Intracellular distribution of 5' nucleotidase in rat liver

The intracellular distribution of 5' nucleotidase was investigated in rat liver by biochemical analysis of cell fractions obtained by differential centrifugation. The enzymatic activity was measured by determination of the inorganic phosphorus liberated from 5' nucleotides. The 5' nucleotidase activity was mainly found in the nuclear and microsomal fractions. An attempt to extract the enzyme from these fractions with Mg(++) ion solutions was unsuccessful. It is concluded that 5' nucleotidase is actually present in the nuclear and microsomal fractions of rat liver cells.     

Rat spleen cytoplasmic nucleotidase: characterization and its physiological significance

1. A cytoplasmic nucleotidase, which preferably hydrolyzed 5'-dUMP, was investigated in rat spleen. 2. Total activity of the nucleotidase increased about 3-fold in the spleen of anemic rat was caused by phenylhydrazine administration. This increase was repressed by the injection of methotrexate, an inhibitor of DNA synthesis. 3. Activities of heme oxygenase or acid phosphatase did not correlate to the change of the nucleotidase activity. 4. The nucleotidase catalyzed dephosphorylation of 3'(5')-dUMP, 3'(5')-dTMP and 3'-UMP more readily, in the presence of Mg2+. Its optimum pH was around 6.0-6.5. It was stimulated by the addition of deoxyinosine. 5. These catalytic properties and tissue distribution of the enzyme, abundant in the thymus, spleen and intestine, were similar to that of 5'(3')-nucleotidase in rat liver [Fritzson P. (1978) Adv. Enzym. Regul. 16, 43-61]. 6. A possible physiological significance of the nucleotidase is in reutilization of preformed pyrimidine nucleotides.     

Distribution of liver plasma membrane 5' nucleotidase as indicated by its reaction with anti-plasma membrane serum

Antiserum against mouse liver plasma membranes was used to investigate the properties and distribution of the surface membrane enzyme 5′ nucleotidase.The antiserum inhibited 5′ nucleotidase but had no effect on alkaline phosphodiesterase, nucleotide pyrophosphatase, or insulin-binding activity.5′ Nucleotidase was purified from mouse liver plasma membranes and the purified enzyme was shown to be inhibited by the antiserum. The membrane-bound and the purified enzyme were both inhibited in a noncompetitive manner.The reaction of the antiserum with 5′ nucleotidase activity of mouse liver plasma membrane “light” and “heavy” subfractions, and of rat liver and pig lymphocyte surface-membrane fractions was investigated. In each case the enzyme was inhibited by the antiserum.Since a protein must be partially exposed on the membrane surface in order to react with its antibody, the results are discussed in terms of the disposition of 5′ nucleotidase within the membrane.     

Activity and subcellular distribution of protein kinase dependent on adenosine 3':5'-monophosphate in liver from normal and adrenalectomized rats.

We have examined whether glucocorticoids control the activity and (or) the subcellular distribution of protein kinase dependent on cyclic AMP (adenosine 3':5'-monophosphate), since they influence cyclic-AMP-dependent responses to other hormones. Protein kinase activity was determined in rat liver homogenates and subcellular fractions, nuclear, large granular, microsomal and supernatant obtained by differential sedimentation in 0.25 M sucrose. 63% of the tissue protein kinase activity detected in absence of cyclic AMP reside in the particulate fractions. Upon addition of exogenous cyclic AMP, protein kinase activity is stimulated 1.8, 1.2, 1.2 and 4.5-fold in nuclear, large granular, microsomal and supernatant fractions, respectively. Under these conditions, 66% of tissue activity are found in the supernatant fraction. The activity sensitive to exogenous cyclic AMP resolves into a major (84%) cytosoluble and a minor (16%) nucleomicrosomal component. The latter activity resists elution with isotonic saline and is increased in the presence of Triton X-100. Three groups of rats were studied: control and adrenalectomized with or without cortisol treatment. In whole liver homogenates, both protein kinase activity detected in absence of exogenous cyclic AMP and sensitivity of the enzyme to cyclic AMP were comparable in all groups. Moreover, the distribution patterns of proteins kinase activity amoung the fractions were essentially the same in all groups of animals, whether or not particles had been treated with Triton X-100. Finally, in cell-free experiments, glucocorticoids alone or in combination with their intracellular receptor did not modify protein kinase activity of rat liver. Thus the results reported do not support the possibility that glucocorticoids influence cyclic AMP-dependent protein kinase in rat liver. Yet, this study provides data, not available before, on subcellular distribution of this enzyme in rat liver.     

