共查询到20条相似文献,搜索用时 31 毫秒
1.
MORPHOGENESIS AND DEVELOPMENT OF MICROBODIES OF HEPATOCYTES OF RATS DURING PRE- AND POSTNATAL GROWTH 总被引:14,自引:11,他引:3
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
In hepatocytes of fetal rats, cytoplasmic organelles identifiable as microbodies appeared, although only a few of them showed nucleoids and most of them generally had an electronlucent appearance due to the low density of their matrices. Some of these microbodies, especially those lacking the nucleoid, showed a substantial connection with granular endoplasmic reticulum (ER), suggesting that microbodies might be formed from granular ER. Agranular tubular profiles projecting from the surface of microbodies were found with a high frequency in fetal and neonatal rats; however, this phenomenon may not provide crucial evidence suggestive of the derivation of microbodies from agranular ER. Growth and maturation of microbodies are considered to be brought about by an enlargement of these organelles, an increase in their matrices, an appearance and enlargement of the nucleoids, and an increase in the enzyme involved. The specific activity of urate oxidase in the isolated nucleoid fraction was significantly lower in the earlier stages of postnatal growth than later. Increases in the enzyme activity per nucleoid (maturation of the nucleoid), in the number of microbodies containing nucleoids (formation of the nucleoid), and in the size of nucleoids (growth of the nucleoid), may contribute to increases in the enzyme activity of the tissues. 相似文献
2.
Synopsis 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--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. 相似文献
3.
Development of radioimmunoassay for thromboxane B2 总被引:3,自引:0,他引:3
A simple method for the preparation of rat liver urate oxidase is described. The enzyme was purified from rat liver homogenate by cell fractionation, detergent treatment, alkali treatment, and affinity chromatography on 8-aminoxanthine-bound Sepharose 4B. This enzyme preparation had a specific activity of 9.1 U/mg of protein and was purified about 1000-fold from the liver homogenate. After sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by staining with Coomassie brilliant blue, this preparation yielded one protein band at a position corresponding to a molecular weight of 33,000. 相似文献
4.
Synopsis The distribution of catalase, amino acid oxidase, -hydroxy acid oxidase, urate oxidase and alcohol oxidase was studied cytochemically in rat hepatocytes. The presence of catalase was demonstrated with the conventional diaminobenzidine technique. Oxidase activities were visualized with methods based on the enzymatic or chemical trapping of the hydrogen peroxide produced by these enzymes during aerobic incubations.All enzymes investigated were found to be present in peroxisomes. Catalase activity was found in the peroxisomal matrix, but also associated with the nucleoid. After staining for oxidase activities the stain deposits occurred invariably in the peroxisomal matrix as well as in the nucleoids. In all experiments the activity of both catalase and the oxidases was confined to the peroxisomes. The presence of a hydrogen peroxide-producing alcohol oxidase was demonstrated for the first time in peroxisomes in liver cells.The results imply that the enzyme activity of the nucleoids of rat liver peroxisomes is not exclusively due to urate oxidase. The nucleoids obviously contain a variety of other enzymes that may be more or less loosely associated with the insoluble components of these structures. 相似文献
5.
Subcellular localization of acyl coenzyme A: dihydroxyacetone phosphate acyltransferase in rat liver peroxisomes (microbodies). 总被引:2,自引:0,他引:2
Upon differential centrifugation of rat liver homogenate, the enzyme acyl-CoA:dihydroxyacetone-phosphate acyltransferase (EC 2.3.1.42) was found to be localized in the light mitochondrial (L) fraction which is enriched with lysosomes and peroxisomes. Peroxisomes were separated from lysosomes in a density gradient centrifugation using rats which were injected with Triton WR 1339. By comparing the enzyme distribution with the distribution of different marker enzymes, it was concluded that dihydroxyacetone phosphate acyltransferase is primarily localized in rat liver peroxisomes (microbodies). Similarly, the enzyme acyl dihydroxyacetone-phosphate:NADPH oxidoreductase (EC 1.1.1.101) was shown to be enriched in the peroxisomal fraction, although a portion of this reductase is also present in the microsomal fraction. 相似文献
6.
