in rat brain of phosphatidylinositol and phosphatidylcholine synthetic and intermembrane transfer activities

Rat brains were dissected into major anatomical regions, including caudate nucleus, cerebellum, inferior and superior colliculi, cortex, hippocampus, olfactory bulb, pituitary gland, pons-medulla, spinal cord and thalamus. Tissue fractionation yielded microsomes and cytosol which were assayed for several phospholipid synthetic enzyme activities; using a vesicle-vesicle system the cytosol fractions were also examined for intermembrane phospholipid transfer activities. For the metabolism of phosphatidylinositol, specific activities were determined for CTP: phosphatidate cytidylyltransferase and CDP-diacylglycerol: inositol phosphatidyltransferase. Regions with high phosphatidylinositol synthetic activity were pituitary gland, pons-medulla, caudate and thalamus. For the metabolism of phosphatidylcholine the measured enzymes were CTP: phosphocholine cytidylyltransferase and CDP-choline: diacylglycerol cholinephosphotransferase. These enzymes showed the highest activity in the colliculi, olfactory bulb, pituitary gland and pons-medulla. The pons-medulla cytosol fraction contained the highest level of phosphatidylinositol transfer activity, while the colliculi and pons-medulla had the highest level of phosphatidylcholine transfer activity. In contrast, the pituitary gland displayed the lowest levels of both phosphatidylinositol and phosphatidylcholine transfer activity. The relationships between synthetic and transfer activities are discussed in terms of regional phospholipid metabolism.     

Developmental changes in malonate-related enzymes of rat brain.

Mitochondria and high-speed supernatant were prepared from rat brain homogenates at 0–50 days of age. The development of malonyl-CoA synthetase, malonyl-CoA decarboxylase, coenzyme A-transferases and acetyl-CoA hydrolase was examined and compared to de novo fatty acid biosynthesis. The specific activity of malonyl-CoA synthetase rose steeply between 6 and 10 days, and this sudden increase coincided with peak specific activity of fatty acid synthetase. Similarly, malonate activation by coenzyme A-transfer from succinyl-CoA increased rapidly at the same time. Transfer of the coenzyme A moiety from acetoacetyl-CoA was only minimal during this period. Brain mitochondria had active malonyl-CoA decarboxylase which showed an almost linear increase of specific activity between 0 and 50 days. Acetyl-CoA resulting from malonyl-CoA decarboxylation underwent enzymatic hydrolysis to acetate and free coenzyme A. Only traces of acetoacetate were recovered. In mitochondria, acetyl-CoA hydrolase increased progressively whereas the cytosolic enzyme had high specific activity at birth which declined slowly during maturation.     

Developmental patterns of sulphate activation and phenolsulphotransferase in rat brain

Abstract— The formation of active suphate has been assayed in developing rat brain, the activity of the enzyme system being maximal at birth and thereafter decreasing gradually. The activity of phenolsuphotransferase, present in rat brain, is minimal at birth and increases gradually to the adult value.     

Alteration of phosphatidylinositol transfer protein during global brain ischemia-reperfusion in gerbils

Phosphatidylinositol transfer proteins (PI-TPs) are responsible for the transport of phosphatidylinositol and other phospholipids. Moreover, these proteins are involved in vesicle transport and in the function of cytoskeleton. Our previous data indicated that brain ischemia affected phosphoinositides metabolism and the level of lipid derived second messengers. In this study, the effect of ischemia-reperfusion injury on the level of PI-TPs and of the role of NMDA receptor stimulation on the alteration of these proteins was investigated during reperfusion after 5 min of forebrain ischemia in gerbils. Some groups of animals were injected intraperitoneally with MK-801, an antagonist of NMDA receptor 30 min before ischemia. The levels of both PI-TP isoforms alpha+beta and separately the alpha-isoform were determined in cytosol and membrane fraction from brain cortex and hippocampus using Western blot analysis. In the cytosolic fractions, the concentration of both isoforms of PI-TP was 2 times higher when compared to the membrane fraction. In brain cortex, PI-TP alpha isoform consist about 32-44% but in hippocampus 72-82% of both isoforms (PI-TP alpha+beta) in cytosolic and membrane fraction respectively. Ischemia-reperfusion had no effect on PI-TPs in brain cortex. However, in hippocampus after 5 min ischemia and during whole reperfusion time up till 7 days the level of PI-TP alpha+beta and PI-TP alpha was significantly higher by about 20-55%, respectively when compared to control. MK-801 eliminated ischemia-reperfusion evoked alteration of PI-TPs. To confirm the role of NMDA receptor in PI-TP alteration additional experiments were carried out on PC-12 cells in culture. The results indicated that activation of NMDA receptor enhances significantly the level of PI-TP alpha. The competitive antagonist of NMDA receptor inhibited this effect. These results indicated that activation of NMDA receptor is connected with PI-TPs alteration and plays an important role in modulation of PI-TPs during ischemia-reperfusion injury that may have important physiopathological consequence.     

