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Using (A14-125I)-insulin as a tracer, insulin proteolytic activity in rat liver was found to be localized both to the cytosol and the endoplasmic reticulum. The membrane-associated activity was highly latent (70-80%). Both cytosolic and particulate activities had similar Km values and Mr of approx. 300 000 by gel filtration. Both were strongly inhibited by diamide (90%), but were unaffected by leupeptin or pepstatin. A comparison of the subcellular distributions with various 125I-isomers of insulin as tracers showed that both particulate and cytosolic activities were highest with (A14-125I)-insulin. 相似文献
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J Esmann J J Voorhees G J Fisher 《Biochemical and biophysical research communications》1989,164(1):219-224
Terminal differentiation of human skin involves the formation of an insoluble cross-linked protein envelope (CLE), which functions as an external barrier. To characterize terminal differentiation in the skin disease psoriasis, we have measured 1) membrane-associated transglutaminase (mTGase) activity, the rate limiting enzyme in the formation of CLE, and 2) the number of CLE in biopsies from normal and psoriatic skin. mTGase activity was increased 5-fold (p less than 0.0001) in psoriatic versus normal skin. Kinetic analysis revealed that the increased activity was due to an elevation in the Vmax of the enzyme. In addition, the number of CLE was 10-fold greater in psoriatic compared to normal skin. The increase in mTGase and CLE in psoriasis is in contrast to the decrease in other markers of terminal differentiation in skin, such as synthesis of specific intermediate filaments, observed in this disease. 相似文献
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Presence of a transglutaminase activity in rat liver lysosomes 总被引:2,自引:0,他引:2
M Juprelle-Soret S Wattiaux-De Coninck R Wattiaux 《European journal of cell biology》1984,34(2):271-274
The intracellular distribution of rat liver transglutaminase has been investigated by centrifugation methods. When measured in presence of Ca++ the enzyme is mainly present in the unsedimentable fraction of the homogenate. When assayed in absence of Ca++, the enzymatic activity exhibits a distribution pattern like that of lysosomal markers, both after differential and isopycnic centrifugation. The enzyme shows the phenomenom of structure linked latency and can be unmasked parallel with acid phosphatase by freezing and thawing. The origin of this transglutaminase is discussed; it is proposed that it is a genuine constituent of lysosomes. 相似文献
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The subcellular localization of superoxide dismutase was investigated in rat liver homogenates. Most of the superoxide dismutase activity is present in the soluble fraction (84%), the rest being associated with mitochondria. No indications for the occurrence of superoxide dismutase in other subcellular structures, particularly in peroxisomes, was found. Mitochondrial activity is not due to adsorption, since the sedimentable activity is essentially latent. Subfractionation of mitochondria by hypo-osmotic shock and sonication shows that half of the mitochondrial superoxide dismutase activity is localized in the intermembrane space, the rest of the enzyme being a component of the matrix space. In non-ionic media the matrix enzyme is, however, adsorbed to the inner membrane, from which it can be desorbed by low (0.04M) concentration of KCl. Superoxide dismutase activity was found in all rat organs investigated. Maximal activity of the enzyme is observed in liver, adrenals and kidney. In adrenals, the highest specific activity is associated with the medulla. 相似文献
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The subcellular localization of the four major low-molecular-weight RNA components, D, C, A and L, was studied in rat liver cells. The cells were fractionated by a non-aqueous technique into a nuclear and a cytoplasmic fraction. The cytoplasm contained 43% of component D, 57% of component C and more than 80% of component L. 相似文献
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1. The subcellular distribution of transglutaminase was investigated by using the analytical approach of differential and isopycnic centrifugation as applied to three organs of the rat: liver, kidney and lung. After differential centrifugation by the method of de Duve, Pressman, Gianetto, Wattiaux & Appelmans [(1955) Biochem. J. 63, 604-617], transglutaminase is mostly recovered in the unsedimentable fraction S and the nuclear fraction N. After isopycnic centrifugation of the N fraction in a sucrose density gradient, a high proportion of the enzyme remains at the top of the gradient; a second but minor peak of activity is present in high-density regions, where a small proportion of 5'-nucleotidase, a plasma-membrane marker, is present together with a large proportion of collagen recovered in that fraction. 2. Fractions where a peak of transglutaminase was apparent in the sucrose gradient were examined by electron microscopy. The main components are large membrane sheets with extracellular matrix and free collagen fibers. 3. As these results seem to indicate that some correlation exists between particulate transglutaminase distribution and those of collagen and plasma membranes, the possible binding of transglutaminase by collagen (type I) and by purified rat liver plasma membrane was investigated. 4. The binding studies indicated that collagen is able to bind transglutaminase and to make complexes with plasma-membrane fragments whose density is higher than that of plasma-membrane fragments alone. Transglutaminase cannot be removed from such complexes by 1% Triton X-100, but can be to a relatively large extent by 0.5 M-KCl and by 50% (w/v) glycerol. 5. Such results suggest that the apparent association of transglutaminase with plasma membrane originates from binding in vitro of the cytosolic enzyme to plasma membrane bound to collagen, which takes place during homogenization of the tissue, when the soluble enzyme and extracellular components are brought together. 相似文献