A correlative ultrastructural and enzymatic study of cotyledonary microbodies following germination of fat-storing seeds 总被引:1,自引:1,他引:0
Prof. Peter J. Gruber Richard N. Trelease Wayne M. Becker Eldon H. Newcomb 《Planta》1970,93(4):269-288
Summary Sunflower, cucumber, and tomato cotyledons, which contain microbodies in both the early lipid-degrading and the later photosynthetic stages of post-germinative growth, were processed for electron microscopy according to conventional procedures and examined 1, 4 and 7 days after germination. Homogenates of sunflower cotyledons were assayed for enzymes characteristic of glyoxysomes and leaf peroxisomes (both of which are defined morphologically as microbodies) at stages corresponding to the fixations for electron microscopy. The particulate nature of these enzymes was demonstrated by differential and equilibrium density centrifugation, making it possible to relate them to the microbodies seen in situ.One day after germination, the microbodies are present as small organelles among large numbers of protein and lipid storage bodies; the cell homogenate contains catalase but no detectable isocitrate lyase (characteristic of glyoxysomes) or glycolic acid oxidase (characteristic of leaf peroxisomes). 4 days after germination, numerous microbodies (glyoxysomes) are in extensive and frequent contact with lipid bodies. The microbodies often have cytoplasmic invaginations. At this stage the cells are rapidly converting lipids to carbohydrates, and the homogenate has high isocitrate lyase activity. 7 days after germination, microbodies (peroxisomes) are appressed to chloroplasts and frequently squeezed between them in the green photosynthetic cells. The homogenate at this stage has substantial glycolic acid oxidase activity but a reduced level of isocitrate lyase. It is yet to be determined whether the peroxisomes present at day 7 are derived from preexisting glyoxysomes or arise as a separate population of organelles. 相似文献
7.
A P Kausch 《European journal of cell biology》1984,34(2):239-247
Microbodies containing bipyramidal crystalline nucleoid inclusions occur within every cortical cell in roots of Yucca torreyi. Reaction product deposition attributable to catalase, glycolate oxidase, and urate oxidase activities are cytochemically localized to Yucca root microbodies and classifies them as unspecialized peroxisomes on the basis of their enzyme complement and tissue origin. Crystalline nucleoids do not stain for glycolate or urate oxidase activities, appearing as negatively-stained inclusions, but are apparently reactive for catalase activity. Development of unspecialized peroxisomes in Yucca roots is consistent with all evidence for glyoxysome and leaf-type peroxisome biogenesis from ER. Dilated ends of ER cisternae accumulate cytochemically detectable glycolate oxidase activity. After considerable dilation, paracrystalline precursors to nucleoids form within the bulge, and the inclusion enlarges to comprise the majority of peroxisomal volume. Peroxisomes that are not attached to ER are observed with high voltage electron microscopy and in serial thin sections, implying that eventually the budding peroxisomes are vesiculated. The functions of these unspecialized peroxisomes are suggested based upon cytochemical detection of their partial enzyme complement and their spatial and developmental timing relationships within developing Yucca root cortical parenchyma cells. 相似文献
8.
Summary An ultrastructural study was made of the cellular sheaths surrounding the sexual organs of five species of algae in the three genera ofCharophyceae: Nitella flexilis, N. mirabilis, Chara brattnii, Tolypella boldii andT. intricata. Microbodies similar in appearance, with crystalline nucleoids, were present in the sheath cells of all five species. The microbodies resembled in size and topographical associations those of other green algae. The hexagonal-shaped crystalloids consisted of parallel arrays of fine tubules of about 15 nm in diameter arranged parallel to the long axis of the crystalloid. In cross sections of the crystalloid, the close packing of the tubules showed hexagonal arrays. The intertubular distance is about 7 nm. At higher magnification there is a suggestion that the walls of these tubules are themselves constructed of smaller tubules. Further electron microscopic observations of diaminobenzidine (DAB)-treated preparations revealed pronounced deposition of reaction product in the microbodies, particularly on the crystalloids. The reaction was completely blocked by the catalase inhibitor, aminotriazole. These results strongly suggest that catalase is involved in this reaction and that catalase is located in the crystalloids. 相似文献
9.