Uptake and phosphorylation of phosphatidylinositol by rat liver nuclei. Role of phosphatidylinositol transfer protein

The incorporation of phosphatidyl[2-3H]inositol ([3H]PI) from vesicles or microsomal membranes into rat liver nuclei is greatly stimulated by phosphatidylinositol transfer protein (PI-TP). The nuclei are able to phosphorylate [3H]PI, with the production of phosphatidylinositol 4-phosphate (PIP). Recovery of tritiated inositol trisphosphate, inositol phosphate, glycerophosphoinositol and inositol, suggests that in isolated nuclei a large set of enzymes of the PI cycle is present, similar to the enzymes involved in the plasma membrane PI cycle. Incubation with [gamma-32P]ATP shows that isolated nuclei are able to phosphorylate endogenous PI to PIP and phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of exogenous PI and detergent the synthesis of PIP is increased, indicating that in nuclei the PI pool is suboptimal for the PI-kinase activity. The present study suggests that PI-TP may be involved in providing substrates for PI metabolism at the nuclear level.     

The hydrolysis of phosphatidylinositol by lysosomal enzymes of rat liver and brain.

1. Lysosomes from rat liver contain two enzymic systems for hydrolysing phosphatidyl-inositol: a deacylation via lysophosphatidylinositol producing glycerophosphoinositol and non-esterified fatty acid, and a phospholipase C-like cleavage into inositol 1-phosphate and diaclygycerol. 2. The separate enzyme systems involved can be distinguished by gel filtration, differential temperature-stability and the inhibitory action of detergents. 3. The enzyme systems both have pH optima at 4.8 and their attack on a pure phosphatidylinositol substrate is inhibited by many bivalent metals including Ca2+ and Mg2+, and cationic drugs. 4. Whereas the deacylation system will attack other glycerophospholipids, the phospholipase C shows a marked specificity towards phosphatidylinositol, although it will also slowly attach phosphatidylcholine with the liberation of phosphocholine. 5. Gel filtration and temperature-stability distinguish the phospholipase C from lysosomal phosphatidic acid phosphatase, but not from sphingomyelinase. 6. Evidence is presented that an EDTA-insensitive phospholipase C degrading phosphatidylinositol is present in rat brain.     

Isolation and sequence of cDNA clones encoding rat phosphatidylinositol transfer protein

Phosphatidylinositol (PtdIns) transfer protein is a cytosolic protein that catalyzes the transfer of PtdIns between membranes. It is expressed in organisms from yeast to man, and activity has been found in all animal tissues examined. Using antibodies prepared against bovine brain PtdIns transfer protein, lambda gt11 rat brain cDNA libraries were screened and several clones isolated. DNA sequence analysis showed that the cDNAs encoded a polypeptide of 271 amino acids with a mass of 31,911 Da. Comparison of the deduced amino acid sequence with N-terminal sequence data obtained for the intact purified bovine brain protein and rat lung phospholipid transfer protein verified that the cDNAs were PtdIns transfer protein clones. The predicted protein shows no significant sequence similarity to other known (phospholipid)-binding proteins. DNA blot hybridization suggests that the rat genome may contain more than one gene encoding PtdIns transfer protein. RNA blot hybridization reveals that the PtdIns transfer protein gene is expressed at low levels in a wide variety of rat tissues; all tissues examined showed a major mRNA component of 1.9 kilobases and a minor component of 3.4 kilobases. The isolation of clones encoding rat PtdIns transfer protein will greatly facilitate studies of the structure and function of PtdIns transfer proteins and their role in lipid metabolism.     

