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The role of catalase in lipid metabolism has been studied by means of a comparison of the turnover characteristics of the major lipid classes in the normal mouse with those of animals in which the catalase activity had been inhibited and blocked by aminotriazole and allylisopropylacetamide. Double isotope ratios were determined in the lipid fractions of several tissues following the injection of labeled glycerol, and a number of significant differences were identified between these treatments. Since catalase is recognized as an integral component of the peroxisomal pathway of fatty acid oxidation, these results may be taken as indicating that interruption of the process of peroxisomal beta-oxidation in this manner cause extensive perturbations of lipid metabolism in the living animal, and these perturbations extend well beyond those tissues where the predominant localization of these organelles occurs. The concept which derives from these data--that of a significant regulatory role of peroxisomes in relation to the overall balance of lipid metabolism in the animal body--is described and discussed.

The multiplicity and subcellular distribution of liver catalase activity have been studied in 10 species of mammals, in order to clarify comparative aspects of the properties of this enzyme. All mammals studied, except mouse, displayed the bulk of liver catalase activity in the cytosol, but the relative proportions of soluble and particulate activity varied quite markedly between species. Sheep, pig, and guinea pig provided the richest sources of supernatant liver catalase; while mouse, rat and pig contained the highest peroxisomal catalase activity. Rabbit, mouse, and rat liver supernatants exhibited 3–4 multiple forms of catalase activity, and displayed an electrophoretic pattern which was distinctly different to that of the aqueous peroxisomal extract. This latter fraction contained a single form of activity of higher anodal migration; a pattern of which was similar to that observed in all fractions of the other species examined. These results may be reconciled with current knowlledge of catalase genetics, subunit structure, and turnover by means of a postulated epigenetic mechanism, involving modifications of the enzyme in rabbit, rat, and mouse liver cytosol.

The effect of ionic strength on the adsorption of aldolase to synthetic thin filaments derived from rabbit skeletal muscle has been investigated by partition equilibrium experiments, the results being interpreted in terms of the intrinsic association constant for the interaction of four sites on aldolase with two sites per filament repeat unit. At physiological ionic strength, values of 10,000 and 2000 m −1 were obtained for this equilibrium constant in the absence and presence, respectively, of calcium ions. Comparison of binding curves obtained with synthetic thin filaments and myofibrils indicated a lesser extent of enzyme adsorption to the myofibrillar system, a difference attributed to the covert nature of many of the potential binding sites on the filaments in the assembly of the myofibril. On the basis of the quantitative information on the effect of ionic strength on the adsorption of aldolase, a case is made for the probable occurrence of the enzyme-filament interaction as a physiologically significant phenomenon in skeletal muscle.

Aspects of the synthesis and degradation of the multiple forms of catalase in mouse liver have been investigated. The kinetics of return of catalase after aminotriazole inhibition indicated a product-precursor relationship between the granular and supernatant pools of the enzyme, and electrophoretic resolution of the individual heteromorphs of catalase during this treatment served to substantiate this relationship and indicated, in addition, that aminotriazole-inhibited catalase may be partially reactivated in the cytosol. Changes in the activity of mouse liver and kidney catalases after the administration of chlorophenoxyisobutyric acid ethyl ester (CPIB) were also monitored. After an initial decline in activity, a rapid increase to an elevated steady state occurred, with an approximately threefold increase in the liver and twofold increase in the kidney. Subcellular fractionation of the livers of CPIB-treated mice showed a massive initial increase in the supernatant pool of catalase, accompanied by a steady decrease in the activity of the peroxisomal pools. Activity increased in the peroxisomal pools at later stages of treatment, but even after a new CPIB-induced steady state was achieved, the supernatant pool of catalase remained grossly elevated. Electrophoresis of the individual heteromorphs of catalase in the supernatant after CPIB treatment showed a predominance of the most cathodal migrating forms, and turnover studies demonstrated that catalase in CPIB-treated animals exhibited a substantially lowered rate of degradation by comparison with normal animals. These results have been discussed in relation to the intracellular sequestration and turnover characteristics of catalase.

Physicochemical investigations of mixtures of aldolase and glyceraldehyde-3-phosphate dehydrogenase in 0.05 m Tris/HCl-0.001 m EDTA, pH 7.5, by frontal gel chromatography as well as by velocity and equilibrium sedimentation have failed to detect an interaction that has been reported to occur between these two enzymes [J. Ovadi, C. Salerno, T. Keleti, and P. Fasella, (1978) Eur. J. Biochem. 90 , 499–503]. Since the previously investigated equilibria should have been readily observed by the present methods, it is suggested that the reported interactions do not reflect association of the native enzymes per se, but rather an artifactual association that has been mediated by the probes introduced into the enzymes to allow their study by fluorescence polarization.

