Peroxisomal Marker Enzyme Research Articles

Subcellular distribution and storage form of rat renal renin.

The subcellular distribution and nature of rat renal renin has been investigated by means of analytical subcellular fractionation and gel filtration on Sephadex G-100. During differential centrifugation, renin activity was recovered mainly in soluble and heavy mitochondrial fractions. On sucrose gradient centrifugation in either a conventional or in a B XIV zonal rotor, renin activity equilibrated at 1.54 M sucrose and was partially resolved from marker enzymes for mitochondria (succinate dehydrogenase), lysosomes (acid phosphatase), plasma membranes (alkaline phosphatase), and peroxisomes (catalase). On gel filtration of the soluble or extracts of the renin-granular fractions on Sephadex G-100, renin activity eluted as a single peak with an apparent molecular weight (MW) of 42,000; no change in activity was found when these fractions were acidified to pH 3.0. When kidney homogenates were prepared in the presence of the proteolytic inhibitor N-ethylmaleimide (NEM, 10 mM), whereas the renin from the granular fractions displayed a MW of 44,000, that from the soluble fraction was apparently higher (69,000). Addition of NEM (10 mM) to the soluble fraction previously shown to contain only the low MW form of renin also resulted in an apparently high MW form of renin. These results indicate that rat renal renin is associated with a mechanically fragile, distinct type of subcellular organelle. Renin within this structure is of the low MW form and is not acid activatable. The soluble fraction, however, contains a factor(s) that, in the presence of NEM, combines with the low MW renin to form a complex of apparently high MW.

Partial purification and immunoreactivity of an 80 000-molecular-weight polypeptide associated with peroxisome proliferation in rat liver.

Hypolipidaemic drugs and industrial plasticizers such as di-(2-ethylhexyl) phthalate, which cause proliferation of hepatic peroxisomes, also cause an increase in an 80000-mol.wt. polypeptide in the liver of rats and mice. This polypeptide has been designated as PPA-80 (PPA, for peroxisome-proliferation-associated; 80 for 80000mol.wt.). The polypeptide PPA-80 was purified to over 90% purity from livers of rats treated with the peroxisome proliferators Wy-14,643, nafenopin, tibric acid and clofibrate by a single-step preparative sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic procedure. The antibodies raised against the PPA-80 polypeptide isolated from livers of rats treated with Wy-14,643 cross-reacted with polypeptide PPA-80 purified from the livers of rats treated with Wy-14,643, as well as from the livers of rats treated with nafenopin, tibric acid and clofibrate. The anti-(polypeptide PPA-80) antibodies did not cross-react with catalase, a marker enzyme for peroxisomes, or with NADPH-cytochrome P-450 reductase, which has the same approximate mol.wt., 80000. The intensity of immunoprecipitin bands formed with microsomal, large-particle and postnuclear fractions from livers of animals pretreated with peroxisome proliferators was significantly greater compared with equal amounts of protein from corresponding fractions obtained from control animals, suggesting that these agents all enhance the synthesis of the same 80000-mol.wt. polypeptide. Although the polypeptide PPA-80 was increased in the postnuclear, large-particle and microsomal fractions of livers of rats pretreated with peroxisome proliferators, the relative abundance of this peptide in the peroxisome-rich light-mitochondrial fraction and its lack in highly purified mitochondrial fractions suggest the localization of this polypeptide in peroxisomes and/or microsomal fraction. Additional studies are needed to establish unequivocally the subcellular localization of the polypeptide PPA-80 and to ascertain if this polypeptide is identical with the multi-functional protein displaying enoyl-CoA hydratase and beta-hydroxyacyl-CoA dehydrogenase activities that was purified by Osumi & Hashimoto [(1979) Biochem. Biophys. Res. Commun.89, 580-584].

