Tissue- and subcellular-distribution of the binding site of [3H]9- methyl-7-bromoeudistomin D, a potent caffeine-like Ca2+ releaser, in rabbits

Tissue and subcellular distribution of the binding site of 3H-labelled 9-methyl-7-bromoeudistomin D ([3H]MBED), a powerful caffeine-like Ca2+ releaser, were investigated in rabbits. The order of specific activities of total homogenates was liver > brain > other tissues. All binding was completely suppressed by 10 mM caffeine, indicating that all [3H]MBED binding sites are modulated by caffeine. [3H]MBED binding sites distributed mainly in membrane fractions rather than soluble fractions in most tissues. In lung and liver, [3H]MBED binding was enriched in microsomes. [3H]MBED may be useful as a probe to investigate the actions of caffeine at the molecular level not only in muscles but also in a variety of tissues including liver, kidney and lung.     

Sigma-1 and sigma-2 sites in rat brain: comparison of regional, ontogenetic, and subcellular patterns

Radioligand binding assay conditions were established for the selective labeling of sigma-1 and sigma-2 sites in membrane homogenates of rat brain. Selective sigma-1 assays were conducted using 5 nM(+)[3H]SKF-10,047 in the presence of 300 nM dizocilpine (MK-801). Selective sigma-2 assays were conducted using 5 nM [3H]DTG in the presence of 1 microM (+)SKF-10,047. Distributions of sigma-1 and sigma-2 binding among brain regions were found to differ. While the brain stem yields the highest level of sigma-1 binding, it yields among the lowest levels of sigma-2 binding. The reverse is true in hippocampal membranes. Different ontogenetic patterns were also observed. Sigma-2 binding decreases substantially during brain development, whereas sigma-1 binding does not vary significantly. Patterns of distribution among subcellular fractions of rat brain homogenates were found to be similar. Both sigma-1 and sigma-2 sites are most enriched in microsomal fractions, and neither is enriched in synaptosomal or mitochondrial fractions. The present results suggest that sigma-1 and sigma-2 sites are distinct entities; they do not appear to be located on a common macromolecule, and they do not represent two different affinity states of a single type of binding site. While the precise subcellular locations of sigma-1 and sigma-2 sites remain to be determined, we conclude that localization of either type of binding site to synaptic regions of plasma membrane or to mitochondria is highly unlikely.     

Myocardial distribution and biotransformation in vitro and in vivo of nicorandil in rats, with special reference to mitochondria

This study reports subcellular localization of nicorandil in the myocardium and metabolism in mitochondria after oral dosing of 3 mg/kg nicorandil to rats. In the in vitro experiments, nicorandil, which was incubated with tissue homogenates (liver, kidney, heart, and small intestine), was metabolized to its denitrated compound, SG-86, and unknown substances. In the absence of a NADPH-generating system in the heart, the metabolic activity existed only in the mitochondrial fraction, but not in cytosolic and microsomal fractions. In the presence of the system, the activity in the mitochondrial fraction became much higher. To examine subcellular distribution of nicorandil in the myocardium, [14C]nicorandil was orally given to rats. Fifteen minutes after oral dosing of 3 mg/kg [14C]nicorandil, of which myocardial concentration reached a peak, nicorandil and SG-86 were found in mitochondrial fractions as well as in cytosolic and microsomal ones of the heart. Electron-microscopic autoradiograms, 15 min after oral dosing of 3 mg/kg [3H]nicorandil to rats, also showed the existence of the silver grains (showing radioactivity) in mitochondria of the heart. We conclude that nicorandil given orally is distributed in mitochondria of the heart, being partly transformed into SG-86, and that the myocardial mitochondria may be a potential site of action of nicorandil, an opener of KATP channels, which have been demonstrated to be present in this subcellular particle.     

