Julius W. Dieckert

Gossypol: Uncoupling of respiratory chain and oxidative phosphorylation☆

Abstract Gossypol produced adverse effects in vitro on rat liver mitochondira. It stimulated mitochondrial respiration at low concentrations, inhibited it at high concentrations; abolished ADP/O and respiration control ratios; reversed inhibition caused by oligomycin; stimulated adenosine triphosphatase activity at low concentrations and inhibited it at high concentrations; and its effect on this enzyme was pH dependent. The possibility that gossypol may exert its toxic effect on poultry and animals by uncoupling respiratory chain-linked phosphorylation is discussed.

Metabolic fate of gossypol: The metabolism of [14C]gossypol in swine

Balance studies designed to obtain information concerning the metabolic fate of gossypol in rats were carried out utilizing two groups of animals. One was fed a basal diet and the other the same diet plus 500 ppm of iron as FeSO4. Single doses of 5 mg each of14C-labeled gossypol (spec. act. 19.8 μCi/mmole) were administered. The animals were maintained in metabolic cages and killed after various periods of time. The data indicate that gossypol was poorly absorbed from the gastrointestinal tract and rapidly eliminated from the animal body. Although the main route for gossypol elimination from the animal was by fecal excretion in both treatments, the percentage of the total activity eliminated via the feces varied and depended on the level of iron supplied in the diet. The results demonstrate that gossypol was least excreted via urine and that urinary excretion of radioactivity was diminished by iron supplementation to the diet. Most of the radioactivity retained was found in the contents of different parts of the gastrointestinal tract. Tissues, the liver, muscle, kidney and blood had the highest radioactivity, with the liver having the highest specific activity. The data also demonstrate that addition of iron to the ration diminishes14C radioactivity in the animal body. This effect might be attributed to the formation of chelates that could not be absorbed through the small intestine. Catalysis of the decarbonylation of gossypol by iron also appears to be a factor.

A paper chromatographic procedure for separating 1-mono-, 1,3-di-, and triglycerides, cholesterol, and cholesteryl esters

SummaryA reproducible paper chromatographic procedure has been developed for resolving a mixture consisting of 1-mono-, 1,3-di-, and triglycerides, cholesterol, and cholesteryl esters. The separation is accomplished, using silicic acid impregnated glass fiber filter paper as the chromatographic medium and various ethyl ether-isooctane mixtures as the solvent systems. A reagent consisting of 1:1 concentrated sulfuric acid: water has been introduced for visualizing the lipides on the chromatogram and for distinguishing between glycerides and cholesterol-containing lipides. Unsaturation in the fatty acid moiety of the mono-, di-, and triglycerides had no detectable effect on the Rf values of these substances. Fatty acid chain length did not affect the Rf of the cholesteryl esters with the solvent systems studied. Increasing the percentage ethyl ether in the solvent system caused an increase in the Rf values of each lipide not moving at the solvent front.

Visualization of protein—SDS complexes in polyacrylamide gels by chilling

Abstract A method is described which gives direct visualization of protein bands in sodium dodecyl sulfate electrophoresis gels. The procedure consists of chilling the gels to 0–4°C and observing the white opaque bands which correspond to the stained protein bands. Ovalbumin can be detected by this method at a minimum concentration of 0.2 μg per mm 2 of gel surface area.

Gossypol: Subcellular localization and stimulation of rat liver microsomal oxidases

14 C-Labeled gossypol was orally administered to rats. The level of gossypol in the liver reached a maximum 1 day after administration, then decreased with time. Although gossypol was not exclusively segregated in any one cell fraction, most of it was found in the microsomal fraction. Gossypol appeared to distribute according to its lipid-soluble property rather than by virtue of its reaction with protein. In rats fed gossypol, the liver microsomal enzymes which catalyze the oxidative demethylation of p -chloro- N -methylaniline HCl (PCMA) increased in activity. A “no effect” dosage for the stimulation of demethylation of PCMA by dietary gossypol was found to be 50 mg/kg. The gossypol stimulation of rat liver microsomal oxidases seems to be due to an increase in enzyme synthesis since it did not alter the K m (6.7 × 10 −3 m ) but increased the maximal velocity of PCMA demethylation (33%). The ability of rat liver microsomal enzymes to dealkylate some carbamate pesticides was increased by the oral administration of gossypol.

