Bertram Sacktor

The respiratory metabolism of insect flight muscle. I. Manometric studies of oxidation and concomitant phosphorylation with sarcosomes

Abstract Oxidation and concomitant phosphorylation in mitochondria (sarcosomes) from flight muscle of the fly, Musca domestica , were characterized by manometric techniques. It was found that the highest respiratory rate was obtained with α-glycerophosphate. With this substrate a Q o 2 twice that for succinate was recorded. P/O values were determined for several substrates. Ratios between 1.0 and 1.7 were observed for α-glycerophosphate and succinate. Ratios greater than 2.0 were found for glutamate and α-ketoglutarate. The oxidative phosphorylation potential of various reaction mixtures was assayed. Replacement of isotonic saline with sucrose in the incubation medium increased the P/O. Fluoride caused a small decrease in respiration when the sarcosomes were isolated in 0.25 M sucrose, but not when 0.25 M sucrose plus ethylenediamine tetraacetate was used. Fluoride had no appreciable effect on phosphorylation except in the experiments with glutamate. With this substrate, inorganic phosphate uptake was markedly inhibited. Malonate lowered the respiratory rate slightly, but the P/O was increased. The optimal hexokinase concentration was determined. The influence of the phosphate acceptor system on oxidative phosphorylation was measured. These preparations showed no respiratory control, for the respiratiory rate was independent of the concentration of adenine nucleotide. Phosphorylation, however, was dependent on the nucleotide. Nucleosides and nucleotides other than adenosine 5-mono-, di-, and triphosphates were not suitable phosphate acceptors. Dinitrophenol uncoupled oxidative phosphorylation. Oxygen uptake was never stimulated. A heterogeneity in the dinitrophenol-sensitive phosphorylation was described. The efficiency of a given concentration of this inhibitor was apparently modified by the substrate metabolized. The high level of α-glycerophosphate oxidation in insects is particularly associated with flight muscle.

Asymmetric distribution of gangliosides in rat renal brush-border and basolateral membranes

Highly enriched brush-border and basolateral membranes isolated from rat renal cortex were used to study the distribution of endogenous gangliosides in the two distinct plasma membrane domains of epithelial cells. These two membrane domains differed in their glycolipid composition. The basolateral membranes contained more of both neutral and acidic glycolipids, expressed on a protein basis. In both membranes, the neutral glycolipids corresponding to mono-, di-, tri- and tetraglycosylceramides were present. The basolateral membranes contained more diglycosylceramide than the brush-border membranes. The major gangliosides found were GM4, GM3, and GD3 with minor amounts of GM1 and GD1a. The latter were identified and quantified by sensitive iodinated cholera toxin binding assays. When the distribution of individual gangliosides was calculated as a percent of total gangliosides, the brush-border membranes were enriched with GM3, GM1 and GD1a compared to the basolateral membranes, which were enriched with GD3 and GM4. The observation of a distinct distribution of glycolipids between brush-border and basolateral membranes of the same epithelial cell suggests that there may be a specific sorting and insertion process for epithelial plasma membrane glycolipids. In turn, asymmetric glycolipid biogenesis may reflect differences in glycolipid function between the two domains of the epithelial plasma membrane.

Respiratory activity of brain mitochondria

Abstract Kinetic studies of respiratory enzymes in mammalian brain showed that greatest respiratory activity of rat brain mitochondria was with α-glycerophosphate. α-Glycerophosphate was oxidized at a rate 50% greater than that obtained with succinate and several fold those obtained with other Krebs cycle intermediates. The oxidation of α-glycerophosphate was inhibited by ethylenediamine tetraacetate (Versene). Mg ions completely reversed this inhibition. The data suggest that the extraordinary rapid rate of α-glycerophosphate oxidation must be considered in the evaluation of brain metabolism.

In situ regulation of glycolysis in tetanized cat skeletal muscle

Abstract The concentrations of metabolites in tetanized cat gastrocnemius muscle, in situ , have been measured to determine the enzymic sites of regulation of glycolysis in mammalian skeletal muscle. Increases of approximately 10-fold in the levels of glucose-6-P and fructose-6-P were found. The concentrations of pyruvate and laetate also increased, but none of the other glycolytic intermediates or citrate accumulated appreciably. The concentration of creatine-P in the tetanized muscle decreased, but those of ATP, ADP, and AMP were maintained at control levels. These findings show that the phosphohexose isomerase reaction is at, or near, equilibrium even during the greatly increased glycolytic flux accompanying contraction, thus precluding it as the rate-limiting step in glycolysis, as has been suggested recently. The marked accumulation of hexosemonophosphate in tetanus and the maintenance of levels of known activators of phosphofructokinase, i.e., ADP, AMP, and fructose-1,6-diP, near those of the unstimulated muscle, suggest that, even during tetanus, phosphofructokinase is functioning at a rate less than optimal and is limiting the rate of glycolysis. Also, the importance of the phosphorylase reaction is demonstrated by the finding that 90% or more of the hexose entering the glycolytic pathway during a 30-second tetanic contraction stems from glycogen rather than from blood sugar. Thus these results are in accord with the concept of glycogenolytic control in skeletal muscle by phosphorylase and phosphofructokinase.