Amodal completion in the absence of image tangent discontinuities

The localization of the low-affinity adenosine binding protein adenotin-1 with respect to distribution in rat organs and subcellular compartments was investigated. Adenotin-1 was characterized by 5'-N-ethylcarboxamido[2,8-3H]adenosine ([3H]NECA) binding and Western blotting. Cytosolic as well as membrane fractions of all tissues contained adenotin-1. Highest levels of membrane-bound adenotin-1 were found in the liver (liver > kidney approximately spleen approximately lung > forebrain approximately cerebellum > fat heart - striated muscle), whereas highest levels of cytosolic adenotin-1 were detected in spleen, liver, lung and fat. Subcellular fractions from rat liver were prepared by differential and density gradient centrifugation. Like the homologous proteins endoplasmin or gp96, adenotin-1 is enriched in the endoplasmic reticulum. Cytosolic and membrane-bound adenotin-1 species are pharmacologically distinct, because in the liver particulate fraction adenotin-1 showed a more rapid binding kinetics, a twofold lower affinity for [3H]NECA (KD 227 nM vs. 105 nM) and a sevenfold higher affinity for 2-chloroadenosine than the cytosolic protein (Ki 1.48 microM vs. 9.25 microM). In rat liver cytosol, two different binding sites were found, which differed in [3H]NECA binding kinetics and displayed a hundredfold difference in their affinity for 2-chloro-5'-N-methylcarboxamidoadenosine (Ki 45.8 nM vs. 4.76 microM). The presence of adenotin-1 in subcellular fractions, as determined by radioligand binding, was confirmed by Western blotting. Adenotin-1 was detected as a 98-kDa band in all rat liver subcellular fractions, which agrees with the molecular mass determined for the purified protein. In the cytosol, a 65-kDa hand was labeled more intensely than the 98-kDa band. This additional band probably represents the pharmacologically distinct species of adenotin-1 found in the cytosol.     

Oxidoreductases and hydrolases as marker enzymes for ultracentrifugation of islets of Langerhans of rats

Using quantitative fluorometric micro methods the presence of glutamate dehydrogenase, acid galactosidase, and acid glucuronidase was detected in pancreatic islets of the rat. Some properties of these enzymes and of malate dehydrogenase, 6-phosphogluconate dehydrogenase, and acid phosphatase were investigated. It has been shown that subcellular fractions of homogenates of islets of Langerhans can be characterized by using glutamate dehydrogenase, 6-phosphogluconate dehydrogenase, and acid hydrolases as marker enzymes for mitochondria, cytosol, and lysosomes, respectively. The degree of contamination from acinar tissue in the islet preparations was calculated from the amylase activity of the homogenates.     

Multiplicity of [3H]1,3-di-o-tolylguanidine binding sites with low affinity for haloperidol in rat brain

Specific binding of [3H]1,3-di-o-tolylguanidine (DTG) was found not only in synaptic membrane fractions but also in subcellular fractions enriched of microsomes, nuclei and mitochondria/myelins, with different sensitivities to displacement by the antipsychotic haloperidol. The highest binding was detected in microsomal fractions followed by, in order of decreasing binding, fractions enriched in nuclei, synaptic membranes, mitochondria/myelins and homogenates. [3H]DTG binding was completely abolished by prior treatment of the synaptic membranes with a low concentration of Triton X-100. [3H]DTG binding reached a plateau within 30 min of the incubation at 2 degree C, whereas raising the incubation temperature to 30 degrees C resulted in marked shortening of the time required to attain equilibrium, without altering the binding at equilibrium. The binding was inhibited by haloperidol in a concentration-dependent manner over a concentration range of 1 nM to 0.1 mM but with a potency more than 100 times weaker than the value reported in the literature, irrespective of the termination method employed and the external proton concentrations. [3H]DTG binding was markedly displaced by a variety of compounds including sigma ligands, benzomorphan opiates and noncompetitive antagonists at the N-methyl-D-aspartate (NMDA) receptor in synaptic membranes of the cortex, hippocampus and cerebellum. However, sigma ligands such as haloperidol, DTG and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine were more potent in displacing [3H]DTG binding in cortical membranes than in hippocampal and cerebellar membranes, while the potencies of the NMDA antagonists were not significantly different from each other among these 3 different central structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