D-aspartate oxidase in rat, bovine and sheep kidney cortex is localized in peroxisomes. 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
D-Aspartate oxidase (EC 1.4.3.1) was assayed in subcellular fractions and in highly purified peroxisomes from rat, bovine and sheep kidney cortex as well as from rat liver. During all steps of subcellular-fractionation procedures, D-aspartate oxidase co-fractionated with peroxisomal marker enzymes. In highly purified preparations of peroxisomes, the enrichment of D-aspartate oxidase activity over the homogenate is about 32-fold, being comparable with that of the peroxisomal marker enzymes catalase and D-amino acid oxidase. Disruption of the peroxisomes by freezing and thawing released more than 90% of the enzyme activity, which is typical for soluble peroxisomal-matrix proteins. Our findings provide strong evidence that in these tissues D-aspartate oxidase is a peroxisomal-matrix protein and should be added as an additional flavoprotein oxidase to the known set of peroxisomal oxidases. 相似文献
10.
Male albino rats (Sprague Dawley) were fed for 2-6 weeks on a diet containing 0.75% clofibrate. Liver cell fractions obtained from these animals were assayed for peroxisomal enzymes. In the cell homogenate the catalase activity was doubled, whereas the activity of urate oxidase was found to be only slightly depressed. The activity of carnitine acetyltransferase increased several times. In liver peroxisomes purified by isopycnic gradient centrifugation the specific activity of urate oxidase decreased appreciably showing that peroxisomes formed under the proliferative influence of clofibrate are not only modified with respect to their morphological characteristics but also to their enzymic equipment. This is also obvious from the changes in peroxisomal carnitine acetyltransferase activity which was enhanced by clofibrate to more than the fivefold amount. In purified mitochondria this enzyme was even more active: clofibrate advances both, the peroxisomal and the mitochondrial moiety of carnitine acetyltransferase. Morphological and cytochemical studies showed an increase in the number of microbodies and as compared to the controls microbodies were lying in groups more frequently. Small particles located closely adjacent to "normal" sized peroxisomes were found particularly after short feeding periods. While the number of coreless microbodies increased studies gave no clear evidence for an increase in marked shape irregularities of the peroxisomes. 相似文献
11.
Aldehyde oxidase (AO) and xanthine oxidase (XO) are cytosolic enzymes that have been involved in some pathological conditions and play an important role in the biotransformation of drugs and xenobiotics. The increasing interest in these enzymes demands for a simple and rapid procedure for their purification. This paper describes for the first time a method that allows simultaneous purification of both enzymes from the same batch of rat livers. It involves few steps, is reproducible and offers high enzyme yields with high specific activities. The rat liver homogenate was fractionated by heat denaturation and by ammonium sulphate precipitation to give a crude extract containing both enzymes. This extract was chromatographed on an Hydroxyapatite column that completely separated AO from XO. Further purification of XO by anion exchange chromatography on a Q-Sepharose Fast Flow column resulted in a highly purified (1200-fold) preparation, with a specific activity of 3.64 U/mg and with a 20% yield. AO was purified about 1000-fold at a yield of 15%, with a specific activity of 3.48 U/mg, by affinity chromatography on Benzamidine-Sepharose 6B. The purified enzymes gave single bands of approximately 300 kDa on a polyacrylamide gel gradient electrophoresis and displayed the characteristic absorption spectra of highly purified enzymes. 相似文献
12.
