Study on chemical species of iodine in human liver

The distribution and chemical species of iodine in various subcellular fractions of human liver were studied by using epithermal neutron activation analysis combined with chemical and biochemical separation techniques, such as gradient centrifugation and gel chromatography. It was found that the total iodine content orders in various subcellular fractions is as follows: nuclei > cytosol > mitochondria > lysosome > microsome. In the lysosomal fraction, iodine is mainly bound to macromolecules, whereas in the nuclei and mitochondrial fractions, mainly with lower-molecular-weight organic compounds. In the cytosol fraction, iodine is combined with three proteins, in which iodine is chiefly bound with mid- and high-molecular-weight proteins.     

Studies on peroxisomes. V. Effect of ethyl p-chlorophenoxyisobutyrate on the centrifugal behavior of rat liver peroxisomes.

After Wistar male rats had been fed on a diet containing 0.25% of ethyl p-chlorophenoxyisobutyrate (CPIB) for 28 days, changes in the enzyme activities and centrifugal behavior of rat liver peroxisomes were investigated. (1) Compared with control rats fed on the basal diet, the catalase [EC 1.11.1.6] activity of rat livers after the administration of CPIB increased about 2.5-fold, while urate oxidase [EC 1.7.3.3] activity did not change significantly. Though D-amino acid oxidase [EC 1.4.3.3] activity markedly decreased to approximately one-sixth of the control, the activity of L-alpha-hydroxy acid oxidase [EC 1.1.3.15], a flavin enzyme like D-amino acid oxidase, was not affected significnatly after the administration of CPIB. (2) When the hepatic cells of CPIB-treated rats were fractionated by differential centrifugation, most of the increase of catalase activity appeared in the supernatant fraction. A decrease in the hepatic D-amino acid oxidase activity of CPIB-treated rats was observed in all the fractions. As for the subcellular distribution of the particle-bound enzymes, the specific activities of both catalase and urate oxidase of CPIB-treated rat livers were higher in the light mitochondrial fraction than in other fractions. (3) Sedimentation patterns in a sucrose density gradient did not show any difference between normal peroxisomers, and CPIB-treated ones. (4) In the case of CPIB-treated rats, studies of their sedimentation patterns by Ficoll density gradient centrifugation showed two main particulate peaks containing both catalase and urate oxidase, although only a single peak was observed in the case of control rats.     

Organization of purine degradation in the liver of a teleost (carp; cyprinus carpio L.). A study of its subcellular distribution

Summary Carp liver was fractionated by differential and density gradient centrifugation and assayed for enzymes of purine catabolism. While urate oxidase is an excusively peroxisomal enzyme, only a very small percentage of the enzymes xanthine oxidase, allantoinase and allantoicase is associated with subcellular or ganelle fractions. There is no general purine catabolizing subcellular compartment.There is some but not yet conclusive evidence for the assumption that urate oxidase is a membrane bound enzyme.     

Analytical study of microsomes and isolated subcellular membranes from rat liver. VII. Distribution of protein-bound sialic acid

Detailed investigations by quantitative centrifugal fractionation were conducted to determine the subcellular distribution of protein-bound sialic acid in rat liver. Homogenates obtained from perfused livers were fractionated by differential centrifugation into nuclear fraction, large granules, microsomes, and final supernate fraction, or were used to isolate membrane preparations enriched in either plasma membranes or Golgi complex elements. Large granule fractions, microsome fractions, and plasma membrane preparations were subfractionated by density equilibration in linear gradients of sucrose. In some experiments, microsomes or plasma membrane preparations were treated with digitonin before isopycnic centrifugation to better distinguish subcellular elements related to the plasma membrane or the Golgi complex from the other cell components; in other experiments, large granule fractions were obtained from Triton WR-1339-loaded livers, which effectively resolve lysosomes from mitochondria and peroxisomes in density gradient analysis. Protein-bound sialic acid and marker enzymes were assayed in the various subcellular fractions. The distributions obtained show that sialoglycoprotein is restricted to some particular domains of the cell, which include the plasma membrane, phagolysosomes, and possibly the Golgi complex. Although sialoglycoprotein is largely recovered in the microsome fraction, it has not been detected in the endoplasmic reticulum-derived elements of this subcellular fraction. In addition, it has not been detected either in mitochondria or in peroxisomes. Because the sialyltransferase activities are associated with the Golgi complex, the cytoplasm appears compartmentalized into components which biogenetically involve the Golgi apparatus and components which do not.     