Developmental Changes in Microbody Enzyme Activities in Germinating Spores of the Fern Pteris vittata

Summary Developmental patterns of glyoxysomel, peroxisomal and phototrophic marker enzymes have been correlated to lipid degradation and photosynthetic activities in germinating spores of Pteris vittata . Degradation of triglycerid-es occurred, after a lag phase of three days, until day 11, whereas after six days polar lipids are synthesized. Concurrent with lipid utilization, the activity of glyoxysomal isocitrate lyase developed, rose to, a maximum and then disappeared. The change from heterotrophic to, photoautotrophic conditions, beginning after day 4, is documented by the synthesis of chlorophyll and protein and by the formation of glyceraldehyde-3-phosphate dehydrogenase. The typically peroxisomal enzyme hydroxypyruvate reductase occurred two days after the beginning of photosynthetic activities. Catalase activity paralleled the increase of isocitrate lyase but did not follow its decline. The data are interpreted in support of the view that glyoxysomes are generated and degraded in the basal cell, whereas peroxisomes are formed independently in the green protonema cells.

Increased β-oxidation of erucic acid in perfused hearts from rats fed clofibrate

1. 1. The metabolism of [14- 14C]erucate and [U- 14C]palmitate has been investigated in perfused heart from rats fed 0.3% clofibrate for 10 days and from control rats. 2. 2. The total uptake of fatty acids in the heart increased in the clofibrate fed group. Clofibrate increased the oxidation of [14- 14C]erucic acid by 100% and the oxidation of [U- 14C]palmitic acid by 30% compared to controls. 3. 3. The chain-shortening of erucate to C 20:1 and C 18:1 fatty acids in the perfused heart was stimulated at least two-fold by clofibrate feeding. 4. 4. The activity of the peroxisomal marker enzyme catalase increased 60%, the activity of cytochrome oxidase increased approx. 16% and the content of total coenzyme A increased 30% in heart homogenates from rats fed clofibrate compared to controls. 5. 5. The isolated mitochondrial fraction from clofibrate fed rats showed an increased capacity for oxidation of palmitoylcarnitine and decanoylcarnitine, while the oxidation of erucoylcarnitine showed little change. 6. 6. It is suggested that clofibrate increases the oxidation of [14- 14C]erucic acid in the perfused heart by increasing the capacity for chain-shortening of [14- 14C]erucate in the peroxisomal β-oxidation system.

Determination of the cross-points of rat liver peroxisomes, peroxisomal core and the core components by cross-partition

The cross-points of rat liver peroxisomes, peroxisomal core and the core components were determined by means of cross-partition in two phase systems. The partitions were carried out in the systems containing 6% (w/w) Dextran T 500 and 6% (w/w) polyethyleneglycol 4000 in sodium salts. The same crosspoint, pH 5.6, was obtained in peroxisomal marker enzymes in light mitochondrial fraction of liver homogenate, such as catalase, d-amino acid oxidase and urate oxidase. The cross-point as determined by cross-partition of purified peroxisomal core was 6.7. The cross-points of urate oxidase and framework protein fractions obtained by alkali treatment on the purified core were 7.8 and 4.2, respectively, and the ratio of the proteins of urate oxidase to framework protein was 2:1. The theoretical value of cross-point of the core calculated from the relationship between the cross-point and protein ratio of each component of the core coincided with the experimental value obtained by this method.

Increased biosynthesis of CoA in the liver of rats treated with clofibrate.

The amounts of CoA in rat liver increased about three fold after treatment with clofibrate. About 10 of total CoA were long‐chain acyl‐CoA in normal rats, and slightly less in clofibrate‐treated animals. The wet weight and protein content of the liver increased by about 25%. From livers of clofibrate‐treated rats, mitochondrial and lysosomal fractions were obtained in increased amounts by conventional tissue fractionation techniques. The supernatants from such livers had rather high activities of marker enzymes for lysosomes and peroxisomes. No changes of the specific activities of the two first enzymes in the degradation pathway for CoA, i.e. acid phosphatase and nucleotide pyrophosphatase, were found in the liver homogenate from clofibrate‐treated rats. In the mitochondrial fraction from such livers, both (contaminating) activities were decreased. The degradation of CoA was increased in the supernatant, however, probably because of an increased leakage of acid phosphatase from lysosomes during the fractionation procedure. The synthesis of CoA from pantothenic acid was estimated by two different methods in vitro: by quantitation of CoA formed and by estimation of the incorporation of [14C]pantothenic acid into CoA. By both methods, CoA synthesis was found to be increased 3–4‐fold in livers from clofibrate‐treated rats. Dephospho‐CoA kinase was found to have a dual localization, with a minor pool in the mitochondrial fraction. This enzyme was only slightly increased in the clofibrate‐treated animals. The activity of pantothenate kinase was more than doubled after clofibrate treatment. This is thought to be the most important factor for the increase of CoA. CoA exerted feed‐back inhibition at this step in liver supernatant from normal and clofibrate‐treated rats.

