Fractions Of Guinea-pig Cerebral Cortex Research Articles

The acylation of lysophosphatidylcholine by subcellular fractions of guinea-pig cerebral cortex

The acylation of lysophosphatidylcholine by isolated subcellular fractions of guinea-pig cerebral cortex has been determined. The microsomal fraction contained the highest acylation activity, in terms of both specific and total activity. In all particulate fractions, including synaptic plasma membrane and mitochondria, there was a high correlation (correlation coefficient r = 0.90; P < 0.001) between acylation and the activity of the microsomal enzyme, NADPH-cytochrome c reductase. No correlation existed between acylation and the activities of ( Na + + K +)-ATPase, acetylcholinesterase or succinate dehydrogenase. Acyl-CoA synthetase and lysophosphatidylcholine/acyltransferase, the individual enzymes responsible for acylation were enriched in the microsomal fraction. The activities of both enzymes in subcellular fractions correlated well with those of NADPH- cytochrome c reductase, with the exception that acylCoA synthetase activity in the mitochondrial fraction was largely independent of endoplasmic reticulum. Neither synaptic plasma membranes nor mitochondria appeared to possess significant amounts of acyltransferase activity. The results indicate that the acylation of lysophosphatidylcholine is confined to the endoplasmic reticulum, and that activity present in the synaptic plasma membrane or mitochondrial fraction is attributable to microsomal contamination.

Calcium transport and proton electrochemical potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart

A method is described for the preparation of ;free' and ;synaptosomal' brain mitochondria from fractions of guinea-pig cerebral cortex respectively depleted and enriched in synaptosomes. Both preparations of mitochondria have a low membrane H(+) conductance, a high capacity to phosphorylate ADP, and a capacity to accumulate Ca(2+) at rates limited by the activity of the respiratory chain. Ca(2+) transport by ;free' brain mitochondria is compared with that of heart mitochondria. The Ca(2+) conductance of ;free' brain mitochondria was at least 20 times that for rat heart mitochondria. Ca(2+) uptake by brain mitochondria increased the pH gradient and decreased membrane potential, whereas little change occurred during the much slower uptake by heart mitochondria. In the presence of ionophore A23187, dissipative Ca(2+) cycling decreased the H(+) electrochemical potential gradient of brain mitochondria from 190 to 60mV, but caused only a slight decrease with heart mitochondria, although the ionophore lowered the pH gradient and increased membrane potential. The Ca(2+) conductance of ;free' brain mitochondria is distinctive in showing a hyperbolic dependency on free Ca(2+) concentration. In the presence of Ruthenium Red, a rapid Na(+)-dependent Ca(2+) efflux occurs. The H(+) electrochemical potential gradient is maintained during this efflux, and membrane potential increases, with both ;free' brain and heart mitochondria. The Na(+) requirement for Ca(2+) efflux appears not to be related to the high Na(+)/H(+) exchange activity but may represent a direct exchange of Na(+) for Ca(2+).

Acylation of lysophosphatidylcholine by subcellular fractions of guinea-pig cerebral cortex: subcellular distributions of acyl-coenzyme A synthetase and acyl-coenzyme A hydrolase [proceedings

Acylation of lysophosphatidylcholine by subcellular fractions of guinea-pig cerebral cortex: subcellular distributions of acyl-coenzyme A synthetase and acyl-coenzyme A hydrolase [proceedings

Inhibition by neomycin of polyphosphoinositide turnover in subcellular fractions of guinea-pig cerebral cortex in vitro.

The addition of 10(-5) M to 10(-3) M neomycin to incubations of subcellular fractions of guineapig cerebral cortex increased the labelling of phosphatidylinositol phosphate and decreased the labelling of phosphatidylinositol diphosphate by [gamma-32P]ATP. The effect was observed in all subcellular fractions tested and depended on the cationic form of the antibiotic. Similar effects on lipid labelling were exerted by related aminoglycosidic antibiotics, by neamine, spermine and poly-L-lysine. Other neomycin fragments, antibiotics, local anesthetics or small polyamines were ineffective. Neomycin also inhibited the enzymatic hydrolysis of 32P-polyphosphoinositides. The addition of the drug to aqueous dispersions of these lipids increased the turbidity and lowered the pH of the suspensions. It is suggested that the effects of neomycin on polyphosphoinositide metabolism result from the formation of an ionic complex between the lipids and the antibiotic.