Tissue distribution, purification and characterization of rat phosphatidylinositol transfer protein

Phosphatidylinositol transfer activity is measured in cytosol fractions prepared from 13 rat tissues; specific activity is highest in brain and lowest in adipose and skeletal muscle. Based upon electrophoretic analysis phosphatidylinositol transfer protein is purified to homogeneity from whole rat brain. The protein has a molecular weight of 36,000 and exists as a mixture of species having isoelectric points of 4.9 and 5.3. In a vesicle-vesicle assay system, the intermembrane transfer rate is greatest for phosphatidylinositol and less by a factor of 2 for phosphatidylcholine; transfer of phosphatidylethanolamine, phosphatidylserine or sphingomyelin is not observed. Using a polyclonal rabbit antibody against bovine phosphatidylinositol transfer protein, immunologic cross-reactivity is noted between the rat protein and other mammalian phosphatidylinositol transfer proteins. A strong correlation is established between a tissue's capacity for phosphatidylinositol transfer and the amount of immunoreactive transfer protein seen in that tissue. Purified phosphatidylinositol transfer protein is capable of transporting newly synthesized phosphatidylinositol molecules from rat brain microsomes to small unilamellar phospholipid vesicles. The results are discussed within the context of cellular phosphoinositide metabolism and the maintenance of the metabolically responsive pool of phosphatidylinositol in the plasma membrane.     

Developmental patterns of galactosyltransferase activity in various regions of rat brain

Abstract: The developmental pattern of glycoprotein-galactosyltransferase activity was determined in the microsomal fractions of three regions of the embryonic rat brain and in parts of the visual system and the cerebellum postnatally. It could be shown that the enzyme activity was highest in the embryonic brain, where regional differences were apparent, and decreased progressively after birth. The enzyme profile in the cerebellum showed no marked postnatal changes.     

On the relationship between the dual specificity of the bovine brain phosphatidylinositol transfer protein and membrane phosphatidylinositol levels

The phosphatidylinositol transfer protein from bovine brain has a remarkable specificity pattern with a distinct preference for phosphatidylinositol (PI) and a low affinity for phosphatidylcholine (PC). In this study we have determined the affinity of PI-transfer protein for PI relative to that for PC by measuring the binding of the fluorescent pyrene-labeled analogs of these phospholipids. From competition binding experiments it was estimated that the transfer protein has a 16-fold higher affinity for PI than for PC. This relative affinity together with the relative abundance of PI and PC, determines what proportion of the protein contains PI (e.g. 65% of the PI-transfer protein in the case of bovine brain). From measuring lipid transfer between donor vesicles consisting of equimolar amounts of PC and PI, and an excess of acceptor vesicles consisting of various ratios of PC and PI, we have observed that the relative rates of the PI-transfer protein-mediated transfer of PI and PC varies between 5 and 20. Kinetic analysis has indicated that PI-transfer protein carrying a PI molecule has different kinetic properties than the PI-transfer protein carrying a PC molecule. It will be discussed that because of the dual specificity, PI-transfer protein is ideally suited for maintaining PI levels in intracellular membranes.     

The phosphatidylinositol kinase of rat brain

1. The presence of a phosphatidylinositol kinase in homogenates of adult rat brain was shown by using labelled ATP or labelled phosphatidylinositol. 2. The kinase was activated by Mg(2+) or Mn(2+) and inhibited by Ca(2+), Cu(2+), K(+), Na(+) and F(-). 3. The detergents sodium deoxycholate, Cutscum and Triton X-100 markedly stimulated the reaction; sodium taurocholate, Tween-20 and cetyltrimethyl-ammonium bromide were less effective. 4. The activity of the enzyme was dependent on SH groups. 5. The subcellular distribution of the kinase in brain resembled that of Na(+)-plus-K(+)-stimulated adenosine triphosphatase and 5'-nucleotidase.     

Bovine brain phosphatidylinositol transfer protein. Selective inhibition by chlorpromazine and other amphiphilic amines

Cationic amphiphilic amines of varied pharmacological activity were evaluated as modulators of the protein-catalyzed, intermembrane transfers of phosphatidylinositol and phosphatidylcholine. The catalytic agent was brain phosphatidylinositol transfer protein; the membrane system consisted of two populations of single bilayer phospholipid vesicles. The majority of the amines tested caused decreases in phospholipid transfer activity with the relative potencies in the following order: chlorpromazine greater than dibucaine greater than propranolol much greater than tripelennamine approximately chloroquine greater than dipyridamole. Concentrations required for 50% inhibition of phosphatidylinositol transfer were 0.24 mM chlorpromazine, 0.46 mM dibucaine, and 0.78 mM propranolol. The phosphatidylcholine transfer activity of this protein was somewhat less sensitive to these compounds. Comparison of chlorpromazine and its quaternary amine analogue, methochlorpromazine, at different pH values indicated that the observed inhibition can be attributed in large part to the charged forms of the amphiphiles. Direct association of methochlorpromazine with egg phosphatidylcholine bilayers was demonstrated by molecular sieve chromatography; no such association of the amphiphile with phosphatidylinositol transfer protein was apparent. Anionic agents, such as indomethacin, phenylbutazone, and tolmetin, were without significant effect on protein-catalyzed phospholipid transfers. Electrostatic interaction between the cationic amines and anionic or zwitterionic phospholipids, forming ion pairs in the lipid bilayers, is suggested as a possible molecular mechanism for the observed inhibition.     

