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Biomedical subjects

J N Kanfer

Publications and source records attributed to J N Kanfer.

At least 37 records · Page 2Linked to original sources
PubMed1995

Phosphatidic acid activation of protein kinase C in LA-N-1 neuroblastoma cells.

Phosphatidic acid (PA), a hydrolytic product of phospholipase D activity, stimulated cytosolic protein kinase C (PKC) activity when LA-N-1 neuroblastoma cells in culture were treated with PA, without translocating the enzyme to the membrane. Treatment of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) translocated and activated PKC in a dogmatic manner. Partially purified PKC activity derived from LA-N-1 neuroblastoma cells was stimulated by PA alone or in the presence of phosphatidylserine or TPA, without affecting [3H]phorbol dibutyrate binding, indicating that the site of action of PA was different from the phorbol ester or diacylglycerol binding site. These results suggest an unorthodox pattern of PKC stimulation mediated by PA which appears to be yet another mode of PA signal transduction.

Binding, Competitive↗
PubMed1995

Amyloid beta protein (25-35) stimulation of phospholipases A, C and D activities of LA-N-2 cells.

[3H]Myristic acid prelabeled LA-N-2 cells were exposed to varying concentrations of amyloid beta protein (25-35), from 20 to 250 micrograms/ml, and the activation of phospholipases A and D estimated. A progressive increase in phosphatidylethanol formation, a measure of phospholipase D activity, and of free fatty acid release, a measure of phospholipase A activity, was observed over a time-course of 60 min. [3H]Inositol prelabeled LA-N-2 cells were exposed to varying concentrations of A beta P, from 20 to 125 micrograms/ml, and phospholipase C activation was measured. There was an increased release of inositol phosphates in the presence of amyloid beta protein as a function of incubation time. The effects of adrenergic, metabotropic amino acid and bombesin antagonists on the A beta P mediated stimulation of phospholipase C activity was investigated. Propranolol, a beta adrenergic antagonist, 7-chloro-kynurenic acid, a metabotropic amino acid antagonist, and [Tyr4-D-Phe12]bombesin, a bombesin antagonist, blunted the A beta P stimulation of phospholipase C activity in [3H]inositol prelabeled LA-N-2 cells. This suggests that amyloid beta protein activation of phospholipase C may be receptor mediated. The phospholipase C inhibitor U 71322 prevented the activation of phospholipase C by A beta P. However, this activation was not effected by tocopherol, propylgallate, or vitamin C.

Amyloid beta-Peptides↗
PubMed1995

Partial purification of a phosphoethanolamine methyltransferase from rat brain cytosol.

The conversion of phosphoethanolamine to phosphocholine requires 3 separate N-methyltransferases. We had previously purified the enzyme catalyzing the last methylation, phosphodimethylethanolamine N-methyltransferase. We have successfully purified the enzyme catalyzing the initial methylation of phosphoethanolamine. A 434 fold purified enzyme from rat brain was obtained by the sequential use of ammonium sulfate fractionation, Q-Sepharose fast flow column chromatography and a omega-aminoethyl agarose column chromatography. The pH optimum was 11 or greater, the Km value for phosphoethanolamine was 167.8 +/- 41.7 microM and the Vmax was 487.3 +/- 85 mmoles/mg/hr. The kinetics for S-adenosyl-methionine, the methyldonor, has characteristics of cooperative binding with a Km of 1.805 +/- 0.59 mM and a Vmax of 16.9 +/- 3.6 mumoles/mg/hr. The activity was stimulated 6 fold by 2.5 mM MnCl2 and inhibited by DZA and S-adenosylhomocysteine. These results reinforce the early in vivo observations which had provided suggestive evidence for the existence of a pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain.

Ammonium Sulfate↗
PubMed1995

Partial purification of two forms of choline kinase and separation of choline kinase from sphingosine kinase of rat brain.