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Subcellular localization of B apoprotein of plasma lipoproteins in rat liver 总被引:23,自引:9,他引:14
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Multispecific antigen-binding fragments (Fab) from rabbit antisera against rat very low density lipoproteins (VLDL) and Fab against rat low density lipoproteins that were monospecific for the B apoprotein were conjugated to horseradish peroxidase. Conjugates were incubated with 6-mum frozen sections from fresh and perfusion-fixed livers and with tissue chopper sections (40 mum thick) from perfusion-fixed livers. In the light microscope, specific reaction product was present in all hepatocytes of experimental sections as intense brown to black spots whose locations corresponded to the distribution of the Golgi apparatus: along the bile canaliculi, near the nuclei, and between the nuclei and bile canaliculi. Perfusion fixation with formaldehyde produced satisfactory ultrastructural preservation with retention of lipoprotein antigenic determinants. In the electron microscope, patches of cisternae and ribosomes of the rough endoplasmic reticulum (ER) and particularly its smooth-surfaced ends, vesicles located between the rough ER and the Golgi apparatus, the Golgi apparatus and its secretory vesicles and VLDL particles in the space of Disse all bore reaction product. The tubules and vesicles of typical hepatocyte smooth ER did not contain reaction product, nor did the osmiophilic particles contained therin. The localization obtained in this study together with other evidence suggests a sequence for the biosynthesis of VLDL that differs in some respects from that proposed by others: (a) the triglyceride-rich particle originates in smooth ER where triglycerides are synthesized; (b) at the junction of the smooth and rough ER the particle receives apoproteins synthesized in the rough ER; (c) specialized tubules transport the particle, now a nascent lipoprotein, to the Golgi apparatus where concentration occurs in secretory vesicles; (d) secretory vesicles move to the sinusoidal surface where the particles are secreted into the space of Disse by fusion of the vesicular membrane with the plasma membrane of the hepatocyte. 相似文献
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Subcellular localization and properties of rat liver adenosine diphosphatase. 总被引:2,自引:0,他引:2
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ADPase (adenosine diphosphatase) was assayed in rat liver homogenates with [beta-32P]ADP as substrate. The activity had a pH optimum of 8.0 and was strongly activated by Mg2+. The intracellular localization was determined by analytical subcellular fractionation with single-step sucrose-density-gradient centrifugation. Selective membrane perturbants were used to enhance the resolution of the various organelles. ADPase was localized to the mitochondria. Mitochondria were isolated by differential centrifugation and subfractionated by selective disruption of the inner and outer membranes. The intramitochondrial localization of ADPase was compared with various marker enzymes and was shown to be concentrated in the outer-membrane fractions. The effects of various inhibitors on the ADPase activity were determined and the possibility that the activity could be due to known enzyme systems was considered. It is concluded that ADP degradation is due to a hitherto unrecognized mitochondrial enzyme. 相似文献
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Transglutaminase and ornithine decarboxylase activities have been assayed at intervals after partial hepatectomy in regenerating liver cells fractionated to obtain nuclear, cytoplasmic-particulate, and cytoplasmic-soluble fractions. Ornithine decarboxylase activity, localized entirely in the cytoplasmic fractions, undergoes a dramatic induction during the first 4 h after partial hepatectomy and remains elevated. This induction is very sensitive to inhibition by cycloheximide and actinomycin D, as previously reported. Transglutaminase activity is localized in both the cytoplasm and the nucleus with the highest specific activity in the nucleus. Nuclear transglutaminase activity approximately doubles in the first 2 h of liver regeneration, apparently as a result of a translocation of enzyme from the cytoplasm to the nucleus. Inhibitor studies indicate that the translocation is not dependent upon protein or RNA synthesis. In the first 2 h, actinomycin D slightly activates transglutaminase activity in the cytoplasmic-particulate and nuclear fractions. Only at 4 h after the onset of regeneration do actinomycin D and cycloheximide show some inhibition of transglutaminase activity indicating de novo synthesis at this time. A broad increase of transglutaminase activity occurs from hours 12–16 to hour 32 after partial hepatectomy in the nuclear and cytoplasmic-particulate fraction. These data suggest the existence of a function for transglutaminase in the nucleus of rat liver cells. 相似文献
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N Gérard J Thirion S Wattiaux-De Coninck R Wattiaux 《Biology of the cell / under the auspices of the European Cell Biology Organization》1992,75(3):253-256