Delta-Aminolevulinic acid synthetase in the heart

The regultion of cardiac delta-aminolevulinic acid synthetase activity was studied in rat heart homogenates. Optimal conditions were determined for the measurement of delta-aminolevulinic acid and an appropriate assay was established for the heart. The activity of cardiac delta-aminolevulinic acid synthetase was determined in rats either fed ad libitum or starved for 24 or 48 h. Marked decreases in delta-aminolevulinic acid synthetase activity were observed in homogenates or mitochondrial fractions prepared from hearts of fasted animals and an explanation for previous findings that the enzyme is undetectable in heart tissue is provided. Dexamethasone treatment was effective in reversing the decreases brought about by fasting but had no effect on the delta-aminolevulinic acid synthetase activity in heart homogenates from fed rats. ACTH treatment had no effect in fed or starved rats. Decreases in delta-aminolevulinic acid synthetase activity induced by fasting were not reversed in homogenates or mitochondrial preparations by succinyl-CoA-generating systems or when alpha-ketoglutarate was substituted for succinate in homogenate preparations. Cardiac delta-aminolevulinic acid dehydratase levels are not altered by fasting. Agents such as allylisopropylacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine, which produce marked increases in hepatic delta-aminolevulinic acid synthetase activity, have no effects on the activity of this enzyme in the heart.     

Relation of renal hemodynamics to metabolism of angiotensin II by the canine kidney

Incubation of brain cortex slices in the presence of glucose resulted in the permeation of about 65% of [14C] mescaline into slices. Of this, about one-third radioactivity was bound with nuclei, mitochondria, microsomes, and ribosomes. Dialysis of subcellular fractions did not markedly reduce the amounts of radioactivity bound to the fractions. The permeation into slices and the binding of mescaline to subcellular fractions were fairly time-dependent, but were inhibited by the presence of potassium cyanide, or by the absence of glucose and by heating to 80 degrees C for 1 min.     

Effect of papain-induced emphysema on the distrubtion of pleural surface pressure

In vivo experiments using 203Pb and radioactively labelled precursors such as [14C] arginine and [3H] tryptophan were performed to identify lead binding components in rat liver. The distribution of lead in 9 tissues and the intracellular distribution in liver and kidney was also investigated. Male rats were injected intravenously with 18 mug of 203Pb/rat and the 203Pb radioactivity was measured in whole tissues as well as in nuclei, mitochondria, lysosomes, microsomes and soluble fractions obtained by centrifugation of liver and kidney homogenates. The subcellular fractions from liver were purified and fractionated into macromolecular components by ultracentrifugation, gel filtration, ion exchange chromatography and solvent extraction. Nuclei were fractionated into membranes, chromatin proteins (histone and residual non-histone proteins) and DNA. Most of the lead was detected in the nuclear membrane fraction bound exclusively to membrane proteins and absent in phospholipids. The intranuclear lead was associated with histone fractions and other basic or very weakly acid proteins as indicated by the incorporation of [14C] arginine and [3H] tryptophan. Lead was present in the chromatographically purified DNA fraction but whether lead was really bound to the nucleic acid was not determined. Mitochondria were fractionated into heavy, soluble and light subfractions representing the inner membranes, the intramitochondrial matrix and the outer membranes respectively. These subfractions contained appreciable quantities of lead. No appreciable lead was present in lipids of the mitochondrial membranes. Significant quantities of lead were associated with the endoplasmic reticulum. Fractionation of microsomes into rough and smooth membranes showed that lead was almost exclusively bound to membranes of rough-surfaced microsomes associated with the heavy rough membrane subfraction. No significant lead was present in the free polysome subfraction or in lipids from the endoplasmic reticulum. More than one lead binding site was identified in the soluble fraction, the high molecular weight components representing the most important lead binding site.     

11 beta-hydroxysteroid dehydrogenases of the choriocarcinoma cell line JEG-3 and their inhibition by glycyrrhetinic acid and other natural substances

