Robert F. Witter

Effect of lysolecithin on oxidative phosphorylation.

Abstract A study was made of the effect of lysolecithin on oxidative phosphorylation by rat liver mitochondria. The lysophosphatide activated Mg-activated ATP-ase and inhibited DNP-activated ATPase. The effect of lysolecithin was studied on oxidative phosphorylation associated with the transfer of electrons: 1) from succinate or β-hydroxybutyrate to oxygen; 2) from succinate or β-hydroxybutyrate to cytochrome c ; 3) from cytochrome c to oxygen. Lysolecithin uncoupled oxidation from phosphorylation in all three systems. The oxidation of succinate or β-hydroxybutyrate was also repressed by higher concentrations of lysolecithin with oxygen or ferricyanide as the terminal oxidant but the respiration was not affected if phenazine methosulfate was used to link to succinic dehydrogenase. On the other hand the rate of oxidation of cytochrome c by oxygen was increased in the presence of lysilecithin. The results obtained indicate that lysolecithin: 1) activates Mg-ATPase; 2) inhibits a phosphorylation reaction common to each of the steps in electron transport; and 3) at higher concentration inhibits some step in electron transport between succinic dehydrogenase and cytochrome c .

The effect of lysolecithin and related compounds on the swelling of isolated mitochondria

Abstract A study was made of the possible role of lysolecithin in the swelling of isolated mitochondria. With the exception of glycolecithin which is not a naturally occuring compound, lysolecithin had a greater destructive effect on the mitochondria than did other purified phospholipids, lipids, surface active agents, phosphate, or succinate. The destructive action of lysolecithin, phosphate, or succinate was decreased but that of surface active agents such as dodecyl sulfate or glycolecithin was not markedly affected by lowering the pH to 6.2 the pH at which the mitochondria were most stable. ATP and Mg were more effective than versene in protecting the mitochondria from the effects of lysolecithin. It was concluded that the formation of lysolecithin might explain the swelling of isolated mitochondria in sucrose solution.

A study of some factors involved in the swelling of isolated mitochondria

Abstract A study was made of the effect of various conditions on the stability of mittochondria isolated from rat liver. The pH of the solution was found to be much more important than the concentration of sucrose in maintaining the stability of these organelles. ATP, ADP, Mg, Ca, versene, and DNP were found to prevent the swelling of isolated mitochondria in hypotonic sucrose at pH 8.0. Calcium was found to have a slight destructive action under hypertonic conditions. The destructive action of phosphate was found to be dependent on hypotonic conditions whereas that of succinate could be observed only in hypertonic suspensions. Although the mitochondria became markedly more unstable to alkaline conditions after short periods of aging at 37° the protective mechanisms utilizing ATP and magnesium are lost more slowly. Little difference in stability was noted among preparations of mitochondria which were round and swollen, “rod” shaped, or well preserved morphologically. Accordingly the alteration in morphology during the process of isolation of the mitochondria probably did not influence the stability of these organelles in the present experiments or in those of others.

Paper chromatography of phospholipides with solvent mixtures of ketones and acetic acid.

Abstract Mixtures of ketones and acetic acid, namely 2-pentanone-acetic acid 30:2, 3-methyl-2-butanone-acetic acid 30:3, 4-methyl-2-pentanone-acetic acid 30:2, and 2, 6-dimethyl-4-heptanone-acetic acid 30:7 were found to give excellent separations of individual phospholipides on paper. It was possible to separate lysolecithin, sphingomyelin, phosphatidyl ethanolamine, lecithin, and phosphatidic acid in a unidimensional chromatogram. The individual phospholipides had to be present at concentrations from 10 to 25 /μg./20 μl. in order to achieve good separations.

The incorporation of [32P]orthophosphate into a specific lipid fraction and into adenine nucleotides of mitochondria during oxidative phosphorylation

Abstract A method for the extraction of mitochondrial lipid using acetone to precipite the mitochondrial lipoprotein is reported. This procedure gave rapid destruction of enzymic activity and high phosphatide recoveries under mild conditions and was used to simultaneously follow the incorporation of [ 32 P]orthophosphate into the adenine nucleotides and the lipid components of mitochondria during oxidative phosphorylation. At 10° the incorporation of [ 32 P]orthophosphate into ATP proceeded rapidly and linearly during the initial minutes of reaction, but leveled off after 10 to 15 min. ADP behaved similarly but the specific activity remained about half that of the ATP. The incorporation into the lipids at 10° occurred almost completely into a very minor fraction; however, this incorporation was not detectable for the first 10 min of reaction, after which it increased rapidly. The conclusion was made that unless this lipid fraction contains a minor but highly active component, it cannot be the precursor of the phosphate moiety of ATP and therefore cannot act as an intermediate in the phosphate esterification reactions during oxidative phosphorylation.

Effects of Dieldrin or DDT in vivo on Alpha-Alanine, Gamma-Amino-butyrate, Glutamine, and Glutamate in Rat Brain

Summary Studies were made of the effects of lethal doses of dieldrin or DDT on the free amino acid pattern of the brains of rats exhibiting severe symptoms of poisoning. Elevated levels of a-alanine and y-aminobutyrate were noted with dieldrin but not with DDT, indicating a different mode of action of the 2 insecticides.

The inhibitory action of human plasma on dinitrophenol uncoupling of oxidative phosphorylation.

Abstract A factor from human plasma has been found and purified about 35-fold which will prevent the uncoupling action of dinitrophenol on the oxidative phosphorylation of rat liver mitochondria. This substance has the properties of a protein, but as yet has not been specifically identified. There appears to be more of this substance present in the plasma of normal individuals than in the plasma of patients with malignant disease.

Oxidation of lecithin and sphingomyelin by tissue preparations.

Summary Liver mitochondria and spleen were found to contain dehydrogenases which attacked lecithin and sphingomyelin and required ATP. Muscle and intestinal mucosa had dehydrogenase activity with lecithin or sphingomyelin if DPN was present whereas under the same conditions homogenates of spleen were active only with sphingomyelin. The dehydrogenase systems were obtained in soluble form from acetone powders of liver mitochondria or intestinal mucosa.

Effect of magnesium chloride and pH on heat inactivation of fatty acid oxidase of mitochondria.

Summary In the reaction mixture alkaline pH 8 and magnesium chloride were the most important factors in prevention of heat inac-tivation of the sorbate, α-ketoglutarate; or malate oxidase of rat liver mitochondria. The most destructive factor was the presence of phosphate. Either magnesium chloride or ATP prevented, and phosphate accelerated, the destruction of mitochondria in hypotonic solution, but magnesium chloride had a specific protective effect on sorbic acid oxidase in addition to its effect of preventing destruction of the mitochondria.

Large Scale Preparation of Liver Mitochondria with the Waring Blendor.

Summary A method is described for the preparation of mitochondria from 50 to 100 g of liver. The tissue is pulped in a special press and homogenized at a reduced motor speed for a short time in the Waring blendor in isotonic sucrose. The mitochondria are isolated by differential centrifugation from isotonic sucrose. The ATP-ase, fatty acid oxidase, and succinoxidase activities of these preparations were shown to be similar to those of mitochondria prepared with a pestle type homogenizer.