Choline kinase of rat brain was purified approximately 200,000 fold using acid precipitation, ammonium sulphate fractionation, Q-Sepharose, Octyl-Sepharose and AH-Sepharose chromatography. The ability of this enzyme to catalyze the phosphorylation of choline, ethanolamine (Etn), monomethylethanolamine (MeEtn), dimethylethanolamine (Me2Etn) and sphingosine was investigated. Choline kinase was separated from sphingosine kinase. The fraction with highly purified choline kinase had four major polypeptides with different molecular masses and possessed activities towards choline, Etn, MeEtn and Me2Etn. Two forms of choline kinase were obtained when the enzymatically active fractions eluted from the Q-Sepharose column were subjected to a horizontal isoelectrofocusing electrophoresis. One form focused around pH 4.7 and is able to phosphorylate choline, Etn, MeEtn and Me2Etn. The other form focused around pH 10 and possessed only choline kinase activity. The latter form of choline kinase did not display classical Michaelis-Menten's mechanism but revealed a positive co-operative pattern for two choline binding sites. This form was purified to apparent homogeneity with a approximate molecular mass of 14.4 kDa.

Animals↗
PubMed1995

Magnetic resonance spectroscopy and its application to aging and Alzheimer's disease.

This is a review of magnetic resonance (MR) spectroscopy and its application to aging and Alzheimer's disease (AD). Examinations of perchloric acid extracts of AD brain tissue by MR spectroscopy reveal elevated levels of phosphomonoesters, phosphodiesters, and glutamate accompanied by reduced levels of N-acetyl-L-aspartate compared with extracts from controls. These metabolicalterations may be an indication of accelerated membrane phospholipid metabolism, glutamate neurotoxicity, and neuronal loss in AD brain that is not seen in normal aging. In vivo 31P MR spectroscopy studies of AD indicate that levels of phosphomonoesters are elevated early in the course of AD, which may be a causative molecular neuropathologic event. In vivo MR spectroscopy is a powerful technique to investigate the molecular neuropathology of the disease, to follow the progression of AD, and to assess the efficacy of experimental therapies.

Adolescent↗
PubMed1994

Modulation of phosphatidylserine synthesis by a muscarinic receptor occupancy in human neuroblastoma cell line LA-N-1.

The incorporation of [3H]serine into lipids, water-soluble metabolites and proteins by the human neuroblastoma cell line LA-N-1 exposed to oxotremorine-M, a muscarinic agonist, was investigated. Oxotremorine-M increased the incorporation of this labelled precursor into phosphatidylserine and proteins in a concentration-dependent manner, with the maximal stimulation at 250 microM. This activation was blunted by 100 microM atropine. There were no detectable changes of the radioactivity in the water-soluble metabolites. Acetylcholine, another muscarinic agonist, slightly decreased the serine incorporation into lipids, but did not affect the protein or water-soluble compartments. Several other muscarinic agonists, including 250 microM pilocarpine, 100 microM McN-A-343 and 1 mM carbachol, did not effect these [3H]serine incorporations. Preincubation of cells with 1 mM oxotremorine M, or 1 mM carbachol, or 1 mM McN-A-343, for 4 h prevented the oxotremorine-M-induced increase of serine incorporation. These observations are consistent with the oxotremorine-M action being mediated by muscarinic-receptor occupancy. The G-protein inhibitor guanosine 5'-[beta-thio]diphosphate (1 mM) and the G-protein activators, guanosine 5'-[gamma-thio]triphosphate (100 microM) and A1F3, prevented the oxotremorine stimulation. The muscarinic agonists, 250 microM oxotremorine-M, 1 mM carbamoylcholine and 500 microM acetylcholine, triggered the accumulation of inositol mono- and di-phosphates by cells that had been prelabelled with myo-[3H]inositol, and this phospholipase C activation was blunted by 100 microM atropine. The protein kinase C inhibitor H7 prevented the oxotremorine-M stimulation of serine incorporation. Over-night exposure of LA-N-1 cells to 100 nM phorbol 12-myristate 13-acetate resulted in a decrease of cytosolic protein kinase C activity, and prevented the oxotremorine-M stimulation of serine incorporation. Neither oxotremorine-M nor acetylcholine caused a redistribution of protein kinase C activity between the cytosol and membrane compartments. In addition, oxotremorine-M did not activate phospholipase D of the LA-N-1 cells.