Mineralocorticoid receptor (MR) selectivity for aldosterone is thought to be exerted by enzymes which inactivate competing glucocorticoids before they bind the receptor. Two different 11 beta-hydroxysteroid dehydrogenases (11 beta-HSD) have been described. 11 beta-HSD-1 is NADP(+)-dependent and has a Km in the micromolar range and bidirectional activity. 11 beta-HSD-2 is NAD(+)-dependent, has a Km in the nanomolar range, exhibits only oxidase activity, and colocalizes with the MR in the kidney, so is likely to serve as the gatekeeper for the MR. We have further characterized 11 beta-HSD activity in JEG-3 cells, a cell line derived from a human choriocarcinoma which was reported to have only the high affinity, NAD(+)-dependent 11 beta-HSD-2. We found that the Km for the conversion of corticosterone to 11-dehydrocorticosterone in intact cells and homogenates was about 16 nM. NAD(+)-dependent corticosterone conversion was equal in the nuclear and mitochondrial fractions and less, but significant, in the microsomal fraction. A high affinity, Km = 40 nM, NADP(+)-dependent enzyme was also found in homogenates. The subcellular distribution of this high affinity activity was greatest in the mitochondria, less in the nuclei, and even less, but still significant, in microsomes. Because of its cofactor dependency, high affinity, and different subcellular distribution, we suggest that this enzyme is neither the 11 beta-HSD-1 nor the 11 beta-HSD-2 and have named it 11 beta-HSD-3. Conversion of 11-dehydrocorticosterone to corticosterone did not occur in intact cells or in homogenates incubated with NADH or NADPH. Enzyme activity in intact cells was inhibited by glycyrrhetinic acid, carbenoxolone, progesterone, 5 beta-dihydroprogesterone, and 5 alpha-dihydroprogesterone, but not bile acids.     

Stable cholinergic-muscarinic and alpha-adrenergic inhibition of rat parotid adenylate cyclase

(-) Isoproterenol-stimulated adenylate cyclase activity of washed at parotid membrane preparations from gland slices previously treated with the alpha-adrenergic agonist, methoxamine, was inhibited approximately 30%. The action of methoxamine required exogenous Ca2+ and was blocked by the alpha-adrenergic blocking agent, phentolamine. Incubation of gland slices with carbamylcholine resulted in a dose-dependent inhibition (50%) of (-) isoproterenol-stimulated adenylate cyclase activity in washed membranes. The action of carbamylcholine was independent of exogenous Ca2+ and was blocked by preincubation with atropine. Cholinergic inhibition of parotid adenylate cyclase was compared to the cholinergic inhibition of adenylate cyclase in dog heart homogenates. While carbamylcholine caused a limited (20-30%) inhibition of basal and (-) isoproterenol-stimulated activities when added to dog heart homogenates, it failed to produce any effect in parotid homogenates prepared in an identical manner. Cholinergic inhibition of parotid adenylate cyclase activity was stable and persisted in washed particulate fractions while the inhibition in dog heart homogenates was reversible by washing. Cholinergic inhibition of adenylate cyclase was markedly dependent on the presence of GTP and was abolished when Mn2+ was subsituted for Mg2+ in both systems. Guanyl-5'-yl imidodiphosphate had little effect on the inhibition in parotid preparations but abolished the inhibition in heart homogenates. It is concluded that, in contrast to the cholinergic action in dog heart homogenates, the exposure of parotid slices to carbamylcholine results in a stable lesion in the adenylate cyclase activity.     

Subcellular distribution of rat brain cortex high-affinity, sodium-dependent, glycine transport sites

The subcellular distribution of the membrane components, present in rat brain cortex homogenates, that interact with glycine in the presence of sodium ions was studied. The distribution in the primary fractions, as per cent of total binding in the homogenate, was: P1 ('nuclear'), 58%; P2 (large granule), 39%; P3 (microsomal), 2%9 Of the subfractions obtained by centrifuging P1 in a linear 0.32--1.5 M sucrose gradient, only the lighter fraction (P1-III) formed by large myelin fragments was enriched in specific binding activity with respect to P1. The pellet formed by purified nuclei had negligible binding, and fractions of intermediate density had a lower activity than P1. Transient exposure of P1-III to 1.5 M sucrose did not diminish its binding ability. Similarly, in the subfractions obtained by centrifuging P1 in a discontinuous sucrose gradient, only the least dense one, P1-A, that is formed exclusively by large myelin fragments, was enriched with respect to P1. The electron microscopy of these fractions is presented. The P2 subfractions, obtained in a linear 2--18% Ficoll gradient, had the following sodium-dependent activity (counts/min/mg protein, fractions being in the order of decreasing density): pellet, 0; P2-I, O; P2-II, 450; P2-III, 1770; P2-IV, 4130; unfractionated P2, 880; P2-IV, the least dense fraction being composed mainly of myelin. With P2 subfractions obtained in a discontinuous sucrose gradient (0.32, 0.8 and 1.2 M sucrose layers), it was also found that sodium-dependent glycine binding was only enriched, with respect to P2, in the myelin fraction P2-A. Glycine binding to purified brain cortex myelin was also found to be very high, while binding to non-myelin membranes, obtained during the purification procedure, was only 0--7% of that seen with myelin. These results suggest that high-affinity glycine binding is located in myelin proper, and possibly also in some other glial plasma membranes, but not in nuclei, mitochondria, endoplasmic reticulum or synaptosomes. The relevance of these findings for interpreting previous reports on high-affinity glycine transport in the central nervous system is analyzed.     