Phospholipase C activity in normal rat mammary tissues and in DMBA-induced rat mammary tumors

Employing phosphatidylinositol as the substrate, phospholipase C (PLC) activity was measured in various cellular fractions derived from DMBA-induced rat mammary tumors and mammary tissues from 12-14 day pregnant rats. In the 2,000g pellet, 10,000g pellet, 100,000g pellet and 100,000g supernatant fractions, PLC activity was more than 5 fold higher in the fractions derived from the neoplastic tissues. This was true whether PLC activity was expressed on the basis of protein content or 5' nucleotidase activities.     

Calcium-activated proteolytic activity in rat liver mitochondria

Soluble extracts from sonicated rat liver mitochondria and rat liver cytosol were each chromatographed on DEAE-cellulose columns, and the fractions assayed for Ca²⁺-activated proteolytic activity using ¹⁴C-casein as a substrate. The mitochondrial preparations were shown to be free of cytosolic and microsomal contamination by the lack of alcohol dehydrogenase activity, a cytosolic marker enzyme, and by a lack of cytochrome P-450 activity, a microsomal marker enzyme. Two peaks of Ca²⁺-activated neutral endoprotease activity were resolved from the mitochondrial fractions. One protease was half-maximally activated with 25 μM Ca²⁺, and the other by 750 μM Ca²⁺. Rat liver cytosol contained only a high Ca²⁺-requiring protease peak. This is the first demonstration of Ca²⁺-activated proteases in mitochondria.     

Inhibition by Orthovanadate of ATP-Dependent Ca2+ Transport in Microsomes Isolated from Rat Liver

A technique employing sucrose-density centrifugation for the enrichment of rat liver microsomes and rat liver plasma membranes in separate subcellular fractions is described. The fractions are enriched in glucose 6-phosphatase and 5′-nucleotidase, respectively, and are free of cytochrome oxidase activity. Vanadate-sensitive Ca²⁺ transport activity (half-maximal inhibition at ～10 μM vanadate, corresponding to ～12 nmol/mg of protein) was detected in only that fraction enriched in microsomal membranes. Inhibition by vanadate of ATP-dependent Ca²⁺ transport is noncompetitive with respect to added Ca²⁺ but competitive with respect to added ATP. Because it inhibits ATP-dependent Ca²⁺ transport in rat liver microsomes but not in rat liver plasma membranes, vanadate becomes a useful tool to distinguish in vitro between these two transport systems.     

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat.

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.     

Association of androgen binding sites with the endoplasmic reticulum of rat ventral prostate

Microsomes from ventral prostate of 24-h castrated rats contain a single set of tissue-specific high-affinity, low-capacity androgen binding sites. These sites are indigenous to the endoplasmic reticulum, as shown by purification procedures associated with marker enzymes and electron microscopic analyses. When prostatic microsomal membranes are separated from plasma membranes using the nuclear or the mitochondrial pellets as the source of fractionation in sucrose gradients, the androgen binding activity is selectively associated with fractions rich in rough endoplasmic reticulum and ribosomes. Eighty-four percent of the total content of Na+/K+ adenosine triphosphatase (ATPase) and only 27% of the total binding capacity were concentrated in fractions rich in smooth-surfaced vesicular membranes, when nuclear suspensions constituted the membrane source. In contrast, the region of the same gradient when enriched in rough endoplasmic reticulum and deficient in plasma membrane content contained 73% of the androgen-binding capacity and only 14% of the ATPase. For fractions collected using mitochondrial suspensions as starting material, the ratio (total glucose-6-phosphatase/total binding capacity) was closer to 1.0 than similar ratios of ATPase/binding capacity, indicating co-sedimentation of binding sites with microsomal membranes and not with plasma membranes. Na+/K+ ATPase, but not 5' nucleotidase, is a valid plasma membrane marker for ventral prostate. Microsomal androgen receptors may constitute a new level of regulation of androgen action in target cells.     

