digitonin and alpha-glycerophosphoric-acid

A rapid fractionation method for determination of metabolite levels in the chloroplast and the extrachloroplast compartment of Chlamydomonas reinhardtii has been developed. Protoplasts containing one large chloroplast were fractionated by passing them through a multilayer gradient containing digitonin, polyacrylamide, and a mixture of silicone oil and bromodecane. Lysis of the plasma membrane and the separation of the chloroplasts from most of the extrachloroplast material was achieved within less than 5 s. The chloroplast enriched fraction was contaminated with 3% fumarase (mitochondria) and 13% phosphoenol pyruvate carboxylase (cytosol). Metabolites of the upper glycolytic chain were detected mainly in the chloroplasts, whereas 2-phosphoglycerate was found only in the extrachloroplast compartment. Analysis of changes in metabolite concentrations after transition to anaerobic conditions in the dark pointed to a regulation of carbohydrate catabolism by chloroplast phosphofructokinase and by cytosolic pyruvatekinase.

Topics: Aerobiosis; Anaerobiosis; Cell Fractionation; Chlamydomonas; Chloroplasts; Cytosol; Darkness; Digitonin; Dihydroxyacetone Phosphate; Fructosephosphates; Glucosephosphates; Glyceraldehyde 3-Phosphate; Glyceric Acids; Glycerophosphates; Glycolysis; Phosphofructokinase-1; Protoplasts; Pyruvate Kinase

1. A permeabilized isolated rat liver cell preparation was developed to achieve selective permeabilization of the cell membrane to metabolites and to allow the assay of mitochondrial overt carnitine palmitoyltransferase (CPT I) activity in situ. By performing the digitonin-induced permeabilization in the presence of fluoride and bivalent-metal-cation sequestrants, it was possible to demonstrate that the activity of other enzymes, which are regulated by reversible phosphorylation, was preserved during the procedure and subsequent washing of cells before assay. 2. CPT activity at a sub-optimal palmitoyl-CoA concentration was almost totally (approximately 90%) inhibited by malonyl-CoA, indicating that mitochondrial CPT I was largely measured in this preparation. 3. The palmitoyl-CoA-saturation and malonyl-CoA-inhibition curves for CPT activity in permeabilized cells were very similar to those obtained previously for the enzyme in isolated liver mitochondria. Moreover, starvation and diabetes had the same effects on enzyme activity, affinity for palmitoyl-CoA and malonyl-CoA sensitivity of CPT I in isolated cells as found in isolated mitochondria. These physiologically induced changes persisted through the cell preparation and incubation period. 4. Neither incubation of cells with glucagon or insulin nor incubation with pyruvate and lactate before permeabilization resulted in alterations of these parameters of CPT I in isolated cells. 5. The results are discussed in relation to the temporal relationships of changes in the activity and properties of CPT I in vivo in relation to the effects of insulin and glucagon on fatty acid metabolism in vivo.

Topics: Acyltransferases; Animals; Carnitine O-Palmitoyltransferase; Cell Separation; Diabetes Mellitus, Experimental; Digitonin; Female; Glucagon; Glycerophosphates; In Vitro Techniques; Insulin; L-Lactate Dehydrogenase; Liver; Palmitoyl Coenzyme A; Permeability; Rats; Rats, Inbred Strains; Starvation

Digitonin has been used to permeabilize bloodstream trypomastigotes of Trypanosoma brucei. Such permeabilized parasites revealed a fully-functional glycolytic pathway which catabolized glucose and some phosphorylated glycolytic intermediates. Glucose-starved bloodstream trypomastigotes revealed saturation kinetics with a glucose Km = 0.6 mM and Vmax = 150 natom O/min per 10(8) for intact parasites; Km = 4 mM and Vmax = 100 natom O2/min per 10(8) for permeabilized parasites. Glucose oxidation in intact parasites was stimulated 40% by addition of 3 micrograms digitonin/10(8) parasites. Higher concentrations of digitonin than this inhibited the glucose oxidation. Ten millimolar phosphoenolpyruvate (PEP) inhibited the rate of O2 consumption by permeabilized trypanosomes respiring on glucose under aerobic conditions by 50%. It is proposed that glucose oxidation is apparently limited by transport across trypanosomal plasma membrane, and phosphofructokinase is regulated by PEP levels. It is concluded that permeabilization of trypanosomes with digitonin might offer a closer physiological condition for the study of the regulation of glycolysis by using glycolytic intermediates and other chemical compounds which would otherwise not be transported across the membrane(s).

Topics: Animals; Cell Membrane Permeability; Digitonin; Dose-Response Relationship, Drug; Glucose; Glucose-6-Phosphate; Glucosephosphates; Glycerophosphates; Glycolysis; Oxygen Consumption; Phosphoenolpyruvate; Trypanosoma brucei brucei