Studies Supporting the Concept of Glyoxyperoxisomes as Intermediary Organelles in Transformation of Glyoxysomes into Peroxisomes

Intermediary forms of microbodies, glyoxyperoxisomes, are most likely synthesized at the stage of replacement of glyoxysomes by peroxisomes during the greening of fatstoring cells of cucumber cotyledons. This hypothesis is supported by three lines of results: 1. The levels of both glycollate oxidase and malate synthase are increased in the microbody fractions upon illumination of cotyledons. 2. Both peroxisomal and glyoxysomal marker enzymes are localized in organelles which exhibit the same buoyant densities. 3. De novo synthesis of malate synthase takes place even when irradiation already leads to a drastic increase of glycollate oxidase. The amount of malate synthase synthesized is almost identical in the light-induced system and in the dark control.

Comparative Investigation of the Action of Several Chlorosis-inducing Herbicides on the Biogenesis of Chloroplasts and Leaf Microbodies

Seedlings of Triticum aestivum L. and Secale cereale L. were grown in the presence of six different (five having different chemical structures) chlorosis-inducing herbicides: aminotriazole and its derivative SDR 5175, haloxidine, Sandoz 6706, fluometuron, and EMD-IT 5914. Concentrations were applied which allowed the leaves to grow normally and to reach normal total amino nitrogen contents but evoked a complete chlorosis (less than 6% chlorophyll). The effects of the herbicides on the accumulation of several chloroplast constituents and on peroxisomal and mitochondrial marker enzyme activities were compared. Wheat and rye, in general, gave very similar results, wheat being more sensitive to unspecific inhibitory effects.In dark-grown plants, the herbicides had no or only minor effects on the rRNA pattern and on enzyme activities of the leaves. In the light, all herbicides applied prevented the accumulation of carotenoids and of chloroplastic rRNA. Consequently, ribulose-1,5-bisphosphate carboxylase activity was virtually absent. After all herbicide treatments in light, the leaves contained only rather low catalase activity. In the presence of aminotriazole and haloxidine, the chloroplast-specific NADP-glyceraldehyde-3-phosphate dehydrogenase and the peroxisomal enzymes glycolate oxidase and hydroxypyruvate reductase had high or even normal activities, as in untreated leaves. In leaves treated with Sandoz 6706, fluometuron, or EMDIT 5914, the activities of the latter three enzymes were, in parallel, only very low. Some herbicides interfered with enzyme activities in vitro, particularly with those of catalase and of glycolate oxidase. Among mitochondrial enzymes, cytochrome c oxidase activity was either unaffected or lower, while fumarase had considerably higher activities in the herbicide-treated, as compared to untreated leaves. The specific effects on peroxisomal enzymes cannot be explained by the hypothesis of herbicide-induced photodestructions in carotene-deficient plastids. Alternative explanations for the genesis of the chlorosis are discussed.

Occurrence of a High Temperature Sensitivity of Chloroplast Ribosome Formation in Several Higher Plants

A specific high temperature-induced deficiency of chloroplast ribosome formation, as indicated by the absence of chloroplast rRNA, has been observed in the leaves of light- or dark-grown seedlings of Avena sativa L., Hordeum vulgare L., and Triticum aestivum L. at certain temperatures between 28 and 34 C. While the growth of the leaves (size, morphology, total amino nitrogen content) was little affected by the elevated temperature, chlorophyll accumulation was strongly inhibited, amounting to only 2 to 20% of its content in 22 C-grown leaves which were used as a reference for normal development. The carotenoid contents were also lower but still reached at least 15 to 20% of the corresponding measurements at 22 C. Ribulose-1,5-bisphosphate carboxylase was absent at the higher temperature while NADP-glyceralde-hydephosphate dehydrogenase reached high activities. For the peroxisomal marker enzyme hydroxypyruvate reductase, 30 to 70% of the activity present in 22 C-grown leaves was found in extracts from high temperature-grown leaves. Fumarase reached 1.5- to 4-fold higher activities at the elevated growth temperature than at 22 C. Leaves of Pisum sativum L. were completely chlorotic and deficient of 70S ribosomes at 33 C but simultaneously suffered from a severe general inhibition of their growth. In Zea mays L., a formation of chlorotic leaves was not observed at elevated temperatures.