In order to clarify the nature of the heterogeneity of mouse liver catalase, the enzyme was purified and characterized by several criteria. Absorption and sedimentation properties provided little indication of significant differences between the mouse liver enzyme and catalases from other mammalian sources which do not display multiplicity. A denotement of the nature of the variformity in mouse liver catalase was provided, however, by the demonstration that the heteromorphs may be interconverted under conditions which favor the addition or removal of sialic acid residues. It was also observed that CMP-sialic acid, together with microsomal extract, protected the supernatant (desialated) pool of catalase from inactivation upon storage; and that the pattern of multiplicity which was exhibited by the purified enzyme on isoelectric focusing, was considerably altered by incubation with neuraminidase. With regard to the individual characteristics of the separate forms of purified mouse liver catalase, significant differences were noticeable in relation to their isoelectric points, specific activities, heme content, and specific binding of [ 14 C]aminotriazole.

The adsorption of aldolase to myofibrils derived from rabbit skeletal muscle has been investigated by partition equilibrium studies at pH 6.8, I = 0.158 M, and the results interpreted in terms of an intrinsic association constant of 410,000 m −1 for the interaction of four sites on aldolase with myofibrillar sites, there being one such site for every 10–12 heptameric repeat units of F-actin-tropomyosin-troponin thin filament. Involvement of the active site of the enzyme in the adsorption process is indicated by the fact that competitive inhibition of the phenomenon by phosphate may be accounted for by an intrinsic association constant of 400 m −1 for the aldolase-phosphate interaction, a value in good agreement with that describing phosphate inhibition of the enzymatic hydrolysis of fructose-1,6-bisphosphate under similar conditions. On the basis of these equilibrium constants plus the aldolase and thin filament contents of muscle, resting muscle is indicated as containing a significant proportion (25–30%) of aldolase in the bound form, with changes in the subcellular distribution of the enzyme being likely during exercise due to the increased concentrations of Ca2+ and fructose-1,6-bisphosphate that then prevail.

A high percentage (generally ~60%) of the aldolase-binding capacity of isolated human erythrocyte membranes was lost either during storage of the membranes at 4 °C in 10 m m imidazole-1 m m MgCl 2 -10 m m 2-mercaptoethanol, pH 6.8, for 24–48 h, or during brief heating at 25–45 °C. In contrast, relatively little (generally ~25%) of the glyceraldehyde-3-phosphate dehydrogenase (G3PDH) binding capacity of the membranes was lost under these same conditions. Binding of both aldolase and G3PDH occurred in (at least) two kinetically distinguishable phases, with a rapidly equilibrating and a slowly equilibrating process being noted. The major portion of the aldolase binding was of a heat-labile rapidly equilibrating type, while the major portion of the G3PDH binding was of a heat-stable slowly equilibrating type. Binding of both aldolase and G3PDH was inhibited by P i in the presence of Mg 2+ , with dianionic P i being the effective species. Elution of peripheral proteins, including erythrocyte actin (Band 5), did not cause a decrease in binding of either enzyme that could be attributed to the loss of peripheral proteins. Binding of G3PDH had a markedly detrimental effect on binding of aldolase, but the reverse was not true. Based on this lack of mutual competition as well as the differences in stability of the aldolase and G3PDH binding sites, it is concluded that these enzymes do not bind to a single molecular feature on the erythrocyte membrane.

As an extension of previous studies on the adsorption of aldolase (EC 4.1.2.13) in nervous tissue, the main features of the subcellular localization of this enzyme in rat brain have been investigated. The major portion of the aldolase activity in homogenates of this tissue was demonstrated to be present in association with the particulate material, and a differential distribution of the AC isoenzymes was evident between the membranes and the cytosol. Some of the enzyme which was associated with the particulate fraction was shown to be occluded rather than absorbed to the membranes. This type of association was evident in the nuclear and mitochondrial fractions, in particular, with the occluded enzyme presenting an isoenzyme content high in C-type activity, and similar to that of the cytosol. The microsomal fraction contained a high proportion of enzyme in the bound form. Isoenzyme analysis of the enzyme in this microsomal fraction revealed a preferential association between the particulate material and A-type aldolase activity. A purified membrane fraction was prepared from the primary microsomal fraction, and identified as the main site of aldolase binding. The significance of the differential binding of aldolase isoenzymes and its localization amongst the subcellular fractions of rat brain have been discussed in relation to the structural and metabolic features of this tissue, and the coupling of energy producing sequences with energy requiring processes.