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A technique for the isolation of intact brush borders from rabbit renal cortex was evaluated. The procedure was monitored by phase and electron microscopy and marker enzymes, i.e. ATP:NMN adenylyl transferase, nuclear; cytochrome oxidase, mitochondrial; beta-glucuronidase, lysosomal; and glucose-6-Pase, microsomal; and indicated an essentially pure preparation of brush borders. The disaccharidase, trehalase, previously reported in renal tubules, was localized uniquely in brush borders. Maltase was also found; the specific activities of the two enzymes in the brush borders were increased 10- to 20-fold. Other disaccharidases, such as sucrase, isomaltase, lactase, and cellobiase, were absent. It is suggested that trehalase and maltase are appropriate candidates for marker enzymes of the renal brush border. Isolated brush borders possessed a ouabain-sensitive (Na(+) + K(+)) ATPase, an oligomycin-insensitive Mg(++) ATPase, and a Ca(++)-activated ATPase. Alkaline phosphatases, dephosphorylating beta-glycero-P, and trehalose-6-P were also present. The specific activities of these enzymes were increased three-to-five fold in the brush-border preparations; however, activities were found in other subcellular fractions of the renal cortex. Hexokinase, although evident in the isolated brush border, was found prominently associated with other membranous fractions. Phosphoglucomutase and UDPG pyrophosphorylase were localized in the soluble fraction of the renal cortex.

1. The ATPase activity of insect mitochondria has been investigated. A comparison was made to determine the distribution and nature of such activity in other isolated fractions of the house fly, Musca domestica L. 2. The ATPase in insect mitochondria is specific in that orthophosphate can be cleaved only from ATP. The Michaelis-Menten constant K(8) = 2.78 x 10(-3)M and V(max.) = 76 micrograms P min.(-1) mg.(-1) dry weight. 3. Mg(++) and Mn(++) activate this enzymatic reaction in mitochondria, but Ca(++) does not. The extent of activation is 60 per cent with the optimal concentration 6 x 10(-4)M. Experiments with combinations of Mg(++) and Mn(++) show that either ion can replace the other and that the effects are additive, depending solely on the final concentration of the combination. Concentrations of Mg, Mn, or Ca ions higher than 6 x 10(-3)M inhibit the enzyme. 4. Fluoride does not inhibit the ATPase of insect mitochondria, whereas azide and chloromercuribenzoate do. The per cent inhibition depends on the concentration of inhibitor. 5. Finely dispersed mitochondrial particles have much greater ATPase activity than intact mitochondria. The possible relationship of this observation to latent ATPase is considered. 6. A magnesium-activated adenylate kinase is present in these mitochondria. The liberated orthophosphate, derived from ADP, is the result of the activity of adenylate kinase followed by the specific ATPase. 7. ATP can be dephosphorylated by enzymes found in the muscle fibrils, and in a "soluble" fraction, as well as in mitochondria. The fibrillar ATPase is Ca(++)-activated. The "soluble" fraction, however, like the mitochondria, is Mg(++)-activated. The "soluble" ATP dephosphorylation mechanism is distinguished from the mitochondrial ATPase in that it is inhibited by fluoride. 8. The "soluble" fraction also contains a magnesium-activated inorganic pyrophosphatase. Fluoride completely inhibits this enzymatic reaction. 9. The possible mechanism of ATP dephosphorylation in the "soluble" fraction is discussed.

Trehalase activity has been localized histochemically within the cortical tubules of the human, rabbit, and mouse kidneys. The specific localization of the enzyme supports the hypothesis that trehalase, in concert with a series of other enzymes which synthesize trehalose from glucose, functions in a mechanism for the reabsorption of glucose from the glomerular filtrate.

The disposition of the surface plasma membrane and its inwardly directed derivatives, corresponding to the T-system of other fibers, and of the corresponding sarcoplasmic reticulum (SR) elements has been examined in a dipteran asynchronous flight muscle (Phormia regina). The presence of uni-axonal neuromuscular junctions within clefts approaching the center of the fiber is described. The most conspicuous SR component is present in the dyads adjoining plasma membrane derivatives, but it is also sparsely represented elsewhere. The accessibility of the plasma membrane-limited compartments within the fiber to the ambient haemolymph, in the living insect, has been investigated by tracing the distribution of ferritin by the circulatory system. The proportion of fiber volume occupied by the T-system and SR components in asynchronous and synchronous muscle is compared and the functional implications of these proportions is discussed.

1. Insoluble and "soluble" forms of cytochrome c oxidase were prepared from house flies by methods described for mammalian tissue. A 4.5-fold purification was accomplished by preparing the enzyme in insoluble form (as compared with a crude water brei). The oxidase in "soluble" form was purified 20.0-fold. 2. The absorption spectrum of the "soluble" preparation from flies was similar to that from mammalian tissue, and indicated the presence of cytochromes a + a(3) and b. In contrast with the corresponding mammalian enzymes these components were not separable by the existing techniques. 3. The Soret peak seen at 440 mmicro and the alpha peak at 600 mmicro were due. primarily to cytochromes a(3) and a, respectively. 4. The oxidase activity of whole house flies was comparable with that of rat abdominal muscle. In both organisms, this activity was inhibited by cyanide to approximately the same extent. 5. The optimum buffer concentration for oxidase activity, as measured spectrophotometrically, was approximately 0.02 to 0.06 M. This is in agreement with results obtained for mammalian enzyme, as measured both manometrically and spectrophotometrically. 6. The present study has demonstrated the following components of the cytochrome system in house flies: b, c, a, and a(3).