The cytochemical demonstration of catalase and D-amino acid oxidase in the microbodies of teleost kidney cells.

The distribution of catalase and D-amino acid oxidase, marker enzymes for peroxisomes, was determined cytochemically in the kidney tubules of an euryhaline teleost, the three-spined stickleback. Catalase activity was localized with the diaminobenzidine technique. The presence of D-amino acid oxidase was determined using H2O2 generated by the enzyme, D-alanine as a substrate, and cerous ions for the formation of an electron-dense precipitate. Both enzymes appeared to be located in microbodies. The combined presence of these enzymes characterizes the microbodies as peroxisomes. Biochemically and cytochemically, no urate oxidase or glycolate-oxidizing L-alpha-hydroxy acid oxidase could be demonstrated. Stereological analysis of the epithelia lining the renal tubules showed that the fractional volume of the microbodies is 5 to 10 times higher in the cells of the second proximal tubules than in the other nephronic segments or the ureter. The fractional volume of the microbodies was similar in kidneys of freshwater and seawater fishes.

Subcellular localization of O-acetylserine sulfhydrylase in spinach leaves.

A combination of differential centrifugation and isopycnic sucrose density gradient centrifugation of extracts from spinach leaves (Spinacia oleracea L.) shows that about 20% of the O-Acetylserine sulfhydrylase are associated with chloroplasts. No appreciable amounts of O-Acetylserine sulfhydrylase band with mitochondrial and peroxisomal marker enzymes.

Préparation et étude analytique comparée des microsomes de foie de chat

Cat liver homogenates have been fractionated by differential centrifugation. Four particulate fractions (1 000 X g, 10 000 X g, and 145 000 X g) and a supernatant have been obtained. The biochemical composition of these fractions has been established from the assay and distribution pattern of 22 enzymatic and chemical constituents including marker enzymes for mitochondria, lysosomes, peroxisomes, plasma membranes, endoplasmic reticulum, Golgi apparatus and cell sap. The microsomal fraction was characterized by a moderate contamination with large cytoplasmic granules and by a low yield in protein and cholesterol. It contained 50 per cent of Golgi complex and about 40 per cent of plasma membranes. Morphological analysis of subcellular fractions was performed and confirmed biochemical results.

Peroxisome development in the metanephric kidney of mouse.

The relationship of enzymatic activity to organelle development and organelle number during differentiation of the metanephric kidney in the mouse was approached from several experimental directions. Biochemical analyses of marker enzymes for peroxisomes (catalase and D-amino acid oxidase), mitochondria (cytochrome oxidase) and lysosomes (acid phosphatase) were performed on kidneys at ages from 17 days prenatal to adult. These data were correlated with a morphometric analysis of populations of peroxisomes and mitochondria in differentiating cells of the proximal tubule. Postnatal development of the metanephric kidney was found to be accompanied by a rapid increase in both the specific activity of catalase and the number of peroxisomes per 100 mu2 in the proximal tubule during the first 4 weeks of postnatal growth. Elaboration of the endoplasmic reticulum (ER) was seen to parallel the increase in number of peroxisomes to which segments of ER were often in close apposition. Extensive interactions between segments of ER and peroxisomes were readily visible in 0.5-mu sections viewed in the high voltage electron microscope. In contrast to peroxisomes, neither mitochondria nor lysosomes followed a similar pattern of net organelle increase, suggesting that a defined population density of mitochondria and lysosomes may exist in the proximal tubule at birth, prior to complete development of the kidney.

Enzyme cytochemistry of Candida albicans.