The Activity of 2',3'-Cyclic Nucleotide 3'-Phosphodiesterase in Rat Tissues

Abstract: The activity of the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was measured in 14 rat tissues and in subcellular fractions of rat liver by a sensitive fluorometric method, using cyclic NADP as substrate. CNP activity in brain (339 μmol/h/mg protein) was fourfold that of the sciatic nerve. The activities in tissues outside the nervous system ranged from a low of 0.42 μmol/h/mg protein in the unwashed red blood cell to a high of 9.96 in the spleen. The activity was highest in tissues containing cells with membranes capable of undergoing transformation and elaboration (spleen and thymus) and low in those in which the cell membranes are morphologically stable (muscle and red cell). The enzyme was found in all major liver subtractions, with the highest activities in the microsomal and nuclear fractions. Despite the large difference in the maximal velocities of CNP in brain and liver, the affinity of the liver enzyme for the substrate ( k _m) was similar to that of brain enzyme. Brain CNP was stable over a 48-h postmortem period.     

The activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase in rat tissues

The activity of the myelin-associated enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) was measured in 14 rat tissues and in subcellular fractions of rat liver by a sensitive fluorometric method, using cyclic NADP as substrate. CNP activity in brain (339 mumol/h/mg protein) was fourfold that of the sciatic nerve. The activities in tissues outside the nervous system ranged from a low of 0.42 mumol/h/mg protein in the unwashed red blood cell to a high of 9.96 in the spleen. The activity was highest in tissues containing cells with membranes capable of undergoing transformation and elaboration (spleen and thymus) and low in those in which the cell membranes are morphologically stable (muscle and red cell). The enzyme was found in all major liver subfractions, with the highest activities in the microsomal and nuclear fractions. Despite the large difference in the maximal velocities of CNP in brain and liver, the affinity of the liver enzyme for the substrate (km) was similar to that of brain enzyme. Brain CNP was stable over a 48-h postmortem period.     

Insulin degradation V. Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane

Glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) inactivates insulin by cleaving its disulfide bonds. The distribution of GSH-insulin transhydrogenase in subcellular fractions of rat liver homogenates has been studied. From the distribution of insulin-degrading activity and marker enzymes (glucose-6-phosphatase and succinate-INT reductase) (INT, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) after cell fractionation by differential centrifugation, the immunological analysis of the isolated subcellular fractions with antibody to purified rat liver GSH-insulin transhydrogenase, and chromatographic analysis (on a column of Sephadex G-75 in 50% acetic acid) of the products formed from ¹²⁵I-labelled insulin after incubation with the isolated subcellular fractions, it is concluded that GSH-insulin transhydrogenase is located primarily in the microsomal fraction of rat liver homogenate. An enzyme(s) that further degrades insulin by proteolysis is located mainly in the soluble fraction; a significant amount of the protease(s) activity is also present in the mitochondrial fraction. The possibility has been discussed that the protease(s) acts upon the intermediate product of insulin degradation, A and B chains of insulin, rather than upon the intact insulin molecule itself.The GSH-insulin transhydrogenase in intact microsomes occurs in a latent state; it is readily released from the microsomal membrane and its activity is greatly increased by treatments which affect the lipoprotein membrane structure of microsomal vesicles. There include homogenization with a Polytron homogenizer, sonication, freezing and thawing, alkaline pH, the nonionic detergent Triton X-100, and phospholipases A and C.     

Alcohol dehydrogenase-coupled spectrophotometric assay of epoxide hydratase activity

A coupled assay was devised for the assay of liver microsomal epoxide hydratase using the ability of alcohol dehydrogenase to transfer electrons from diols to NAD⁺: epoxide hydratase activity was continuously monitored at 340 nm. Rates of hydrolysis of octene-1,2-oxide and styrene-7,8-oxide measured utilizing this assay were similar to those determined using gas-liquid chromatography and radiometric thin-layer chromatography, respectively. The assay was used to examine the ability of rat liver microsomes and highly purified rat liver microsomal epoxide hydratase fractions to hydrolyze 15 other epoxides.     

