The molecular organization of nerve membranes : VI. The separation of axolemma from schwann cell membranes of giant and retinal squid axons by density gradient centrifugation.

Plasma membranes were isolated from two types of squid nerves which have morphologically, different ratios of axolemma/Schwannlemma (A/S). These membranes were studied by means of differential and density gradient centrifugation.Thoroughly dissected giant axons were used as membrane source having low A/S ratio. Retinal fibers were used as membrane source with high A/S ratio. A similar procedure for the isolation of the plasma membranes was used for both types of squid axons.Differential centrifugation showed that at 1,500×g, the yield of membrane enzymes (Na, K-ATPase and NADH-ferricyanide oxidoreductase) from giant axon homogenates was 2 to 5 times greater than from retinal nerve homogenates, but at 105,000×g the opposite was the case, the yield from retinal axons being about two times greater. Thus, the major part of the membrane material from the retinal nerve seems to be less dense than the membrane material from giant axons.The behavior of the 105,000×g fraction from both types of fibers was studied by determining protein Na, K-ATPase, and NADH-oxidoreductase along a lineal sucrose gradient (10 to 40%; centrifuged at 40,600×g for 90 min). By any of the three measurements, retinal axons yielded a greater amount (2:1) of plasma membranes sedimenting at low sucrose concentration (16 to 25%) as compared to that observed at high sucrose concentration (35 to 38%). Giant axons, on the contrary, yielded a higher proportion of membranes (2.5:1) sedimenting at high sucrose concentrations (over 40%).The experimental data indicate that a different cellular origin can account for the behavior of nerve membranes along lineal gradient centrifugation. The membranes floating at low sucrose concentration ("light membranes") can be tentatively ascribed to the axolemma; the membranes found at high sucrose concentration ("heavy membranes") to the Schwannlemma and basement membranes.In accord with their high A/S morphological ratio, squid retinal axons yielded 5 times more light membranes (axolemma) than dissected giant axons.

The molecular organization of nerve membranes : IV. The lipid composition of plasma membranes from squid retinal axons.

The lipid content and composition from an axolemma-rich preparation isolated from squid retinal axons was analyzed.The lipids, which accounted for 45.5% of the dry weight of this membrane, were composed of 22% cholesterol, 66.7% phospholipids and 5.2% free fatty acids. The negatively charged species phosphatidyl ethanolamine (37%), phosphatidyl serine (10%) and lysophosphatidyl ethanolamine (4%) made up 51% of the phospholipids. The amphoteric phosphatidyl choline and sphingomyelin accounted for 39% and 4%, respectively.The relative distribution of fatty acids in each of the isolated phospholipids was studied. The most remarkable feature of these phospholipids was the large proportion of long-chain polyunsaturated fatty acids. The 22∶6 acyl chain accounted for 37% in phosphatidyl ethanolamine, 21.7% in phosphatidyl choline, 17.5% on phosphatidyl serine and 20.3% in sphingomyelin (all expressed as area %).The molar fraction of unsaturated fatty acids reached 65% in phosphatidyl ethanolamine and 42.0 and 44.8% in phosphatidyl choline and phosphatidyl serine, respectively. The double bond index in these species varied between 1.0 and 2.6.The lipid composition of the axolemma-rich preparation isolated from squid retinal axons appears to be similar to other excitable plasma membranes in two important features: (a) a low cholesterol/phospholipid molar ratio of 0.61; and (b) the polyunsaturated nature of the fatty acid of their phospholipids.This particular chemical composition may contribute a great deal to the molecular unstability of excitable membranes.

The molecular organization of nerve membranes : V. Properties of mono- and bimolecular films formed with lipids isolated from an axolemma-rich preparation from squid retinal axons.

The formation and properties of mono- and bimolecular films of total lipids extracted from an axolemma-rich preparation from retinal axons of the squid are described.The bilayers formed with α-tocopherol showed resistance values of 10(7) Ω cm(2) indicating their low ionic permeability. These membranes were stable for 30 to 60 min and they behave as an ohmic resistance in the range of -120 to +120 mV.The films formed with these lipids did not discriminate between Na(+) and K(+) and showed a slight selectivity for Cl(-) as compared with cations, indicating a behavior as a rather neutral barrier.The pressure-area characteristics of monolayers built with the phospholipids fraction gave an area of 79 Å(2)/molecule at a pressure of 10 dynes/cm. This expanded molecular area can be accounted for by the unsaturated fatty acid chains of polyenoic structure attached to these phospholipid molecules. The fraction containing 81% cholesterol presented an area of 29 Å(2)/molecule at 10 dynes/cm.Monolayers of the total lipids displayed an area of 51.7 Å(2)/molecule at a pressure of 10 dynes/cm. These findings indicate that the phospholipids, when mixed with cholesterol and free fatty acids, formed rather condensated films. Cholesterol might contribute significantly to increase the cohesive forces in the film and hence to its stability.The expanded films given by the phospholipids extracted from nerve membrane also indicate that they have a low transition temperature; their unusual unsaturated aliphatic chains might be in a special high mobile condition. Their behavior might be important for the position and order of polar groups in an excitable membrane.

Sodium movements in perfused squid giant axons. Passive fluxes.

Sodium movements in internally perfused giant axons from the squid Dosidicus gigas were studied with varying internal sodium concentrations and with fluoride as the internal anion. It was found that as the internal concentration of sodium was increased from 2 to 200 mM the resting sodium efflux increased from 0.09 to 34.0 pmoles/cm(2)sec and the average resting sodium influx increased from 42.9 to 64.5 pmoles/cm(2)sec but this last change was not statistically significant. When perfusing with a mixture of 500 mM K glutamate and 100 mM Na glutamate the resting efflux was 10 +/- 3 pmoles/cm(2)sec and 41 +/- 10 pmoles/cm(2)sec for sodium influx. Increasing the internal sodium concentration also increased both the extra influx and the extra efflux of sodium due to impulse propagation. At any given internal sodium concentration the net extra influx was about 5 pmoles/cm(2)impulse. This finding supports the notion that the inward current generated in a propagated action potential can be completely accounted for by movements of sodium.