Choline metabolism in the cerebral cortex of guinea pigs. Phosphorylcholine and lipid choline.

1. The labelling of phosphorylcholine and choline-containing phospholipids in the subcellular fractions of guinea-pig cerebral cortex after the intraventricular injection of [N-Me-(3)H]choline into conscious animals has been studied. Special emphasis was placed upon the synaptosome fraction and early time-periods after administration. 2. The labelling of phosphorylcholine was rapid compared with that of phospholipid and was confined to two distinct subcellular fractions: the soluble cytoplasmic fraction and the synaptosome fraction. Most of the labelled phosphorylcholine of the synaptosome fraction was readily released by osmotic rupture indicating location in the nerve-ending cytoplasm. The two pools of phosphorylcholine had similar specific radioactivities at all observed times. 3. (3)H-labelled phospholipid was found in all membranous fractions. The labelling was confined to choline-containing phospholipids, notably phosphatidylcholine. 4. The labelling of the different membranous fractions was similar. 5. The half-life of the choline-containing phospholipids in the synaptic vesicle fraction was very much greater than the acetylcholine in this fraction. 6. Evidence is presented that synthesis de novo of phosphatidylcholine at nerve terminals occurs in vivo.

The effects of a hemicholinium analogue, HC-15, on neuromuscular transmission

The bisected hemicholinium molecule HC-15, which is exactly half the HC-3 molecule, was investigated for neuromuscular blocking activity and its action compared to the effect of the parent compound. HC-15 was shown to inhibit neuromuscular transmission by both a pre- and a post-junctional action. Large doses of the compound produced a post-junctional, nondepolarizing block whereas after smaller doses a slowly developing block occured selectively in rapidly stimulated muscles which was not antagonized by anticholinesterase drugs. HC-15 inhibited both acetylcholinesterase and cholinesterase but was much less powerful than physostigmine and neostigmine in this respect. HC-15 inhibited acetylcholine (ACh) systhesis by mitochondrial (P 2) fractions of guinea-pig cerebral cortex suspended in Tris buffer but had no effect on ACh synthesis by P 2 fractions homogenized in Triton X-100 where the membranes surrounding the choline acetyltransferase (ChAc) enzyme are broken down. HC-15 therefore appears to inhibit ACh synthesis in a similar manner to HC-3 by preventing choline transport to the intracellular sites of acetylation. In the absence of choline HC-15 and HC-3 were acetylated by ChAc. HC-3 has a higher rate of acetylation than HC-15 and this effect parallels the greater activity of HC-3 on neuromuscular transmission. The possibility is suggested that the pre-junctional blocking action of both HC-15 and HC-3 may be in part due to their incorporation into cholinergic nerve endings and their subsequent release as false inactive neurotransmitters.

Effects of some polymethylene bis(hydroxyethyl) dimethylammonium compounds on acetylcholine synthesis.

1. The effects of some polymethylene bis(hydroxyethyl)dimethylammonium compounds have been studied on acetylcholine (ACh) synthesis and as substrates for choline acetyltransferase (ChAc).2. The decamethylene analogue (C(10)-dichol) inhibited ACh synthesis by mitochondrial (P(2)) fractions of guinea-pig cerebral cortex suspended in Tris buffer but had no effect on ACh synthesis by P(2) fractions when the membranes surrounding the ChAc enzyme were broken down by homogenization in Triton X-100.3. C(10)-Dichol was acetylated by ChAc almost to the same extent as choline. The initial rate of acetylation, at a concentration of 10(-3)M, was more rapid than for choline; however, the apparent Michaelis-Menten constant for C(10)-dichol was greater than the K(m) for choline, showing a loewr affinity for the ChAc enzyme.4. All the dicholine compounds were acetylated to some extent by ChAc and the rate of acetylation increased with an increase in the length of the methylene chain between the two quaternary nitrogen atoms in each dicholine molecule.5. The rate of acetylation of the dicholine compounds paralleled the activity of these analogues at the prejunctional site at the neuromuscular junction. The possibility is suggested that part of the pharmacological activity of these compounds may be due to their incorporation into cholinergic nerve endings followed by acetylation by ChAc before subsequent release as a false transmitter.

The distribution of gangliosides in subcellular fractions of guinea-pig cerebral cortex.

The distribution of gangliosides in subcellular fractions of guinea-pig cerebral cortex.