Cyril L. Moore

Peroxisomal and mitochondrial defects in the cerebro-hepato-renal syndrome.

The cerebro-hepato-renal syndrome is a rare familial malady with cerebral, renal, and skeletal abnormalities, severe hypotonia, cirrhosis, iron and lipid storage, and death within 6 months. Correlated electron microscopic, histochemical, and biochemical studies demonstrate defects in two oxidative organelles. Peroxisomes cannot be found in hepatocytes and renal proximal tubules. In hepatocytes and cortical astrocytes, mitochondria are distorted in their appearance and glycogen stores are increased. Oxygen consumnption of brain and liver mitochondrial preparations with succinate and with substrates reducing nicotinamide adenine dinucleotide is markedly diminished, but the consumption is normal with ascorbate and tetramethylphenylenediamine, which suggests a defect in electron transport prior to the cytochromes. Histochemical studies of mitochondrial oxidation point to a defect between the succinate dehydrogenase flavoprotein and coenzyme Q, possibly in the region of nonheme iron protein.

Specific inhibition of mitochondrial Ca++ transport by ruthenium red☆

Abstract The ability of rat liver mitochondria to transport calcium ions has been found to be inhibited specifically by the dye ruthenium red. Since this dye reacts specifically with mucopolysaccharides, and since energy conservation is not inhibited by this dye, it is concluded that mucopolysaccharides (in the form of mucoproteins or muco or glycolipids) are at the active center of the sites of mediation of mitochondrial Ca++ transport.

Mitochondrial DNA in Yeast and Some Mammalian Species

Yeast DNA, in a cesium chloride density gradient, shows a minor or satellite band with a density lower than that of the main nuclear component. The DNA isolated from purified mitochondria of yeasts corresponds in density to this satellite band. In solution, this DNA more easily undergoes renaturation as compared to DNA from cell nuclei. The ease of this renaturation is presumably due to a homogeneity greater than that of nuclear DNA. Mitochondrial DNA isolated from several mammalian species has the same or higher density than nuclear DNA, but differs in its ready renaturability.

The effect of hexachlorophene on the respiration of brain and liver mitochondria

Abstract Hexachlorophene (HCP) in micromolar concentrations increased the rates of respiration of rat liver or calf brain mitochondria utilizing succinate or glutamate plus malate as substrate. Higher concentrations of HCP produced a stimulation of respiration followed by inhibition. When respiration in the presence of ADP had been inhibited by oligomycin, HCP was still capable of stimulating respiration. These observations are typical for uncouplers of oxidative phosphorylation.

PEROXISOMAL ABNORMALITIES IN METABOLIC DISEASES

Two congenital metabolic disorders that can be thought of as peroxisomal diseases (14-17) may provide clues to the function of peroxisomes in mammalian cells. One, acatalasemia, is an enzymatic defect in mice that have been selectively bred for this trait (11). The second, the cerebrohepatorenal syndrome of Zellweger, is a rare familial human malady characterized by severe muscle weakness, hepatomegaly, cerebral, renal, facial and skelet al abnormalities and death within 6 months (see References 24 and 27 for reviews).

Oxidative Phosphorylation in Immature Rat Brain Mitochondria

A micromethod for the polarographic measurement of oxygen consumption is used to study ADP-dependent respiration in mitochondria isolated from regions of the developing rat brain and liver. Mitochondr

Purification and properties of two hexokinases from beef brain

Abstract Two hexokinases have been isolated from beef brain. One (SBH) was isolated from the soluble particle-free high-speed supernatant solution obtained from homogenates of gray matter. The other hexokinase (PBH) was extracted from the acetone desiccated particulate (mitochondrial) fraction of the same homogenates. The enzymes were found to have (a) different temperature stabilities; (b) different sensitivities to oxygen, PBH being inactivated upon aerobic dialysis or chromatography; (c) different chromatographic behavior of DEAE cellulose; but (d) similar pH sensitivities. Kinetically, the enzymes have similar K m values for ATP, (varying in the same direction with two substrates). The K m values for all hexoses tried were different for both enzymes. However, there was an inhibition of PBH but not of SBH at high mannose concentration. Glucose 6-phosphate inhibited both enzymes uncompetitively with respect to glucose. With PBH G-6-P was competitive with respect to ATP, while with SBH, it acted as a mixed inhibitor with respect to ATP. With SBH ADP acted as a competitive inhibitor with respect to ATP, while with PBH, the ADP inhibition with respect to ATP was noncompetitive. PMB inhibition of both enzymes was prevented by glucose. ATP afforded slight protection of SBH but seemed to potentiate the PMB inhibition of PBH.

The isolation of cerebral neurons with partial retention of processes.

A novel tissue disaggregation technique has been devised which permits the isolation of neurons with fairly extensive processes attached. Cortex is dissociated by aspiration through nozzles of decreasing size followed by agitation on a vortex mixer, rather than by the usual technique of forcing tissue through sieves. After each aspiration step, dissociated cells are separated from undisrupted tissue by coarse filtration and the latter is subjected to repeated treatment. This prevents unnecessary trauma to the free cells. After disruption is complete, small pieces of undisrupted tissue are removed from the cell suspension by floating on the foam created by degassing the suspension under vacuum. Cells are purified by conventional velocity-gradient centrifugation. This procedure has been applied successfully to fresh rat brain, with or without a preincubation with trypsin, frozen human brain and frozen bovine brain. The cell yields from rat brain were comparable to or better than, those obtained by other procedures (37 X 10(6) cells/g brain) while the purity was comparable. Cell yields from human brain were similar to those from rat brain but the purity was lower. The lowered particle purity of human and bovine cells can probably be attributed to the conditions of storage of the tissue and to trapping of free nuclei in the meshwork of dendritic processes. Values are given for the amount of protein, RNA and DNA per cell.

A micro-method for the study of oxidative phosphorylation

Abstract 1. 1. A method is described for polarographic measurement of oxidative phosphorylation in mitochondria isolated from very small quantities of rat brain and liver. Measurements of O2 consumption are made with an O2 cathode and a standard polarization circuit using a reaction chamber with a volume of 40 μl. 2. 2. The characteristics of this system are described. They are similar to the characteristics of polarographic systems requiring much larger quantities of mitochondria. 3. 3. Oxidative phosphorylation was measured in mitochondria isolated from as little as 100 mg of tissue. Respiratory control ratios and ratios of phosphate acceptor to O2 consumption (ADP/O) obtained are comparable to those measured in much larger volumes of mitochondria as reported in the literature.

Accessibility of the mitochondrial kynurenine hydroxylase reaction to exogenous and endogenous pyridine nucleotides

Abstract The outer and inner membranes of rat liver mitochondria were separated, and it was confirmed that the kynurenine hydroxylase is located in the outer membrane. In intact mitochondria the kynurenine hydroxylase could be supported by exogenous NADPH. NADPH generated from endogenous mitochondrial NADP + by the isocitric dehydrogenase reaction could not be used for the kynurenine hydroxylase reaction unless the mitochondria were solubilized by detergent. Conversely, NADP + generated by the kynurenine hydroxylase from exogenous NADPH could not be reduced by the intramitochondrial isocitric dehydrogenase unless the mitochondria were solubilized. These results provide direct evidence of a permeability barrier for pyridine nucleotides between the outer mitochondrial membrane and the inner membrane compartment.