Bovine brain phosphatidylinositol transfer protein. Effects of pH, ionic strength and lipid composition on transfer activity

Phosphatidylinositol and phosphatidylcholine are transferred between bilayer membranes in the presence of a specific phosphatidylinositol transfer protein isolated from bovine brain. The effects of pH, ionic strength and lipid composition on the rate of transfer of these phospholipids between small unilamellar vesicles have been investigated. At low ionic strength, phosphatidylinositol transfer between vesicles prepared from phosphatidylcholine and 5 mol% phosphatidylinositol was maximal at about pH 5 and moderately dependent on hydrogen ion concentration in more alkaline regions. A similar dependence on pH was noted for phosphatidylcholine transfer between membranes containing phosphatidylcholine or mixtures of phosphatidylcholine and 5 mol% phosphatidylinositol, phosphatidic acid, phosphatidylglycerol, phosphatidylethanolamine or stearylamine. The rate of transfer between anionic vesicles was somewhat higher than that between neutral or cationic vesicles. At higher ionic strength the transfer reactions in neutral and alkaline regions were less sensitive to pH. Phospholipid transfers between vesicles containing 5 mol% of anionic lipid increased sharply as ionic strength decreased below 0.1. In contrast, phosphatidylcholine transfer between membranes which contained only zwitterionic phospholipids or 5 mol% stearylamine was unaffected by variations of ionic strength. Irrespective of the lipid composition of membranes, pH affected both the apparent Km and Vmax, while ionic strength generally affected the apparent Vmax. These results indicate a significant role of electrostatic interactions in the phospholipid transfer catalyzed by phosphatidylinositol transfer protein.     

Mature rat testis contains a high molecular weight species of phosphatidylinositol transfer protein

Immunoblot analysis of a rat testis cytosol fraction revealed two proteins which reacted with a polyclonal rabbit antibody to bovine phosphatidylinositol transfer protein. These two proteins were separated by anion exchange and molecular sieve column chromatographic procedures and shown to catalyze the transfer of phosphatidylinositol and phosphatidylcholine between populations of small unilamellar vesicles. One protein was identified as the phosphatidylinositol transfer protein detectable in 16 other rat tissues and many eukaryotic species; the other phosphatidylinositol transfer protein was unique to testis. The molecular masses of the proteins, determined under denaturing electrophoretic conditions, were 35 and 41 kDa, respectively. When testis was examined in animals from birth to six weeks of age, the 35-kDa protein was present throughout, while the 41-kDa protein first appeared during week 4 and increased to adult levels by week 6; a small yet significant increase in tissue phosphatidylinositol transfer activity accompanied this expression of the testis-specific protein. Selective destruction of Leydig cells by ethylene dimethanesulfonate did not cause any detectable loss of the 41-kDa phosphatidylinositol transfer protein. The structural and catalytic relationships between the two testicular phosphatidylinositol transfer protein species remain to be elucidated.     

L-alpha-glycerylphosphorylcholine inhibits the transfer function of phosphatidylinositol transfer protein alpha

Phosphatidylinositol transfer protein alpha (PITP-alpha) is a bifunctional phospholipid transfer protein that is highly selective for phosphatidylinositol (PtdIns) and phosphatidylcholine (PtdCho). Polar lipid metabolites, including L-alpha-glycerylphosphorylcholine (GroPCho), increasingly have been linked to changes in cellular function and to disease. In this study, polar lipid metabolites of PtdIns and PtdCho were tested for their ability to influence PITP-alpha activity. GroPCho inhibited the ability of PITP-alpha to transfer PtdIns or PtdCho between liposomes. The IC(50) of both processes was dependent on membrane composition. D-myo-inositol 1-phosphate and glycerylphosphorylinositol modestly enhanced PITP-alpha-mediated phospholipid transfer. Choline, phosphorylcholine (PCho), CDP-choline, glyceryl-3-phosphate, myo-inositol and D-myo-inositol 1,4,5-trisphosphate had little effect. Membrane surface charge was a strong determinant of the GroPCho inhibition with the inhibition being greatest for highly anionic membranes. GroPCho was shown to enhance the binding of PITP-alpha to anionic vesicles. In membranes of low surface charge, phosphatidylethanolamine (PtdEtn) was a determinant enabling the GroPCho inhibition. Anionic charge and PtdEtn content appeared to increase the strength of PITP-alpha-membrane interactions. The GroPCho-enhanced PITP-alpha-membrane binding was sufficient to cause inhibition, but not sufficient to account for the extent of inhibition observed. Processes associated with strengthened PITP-alpha-membrane binding in the presence of GroPCho appeared to impair the phospholipid insertion/extraction process.     