The respiratory metabolism of insect flight muscle. III. Low-temperature spectra of the cytochromes of flight muscle sarcosomes.

1. n1. The spectral properties of the pigments of sarcosomes isolated from the flight muscle of the fly, as recorded when samples are cooled in liquid nitrogen, reveal differences in cytochrome content from that observed with heart muscle or liver mitochondrial preparations. n n2. n2. Fly sarcosomes or saline-washed particles prepared from these sarcosomes do not have an enzymically reducible cytochrome c1, as characterized in mammalian mitochondria by an absorption band at 554 mμ. n n3. n3. The appearance of an absorption band at 551 mμ, distinct from cytochrome c, is shown in preparations of saline-washed particles. In addition, a pigment with an α-absorption band maximum at 555 mμ is observed when pigments are reduced enzymically in the presence of antimycin A or when the pigments are reduced by sodium dithionite. n n4. n4. The low-temperature spectrum of reduced cytochrome c, isolated and purified from the flight muscle of the fly, is presented and the similarity to the spectrum of reduced cytochrome c purified from heart muscle is demonstrated. n n5. n5. A respiratory chain for insect sarcosomes is postulated.

The role of the α-glycerophosphate cycle in the control of carbohydrate oxidation in heart and in the mechanism of action of thyroid hormone

Abstract 1. 1. The hypothesis that thyroid hormone regulates the rate of operation of the α-glycerophosphate cycle, affects the availability of NAD + , and controls the metabolism of carbohydrate was tested utilizing isolated, perfused rat hearts. Hyperthyroid hearts showed a 300% increase and hypothyroid hearts an 80% decrease in specific activity of mitochondrial α-glycerophosphate dehydrogenase. Lactate, pyruvate, and glucose plus insulin were perfused individually through hyper- and hypothyroid heart matched to control hearts of the same weight, and the uptakes of the respective substrates were corrected for differences in heart rates. Control hearts utilized lactate at half the rate as that found for pyruvate. Hyperthyroidism increased the rate of lactate uptake to the same level as that of pyruvate. The thyroid status of the animals had no effect on the rate of uptake of pyruvate from the perfusate. Hypothyroidism did not affect the rate of lactate utilization. On the basis of triose uptake during perfusion with glucose plus insulin, glucose uptake was the same as pyruvate uptake in control hearts, was unaffected by hyperthyroidsm, adn was increased slightly in hypothyroid hearts. The concentration of glycogen in hyperthyroid hearts was 38% that in control hearts but it increased to normal levels after perfusion with glucose. The concentrations of extramitochondrial pyridine nucleotide redox metabolites in cardiac tissue were not significantly altered by the thyroid status of the rats. These data indicate that a limitation on lactate oxidation, present in normal hearts, is removed in hyperthyroidism and provide evidence consistent with the operation of the α-glycerophosphate cycle in cardiac tissue and with the significance of this cycle, regulated by thyroid hormone, in the control of carbohydrate metabolism.

Investigations on the mitochondria of the housefly, Musca domestica L. II. Oxidative enzymes with special reference to malic oxidase.

Summary The oxidation of carbohydrate intermediates, with special emphasis on malic acid, was investigated with mitochondria isolated from the housefly, Musca domestica L. It was found that these sarcosomes have the following enzymatic activities: malic dehydrogenase; DPNH 2 oxidase; cytochrome c oxidase; malic- and succinic-cytochrome c reductase. Augmenting the mitochondria with DPN or cytochrome c increased the malic-cytochrome c reductase and DPNH 2 oxidase activities, respectively. A comparative study of these mitochondria and whole thoracic homogenates indicated that the above enzymatic activities, except malic dehydrogenase, are apparently confined to mitochondria. This study provides additional evidence that, in spite of some specific variations, insect sarcosomes possess many of the characteristic properties of mammalian mitochondria.

Alteration of kidney brush border membrane maltase in aging rats

The specific activities of membrane-bound maltase (alpha-d-glucoside glucohydrolase, EC 3.2.1.20) in renal cortex homogenates and isolated brush border membranes of senescent rats decreased about 30% compared to the specific activities of the enzyme from young adult animals. The decline was gradual and concomitant with the aging process. When the enzymes from rats of 25 and 6 months of age were solubilized and purified to homogeneity the same decrement with age was found, 32.5 and 46.1 units/mg of protein, respectively. This finding suggests that the decrease in maltase activity with age results from an alteration in the enzyme per se, rather than from a change in the enzymes membrane environment, which was reflected secondarily as a loss in activity. Recoveries of enzyme activity and protein and fold-purification were similar for young and old maltase, indicating that the age-related difference in specific activities of the pure enzymes was not due to the selective purification of an altered species of enzyme. The age-associated difference in activity was not attributable to the presence of proteolytic activity in the homogenate nor to the presence of an activator in the young or an inhibitor in the old kidney. The pure enzymes from young adult and aged animals did not differ in molecular weight, electrophoretic mobility, amino acid composition and Km value. Circular dichroism spectra revealed that both the young and old enzymes contained beta-structure. However, the old enzyme had more helical structure than did the young enzyme, suggesting a conformational alteration with age.