The application of a new preparation method for demonstrating the activities of hydrolytic and oxidative enzymes in Candida albicans is reported. The problem of inadequate penetration of fixatives into yeast cells has been solved by sectioning solidified pellets of the cells in the presence of glutaraldehyde, a procedure that yields a fairly well preserved ultrastructure and sufficient enzyme activities. The subcellular distribution of most specific and nonspecific phosphatases and of peroxidases is at variance with that found in mammalian cells. The activities toward beta-glycerophosphate, p-nitrophenylphosphate, adenosine triphosphate, adenosine monophosphate, thiamine pyrophosphate and glucose 6-phosphate are almost exclusively confined to the central vacuolar apparatus. Oxidative and peroxidative activities are demonstrated only in mitochondria. Specific marker enzymes for endoplasmic reticulum, plasmalemma, Golgi apparatus and peroxisomes in C. albicans are not found. The possible function of the various subcellular organelles in relation to their enzymatic content is discussed.

ISOLATION AND CHARACTERIZATION OF MICROBODIES FROM CANDIDA TROPICALIS PK 233 CELLS GROWN ON NORMAL ALKANES

Numerous microbodies developed at the logarithmic phase of growth in cells of Candida tropicalis, when cultures were grown in a medium containing normal alkanes. From the homogenate of protoplasts prepared from 16-hr cells of Candida, biologically active microbodies were isolated by discontinuous sucrose density gradient centrifugation. The fractions recovered in the middle and lower-density regions of the gradient were associated with substantially high activities of three peroxisomal marker enzymes, catalase, D-amino acid oxidase, and urate oxidase, while a mitochondrial marker enzyme, cytochrome oxidase, was only slightly detected. Electron microscopic studies revealed that these fractions were largely comprised of morphologically well-preserved microbodies with a smaller number of more or less degraded organelles, but were virtually devoid of mitochondria. The microbody fractions contained an appreciable amount of DNA.

Progesterone-induced lysis of rat kidney lysosomes as studied by changes in light-absorbance.

1. A rat kidney lysosomal fraction was prepared by the method of Maunsbach (1966) and characterized by its content of representative marker enzymes for lysosomes, mitochondria, peroxisomes and endoplasmic reticulum. 2. It was shown that both pH-dependent and progesterone-induced lysis lead to a decrease in the E(520) of suspensions of this preparation. This decrease parallels quantitatively and temporally the release of soluble acid phosphatase. 3. It is suggested that E(520) measurements are a valid method for the continuous measurement of changes in lysosomal integrity. 4. As an example, results are included which demonstrate the ability of Zn(2+) to stabilize lysosomes against spontaneous and progesterone-induced lysis.

Analytical study of microsomes and isolated subcellular membranes from rat liver. II. Preparation and composition of the microsomal fraction.

Liver homogenates have been submitted to quantitative fractionation by differential centrifugation. Three particulate fractions: N (nuclear), ML (large granules), and P (microsomes), and a final supernate (S) have been obtained. The biochemical composition of the microsomal fraction has been established from the assay and distribution pattern of 25 enzymatic and chemical constituents. These included marker enzymes for mitochondria (cytochrome oxidase), lysosomes (acid phosphatase and N-acetyl-beta-glucosaminidase), and peroxisomes (catalase). The microsomal preparations were characterized by a moderate contamination with large cytoplasmic granules (only 6.2% of microsomal protein) and by a high yield in microsomal components. Enzymes such as glucose 6-phosphatase, nucleoside diphosphatase, esterase, glucuronyltransferase, NADPH cytochrome c reductase, aminopyrine demethylase, and galactosyltransferase were recovered in the microsomes to the extent of 70% or more. Another typical behavior was shown by 5'-nucleotidase, alkaline phosphatase, alkaline phosphodiesterase I, and cholesterol, which exhibited a "nucleomicrosomal" distribution. Other complex distributions were obtained for several constituents recovered in significant amount in the microsomes and in the ML or in the S fraction.