Hartmannella culbertsoni: enzymatic, ultrastructural, and cytochemical characteristics of peroxisomes in a density gradient

Isopycnic density gradient centrifugation techniques demonstrated that catalase (EC 1.11.1.6) and urate oxidase (EC 1.7.3.3) had similar distribution patterns with a peak at equilibrium density 1.22 suggesting that both enzymes were associated with a single population of subcellular particles. Catalase (EC 1.11.1.6) was shown cytochemically to be associated with peroxisomes in the sediment of the catalase-rich fractions. Protein showed a bimodal distribution with a soluble peak at density 1.10 and a particulate peak at density 1.20. The particulate protein peak corresponded to the mitochondrial peak. Acid phosphatase (EC 3.1.3.2) had an equilibrium density of 1.10. Acid phosphatase (EC 3.1.3.2) localization and ultrastructural examination of the acid phosphatase-rich fraction revealed that activity was associated with vacuoles. No primary lysosomes were identified.     

Localization of dolichol in the lysosomal fraction of rat liver

The distribution of dolichol and/or dolichol esters in subcellular fractions prepared from a rat liver homogenate has been investigated. After saponification of the various fractions dolichol was isolated and quantitated by high performance liquid chromatography in three systems. The degree of purity of the subcellular preparations was examined by marker enzymes and by electron microscopy. Using differential centrifugation it was found that the level of dolichol was highest in the mitochondria-lysosome fraction. Upon further resolution of this fraction by sucrose density centrifugation it was found that the majority of the dolichol was associated with the lysosome-rich fraction. In contrast, the mitochondrial fraction had only a low level of dolichol. This novel observation was confirmed by the finding that dolichol was greatly enriched in a highly purified lysosome fraction preparations isolated by Metrizamide density centrifugation. The enrichment of dolichol in this purified preparation paralleled the observed enrichment of the lysosomal enzyme activity in this fraction. All of these data suggest that the majority of cellular dolichol and/or dolichol esters is localized in the lysosome fraction. The significance of this finding in relation to the metabolism of dolichol is discussed.     

STUDIES ON ISOLATED CELL COMPONENTS : XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.     

Subcellular Distribution of Enzymes in Ochromonas malhamensis*

SYNOPSIS. The activity and distribution of 7 enzymes in Ochromonas malhamensis were studied. Subcellular organelles were separated by centrifugation at 648,000 g min to precipitate the larger particles; the resulting supernatant was centrifuged at 5,560,000 g min to separate the microsomal fraction from the supernatant. Sixty-four percent of the cytochrome oxidase (1.9.3.1 ferrocytochrome c:oxygen oxidoreductase, 81% of the catalase (1.11.1.6 hydrogen-peroxide: hydrogen-peroxide oxidoreductase) and 70% of the urate oxidase (1.7.3.3 urate:oxygen oxidoreductase) activity was associated with the larger particles, altho only 20% of the total protein was found in this fraction. Three acid hydrolases, cathepsin (3.4.4.9 cathepsin C, acid phosphatase (3.1.3.2 orthophosphoric monoesterphosphohydrolase) and acid ribonuclease (2.7.7.17 ribonucleate nucleotido-2′-transferase) were found mostly in the supernate (50-60%, yet their latency and their similar subcellular distribution indicated the presence of lysosomes. After 2.5 hr centrifugation in a sucrose density gradient (ρ= 1.08–1.25, the acid hydrolases showed a broad distribution which differed greatly from cytochrome oxidase associated with mitochondria. Catalase, which could not be separated from cytochrome oxidase by centrifuging on this gradient, had a different distribution after centrifugation on a kinetic gradient. Urate oxidase had a similar distribution to catalase and both these enzymes were latent, indicating the presence of peroxisomes.     

Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain.

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Subcellular localization of oestrogen-induced uterine peroxidase.

The distribution of oestrogen-induced peroxidase in the resuspended 8000g pellet of rat uterine homogenates was examined by centrifugation in a sucrose density gradient. Within 10h of treatment with oestradiol, peroxidase activity was found in a region devoid of catalase or urate oxidase (peroxisomal markers) which did not overlap the fractions containing succinate dehydrogenase (mitochondrial marker) or acid phosphatase (lysosomal marker). The induced uterine enzyme was localized in reticular membrane-bound vesicles with isopycnic density of 1.28g/ml from which it could be released by treatment with detergent.     

Release of the phophodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers.In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mictochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes.Activator was released from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles.The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

The distribution of protein-bound N-acetylneuraminic acid in subcellular fractions of rat brain

Protein-bound N-acetylneuraminic acid and hexosamine, including the sialomucopolysaccharides, occur mainly in the least dense particles sedimented in the microsomal fraction from rat whole brain. Particles rich in protein-bound N-acetylneuraminic acid and hexosamine are also found in the subcellular fraction separated as a layer between 0.8m- and 1.2m-sucrose after centrifuging the crude mitochondrial preparation in a density gradient. This distribution is similar to that of the gangliosides and suggests an association of all of these substances in the same subcellular structures. It is postulated that the sialomucopolysaccharides, as well as the gangliosides, are components of cell membranes. Evidence is presented that indicates that there are quantitative differences between distribution of the gangliosides on the one hand, and protein-bound N-acetylneuraminic acid and hexosamine on the other. The ratio of protein-bound N-acetylneuraminic acid (and hexosamine) to gangliosidic N-acetylneuraminic acid (and hexosamine) present in individual subcellular fractions obtained by density-gradient centrifugation tends to increase with increasing particle density. Exposure of the crude mitochondrial fraction to osmotic ;shock' before density-gradient centrifugation causes a shift of the protein-bound N-acetylneuraminic acid and gangliosides to the less dense fractions. In some experiments, a selective shift of the protein-bound N-acetylneuraminic acid was observed.     

Sucrose density gradient distribution of mitochondrial protein and enzymes from preclimacteric and climacteric pears

Mitochondrial fractions isolated from pears (Pyrus communis L.) at the climacteric minimum and peak were subjected to sucrose density gradient centrifugation. The distribution of protein and specific activities of 3 enzymes from this mitochondrial fraction were investigated.

Cytochrome oxidase specific activity remained associated with the particulate fraction and increased slightly during the period in which respiration of the whole fruit reached its climacteric peak. Catalase and acid phosphatase specific activity was associated with both the particulate and the least dense region of the gradient and decreased with postharvest ripening.

Evidence for several differences between the subcellular behavior of catalase and acid phosphatase from pear tissue compared to their counterparts isolated from mammalian cells is discussed. A general shift of maximum specific enzymic activities and protein distribution to lighter regions of the density gradient occurs with ripening, suggestive of diminution in size or density of intracellular particles.

     

Different localization of receptors for omega-conotoxin and nitrendipine in rat brain

The bindings of radioiodinated omega-conotoxin GVIA and [3H]-nitrendipine to subcellular fractions of rat brain were examined. The results indicated that omega-conotoxin binding site was mainly present in the mitochondrial fraction, whereas nitrendipine binding site was rich in the mitochondrial but also present in the post-mitochondrial fraction. Fractionation of the mitochondrial fraction on a sucrose density gradient centrifugation showed that the both binding sites were localized in the heavy synaptosomal fraction. These results strongly suggest that the N- and L-type voltage-sensitive calcium channels have different localizations.     