The subcellular distribution of acid carboxypeptidase was investigated in rat liver, normal human skin (CRL 1501) and lung (WI-38) fibroblasts, galactosialidosis skin fibroblasts (GM 00806) and transformed lung fibroblasts (WI-38 VA 13). Results of differential and isopycnic centrifugations and osmotic activation experiments clearly indicate that the enzyme is located in lysosomes, in agreement with observations suggesting that carboxypeptidase is the protective protein of the 'Galjaard complex' which is defective in galactosialidosis. 相似文献
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R. Kennedy Keller Grant D. Rottler Nancy Cafmeyer W. Lee Adair 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,719(1):118-125
When purified subcellular fractions were prepared from rat liver and assayed for dolichol kinase activity using pig liver dolichol as a substrate, the microsomes were found to contain the highest specific activity and greater than 75% of the total actvity. With regard to substrate specificity, the microsomal enzyme showed a marked preference for saturation of the α-isoprene: dolichol-16 and -19 were 2.5-fold more active than the corresponding polyprenols. For a given class of prenol, the 16 and 19 isoprenologs exhibited similar activity, whereas the 11 isoprenolog appeared less active. The enzyme was twice as active against the naturally occurring polyprenol-16 (α-cis-isoprene) compared to synthetic α-trans-polyprenol-16. Taken together, the data indicate that the α-isoprene specificity follows the order: saturated>cis>trans. In addition, all-trans-2,3-dihydrosolanesol was not a substrate, suggesting that at least one cis isoprene residue is required. 相似文献
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Subcellular localization of ribonucleotide reductase in Novikoff hepatoma and regenerating rat liver 总被引:1,自引:0,他引:1
H L Elford 《Archives of biochemistry and biophysics》1974,163(2):537-543
The subcellular localization of ribonucleotide reductase was ascertained in Novikoff heptoma and normal and regenerating rat tissue. Over 90% of the cellular ribonucleotide reductase is found to be associated with a membrane fraction derived from the postmicrosomal supernatant after centrifugation at 78,000g for 18 hr which bands at 1.3 m sucrose in a discontinuous sucrose gradient. The properties of this particular ribonucleotide reductase are similar to those reported for mammalian ribonucleotide reductase. This membrane fraction, which contains ribonucleotide reductase, had been previously shown to contain a DNA polymerase whose activity is related to cell proliferation. The association of these two enzymes involved in DNA synthesis leads to the suggestion that there may exist a complex of enzymes involved in deoxynucleotide and DNA synthesis in this membrane fraction. 相似文献
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Subcellular distribution of the glutaminesynthetase activity (EC 6.3.1.2) is studied in muscle tissue and liver of carp. It is established that this activity is distributed by subcellular fractions analogously to the glutamatedehydrogenase activity--enzyme-marker activity of mitochondria. An additional evidence for the association of the two given enzymes is obtained during equilibrium density-gradient centrifugation of mitochondria on 30-60% gradient of sorbitol. Comparison of the obtained results with data from literature testifies to a coincidence of the subcellular localization of the processes of ammonia formation and binding into glutamine, thus confirming a view of the leading role of glutaminesynthetase in ammonia detoxication in carps. 相似文献
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Subcellular localization of the enzymes of cholesterol biosynthesis and metabolism in rat liver 总被引:11,自引:0,他引:11
M P Reinhart J T Billheimer J R Faust J L Gaylor 《The Journal of biological chemistry》1987,262(20):9649-9655
We have used isopycnic density gradient centrifugation to study the distribution of several rat liver microsomal enzymes of cholesterol synthesis and metabolism. All of the enzymes assayed in the pathway from lanosterol to cholesterol (lanosterol 14-demethylase, steroid 14-reductase, steroid 8-isomerase, cytochrome P-450, and cytochrome b5) are distributed in both smooth (SER) and rough endoplasmic reticulum (RER). The major regulatory enzyme in the pathway, hydroxymethylglutaryl-CoA reductase, also was found in both smooth and rough fractions, but we did not observe any associated with either plasma membrane or golgi. Since cholesterol can only be synthesized in the presence of these requisite enzymes, we conclude that the intracellular site of cholesterol biosynthesis is the endoplasmic reticulum. This is consistent with the long-held hypothesis. When the overall pathway was assayed by the conversion of mevalonic acid to non-saponifiable lipids (including cholesterol), the pattern of distribution obtained in density gradients verified its general endoplasmic reticulum localization. The enzyme acyl-CoA-cholesterol acyltransferase which removes free cholesterol from the membrane by esterification, was found only in the rough fraction of endoplasmic reticulum. In addition, when the RER was degranulated by the addition of EDTA, the activity of acyl-CoA-cholesterol acyltransferase not only shifted to the density of SER but was stimulated approximately 3-fold. The localization of these enzymes coupled with the stimulatory effect of degranulation on acyl-CoA-cholesterol acyltransferase activity has led us to speculate that the accumulation of free cholesterol in the RER membrane might be a driving factor in the conversion of RER to SER. 相似文献