Glutamate dehydrogenase activity in the structures of the rabbit brain limbic system

The activity of glutamate dehydrogenase (EC 1.41.1.3) is studied in homogenates and subcellular fractions of five limbic structures: regio superior, regio inferior of hippocampus, fascia dentata, septum and corpora mamillaria. The lowest activity of the enzyme is found in regio superior of hippocampus. 80% of the total enzyme activity of primary fractions is found in "crude" mitochondria. After centrifugation of the latter within the linear sucrose density gradient the distribution of the enzume activity is similar for different structures and the highest activity is found in the region of sucrose molarity from 1.44 up to 1.50 M which corresponds to the mitochondria distribution region. 50% of the total found activity is in the fraction enriched by mitochondria, 30% is in the fraction enriched by nerve endings with the high activity of glutamate decarboxylase. It was found for different fractions that 1 mM of ADF with 0.2 mM NAD-H+ produces about 10-fold increase in the enzyme activity. Pyridoxal-5'-phosphate inhibits the enzyme from inactivation. The results are discussed in connection with the possible role of pyridoxal-5'-phosphate in regulation of the glutamate dehydrogenase activity in vivo.     

Specificity studies of particulate binding sites for steroid hormones in subcellular fractions of the porcine corpus luteum

We have studied the binding of a number of radiolabelled steroids and lipophilic substances to porcine corpus luteum (CL) particulate fractions. Following preincubation of CL homogenates with radiolabelled progesterone or pregnenolone prior to fractionation on continuous sucrose density gradients, a broad peak of binding was observed associated with a particulate fraction of buoyant density 1.05-1.10 g/cm3. Progesterone content also peaked at a similar buoyant density (1.06-1.12 g/cm3). Pretreatment of luteal homogenates with digitonin perturbed the buoyant density of the progesterone-binding particulate fraction to 1.10-1.14 g/cm3 and sharpened the binding peak. Progesterone content was also perturbed to a similar extent by digitonin pretreatment, without release of the steroid. Oestrogens were also sequestered by this fraction, but steroid precursors (cholesterol, cholesterol ester), corticosteroids (cortisol, corticosterone), sterol conjugates (oestrone sulphate, pregnanediol glucuronide) and other lipophilic substances (arachidonic acid, phospholipid, prostaglandins E1, E2 and F2 alpha) were not bound. Androgens were bound weakly by fractions from control gradients but, in the presence of digitonin, significant binding could be demonstrated. Radiolabelled steroids were shown to interact directly with luteal membrane fractions, rather than interacting first with cytosolic steroid receptors which then bound to membranes. Furthermore, [3H]progesterone was not bound by porcine granulosa cell particulate fractions. These observations suggest that this fraction may be involved in sequestration or packaging of progesterone for secretion by the luteal cell.     