Subcellular distribution of steroid delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the rat epididymis during sexual maturation

The curve of the specific activity of rat epididymal nuclear delta 4-5 alpha-reductase is bell shaped as a function of age, whereas that of cytoplasmic 3 alpha-hydroxysteroid dehydrogenase does not change significantly with age. The present study examines the subcellular distribution of delta 4-5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase in the caput-corpus and cauda epididymidis during development. A 5-step discontinuous sucrose gradient was developed for fractionation of epididymal homogenates. By using enzyme markers specific for different subcellular organelles, the five different subcellular fractions obtained were shown to be of cytoplasmic, microsomal, mitochondrial, nuclear and spermatozoal origin. 3 alpha-Hydroxysteroid dehydrogenase activity was associated only with the cytoplasmic fraction. The activity of the enzyme did not change significantly with age in either the caput-corpus or cauda epididymidis. delta 4-5 alpha-Reductase activity was found in fractions containing microsomal and nuclear markers. delta 4-5 alpha-Reductase activity in the nuclear fraction of the caput-corpus epididymidis was evident in the youngest age group (Day 25), increased 4-fold and peaked in the next age group (Day 35), and declined with each successive age group: Day 45 (60% of maximum), Day 60 (20% of maximum), Day 75 (15% of maximum) and Day 105 (10% of maximum). In contrast, microsomal delta 4-5 alpha-reductase activity increased successively from Day 25 to Day 105; enzyme activity doubled between these two ages. The ratio of nuclear to microsomal delta 4-5 alpha-reductase activity from the caput-corpus epididymidis thus changed markedly with age: Day 25:1.32; Day 35:3.76; Day 45:2.44; Day 60:1.03; Day 75:0.41; and Day 105:0.21. In the cauda epididymidis nuclear delta 4-5 alpha-reductase activity was only evident at Day 35 and Day 45; in microsomal fractions, activity was first found at Day 35 and did not subsequently change with age. These results demonstrate that: 1) epididymal 3 alpha-hydroxysteroid dehydrogenase activity is found only in the cytoplasmic fraction; 2) delta 4-5 alpha-reductase activity is found in nuclear and microsomal fractions; and 3) the subcellular distribution of delta 4-5 alpha-reductase activity changes markedly with age and epididymal section, suggesting differential regulation of nuclear and microsomal delta 4-5 alpha-reductase activities.     

Thiol-protein disulphide oxidoreductases. Assay of microsomal membrane-bound glutathione-insulin transhydrogenase and comparison with protein disulphide-isomerase.

1. Inhibition of endogenous microsomal NADPH oxidase by CO enables membrane-bound glutathione-insulin transhydrogenase (EC 1.8.4.2) to be assayed conveniently by a linked assay involving NADPH and glutathione reductase (EC 1.6.4.2). 2. The specific activity of the enzyme in rat liver microsomal preparations is of the order of 1 nmol of oxidized glutathione formed/min per mg of membrane protein. 3. The specific activity of the enzyme is comparable in rough and smooth microsomal fractions, and the activity is not affected by treatment with EDTA and the removal of ribosomes from rough microsomal fractions. 4. Membrane-bound glutathione-insulin transhydrogenase is not affected by concentrations of deoxycholate up to 0.5%, whereas protein disulphide-isomerase (EC 5.3.4.1) is drastically inhibited. 5. On these grounds it is concluded that, in rat liver microsomal fractions, glutathione-insulin transhydrogenase and protein disulphide-isomerase activities are not both catalysed by a single enzyme species.     

A new histo- and cytochemical method for demonstration of cyclic 3',5'-nucleotide phosphodiesterase activity in retinal rod photoreceptor cells of the rat

Cyclic 3',5'-mononucleotide phosphodiesterase (cyclic nucleotide PDEase) activity was studied histo- and cytochemically in the retinal rod photoreceptor cells of the rat by means of a newly developed technique utilizing the intrinsic 5' nucleotidase activity instead of an exogenous 5' nucleotidase source (snake venom). Cyclic GMP and was used as a substrate, the intense activity of phosphodiesterase (PDEase) was distributed over the entire rod outer segments; reaction product was observed on the plasmalemma and on the disk membranes of the outer segments. A slight reaction was also observed on the plasmalemma of the inner segments. However, no precipitate was found in the perinuclear and synaptic regions of the rod photoreceptors. In contrast, when cyclic AMP was utilized as a substrate, a moderate reaction was seen in the synaptic region of the plexiform layer. The intensity of the reaction in the outer segments was much reduced in comparison to the results with cyclic GMP. The enzyme activity was almost completely inhibited by 2 mM 3-isobutyl-1-methylxanthine (IBMX) or 2 mM theophylline, which were potent inhibitors of PDEase. To confirm the propriety of our new cytochemical method, the localization of 5' nucleotidase was also studied utilizing 5' AMP or 5' GMP as substrates. In contrast to the activity of cyclic nucleotide PDEase, the activity of 5' nucleotidase was distributed on all membranes of the photoreceptors from the synaptic outer plexiform layer to the tip of outer segments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue and subcellular distribution of 3H-dioxane in the rat and apparent lack of microsome-catalyzed covalent binding in the target tissue