Paper chromatography of phospholipides on silicic acid impregnated glass fiber filter paper.

Summary1. A reasonably reproducible method has been developed for separating lysolecithin, lecithin,, sphingomyelin, and phosphatidylethanolamine, using glass fiber filter paper impregnated with silicic acid as the chromatographic medium and 1:1 methanol:ethyl ether as the developing solvent.2. Hot sulfuric acid was used to locate the chromatographed compounds on the chromatogram. The advantages and disadvantages of this reagent and the other spot test reagents used were discussed.3. No separation between the unsaturated and saturated counterparts of lysolecithin or lecithin was obtained.

Isolation and partial characterization of manganin, a new manganoprotein from peanut seeds

Abstract Manganin, a new manganoprotein, has been isolated from the seeds of Virginia-type peanuts. The preparation was homogeneous by ultracentrifugal analysis and cellulose polyacetate strip electrophoresis. A minimum molecular weight of 56,300 for manganin was found based on the Mn content. The molecular weight, obtained using the methods of ultracentrifugal analysis of Yphantis and Ehrenberg, was found to be 57,900 and 56,800, respectively. The close correlation between the minimum molecular weight based on the Mn content and the molecular weights obtained from ultracentrifugal analysis indicates that manganin contains one atom of Mn per molecule. The amino acid composition of manganin is distinctly different from that reported for arachin and conarachin, the major globulins of peanut seeds.

Types and Molecular Weights of Subunits of Arachin-P6

Abstract Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that arachin-P6 contained nine types of subunits. The subunits were designated K15 (15, 069), K17 (17, 334), K20 (20, 000), K22 (21, 750), K24 (23, 557), K29 (29, 019), K37 (37, 161), K41 (41, 326) and K43 (43, 104) and molecular weight of each is given in parenthesis following its name. This observation was qualitatively comparable to the results of electrophoretic analysis of arachin-P6 conducted in the presence of urea.

The influence of dietary fat on the glyceride structure of animal fat.

Summary and ConclusionsIn order to determine the glyceride structure of a representative mammal and bird, rats and chicks were raised on an essentially fat-free ration, and the percentage of saturated triglycerides in their neutral fat was determined by an isotope dilution procedure.In order to determine the influence of ingested fat, second groups were fed the fat extracted from the animals in the first group, at the 20% level. Third groups were fed cottonseed oil, which has “even” distribution of its fatty acid. It was found that:1.The glyceride structure of endogenous rat fat conforms to the “random” type distribution.2.Ingested fat appears to be digested and resynthesized by the rat according to “even” type distribution, or, at least, in a manner which tends to distribute the fatty acids.3.Chicks tend to produce simple or “mono-acid” glycerides (8) in which the percentage of trisaturated glycerides is higher than expected for random distribution.4.It is suggested that the findings can be explained by a selective affinity of the esterifying enzyme system for saturated acids and for the 1-position on the glyceride molecule. In the case of the bird its higher body temperature may increase the speed of the reaction on the 2- and 3-positions of the glycerol sufficiently to account for an increase in tri-saturated glycerides above that required by random distribution.

Synthesis of mucosa and lymph phospholipide during fat absorption.

Summary and Conclusion Comparison of the relative specific activities of the glycerol of lymph triglycerides and phospholipides from previous studies, and of intestinal mucosa phospholipides from new studies demonstrated that resynthesized triglycerides must be the precursors of mucosa and lymph phospholipides, rather than the converse.