Mitochondria from flight muscle of aging blowflies, Phormia regina, were examined morphologically and biochemically with the electron microscope. An age-dependent degeneration of the mitochondria that is characterized, in part, by the reorganization of the inner membrane into myelin-like whorls has been found. The concentric rings increase in size and number, eventually replacing the normal cristal conformation. Glycogen rosettes are frequently seen in the center of the whorl and may represent the intrusion into the mitochondria of the glycogen in the cytoplasmic matrix of the muscle. The degenerating mitochondria are not associated with lysosomal activity, as indicated by the absence of acid phosphatase. An intense acid phosphatase activity is noted, however, in the dyad, comprising elements of the T system and sarcoplasmic reticulurn. Cytochrome oxidase is active in the ultrastructurally intact portion of the mitochondrion but activity is not evident in that part of the mitochondrion that has undergone morphological change. Thus, the ultrastructural degradation of the mitochondria is correlated with a decrease in biochemical function. This suggests a correspondence between a decrease in the bioenergetic capacity of the flight muscle and a decline in the ability of the aged insect to fly.

The effect of different temperatures on the biochemical activity and morphology of insect flight muscle mitochondria was examined. It was found that respiration and phosphorylation have the same thermal response at temperatures of 25 degrees C. and below. The energy of activation for both systems is approximately 12,300 calories. Oxidation and phosphorylation can be uncoupled effectively by temperature, for at temperatures above 25 degrees C. there is more rapid heat inactivation of phosphorylation. This is evident from reduced P/O values as well as from morphological deterioration in the mitochondrial population. The thermal response of both this sarcosomal enzyme system and the respiration in the living fly are quantitatively similar.

It has been found that mitochondria isolated from the flight muscle of the housefly, Musca domestica, are capable of effecting oxidative phosphorylation. A systematic investigation of the factors which regulate this coupling was undertaken. It was found: 1. The molarity of the isolation medium had considerable influence on the morphology of the mitochondria. These physical alterations were associated with changes in oxidation, phosphorylation, and ATPase activity. 2. In addition to an optimum isolation medium, the normal morphology of the mitochondria needed to be further stabilized by serum albumin. 3. A "latent" ATPase activity in insect mitochondria was demonstrated. An inverse relationship was found between oxidative phosphorylation and ATPase activity. 4. Oxygen consumption and the uptake of phosphate were linear with respect to time. 5. A respiratory substrate was necessary for phosphorylation and for maintenance of spatially organized mitochondria. 6. No differences in oxygen uptake were found in the presence or absence of inorganic phosphate. 7. Magnesium was required for optimal oxidative phosphorylation. Calcium and manganese inhibited both respiration and phosphorylation. 8. The addition of cytochrome c had no effect on either oxygen or phosphate uptake. 9. ATP, ADP, or AMP were capable of participating in oxidative phosphorylation, but the glucose-hexokinase trapping system was necessary. 10. Fluoride inhibited the phosphorylation of AMP, but increased P/O when ATP was used. This stimulation was not due to the inhibition of ATPase. 11. Neither arginine nor creatine was phosphorylated. 12. The addition of other isolated fractions of flight muscle to the mitochondrial system had no appreciable effect on respiration or phosphorylation.

The biochemical properties of insect flight muscle were investigated to ascertain the mechanisms whereby energy is made available for the contractile processes. It was found: 1. The endogenous respiration of muscle homogenates was diminished by starving the flies. The substrate for this respiration was probably glycogen. 2. To obtain the maximal rate of oxidation of glucose, the homogenate had to be fortified with inorganic phosphate, Mg ions, ATP, and cytochrome c. The nucleotides, AMP and ADP, were not as effective as ATP. The addition of DPN or TPN was not necessary for this system. 3. Flight muscle homogenates oxidized glycogen, some sugars, and amino acids, as well as the intermediates of the glycolytic and tricarboxylic acid cycles. Other evidence demonstrated the substrate specificity of the muscle. 4. By centrifugation, the muscle homogenate was divided into two fractions: one, a soluble fraction representing the sarcoplasm; the other, the particulate fraction which contained the fibrils and the sarcosomes. 5. The particulate fraction, alone, oxidized all the citric acid cycle intermediates, alpha-glycerophosphate, phosphopyruvate, and the amino acids, glutamic, proline, and cysteine. Regardless of the substrate, no oxygen uptake was found with the sarcoplasm by itself. 6. A recombination of the sarcoplasm and the particulate component was required for the oxidation of glycogen, the hexoses, and all the phosphorylated intermediates of glycolysis, except phosphopyruvate. 7. Isolated mitochondria accounted for all the enzymatic activity of the particulate fraction. These results demonstrate that the enzymes of intermediate metabolism are localized in the sarcoplasm or sarcosomes. The third cytological entity, the myofibrils, plays no role in the energy-providing scheme. From a functional viewpoint, the sarcoplasm and the mitochondria, in combination, furnish the energy for the actomyosin contraction. The results are discussed in relation to analogous findings in other insects and vertebrates.