Acetylcholine↗
PubMed1994

Phospholipase D activity of isolated rat brain plasma membranes.

With [14C]oleate-labeled phosphatidylcholine as a substrate for phospholipase D the hydrolytic activity was measured by phosphatidic acid formation and the transphosphatidylation activity was measured by the phosphatidylethanol formed in the presence of ethanol. The pH optimum was 6.5 with dimethylglutarate as the buffer. EGTA inhibited the transphosphatidylation activity to a greater extent than the hydrolytic activity. In contrast CaCl2, BaCl2, MgCl2 and SrCl2 stimulated the hydrolytic activity without effecting the transphosphatidylation activity. BeCl2 another member of the group IIa transition metals was a very potent inhibitor of both the hydrolytic and transphophatidylation activity. GTP gamma S, an activator of G protein-mediated events, was an inhibitor of both activities.

Animals↗
PubMed1994

Incorporation of [3H]ethanolamine into acetylcholine by a human cholinergic neuroblastoma clone.

Human neuroblastoma cholinergic LA-N-2 cells were used as an experimental model to test the possibility that the methylation of phosphoethanolamine (PEtn) to phosphocholine (PCho) and free choline (Cho) (Andriamampandry et al. 1989) could contribute to acetylcholine (AcCho) synthesis. LA-N-2 cells were incubated with [3H]Cho for 90 min and 22.7% of the radioactivity was present in PCho, 18.5% in free Cho and 4.8% as AcCho. The ratio of Cho/AcCho, however, was of about 1 after 16 hours of incubation. The incorporation of 10 microM [3H]ethanolamine (Etn) into MeEtn, PMeEtn, PMe2Etn and their corresponding phospholipids was reduced in cells incubated in medium containing 7.2 microM choline as compared to cells incubated in medium devoid of choline indicating that the lack of Cho from the incubation medium stimulated the conversion of PEtn to Cho water soluble derivatives. Incubation of LA-N-2 cells with [3H]Etn led to the labelling of [3H]AcCho. Cultures incubated in parallel with [3H]Cho showed that roughly 10% of [3H]AcCho obtained after 16 hrs of incubation with the Cho label derived from [3H]Etn. The synthesis of Cho and AcCho from Etn may be enhanced after cellular differentiation induced by the growth of the cells in the presence of retinoic acid (RA). The results indicate that the methylation of [3H]Etn and/or of [3H]PEtn may be used by cholinergic neurons as precursor for AcCho.

Acetylcholine↗
PubMed1994

Purification and properties of a glycerophosphocholine phosphodiesterase from bovine brain myelin.

An enzyme releasing phosphocholine from glycerophosphocholine was purified to apparent homogeneity based upon SDS-PAGE. The enzyme was liberated from lyophilized bovine myelin by differential detergent extraction and final purification was accomplished with Q-Sepharose Fast Flow chromatography yielding an apparently homogeneous protein. The molecular mass based upon PAGE was approximately 14 kDa. The enzyme was also capable of releasing p-nitrophenol from p-nitrophenyl-phosphocholine. Maximal activity was obtained with 0.2 mM ZnCl2 or 1 mM CoCl2. p-Nitrophenylphosphocholine and phosphocholine were competitive inhibitors of glycerophosphocholine hydrolysis with Ki's of 0.028 mM and 0.03 mM respectively. Glycerophosphocholine and phosphocholine were competitive inhibitors of p-nitrophenylphosphocholine hydrolysis with Ki's of 0.5 mM and 1.75 mM respectively.

Animals↗
PubMed1994

The conversion of ethanolamine and of its metabolites to choline in human neuroblastoma clones: effect of differentiation induced by retinoic acid.

The sequential methylation of ethanolamine (Etn) or phosphorylethanolamine to the corresponding choline (Cho) derivatives was studied in both undifferentiated and retinoic acid (RA) differentiated human neuroblastoma clones LA-N-1 and LA-N-2. Conversion of Etn derivatives to the respective Cho metabolites was low in both cell types. However, after treatment of the cultures with ethanol or RA, the methylation of phosphoryl-Etn was stimulated while that of phosphatidyl-Etn was severely reduced in both cholinergic LA-N-2 and catecholaminergic LA-N-1 cells.