Improved recovery of rat liver fractions enriched in lysosomes by specific alteration of the sedimentation properties of mitochondria

A method for the preparation of lysosomes from rat liver is presented. The procedure requires only standard equipment and is completed within less than 3 h. Homogenization and differential centrifugation were performed at pH 7.4 in isotonic potassium phosphate-buffered sucrose medium. The addition of potassium phosphate, at the concentration used (10 mM), accelerated the sedimentation rate of mitochondria without altering that of lysosomes resulting in the decrease in the mitochondrial contamination of the final pellet. Further purification was achieved by isopycnic centrifugation in 45% isotonic Percoll performed in an angle rotor. Lysosomal fractions representing 51.5% of the original population were recovered over a density range of 1.09 to 1.15 g/ml. The most purified fraction (37-fold purified) contained 25.3% of lysosomal beta-N-acetylglucosaminidase, and only 0.9% of mitochondrial monoamine oxidase and 0.6% of peroxisomal urate oxidase original activities. It was practically devoid to endoplasmic reticulum contamination.     

The subcellular location in rat kidney of the peripheral benzodiazepine acceptor

In rat kidney high-affinity binding sites for [3H]Ro-5-4864 and [3H]PK-11195 with the properties of the peripheral-type acceptor were found enriched in mitochondrial (M) and light-mitochondrial-lysosomal (L) fractions on differential centrifugation. When the combined M and L fractions were subjected to sucrose density gradient centrifugation, these binding sites were found enriched at a density of 1.155 g/ml coincident with a population of light mitochondria, whereas a population of heavier mitochondria (rho = 1.175 g/ml) had few or no binding sites. Transmission electron microscopy showed that whereas the heavier mitochondria appeared highly pure and intact, the lighter mitochondria appeared less intact and to be contaminated with vesicular structures. After fractionation of the light mitochondria and vesicles by centrifugation, both fractions showed the same ratio of [3H]Ro-4864 binding sites to monoamine oxidase activity consistent with the vesicles being of mitochondrial outer-membrane origin. Digitonin pre-treatment had no effect on the density of acceptor-rich fractions on sucrose density gradient centrifugation. However, pretreatment with succinate/iodophenylnitrophenylphenyltetrazolium (INT) perturbed equally the density of acceptor-rich fractions and mitochondrial marker enzymes. When mitochondrial fractions were subjected to sonication prior to density gradient centrifugation the binding sites were now found highly enriched in a much lighter fraction coincident with the monoamine oxidase activity and thus consistent with being outer-membrane vesicles. When a mitochondrial fraction was subjected to hypotonic treatment before assay no evidence for activation/unmasking of binding sites was found. The hypotonic treatment did not release any inhibitor of the binding sites. These results are consistent with the peripheral benzodiazepine acceptor having an outer-membrane location on a sub-population of rat kidney mitochondria. Those mitochondria showing high levels of the acceptor are either light mitochondria or appear more susceptible to osmotic damage than those mitochondria in which the acceptor is absent or at low levels.     

Isolation and characterization of subcellular membranes from canine stomach smooth muscle

Subcellular membrane fractions were isolated from the circular muscle of the corpus of canine stomach by differential and isopycnic sucrose density gradient centrifugation. Differential centrifugation gave a mitochondrial fraction enriched (fourfold) in cytochrome c oxidase and a microsomal fraction enriched (fourfold) in 5'-nucleotidase and NADPH-cytochrome c reductase over postnuclear supernatant. On the basis of a study using continuous gradient, a discontinuous sucrose density gradient was prepared to yield F1 to F5 fractions. The F3 fraction at the interface of 18-32% (w/w) sucrose was maximally enriched (13-fold) in 5'-nucleotidase. The fraction contained very low levels of cytochrome c oxidase but did contain NADPH-cytochrome c reductase (eightfold enrichment). The F4 fraction, at the interface of 32-40% (w/w) sucrose, was maximally enriched in NADPH-cytochrome c reductase (12-fold) and cytochrome c oxidase (6-fold). The distribution of the azide-insensitive. ATP-dependent Ca2+ uptake correlated very well with that of 5'-nucleotidase but less well with NADPH-cytochrome c reductase and not at all with cytochrome c oxidase. Sodium azide and ruthenium red inhibited the ATP-dependent Ca2+ uptake by the mitochondrial fraction and postnuclear supernatant, but not by the F3 fraction. ATP-dependent Ca2+ uptake by the F3 fraction was inhibited by calcium ionophores A23187 and ionomycin, but not by the sodium ionophore, monensin. These results are consistent with the hypothesis that the plasma membrane plays a major role ih regulating intracellular Ca2+ concentration in canine corpus circular muscle.     