Effect of phytochrome on development of catalase activity and isoenzyme pattern in mustard (Sinapis alba L.) seedlings

In contrast to an earlier publication (Drumm et al., Cytobiol. 2, 335, 1970), a definite enhancement by phytochrome of the catalase level in mustard (Sinapis alba L.) cotyledons can be demonstrated. This response can be obtained either with continuous far-red light or with short red pulses, the effect of which is reversible by short far-red pulses. From the comparison of the time courses of catalase activity with the time courses of glyoxysomal (isocitrate lyase) and peroxisomal (glycolate oxidase, glyoxylate reductase) marker enzymes in dark grown and far-red irradiated cotyledons, there appears to be a close relationship between the catalase present in darkness and glyoxysomes and between the phytochrome-stimulated portion of total catalase and peroxisomes, respectively.The isoenzyme pattern of catalase shows 3 strong and several weaker bands in dark grown cotyledons. Irradiation with white or far-red light leads to a more complex pattern with at least 12 detectable bands. The isoenzymes increased by light supplement rather than replace the isoenzymes present in darkness. This is true also in cotyledons and true leaves of white light grown plants which do not possess glyoxysomes. In the hypocotyl of the seedling, catalase formation is depressed by far-red light and no change in the isoenzyme pattern is observed.It is concluded that the development of peroxisomes in the cotyledons is specifically controlled by phytochrome and that this subcellular differentiation also involves the control of catalase, a marker enzyme for both glyoxysomes and peroxisomes. The implications of these results with respect to the developmental origin of peroxisomes in cotyledons of fat-storing, potentially photosynthetically active cotyledons is discussed.

The Occurrence of Glycolate Dehydrogenase and Glycolate Oxidase in Green Plants

Homogenates of various lower land plants, aquatic angiosperms, and green algae were assayed for glycolate oxidase, a peroxisomal enzyme present in green leaves of higher plants, and for glycolate dehydrogenase, a functionally analogous enzyme characteristic of certain green algae. Green tissues of all lower land plants examined (including mosses, liverworts, ferns, and fern allies), as well as three freshwater aquatic angiosperms, contained an enzyme resembling glycolate oxidase, in that it oxidized l- but not d-lactate in addition to glycolate, and was insensitive to 2 mm cyanide. Many of the green algae (including Chlorella vulgaris, previously claimed to have glycolate oxidase) contained an enzyme resembling glycolate dehydrogenase, in that it oxidized d- but not l-lactate, and was inhibited by 2 mm cyanide. Other green algae had activity characteristic of glycolate oxidase and, accordingly, showed a substantial glycolate-dependent O(2) uptake. It is pointed out that this distribution pattern of glycolate oxidase and glycolate dehydrogenase among the green plants may have phylogenetic significance.Activities of catalase, a marker enzyme for peroxisomes, were also determined and were generally lower in the algae than in the land plants or aquatic angiosperms. Among the algae, however, there were no consistent correlations between levels of catalase and the type of enzyme which oxidized glycolate.

Untersuchungen zur Funktions�nderung der Microbodies in den Keimbl�ttern von Helianthus annuus L.

The enzyme patterns in sunflower cotyledons indicate that the glyoxysomal function of microbodies is replaced by the peroxisomal function of these organelles during the transition from fat degradation to photosynthesis. The separation of the microbody population into glyoxysomes and peroxisomes during this transition period is reported. The mean difference in density between the activity peaks of glyoxysomal and peroxisomal marker enzymes on a sucrose gradient was calculated to be 0.007±0.004 g/cm3 and turned out to be significant (t=7.8>4.04=t 5;0.01). The activity peak of catalase coincides with that of isocitrate lyase in early stages of development, but shifts to the activity peak of peroxisomal marker enzymes during the transition period. No isozymes of the catalase could be detected by gel electrophoresis in the microbodies with the two different functions. During the rise of the peroxisomal marker enzymes no synthesis of the common microbody marker, catalase, could be demonstrated using the inhibitor allylisopropylacetamide. Using D2) for density labeling of newly-formed catalase, no difference is observed between the density of catalase from cotyledons grown on 99.8% D2O during the transition period and the density of enzyme from cotyledons grown on H2O. The activity of particulate glycolate oxidase is reduced 30–50% by allylisopropylacetamide, but is not affected by D2O. The chlorophyll formation in the cotyledons is strongly inhibited by both substances.