Diffusion in the aqueous compartment.

Measurements of diffusion of molecules in cells can provide information about cytoplasmic viscosity and structure. In a series of studies electron-spin resonance was used to measure the diffusion of a small spin label in the aqueous cytoplasm of mammalian cells. Translational and rotational motion were determined from the same spectra. Based on measurements made in model systems, it was hypothesized that calculations of the apparent viscosity of the cytoplasm from both rotational and translational motion would distinguish between the effects of viscosity and structure on diffusion. The diffusion constant measured in several cell lines averaged 3.3 X 10(-6) cm2/s. It was greater in growing cells and in cells treated with cytochalasin B than in quiescent cells. The viscosity of the cytoplasm calculated from the translational diffusion constant or the rotational correlation time was 2.0-3.0 centipoise, about two to three times that of the spin label in water. Therefore, over the dimensions measured by the technique, 50-100 A, solvent viscosity appears to be the major determinant of particle movement in cells under physiologic conditions. However, when cells were subjected to hypertonic conditions, the translational motion of the spin label decreased threefold, whereas the rotational motion changed by less than 20%. These data suggest that the decrease in cell volume under hypertonic conditions is accompanied by an increase in cytoplasmic barriers and a decrease in the space between existing cytoplasmic components without a significant increase in viscosity in the aqueous phase. In addition, a comparison of reported diffusion values of a variety of molecules in water and in cells indicates that cytoplasmic structure plays an important role in the diffusion of proteins such as bovine serum albumin.

Viscosity of cellular protoplasm.

The protoplasmic viscosity was studied by using a small spin label having high permeability and broad solubility properties and nickel chloride as an extracellular spin-subtracting agent to localize signal inside cells. The viscosity is variable and in some cells is many times that of water or phospholipids, suggesting that lateral diffusion in biological membranes is important to cell function.

Spin label evidence for the role of lysoglycerophosphatides in cellular membranes of hibernating mammals.

The phospholipid composition of ground squirrel heart muscle changes during hibernation: more lysoglycerophosphatides are found in the hibernating state than in the active state. Phase transitions inferred from spin label motion occur in the usual manner typical of mammalian mitochondria for the mitochondria and mitochondrial lipids from active squirrels. However, a conspicuous absence of a spin label-detectable phase transition is observed in equivalent preparations from hibernating animals. The addition of lysolecithin to preparations from active squirrels removes the break and induces a straight line in the Arrhenius plot. The lack of a spin label-detectable phase transition in hibernating animals, therefore, is attributed to an increased content of lysoglycerophosphatides present in the phospholipids during hibernation.

In vitro biosynthesis of fatty acids inDrosophila melanogaster.

A new in vitro technique, utilizing ruptured larvae ofDrosophila melanogaster, was employed to study the incorporation of(3)H-acetate into long-chain fatty acids. Preparative gas-liquid chromatography and scintillation spectroscopy were used to determine the relative activity of each fatty acid from total lipid extracts. Quantitative changes were observed in the distribution of label during the course of the incubation times, which ranged from five minutes to nine hours. All fatty acids which were observed to incorporate acetate in previous in vivo studies also showed incorporation of label under these in vitro conditions. It is concluded that this system may be useful for studying aspects on insect metabolism for short intervals of time.

Temperature-induced phase transitions in nematode lipids and their influence on respiration.

Temperature-induced phase transitions estimated by electron spin resonance (ESR) technique were ohscrved in the lipids of several nematode species. In both Meloidogyne javanica and Caenorhahditis elegans, there was a phase transition in their phospholipids from a liquid-crystalline state to a solid gel state at about 10 C. Aphelenchus avenae also had a phase transition, but at about 20 C. With this species, the spin-label motion parameters indicated the transition was from the liquid-crystalline state below 20 C to a more liquid or disordered state above 20 C. Anguina tritici and Meloidogyne hapla, in contrast, had no phase transitions over the entire temperature range studied. Each phase transition detected by ESR was reflected in the respiratory rates of the nematodes, and the temperature of the transition coincides with the environmental adaptation of these species.