digitonin and Starvation

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

To evaluate published indications that about 25% of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), is located in mitochondria of adult rat liver, cell fractionations were conducted with hepatocytes isolated from rats that were fed ad libitum or starved for 2 days. Hepatocytes were exposed to digitonin for 10 s, and the released materials were separated from residual cell structures by centrifugation through a layer of brominated hydrocarbon. In addition to PEPCK, activities of 9 other enzymes were measured in the untreated cells and with good recovery in the two fractions obtained with digitonin treatment. By comparison with the release of marker enzymes for the cytosol and mitochondria, the subcellular distribution of PEPCK was determined. With cells from either fed or 2-day-starved rats, this enzyme was released exactly like lactate dehydrogenase and within 2-3% of phosphoglycerate kinase and pyruvate kinase. These results indicate that, even after induction by starvation, at least 97% of PEPCK activity is located in the cytosol of rat liver.

Topics: Animals; Aspartate Aminotransferases; Cell Fractionation; Cytosol; Digitonin; L-Lactate Dehydrogenase; Liver; Male; Mitochondria, Liver; Phosphoenolpyruvate Carboxykinase (GTP); Phosphoglycerate Kinase; Pyruvate Kinase; Rats; Rats, Inbred Strains; Starvation

The release of carnitine palmitoyltransferase (CPT) activity from rat liver mitochondria by increasing concentrations of digitonin was studied for mitochondrial preparations from fed, 48 h-starved and diabetic animals. A bimodal release of activity was observed only for mitochondria isolated from starved and, to a lesser degree, from diabetic rats, and it appeared to result primarily from the enhanced release of approx. 40% and 60%, respectively, of the total CPT activity. This change in the pattern of release was specific to CPT among the marker enzymes studied. For all three types of mitochondria there was no substantial release of CPT concurrently with that of the marker enzyme for the soluble intermembrane space, adenylate kinase. These results illustrate that the bimodal pattern of release of CPT reported previously for mitochondria from starved rats [Bergstrom & Reitz (1980) Arch. Biochem. Biophys. 204, 71-79] is not an immutable consequence of the localization of CPT activity on either side of the mitochondrial inner membrane. Sequential loss of CPT I (i.e. the overt form) from the mitochondrial inner membrane did not affect the concentration of malonyl-CoA required to effect fractional inhibition of the CPT I that remained associated with the mitochondria. The results are discussed in relation to the possibility that altered enzyme-membrane interactions may account for some of the altered regulatory properties of CPT I in liver mitochondria of animals in different physiological states.

Topics: Acyltransferases; Animals; Carnitine O-Palmitoyltransferase; Diabetes Mellitus, Experimental; Digitonin; Female; Intracellular Membranes; Malonyl Coenzyme A; Mitochondria, Liver; Rats; Rats, Inbred Strains; Starvation

Three isolation procedures were used to test the labilization of the lysosomes after adaptation of the animals to seawater, starvation or acute and chronic treatments with 2,4,5-T, an organochlorine pesticide. The lysosomes from gill and liver had different properties with respect to their resistance to osmotic and mechanical shocks, or treatments with digitonin. Starvation induced a significant labilization of the lysosomes in liver, but not in gill. Salinity changes were without effect on the stability of the gill lysosomes, but induced an increase of the specific activity of the lysosomal enzyme beta-acetylglucosaminidase. Acute treatments with 2,4,5-T increased lysosomal fragility in the gill but not in the liver, while chronic treatments with this herbicide increased lysosomal lability in the two tissues. Liver lysosomes were much more susceptible to in vitro treatments with 2,4,5-T than gill lysosomes. The results are discussed with respect to the different functions of gill and liver, their exposure to the environment, and the possible discrimination between different lysosomal populations.

Topics: 2,4,5-Trichlorophenoxyacetic Acid; Acetylglucosaminidase; Animals; Digitonin; Gills; Liver; Lysosomes; Osmolar Concentration; Starvation; Trout

The susceptibility of hepatocytes to the deleterious effects of cadmium (10-100 microM Cd), as evidenced by SH-group content and cell membrane permeability for Trypan Blue (TB), was shown to be strongly dependent on the dietary condition of the donor animal. Although Cd stimulated lipid peroxidation (LPO) equally well in "starved" as in "fed cells", SH-group content and TB-exclusion decreased markedly only in "starved" cells exposed to Cd. Experiments with methionine + serine and/or the antioxidant (+)-cyanidanol-3 suggested that the Cd-dependent LPO proceeded independent of the decrease in SH-group content or TB-exclusion.

Topics: Animals; Cadmium; Digitonin; Female; Glutathione; In Vitro Techniques; Liver; Methionine; Rats; Rats, Inbred Strains; Serine; Starvation; Sulfhydryl Compounds

Topics: Animals; ATP Citrate (pro-S)-Lyase; Digitonin; In Vitro Techniques; Kinetics; Mitochondria, Liver; Rats; Starvation; Temperature

Conditions were determined for rapid separation of cytosolic and mitochondrial compartments by digitonin fractionation of rat hepatocytes. The minimum time required for separation of mitochondrial and cytosolic enzyme markers decreased rapidly with increasing temperature. Kyro EOB, a non-ionic detergent, increases the release of cytosolic enzymes, particularly at lower temperatures. Experimental procedures are described for greater than 90% release of cytosolic enzymes and less than 2% release of mitochondrial enzymes in 3s. By using appropriate concentrations of digitonin and Kyro EOB in a fractionation medium maintained at 1 degrees C and a minimum time of exposure to the medium, nearly separate patterns of release were obtained for enzyme markers for the cytosol, mitochondrial matrix and mitochondrial intermembrane space. The distribution of enzymes that exist in more than one of these compartments was quantified by comparing their rates of release with those of marker enzymes. The cytosol/mitochondrial-matrix distributions for such enzymes in hepatocytes from starved rats were 16%/84% for aspartate aminotransferase, 34%/66% for fumarase and 77%/23% for ATP citrate lyase. In hepatocytes from rats that were induced to synthesize ATP citrate lyase by starvation and re-feeding, the ratio had increased to 95%/5%. The maximum cytosol/intermembrane-space ratio for adenylate kinase was 8%/92%. A procedure is also described for treating commercial digitonin that increases its solubility in water from about 1mg/ml to more than 800mg/ml.

Topics: Animals; Cell Fractionation; Detergents; Digitonin; In Vitro Techniques; Liver; Male; Organic Chemicals; Rats; Starvation; Subcellular Fractions; Temperature

Topics: Adenine Nucleotides; Alanine; Animals; Cytosol; Digitonin; Male; Mitochondria, Liver; Oleic Acids; Phosphates; Rats; Starvation; Thermodynamics