Using ³H-dioxane, the distribution of dioxane among a number of tissues and various subcellular fractions of rat liver was studied. At various times after i.p. injection, dioxane was found to distribute more or less uniformly among various tissues (liver, kidney, spleen, lung, colon and skeletal muscle), consistent with its polar/nonpolar nature. Studies of the nature of dioxane binding, however, revealed that the extent of “covalent” binding (as measured by incorporation into lipid-free, acid-insoluble tissue residues) was significantly higher in the liver (the main carcinogenesis target tissue), spleen and colon than that in other tissues. Investigations of the subcellular distribution in liver indicated that most of the radioactivity was in the cytosol, followed by the microsomal, mitochondrial and nuclear fractions. The binding of dioxane to the macromolecules in the cytosol was mainly noncovalent. The percent covalent binding was highest in the nuclear fraction, followed by mitochondrial and microsomal fractions and the whole homogenate. Pretreatment of rats with inducers of microsomal mixed-function oxidases had no significant effect on the covalent binding of dioxane to the various subcellular fractions of the liver. There was no microsome-catalyzed $\text{in}$$\text{vitro}$ binding of ³H- or ¹⁴C-dioxane to DNA under conditions which brought about substantial binding of ³H-benzo[a]pyrene.     

INCORPORATION IN VIVO OF RADIOACTIVE LEUCINE INTO NEURONAL AND GOAL NUCLEAR PROTEINS OF RAT BRAIN

Purified neuronal and glial nuclei were separated from rat brain cells. The fraction rich in neuronal nuclei contained 68 ± 9 per cent neuronal nuclei and the fraction rich in glial nuclei contained 89 ± 6 per cent glial nuclei. The fraction rich in neuronal nuclei isolated from cells of adult rat brain incorporated l -[4,5-³H]leucine into TCA-insoluble material at a rate comparable to those of the microsomal and the soluble fractions of the brain, and at a much higher rate than the fraction rich in glial nuclei. The proteins soluble in buffered-saline, the acid-soluble deoxyribonucleoproteins, and the residual proteins of the neuronal nuclei are apparently the proteins which account for the higher specific activity of neuronal proteins compared with glial nuclear proteins. In liver and kidney, the incorporation of [³H]leucine into nuclear proteins was lower than into other subcellular fractions from the same organs.     

Binding of S-adenosylhomocysteine to various domains of the plasma membrane and to the endoplasmic reticulum from rat liver: Relation between binding and phospholipid methyltransferase activity

S-Adenosylhomocysteine (AdoHcy) binding to various membrane fractions of rat liver was determined at pH 7.4, using an oil centrifugation technique. The highest binding activity was found in the heavy microsomal (M-H) fraction enriched in endoplasmic reticulum, but high binding activity was also observed in the light microsomal fractions enriched in blood sinusoidal membranes (M-L fraction), and the heavy nuclear fraction (N-H fraction) containing the contiguous area. A substantial portion of AdoHcy binding activity in the M-L fraction may be ascribed to contamination of this fraction with endoplasmic reticulum, as indicated by the distribution of NADPH cytochrome c reductase activity. Binding activity was low in the light nuclear (N-L) fraction corresponding to the bile canaliculi. Phospholipid methyltransferase activity was determined in the same membrane fractions under similar conditions (pH 7.4), and in the absence and presence of added phospholipids. The distribution of the enzyme activity was dependent on the presence of exogenous phospholipids, and grossly similar to AdoHcy binding, the highest activities being observed in the M-H and the M-L fractions. The N-H fraction, rich in AdoHcy-binding activity, demonstrated, however, a very low phospholipid methyltransferase activity. It is concluded that AdoHcy-binding activity is not confined to the plasma membranes, and a major fraction of the binding activity resides on membranes derived from the endoplasmic reticulum. Also, the present results add to previous data suggesting that phospholipid methyltransferase does not totally account for the AdoHcy-binding sites on rat liver membranes.