A B S T R A C T As a highly reactive substance produced in biological systems by the one-electron reduction of oxygen, superoxide (02-) seemed a likely candidate as a bactericidal agent in leukocytes. The reduction of cytochrome c, a process in which 02-may serve as an electron donor, was found to occur when the cytochrome was incubated with leukocytes. 02-was identified as the agent responsible for the leukocyte-mediated reduction of cytochrome c by the demonstration that the reaction was abolished by superoxide dismutase, an enzyme that destroys 02-, but not by boiled dismutase, albumin, or catalase.Leukocyte 0i-production doubled in the presence of latex particles. The average rate of formation of 027 in the presence of these particles was 1.03 nmol/107 cells per 15 min. This rate, however, is only a lower limit of the true rate of 02-production, since any 02-which reacted with constituents other than cytochrome c would have gone undetected. Thus, 02-is made by leukocytes under circumstances which suggest that it may be involved in bacterial killing.

A B S T R A C T Recent evidence has suggested that a particulate 02-forming system is responsible for the respiratory burst in activated neutrophils. The respiratory burst is normally a transient event, lasting only 30-60 min. To investigate the mechanism by which the btrst is terminated, we examined the 02-forming activity of neutrophil particles as a function of time in the presence and absence of agents known to affect the function of intact cells. Measurements of the°2-forming capacity of the particles against time of exposure of neutrophils to opsonized zymosan, a potent stimulating agent, revealed a rapid fall in activity when exposure was continued beyond 3 min. Exposure to zymosan under conditions in which the myeloperoxidase system was inactive (i.e., in the presence of myeloperoxidase inhibitors, or in the absence of oxygen) resulted in a substantial increase in the initial 02-forming activity of particles from the zymosan-treated cells, but did not prevent the sharp fall in activity seen when zymosan exposure exceeded 10 min. The fall in activity was, however, prevented when activation took place in the presence of cytochalasin B (1.5 ,ugml), an agent thought to act largely by paralyzing the neutrophil through an interaction with its microfilament network.We conclude from these findings that the termination of the respiratory burst results at least in part from the inactivation of the particulate 02-forming system. This inactivation involves at least two processes which probably act simultaneously. One is the destruction of the system through the action of myeloperoxidase. The other appears to require active cell motility and is independent of oxygen. The current view holds that the 02-forming system of the neutrophil is located in the plasma membrane. It may be that the second process involves the internalization and degradation of this membrane-bound system.

Particulate fractions from normal human granulocytes preactivated with opsonized zymosan were found to catalyze superoxide production in the presence of reduced pyridine nucleotides. Similar preparations from three patients with X-linked chronic granulomatous disease produced no detectable superoxide. The failure to produce superoxide was not due to an inhibitor, since cell-free preparations from the patients' granulocytes had no effect on superoxide production by normal particles. Particles from the mothers of two of the patients produced superoxide at diminished rates; superoxide production by particles from the third mother was normal. These findings suggest that chronic granulomatous disease represents either a defect in a pyridine nucleotide-dependent superoxide-forming oxidase or a lesion in the apparatus responsible for activating the oxidase.

Thyroid hormone administered in vivo increased carrier-mediated (atractyloside-sensitive) ADP uptake by rat liver mitochondria. 3 Days after a single large dose of triiodothyronine (20 ;g/100 g of body weight), mitochondrial uptake of ADP measured at 60 was 2.35 4 0.17 nmol/min per mg of protein, compared with an uptake of 1.81 i 0.19 nmol/min per mg of protein in mitochondria from untreated rats (P < 0.025). Cyanide (1.33 mM) had no effect on ADP uptake by mitochondria from either untreated or triiodothyronine-treated animals. Uptake of ADP by mitochondria from thyroidectomized rats treated with thyroxine for 7 days was 2.89 4 0.40 nmol/min per mg in mitochondria from thyrotoxic rats (20 ug of thyroxine per 100 g per day) and 1.98 -0.22 nmol/min per mg in mitochondria from euthyroid rats (2 ug of thyroxine per 100 g per day) (P < 0.025). Mitochondria from both untreated and thyroid hormone-treated rats displayed a highly significant linear correlation between ADP uptake and ADP-dependent (i.e., state 3 minus state 4) oxygen consumption. There was, however, no difference in respiratory control ratios between mitochondria from euthyroid and thyrotoxic animals. Administration of dinitrophenol (2 mg/100 g) also stimulated carrier-mediated ADP uptake, but respiratory control of mitochondria from dinitrophenol-treated animals was virtually abolished. Triiodothyronine in vitro, at concentrations of 100 and 0.1 nM, appeared to inhibit rather than stimulate the uptake of mitochondrial ADP. The relationship between these observations and the clinical manifestations of thyrotoxicosis is discussed from the point of view of the possible effects of increased mitochondrial ADP transport on oxidative phosphorylation and adenosyl nucleotide metabolism.It has long been known that an increase in metabolic rate follows administration of a sufficient dose of thyroid hormone. Animals treated with large doses of thyroxine manifest an increased rate of oxygen consumption, a rise in body temperature, and a loss in weight, all consequent to an increase in the rate of oxidation of substrate (1, 2). Since most substrate oxidation takes place in mitochondria, many studies on the mechanism of action of thyroid hormone have involved these organelles. These studies have shown, for example, that * To whom to address correspondence. thyroid hormone increases the rate of mitochondrial oxidation of succinate, glutamate, ,-hydroxybutyrate, and isocitrate (3); the activity of the electron transport chain is increased without an increase in the concentrations of the chain components (4); and there is an increase in the activity of membrane-bound a-glycerophosphate dehydrogenase (5).In addition, thyroid hormone affects mitochondrial swelling (6, 7) and respiratory control (8), but these effects are only observed with rather large doses of hormone.The mitochondrion is composed of an outer membrane, an inner membrane, and two spaces: the intermembrane space, which lies between the membranes, and the matrix, which is enclosed within the inner me...