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The regulation of the activity of blowfly flight-muscle phosphorylase b kinase by P(i) and Ca(2+) was studied, and the actions of these effectors on the kinases from insect flight and rabbit leg muscles were compared. Preincubation of blowfly kinase with P(i) increased activity severalfold. The effect was concentration-dependent, with an apparent K(m) of about 20mm, and time-dependent, requiring at least 10min for maximal activation. Neither ATP nor cyclic AMP was needed, suggesting that a protein kinase may not be involved. Maximal activation of the insect kinase required Mg(2+) in addition to P(i). The apparent K(m) for Mg(2+) was 3mm. Rabbit leg-muscle phosphorylase b kinase was slightly inhibited, rather than stimulated, by P(i), and was strongly inhibited by K(+), Na(+) and Li(+). At physiological concentrations, Ca(2+) activated the phosphorylase b kinases from both blowfly flight and rabbit leg muscles. However, the responses to Ca(2+) of the enzymes from the two tissues were different. The mammalian kinase had virtually no activity in the absence of Ca(2+), and showed a large increase in activity over a narrow range of Ca(2+) concentrations. Flight-muscle kinase had appreciable activity in the absence of Ca(2+), and had a smaller increase over a wide range of Ca(2+) concentration. The concentrations of Ca(2+) required for half-activation were 0.1 and 1mum for the blowfly and rabbit enzymes respectively. The pH-activity profiles of the non-activated, phosphate- and Ca(2+)-activated kinase revealed considerable enhancement of activity with little, if any, increase in the ratio of activities at pH6.8 to those at 8.2. These results are discussed in relation to the mechanism coupling contraction to glycogenolysis and to the biochemical distinction between asynchronous and synchronous types of muscle.

Glycogen from flight muscle of the blowfly, Phormia regina, has been characterized ultrastructurally and biochemically. In situ, glycogen is in the form of rosettes, which vary in size with diameters of up to 0.1 micro. Sedimentation analysis of pure glycogen, isolated by mild buffer extraction, reveals a polydisperse molecular weight spectrum, with larger particles having molecular weights of 100 million. Treatment of native glycogen with alkali, under conditions usual for the extraction of the polysaccharide from tissues, results in a 5- to 10-fold reduction in molecular weight, as well as a chemical alteration of the molecule. Flight muscle phosphorylase has a lower affinity for native than for alkali-treated glycogen. The maximum velocity of the enzyme is also lower with native substrate. The apparent K(m) for inorganic phosphate is higher with native glycogen as cosubstrate. These kinetic differences with native and partially degraded glycogen demonstrate the importance of using the natural substrate in studies of biochemical control mechanisms.

The rate of pyruvate oxidation by mitochondria from blowfly flight muscle decreased in the presence of tris (hydroxymethyl) aminomethane (Tris). An increase in the rate of mitochondrial swelling was concomitant with the loss of pyruvate oxidation. These changes were prevented by bovine serum albumin, adenosine triphosphate, and magnesium ions, factors required for mitochondrial contraction. Proline, but not glutamate or malate, restored the rate of pyruvate oxidation to original values. These findings suggest that mitochondrial swelling leads to leakage of intramitochondrial intermediates of the Krebs cycle, accounting for the decrease in the rate of pyruvate oxidation. Exogenous proline penetrates the mitochondrial membrane and is rapidly oxidized, via glutamate, forming intramitochondrial precursors of oxaloacetate. Malate and glutamate were ineffective because of the selective permeability of the mitochondrial membrane.

Exogenous reduced diphosphopyridine nucleotide was not oxidized by mitochondria but by cytoplasmic hydrogen-accepting systems catalyzed by lactic, alpha-glycerophosphate, and malic dehydrogenases. The three enzymes were found in all tissues of the mouse; the amount of activity of each enzyme differed in the various tissues, however. It is suggested that each tissue has a unique pattern for oxidation of extramitochondrial diphosphopyridine nucleotide.

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.

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.