Compartmentation of the type I regulatory subunit of cAMP-dependent protein kinase in cardiac ventricular muscle

The species-dependent compartmentation of type I cAMP-dependent protein kinase (PKA I) and its dissociated regulatory subunit (RI) was examined in the heart by biochemical and immunohistochemical means. PKA I and RI were resolved from type II cAMP-dependent protein kinase and its regulatory subunit by DEAE-Sephacel chromatography of the supernatant and Triton X-100 soluble particulate fractions of heart homogenates. The relative amounts of holoenzymes and subunits were determined by cAMP-binding, protein kinase, 8-N3-[32P]cAMP photoaffinity labeling, and Western blot assays. Rat, rabbit, and guinea pig hearts all contained PKA I to varying degrees, but only in the supernatant fractions. Significant amounts of dissociated RI were found in the supernatant fractions, and to a lesser extent the particulate fractions, of these species. In contrast, though no PKA I was detected in the supernatant or particulate fractions of pig and beef heart, half of the cAMP-binding activity in the particulate fraction was attributed to RI. The results suggest that RI may associate with membrane fractions when it is not associated with the PKA catalytic subunit. Immunohistochemical studies of tissue sections from pig, beef, and rat cardiac ventricle using antibodies directed against RI also revealed species-dependent localization of RI. Cardiac myocyte intercalated discs were stained in pig and beef sections with additional sarcolemmal staining in beef sections. Rat ventricle, which contained large amounts of supernatant PKA I, showed nuclear staining. The localization of RI to cardiac myocyte intercalated discs and sarcolemma in certain species suggests a role(s) for this subunit in mediating cAMP-regulated events in these regions.     

Effects of chronic U50,488H treatment on binding and mechanical responses of the rat hearts

The effects of chronic injection of U50,488H (trans-3,4-dichloro-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeacetamidel++ +), a selective kappa opioid agonist, on the properties of the binding sites of tritiated U69593 [(5 alpha,7 alpha,8 beta)-(-)-N-methyl-N-(7-(1-pyrrolidinyl)-1-oxaspiro (4,5)dec-8-yl)benzeneacetamide], another selective kappa opioid agonist, and mechanical responses to U50,488H of the heart were studied. Rats received injection twice a day with U50,488H for 4 days. Binding studies on the crude membrane homogenates revealed that there was no change in maximum binding, but a significant increase in Kd after the treatment, indicating that the number of kappa binding sites remained unchanged whereas the affinity of the binding sites to kappa-agonist decreased. The study on the mechanical responses to U50,488H in the isolated perfused heart preparation showed that although the agonist at 10(-6) M caused MR2266 reversible reductions in heart rate and force of contraction as well as ventricular ectopic beat in the heart of rats in the control group, its effects were absent in the U50,488H-treated group, indicating the development of tolerance to the mechanical effects of U50,488H on the heart. The results indicate that the development of tolerance to the mechanical effects of a kappa-agonist after chronic treatment with the agonist was not accompanied by down-regulation, but only a slight and significant reduction in affinity of kappa binding sites in the rat heart.     

A novel muscle protein located inside the terminal cisternae of the sarcoplasmic reticulum

An immunofluorescence study of adult rat muscle tissues with a polyclonal antibody against the RGD-directed fibronectin receptor of Friend's erythroleukemia cells (alpha5beta1-integrin) unexpectedly revealed a pattern of intracellular antigen distribution. Western blotting analysis of rat and rabbit membrane fractions indicated that the antibody recognizes a 167-kDa protein expressed both in heart and in skeletal muscle (relative abundance: heart > slow muscle > fast muscle), but not in liver and kidney. The 167-kDa protein did not show altered electrophoretic mobility upon reduction and failed to bind several lectins, including wheat germ agglutinin. A study of its subcellular distribution in rabbit skeletal muscle revealed that the 167-kDa protein is mostly associated with the terminal cisternae of the sarcoplasmic reticulum (SR) and, to a smaller extent, with the sarcolemma, while it is absent in the longitudinal tubules of the SR. The 167-kDa protein is not an integral membrane protein since it can be extracted at pH >/=10. This protein can be proteolytically cleaved only in the presence of detergent, indicating that it resides on the luminal side of the SR. The 167-kDa protein could be resolved from the closely spaced sarcalumenin and histidine-rich protein by column chromatography followed by detergent dialysis and two-dimensional gel electrophoresis. The N terminus and the internal sequences did not match any known sequence in protein and DNA data bases, indicating that the 167-kDa protein is a novel muscle protein selectively localized to the SR. Integrins from rat kidney fibroblasts were not recognized by either (i) a polyclonal antiserum against the purified 167-kDa protein or (ii) the anti-alpha5beta1-integrin antiserum after affinity purification onto the 167-kDa protein. These data indicate that the 167-kDa protein is not immunologically cross-reactive with integrins, despite its reaction with a polyclonal anti-integrin antibody.     