The association between phosphatidylinositol phosphodiesterase activity and a specific subunit of microtubular protein in rat brain

1. Supernatant proteins from rat brain were separated into two fractions containing phosphatidylinositol phosphodiesterase activity by chromatography on DEAE-Sephadex A-50. 2. The first fraction sediments in linear sucrose density gradients in two bands corresponding to molecular weights of 66000 and 36000. There was presumptive evidence that the lighter protein constituted the monomeric form of the enzyme. The second fraction sediments predominantly as a single protein of molecular weight 86000. 3. Treatment of rat brain supernatant with [(3)H]colchicine abolished the second DEAE-Sephadex peak and removed the lighter protein from the first peak. These proteins emerged in the same position as the protein binding [(3)H]colchicine at high salt concentration; phospholipase activity was recovered from linear sucrose density gradients in positions corresponding to molecular weights 88000 and 43000, together with an aggregate of molecular weight 140000. Electrophoresis on sodium dodecyl sulphate-urea-polyacrylamide gels of this fraction revealed only three proteins: the alpha and beta-subunits of microtubular protein, of molecular weights 56000 and 52000 respectively, and a protein of molecular weight 38000. 4. A sample of microtubular protein from mouse, labelled in vivo with [(3)H]proline and (32)P(i), was added to rat brain supernatant together with an equal amount of the same microtubular protein treated with cyclic AMP and [gamma-(32)P]ATP and the mixture subsequently characterized by ion-exchange chromatography. Some phospholipase activity characteristic of the second peak from DEAE-Sephadex was associated with one fraction of added microtubular protein. This fraction was identified on the basis of the (3)H:(32)P ratio as the beta subunit of the protein treated with ATP and cyclic AMP. The subunit of added microtubular protein untreated with nucleotides was not associated with phospholipase activity.     

Heterogeneity of the calcium-dependent phosphatidylinositol phosphodiesterase in rat brain

1. The Ca²⁺-dependent phosphatidylinositol phosphodiesterase (phospholipase C-type) from the cytosolic supernatant of rat brain was active against exogenous [³²P]-phosphatidylinositol from pH5.0 to pH8.5. However, the activity in the range pH7.0–8.5 could not be recovered after precipitation with (NH₄)₂SO₄; most of the enzyme activity was recovered in the 30–50% fraction and showed a single sharp pH optimum at 5.5. 2. The cytosolic supernatant was analysed by isoelectric focusing on acrylamide gels, and assay at pH5.5. Four peaks of phosphodiesterase activity were found at pI ranges 7.4–7.2, 6.0–5.8, 4.8–4.4 and 4.2–3.8. 3. The cytosolic supernatant was also applied to a chromatofocusing column, and again assayed at pH5.5. Four peaks were eluted: minor, but consistent, activity at the beginning of the elution with a pI of near 7.2 or above; a second peak at pH6.0–5.85; a third broad peak with a wide range pH5.3–4.2; and a fourth peak, which was eluted by washing the column with 1m-NaCl, suggesting an isoenzyme with a pI below 4.0 (supported by the result of the isoelectric focusing). 4. If all the chromatofocusing fractions were assayed at pH7.0 or 8.0 (at 1mm-Ca²⁺), only a single sharp peak was detected, with a pI of 4.6–4.8. This peak disappeared on (NH₄)₂SO₄ fractionation (30–50%) of the cytosolic supernatant, whereas the four peaks with activity at pH5.5 were virtually unaffected. 5. The four activities (assayed at pH5.5) separated by chromatofocusing produced inositol 1:2-cyclic monophosphate, inositol 1-monophosphate and diacylglycerol as enzymic products. 6. We conclude that the Ca²⁺-dependent phosphatidylinositol phosphodiesterase exhibits considerable heterogeneity, both with respect to pH optima of activity, and its isoelectric properties.