Biological Transport↗
PubMed1993

Glycerophosphorylcholine phosphocholine phosphodiesterase activity during the differentiation of glial progenitor cells.

O-2A progenitor cells were grown in medium containing either 1% or 10% fetal calf serum (FCS) for 4 weeks. The cells in 1% FCS were 75% oligodendrocytes by 3 weeks in culture. The cell population was so overgrown with astrocytes in the 10% medium that an accurate estimate of cell number could not be made. The activities of glycerophosphorylcholine phosphocholine phosphodiesterase (GPC-PC-PdE), p-nitrophenylphosphorylcholine phosphodiesterase (pNPPC-PC-PdE), and ceramide UDP galactose galactosyl transferase (CGalT) were barely detectable in the cells grown in 10% FCS. The activities of these 3 enzymes were low in the cells grown in 1% FCS for the first 2 weeks and then all 3 increased manyfold. These observations reinforce the evidence previously accrued showing that these two phosphodiesterase activities (GPC-PC-PdE and pNPPC-PC-PdE) are markers of oligodendroglial cells as well as myelin. In contrast, glycerophosphorylcholine choline phosphodiesterase (GPC-C-PdE) activities were present in cells grown in both 1% and 10% FCS.

Animals↗
PubMed1993

Alterations of selected enzymes of phospholipid metabolism in Alzheimer's disease brain tissue as compared to non-Alzheimer's demented controls.

Previous studies have demonstrated elevated brain levels of phosphomonoesters in early stages of Alzheimer's disease and elevations of phosphodiesters later in the disease. In addition, preliminary quantitative analyses of the phospholipids of Alzheimer's brain reveals either decreases in some phospholipids or elevations followed by decreases in others. This study quantitated the activities of selected enzymes involved in phospholipid and choline metabolism and demonstrated elevated glycerol-3-phosphorylcholine phosphodiesterase and decreased choline kinase activities in Alzheimer's disease brain. The former could provide an enzymatic mechanism for the increased phosphorylcholine found in Alzheimer's disease brain.

Aged↗
PubMed1993

Possible involvement of the base exchange enzymes in the phospholipid metabolism in LAN-2 cells.

The incorporation of [14C] choline, [14C] ethanolamine, and [14C] serine by LAN-2 cells into their corresponding phospholipids was investigated in the presence or absence of TPA. The presence of TPA increased the amount of radioactivity incorporated into the phospholipids with a corresponding decrease in the amount of radioactivity in the cytosolic compartment compared to control cultures. There were no differences between TPA-exposed and control cells in the distribution of radioactivity in free choline, phosphorylcholine or CDP-choline of [14C] choline labeled cells. This indicates that the increased lipid labeling was not accompanied by enhanced labeling of the intermediates of the de novo pathway. These results suggest that a choline base exchange enzyme was stimulated in TPA exposed cells. In addition, the enhanced incorporation of serine by TPA into its corresponding phospholipid implies the stimulation of the serine base exchange enzyme which is responsible for phosphatidylserine synthesis in mammals. These observations suggest a

Choline↗
PubMed1993

Modulation of the serine base exchange enzyme activity of rat brain membranes by amphiphilic cations and amphiphilic anions.

The biosynthesis of phosphatidylserine in mammalian tissues is catalyzed by the serine base exchange enzyme. The activity of this membrane-bound enzyme can be manipulated by amphiphiles. Amphiphilic cations, such as oleylamine, W-7, chlorpromazine, and didodecyldimethylamine, stimulate the serine base exchange activity. Amphiphilic anions, such as bis(2-ethylhexyl) hydrogen phosphate and cholesterol sulfate, inhibit the serine base exchange activity. These effects are more pronounced at pH 7.0 than at the pH optimum of 8.5 for this enzyme. Both the stimulators and the inhibitors alter the Vmax values without changing the Km value for serine, suggesting that their mechanism of action is related to interactions of the membrane-bound cosubstrate, phosphatidylethanolamine, with the membrane-bound enzyme. The optimal concentration of stimulator varies with the amount of membrane protein present; however, supraoptimal concentrations cause inhibitions. It is proposed that the amphiphilic cations enhance the interaction of the phosphorylethanolamine moiety of the membrane-bound cosubstrate with the enzyme and the amphiphilic anions interfere with such an interaction. Some of the pharmacological properties of these amphiphilic cations, employed clinically as antidepressants, may be mediated by modulation of the serine base exchange enzyme activity.