Ethanol administration alters the proteolytic activity of hepatic lysosomes

Protein accumulation in liver cells contributes to alcohol-induced hepatomegaly and is the result of an ethanol-elicited deceleration of protein catabolism (Alcohol Clin Exp Res 13:49, 1989). Because lysosomes are active in the degradation of most hepatic proteins, the present studies were conducted to determine whether ethanol administration altered the proteolytic activities of partially purified hepatic lysosomes. Rats were fed liquid diets containing either ethanol (36% of calories) or isocaloric maltodextrin for periods of 2-34 days. Prior to death, all animals were injected with [3H]leucine to label hepatic proteins. Rats subjected to even brief periods of ethanol feeding (2-8 days) exhibited significant hepatomegaly and hepatic protein accumulation compared with pair-fed control animals. Crude liver homogenates and isolated lysosomal-mitochondrial and cytosolic subfractions were incubated at 37 degrees C, and the acid-soluble radioactivity generated during incubation was measured as an index of proteolysis. At neutral pH, in vitro protein breakdown in incubated liver homogenates and subcellular fractions from control and ethanol-fed rats did not differ significantly. The extent of protein hydrolysis increased when samples were incubated at pH 5.5, which approximates the pH optimum for catalysis by lysosomal acid proteases. Under the latter conditions, partially purified lysosomes from control animals had 2-fold higher levels of proteolysis than corresponding fractions from ethanol-fed rats. The difference in proteolytic capacity appeared to be related to a lower latency and a higher degree of fragility of lysosomes from ethanol-fed rats at the acidic pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibition in vivo of lipoprotein lipase (clearing-factor lipase) activity by triton WR-1339

1. Lipoprotein lipase activity was measured in heart homogenates and in heparin-releasable and non-releasable fractions of isolated perfused rat hearts, after the intravenous injection of Triton WR-1339. 2. In homogenates of hearts from starved, rats, lipoprotein lipase activity was significantly inhibited (P less than 0.001) 2h after the injection of Triton. This inhibition was restricted exclusively to the heparin-releasable fraction. Maximum inhibition occurred 30 min after the injection and corresponded to about 60% of the lipoprotein lipase activity that could be released from the heart during 30 s perfusion with heparin. 3. Hearts of Triton-treated starved rats were unable to take up and utilize 14C-labelled chylomicron triacylglycerol fatty acids, even though about 40% of heparin-releasable activity remained in the hearts. 4. It is concluded that Triton selectively inhibits the functional lipoprotein lipase, i.e. the enzyme directly involved in the hydrolysis of circulating plasma triacylglycerols. 5. Lipoprotein lipase activities measured in homogenates of soleus muscle of starved rats and adipose tissue of fed rats were decreased by 25 and 39% respectively after Triton injection. It is concluded that, by analogy with the heart, these Triton-inhibitable activities correspond to the functional lipoprotein lipase.     

Equilibrium-dialysis studies of the interaction between cholic acid and 100000g-supernatant preparations from the rat liver

1. The binding of cholic acid to 100000g supernatants from rat livers was investigated by equilibrium dialysis and gel-exculsion chromatography. 2. Supernatants were found to contain at least two classes of binding site for cholic acid. 3. These recptor molecules are probably proteins since incubation with proteolytic enzymes resulted in complete loss of cholic acid binding. 4. Supernatants were added to columns of Sephadex G-75, and two groups of fractions were shown to bind cholic acid. One of these contained low-affinity binding sites and the other contained both low- and high-affinity binding sites. 5. Feeding cholestyramine had no effect on cholic acid binding. 6. Increased cholic acid binding occurred after injection of phenobarbitone. There was an increase in the amount of the low-affinity component but no change in the high-affinity component. 7. The dissociation constants of the binding of cholic acid suggest that the binding proteins may be involved in bile acid transport.