I955 more acidic solutions, with pH < 7 0, a few seconds elapsed before equilibrium was attained. Taking into account the ionization of the haem-linked group on MetMb and the higher oxidation state, the variation of Kb,. with pH is shown to confirm the conclusion that 2 moles of H+ are liberated/mole of acidic MetMb. Using 6-1 for the pK of the group in MetMb as established in other studies, the results give a pK of 75 for the group in the higher oxidation state at 200 and I = 0 04. 3. The variation of KRb, with temperature gives AHO = 10-0 ± 2*0 kcal./g.mol.: if the ionization of the haem-linked group is allowed for, the value 9*0 ± 1.0 kcal./g.mol. is obtained. 4. The dependence of Kob. on ionic strength is in accord with a change in charge from + 1 on MetMb to zero on the higher oxidation state. 5. The results are shown to favour the ferryl ion structure, or an isomer of this structure, for the higher oxidation state. The isomeric structures would, in general, require the presence of another ionizing group in myoglobin, but no evidence for such an ionization could be found. With other direct evidence favouring the ferryl ion structure this is to be preferred, and the higher oxidation state may provisionally be named ferrylmyoglobin. We gratefully acknowledge grants from the Department of Scientific and Industrial Research, and the Medical Research Council, held by one of us (D.H.I.) during the course of this investigation.

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Purified rat liver peroxisomes contain a cyanide-insensitive fatty acyl-CoA oxidizing system that uses 02 and NAD as electron acceptors. The system was detected by the ability of added palmitoylCoA to elicit 02 consumption, H202 production, and Ordependent NAD reduction. The activity of this system is increased approximately one order of magnitude in rats treated with clofibrate, a hypolipidemic drug known to cause peroxisomal proliferation. The possibility that rat liver peroxisomes may be involved in lipid metabolism is suggested by the fact that clofibrate*, which decreases serum lipid levels in man (1, 2) and animals (3, 4) and is used clinically for this purpose, also causes a striking increase in hepatic peroxisomes in several species (5, 6). Whether these two effects are related is unknown. Such a possibility was once denied (7), but is supported by the recent observation that two other hypolipidemic drugs, structurally unrelated to clofibrate, also induce proliferation of hepatic peroxisomes (8).Peroxisomes are characterized by their content of H202-producing oxidases and catalase (9). Curiously, although the number of electron microscopic images of peroxisomes increases as much as four to ten times upon clofibrate treatment (5, 6, 10), catalase activity increases only by 30-140% (5, 7, 10), and the known oxidases are either unchanged or decline (5,(10)(11)(12). This raises the intriguing possibility that the increase in protein in peroxisomes during clofibrate treatment might be due, at least in part, to the synthesis of lipid-metabolizing enzymes.Cytoplasmic organelles from castor bean endosperm, glyoxysomes, related to animal peroxisomes by their content of catalase and oxidases, have been shown by Beevers and coworkers (13) to contain enzymes capable of catalyzing fl-oxidation and the glyoxylate cycle reactions, thus playing a central role in the conversion of fat to carbohydrate at germination. The glyoxysomal system of fatty acyl-CoA oxidation resembles that of mitochondria except that the first dehydrogenase transfers its electrons to 02, producing H202 (14).These facts have encouraged us to look for a system of the type pictured in Fig. 1 in rat liver peroxisomes. This paper presents our first results, which establish that fatty acyl-CoA oxidizing activity is present in peroxisomes, and is strikingly increased by treatment of the animals with clofibrate. METHODS Palmitoyl-CoA oxidation was assayed by its ability to elicit the reduction of NAD, or in some cases that of oxygen to H202.NAD reduction was followed spectrophotometrically at 340 nm in a reaction volume of 1.0 ml containing 30 mM potassium phosphate buffer, pH 7.4, 0.2 mM NAD, 0.1 mM CoA, 12 mM dithiothreitol, 0.15 mg/ml of bovine serum albumin, and 0.01% Triton X-100. The temperature was maintained at 37'. Enzyme was added to duplicate cuvettes, and the reaction was started by the addition of palmitoyl-CoA to one of the cuvettes (10 nmol, unless otherwise indicated). The initial reaction rate was measured and expressed in Mmol o...

Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L-a-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage.

The response of rat liver lysosomes to an intraperitoncal injection of glucagon has been evaluated from studies on the mechanical fragility, osmotic sensitivity, and sedimentation properties of these subcellular particles. It has been found that about ~ hr after the injection of glucagon the hepatic iysosomes exhibit a fairly sudden increase in their sensitivity to mechanical stresses and to exposure to a decreased osmotic pressure. At the same time, their sedimentation properties undergo complex changes characterized mainly by a significant increase in the sedimentation coefficient of a considerable proportion of the total particles. In addition, glucagon causes an increase in the proportion of slowly scdimenting particles, with the result that the distribution of sedimentation coefficients within the total population tends to becomc bimodal. The latter change is more pronounced for acid phosphatase, less so for cathepsin D, and barely detectable for acid deoxyribonuclease. All these modifications are maximal between 45 and 90 min after injection and regress to normal within approximately 4 hr. With the exception of the increase in thc slow component, for which no explanation can be advanced at the present time, they are consistent with the hypothesis that giucagon causes an increase in lysosomal size, and may be related to the autophagic-vacuole formation known to occur after giucagon administration.

In the course of an investigation aimed at characterizing hepatic glucose 6-phosphatase, the unrelated acid phosphatase of rat liver was serendipitously observed to be latent and particle-bound in freshly prepared homogenates. Experiments designed to elucidate this intriguing finding led, 50 years ago, to the discovery of lysosomes. This could not have happened if strict adherence to a previously set programme had been mandatory.

Conflicting reports exist in the literature with respect to the intracellular localization of catalase, D-amino acid oxidase and monoamine oxidase in liver. Studies performed by a number of different authors indicate that catalase is associated partly with cytoplasmic particles and partly with the supernatant fraction, the ratio of particulate to soluble activity depending on the species and sex of the aniimal and on the nature of the medium adopted for preparing the homogenate (Ludewig &

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