Amines↗
PubMed1993

Activation of phospholipases D and A by amphiphilic cations of cultured LA-N-2 cells is G protein- and protein kinase C-independent.

Several amphiphilic cations such as mepacrine, desipramine, didodecyldimethylamine, chlorpromazine, oleylamine and W-7 activated the phospholipase D (PLD) activity of cultured LA-N-2 cells. These compounds, except for oleylamine, provoked the release of fatty acids, suggesting phospholipase A activation. Melittin, a PLA2 stimulator, caused the robust release of the free fatty acids but was a poor PLD activator. Although PLD could be activated by GTP gamma S, the stimulation by these amphiphilic cations was not abolished by GDP beta S, an inhibitor of G protein function. There was no change in the PLD activation by these amphiphilic cations by DiC8, a PKC activator, or by H-7, a PKC inhibitor or in PKC down-regulated cells.

Amines↗
PubMed1992

Properties of a partially purified phosphodimethylethanolamine methyltransferase from rat brain cytosol.

The presence of cytosolic S-adenosylmethionine-dependent N-methyltransferase(s) activity(ies) capable of converting phosphoethanolamine into phosphocholine has been recently demonstrated in the rat brain. At least two enzymes are involved in the methylation of phosphoethanolamine to phosphocholine and these are separable by ammonium sulphate fractionation. The enzyme catalysing the last step of this methylation process is present in the 50-80% ammonium sulphate fraction. A 220-fold purified enzyme has been obtained with sequentially employed Q-Sepharose fast flow and octyl-Sepharose CL4B column chromatography. The maximum enzyme activity was at pH 9.5. The Km values for S-adenosylmethionine, the methyl donor, and phosphodimethylethanolamine, the methyl acceptor, were 125 microM and 750 microM respectively. This phosphodimethylethanolamine N-methyltransferase was found to be calcium-dependent, with a 4-fold increase in activity at 0.5 mM-CaCl2. S-Adenosylhomocysteine at 0.5 mM caused 100% inhibition of the activity. The effects of various structural analogues on the phosphodimethylethanolamine N-methyltransferase activity were also investigated and these results suggest that the enzyme is specific to the substrate. These results provide evidence for the existence of the pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain cytosol.

Ammonium Sulfate↗
PubMed1992

Oleoylamine and sphingosine stimulation of phosphatidylserine synthesis by LA-N-2 cells is protein kinase C independent.

The presence of sphingosine and oleoylamine in the culture medium of LA-N-2 cells stimulated the incorporation of [3H]serine into its corresponding phospholipid, phosphatidylserine (PtdSer). The optimum stimulation for sphingosine and oleoylamine were 50 microM and 100 microM, respectively. Oleoylamine increased the incorporation of [3H]serine over 6-fold while sphingosine increased the incorporation of [3H]serine over 2.5-fold. The amount of radioactivity found in water-soluble components and in protein was similar to that found with control LA-N-2 cells. The incorporation of [3H]choline and [3H]ethanolamine into their corresponding phospholipids were decreased in the presence of either oleoylamine or sphingosine. A protein kinase C (PKC) activator, DiC8, and a PKC inhibitor, H7, did not influence the enhanced phosphatidylserine formation by sphingosine and oleoylamine. In addition, there were no differences in the stimulatory effect of sphingosine and oleoylamine discernable between PKC down-regulated cells or controls. These observations indicate that this oleoylamine and sphingosine mediated enhanced phosphatidylserine synthesis is PKC-independent.

Amines↗