Growth of Candida famata and Trichosporon cutaneum on uric acid as the sole source of carbon and nitrogen was associated with the development of a number of microbodies in the cells. Cytochemical staining experiments showed that the organelles contained urate oxidase, a key enzyme of uric acid metabolism, and catalase. Transfer of cells, precultured on glucose or glycerol, into uric acid-containing media indicated that these microbodies originated from the organelles, originally present in the inoculum cells, by growth and division. In urate-grown C. famata the microbodies were frequently observed in large clusters; in both organisms they existed in close association with mitochondria and strands of ER. The organelles lacked crystalline inclusions. In freeze-fractured cells their surrounding membranes showed smooth fracture faces.Exposure of urate-grown cells to glucose-excess conditions led to a rapid inactivation of urate oxidase activity but catalase was only slightly inactivated. Glucose-induced enzyme inactivation was not associated with the degradation of the microbodies present in the cells. Similarly, repression of urate oxidase synthesis by ammonium ions also did not lead to the degradation of peroxisomes. 相似文献
13.
In prior studies, we employed a crude amyloglucosidase preparation, in conjunction with glucose oxidase reagent, to determine total carbohydrate in liver and muscle homogenates by a two-step procedure. Glycogen content was determined by subtracting tissue glucose (determined separately). By use of a purified amyloglucosidase, we have now verified the accuracy of two-step assay of rat liver homogenate with crude amyloglucosidase. The reliability of glucose oxidase detection of glucose in amyloglucosidase hydrolysates was established by comparing results with those obtained with hexokinase/glucose 6-phosphate dehydrogenase reagent. The availability of purified amyloglucosidase made it feasible to assay total carbohydrate content of homogenate in a single step, by incubating aliquots in the presence of both amyloglucosidase and glucose oxidase reagents. Results obtained with this one-step assay agreed with those of two-step analysis. To our knowledge, this is the first report of direct enzymic assay of total carbohydrate in rat liver homogenate, in a single step. 相似文献
14.
Chara fragilis possesses microbodies with a remarkably large size of up to 2 µm in diameter . Many of the organelles contain huge nucleoids of amorphous material or paracrystalline inclusions. After isolation of the organelles by gradient centrifugation the specific density of the microbodies was determined to be 1.25 g cm−3 . Catalase, glycolate oxidase and hydroxypyruvate reductase as well as enzymes of the fatty acid β -oxidation pathway were demonstrated to be constituents of the microbodies in Chara indicating that they are similar to those in green leaves. The data obtained are in agreement with the view that the Charophyceae and especially the algae in the subgroup of Charales are very closely related to the land plants. 相似文献
15.
The regulation of rat liver xanthine oxidase. Involvement of thiol groups in the conversion of the enzyme activity from dehydrogenase (type D) into oxidase (type O) and purification of the enzyme 总被引:19,自引:4,他引:15
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
1. The ;xanthine oxidase' activity of rat liver supernatant, most of which behaves as an NAD(+)-dependent dehydrogenase (type D) can be rapidly converted into an oxidase (type O) by thiol reagents such as tetraethylthiuram disulphide, copper sulphate, 5,5'-dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide and p-hydroxymercuribenzoate. Treatment with copper sulphate, if prolonged, leads to almost complete inactivation of the enzyme. The effect of these reagents is prevented by dithioerythritol, and in all cases but that of N-ethylmaleimide is reversed by the same thiol. 2. Dithioerythritol prevents and reverses the conversion of xanthine oxidase from type D into type O brought about by storage of rat liver supernatant at -20 degrees C, preincubation under anaerobic conditions, treatment with carbon or with diethyl ether, and reverses, but does not prevent, the conversion obtained by preincubation of the whole liver homogenate. 3. Conversion of the enzyme from type D into type O is effected by preincubation of rat liver supernatant with the sedimentable fraction from rat liver but not from chick or pigeon liver. The xanthine dehydrogenase activity of chick liver supernatant is not changed into an oxidase by preincubation with the sedimentable fraction from rat liver. 4. The enzyme activity of rat liver supernatant is converted from type D into type O during purification of the enzyme: the purified enzyme can be reconverted into type D by dithioerythritol. 5. The enzyme appears as an oxidase in the supernatant of rat heart, intestine, spleen, pancreas, lung and kidney. The enzyme of all organs but intestine can be converted into a dehydrogenase by dithioerythritol. 相似文献
16.
Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm3 which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [14C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm3, whereas the original glyoxysomes appeared at density 1.24 g/cm3. Electron microscopy showed that the fraction at 1.21–1.22 g/cm3 was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance. 相似文献
17.
Immunocytochemical localization of urate oxidase, fatty acyl-CoA oxidase, and catalase in bovine kidney peroxisomes 总被引:2,自引:0,他引:2
N Usuda M I Usman M K Reddy T Hashimoto J K Reddy M S Rao 《The journal of histochemistry and cytochemistry》1988,36(3):253-258
We investigated the localization of urate oxidase, peroxisomal fatty acyl-CoA oxidase, and catalase in bovine kidney by immunoblot analysis and protein A-gold immunocytochemistry, using the respective polyclonal monospecific antibodies raised against the enzymes purified from rat liver. By immunoblot analysis, these three proteins were detected in bovine kidney and bovine liver homogenates. Subcellular localization of these three enzymes in kidney was ascertained by protein A-gold immunocytochemical staining of Lowicryl K4M-embedded tissue. Peroxisomes in bovine kidney cortical epithelium possessed crystalloid cores or nucleoids, which were found to be the exclusive sites of urate oxidase localization. The limiting membrane, the marginal plate, and the matrix of renal peroxisomes were negative for urate oxidase staining. In contrast, catalase and fatty acyl-CoA oxidase were found in the peroxisome matrix. These results demonstrate that, unlike rat kidney peroxisomes which lack urate oxidase, peroxisomes of bovine kidney contain this enzyme as well as peroxisomal fatty acyl-CoA oxidase. 相似文献
18.
Bacterial DNA is largely localized in compact bodies known as nucleoids. The structure of the bacterial nucleoid and the forces that maintain its DNA in a highly compact yet accessible form are largely unknown. In the present study, we used urea to cause controlled unfolding of spermidine nucleoids isolated from Escherichia coli to determine factors that are involved in nucleoid compaction. Isolated nucleoids unfolded at approximately 3.2 M urea. Addition of pancreatic RNase reduced the urea concentration for unfolding to approximately 1.8 M urea, indicating a role of RNA in nucleoid compaction. The transitions at approximately 3.2 and approximately 1.8 M urea reflected a RNase-sensitive and a RNase-resistant restraint to unfolding, respectively. Removal of the RNase-sensitive restraint allowed us to test for roles of proteins and supercoiling in nucleoid compaction and structure. The remaining (RNase-resistant) restraints were removed by low NaCl concentrations as well as by urea. To determine if stability would be altered by treatments that caused morphological changes in the nucleoids, transitions were also measured on nucleoids from cells exposed to chloramphenicol; the RNase-sensitive restraint in such nucleoids was stabilized to much higher urea concentrations than that in nucleoids from untreated cells, whereas the RNase-resistant transition appeared unchanged. 相似文献
19.
Charles Kuhn 《Cell and tissue research》1968,90(4):554-562
Summary By electron microscopy, the parenchymal cells of the perianal glands of dogs contain granules which have the morphological features of microbodies (peroxisomes) including marginal plates and, occasionally, dense nucleoids. Like microbodies, they are occasionally attached to the endoplasmic reticulum. Histochemical evidence is presented suggesting that they contain at least one of the peroxisomal enzymes, L--hydroxy acid oxidase. The granules of a perianal gland adenoma showed abnormal morphologic variations.Mrs. Murtie Still, Mrs. Bertha McClure and Mr. Bob White gave valuable technical assistance. 相似文献
20.
Susana Valenciano J. Ramón De Lucas A. Pedregosa Inmaculada F. Monistrol F. Laborda 《Archives of microbiology》1996,166(5):336-341
Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced β-oxidation pathway showed the
presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase.
After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the β-oxidation enzymes, isocitrate
lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was
associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies
(peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase.
The β-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase
activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans.
Received: 8 March 1996 / Accepted: 5 August 1996 相似文献