Formation of chenodeoxycholic acid from 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid by rat liver peroxisomes

The oxidation of the side chain of 3 alpha, 7 alpha-dihydroxy-5 beta-cholestanoic acid (DHCA) into chenodeoxycholic acid has been studied in subcellular fractions of rat liver. The product was separated from the substrate by high pressure liquid chromatography and identified by gas-liquid chromatography-mass spectrometry. The highest specific rate of conversion was found in the heavy (M) and the light (L) mitochondrial fractions with the highest enrichment in the L fraction. Washing the M fraction reduced the side chain cleavage activity by 90%. The peroxisomal marker enzyme urate oxidase was reduced to the same extent. The activity found in the M fraction may thus be due to peroxisomal contamination. After centrifugation of the L fraction on a Nycodenz density gradient, the highest specific activity for side chain cleavage of DHCA (31 nmol X mg-1 X h-1) was found in the fraction with the highest peroxisomal marker enzyme activity. This fraction also catalyzed conversion of 3 alpha,7 alpha,12 alpha-5 beta-cholestanoic acid (THCA) into cholic acid at the highest rate (32 nmol X mg-1 X h-1). The peroxisomal oxidation of DHCA into chenodeoxycholic acid required the presence of ATP, CoA, Mg2+, and NAD in the incubation medium. The reaction was not inhibited by KCN. It is concluded that rat liver peroxisomes contain enzymes able to catalyze the cleavage of the side chain of both DHCA and THCA. The enzymes involved are similar to, but not necessarily identical to, those involved in the peroxisomal beta-oxidation of fatty acids.     

THE SUBCELLULAR LOCALIZATION OF ACETYLCHOLINESTERASE AND ITS MOLECULAR FORMS IN PIG CEREBRAL CORTEX

Abstract— The distribution of acetylcholinesterase among the subcellular fractions of pig cerebral cortex was determined. The crude mitochondrial and microsomal fractions obtained by differential centrifugation accounted for 75% of the enzyme, with the remainder divided between the crude nuclear and soluble fractions.
The occurrence and distribution of the multiple molecular forms of AChE was the same in all four fractions with the dominant species of molecular weights 350,000, 270,000 and 60,000. Further purification of the mitochondrial fraction by density gradient centrifugation gave a series of membrane fractions with very similar multiple forms. The one possible exception was the fraction containing the purified synaptosomal membranes where one band of mol wt 270,000 predominated, although the other molecular weight entities were present. The electrophoretic pattern of AChE present in the fractionated microsomes was the same as in the crude preparation. The content and pattern of the multiple molecular forms of AChE was therefore the same in all fractions of pig brain, apart from that containing the purified synaptosomal membranes.     

Studies on isolated subcellular components of cat pancreas. I. Isolation and enzymatic characterization.

Pancreas of the cat was fractionated into its subcellular components by centrifugation through an exponential ficoll-sucrose density gradient in a zonal rotor. This enables a preparation of four fractions enriched in plasma membranes, endoplasmic reticulum, mitochondria and zymogen granules, respectively. The first fraction, enriched by 9- to 15-fold in the plasma membrane marker enzymes, hormone-stimulated adenylate cyclase, (Na+K+)-ATPase, and 5'-nucleotidase, is contaminated by membranes derived from endoplasmic reticulum but is virtually free from mitochondrial and zymogen-granule contamination. The second fraction from the zonal gradient shows only moderate enrichment of the above marker enzymes but contains a considerable quantity of plasma membrane marker enzymes and represents mostly rough endoplasmic reticulum. The third fraction contains the bulk of mitochondria and the fourth mainly zymogen granules as assessed by electron microscopy and marker enzymes for both mitochondria and zymogen granules, namely succinic dehydrogenase, trypsin and amylase. Further purification of the plasma membrane fractions by differential and sucrose step-gradient centrifugation yields plasma membranes enriched 40-fold in basal and hormone-stimulated adenylate cyclase and (Na+K+)-ATPase.