Biochemical and morphological characterization of subcellular fractions isolated from rabbit colon muscle.

From a homogenate of rabbit colon muscle subcellular fractions were isolated by differential centrifugation. The crude microsomal fraction could be separated into subfractions, a fraction of vesicular microsomes at 35% sucrose, a fraction containing sarcolemma, mitochondrial fragments and microsomal vesicles at 35--45% sucrose and a small protein fraction at 45--55% sucrose. Their biochemical properties and their morphological characterization were investigated. The cholesterol and the phospholipid content was equally distributed between the microsomal fractions 35% and 35--45% while the RNA was localized to the mitochondria and the microsomal fraction 35%. The enzyme cytochrome c oxidase was found to be concentrated in the mitochondria while a high contamination was found in the microsomal fractions 35--45%. The NADH-oxidase activity was highest in the 35% fraction and the 5'-nucleotidase activity in the 40,000 X g supernatant. The microsomal subfractions contained the enzymes ATPase, adenylate cyclase and phosphodiesterase. In the 35% fraction Ca stimulated the hydrolysis of ATP. The binding of [3H]-ouabain and the incorporation of [3H]-leucine was most pronounced in the 35% fraction. In a K+-free Krebs Ringer medium the binding of the glucoside was stimulated in all the fractions. From these results we concluded that the fraction 35% sucrose may be mainly derived from the endoplasmic reticulum and the plasma membrane while the 35--45% originates from the plasma membrane, mitochondria and to a lesser extent the endoplasmic reticulum.     

Some properties of Ca-binding microsomal subfractions isolated from rabbit colon muscle

From a homogenate of rabbit colon smooth muscle a microsomal fraction was isolated, which was divided into subfractions by centrifugation on a discontinuous sucrose gradient. The Ca-binding properties of the subfractions were investigated under different conditions. In the presence of 0.35 mM ATP the Ca binding of the fractions amounted to 4--8 nmol/mg protein. The 35% fraction bound more Ca per mg protein than the 35--45% fraction. The Ca accumulation was comparatively higher both in the presence of 5 mM ATP and in the presence of 5 mM oxalate. The two fractions showed about the same sensitivity for oxalate. This substance stimulated the Ca uptake at 5 mM but not at lower concentrations. The amount and the rate of Ca binding were more dependent on variations in the exogenous ATP concentration in the 35% fraction than was the case for the 35--45% fraction. The Ca binding was completely inhibited by salyrgan when the microsomal fractions were pretreated with this agent. Sodium azide did not influence the Ca-binding capacity of the fractions. It is suggested that the microsomal subfractions of the rabbit colon muscle represent physiologically important parts of the Ca sequestering system of the muscle, since Ca binding takes place at Ca- and ATP-concentrations which are believed to be present in the myoplasm.     

Cyclic AMP and Ca-binding in Microsomal Fractions Isolated from Rabbit Colon Smooth Muscle

Abstract From a homogenate of rabbit colon muscle two ATP dependent Ca-accumulating microsomal fractions were isolated by differential centrifugation on a sucrose density gradient at 35 % and 35-45 % sucrose. Adenylate cyclase and phosphodiesterase activities were found in the fractions. The Ca-accumulation and the ATPase activity of these fractions were stimulated by cyclic AMP (10-⁵ M) at an ATP concentration of 0.35 mM ATP. In the presence of higher concentrations of ATP (5 mM) cyclic AMP had no effect on the Ca-binding. The higher concentration of ATP markedly increased the cyclic AMP formation in relation to the activity found at the lower concentration of ATP. Isoprenaline (2 × 10-⁶ M) stimulated the Ca-accumulation in the 35-45 % fraction and increased the hydrolysis of ATP. These effects were absent in the fraction isolated at 35 % sucrose. In the former fraction isoprenaline also stimulated the adenylate cyclase activity at 0.35 mM but not at 5 mM ATP. Both the effect of isoprenaline on the Ca-binding and the adenylate cyclase activity were inhibited by the adrenergic β-receptor blocking agent sotalol. In the 35-45 % fraction papaverine (1 × 10-³ M) stimulated the Ca-accumulation and inhibited the phosphodiesterase activity. It is suggested that cyclic AMP and agents which influence the cyclic AMP metabolism in the microsomes may have a regulatory role on the Ca-binding of the microsomes.     

Cyclic AMP and Ca-binding in microsomal fractions isolated from rabbit colon smooth muscle.

From a homogenate of rabbit colon muscle two ATP dependent Ca-accumulating microsomal fractions were isolated by differential centrifugation on a sucrose density grandient at 35% and 35-45% sucrose. Adenylate cyclase and phosphodiesterase activities were found in the fractions. The Ca-accumulation and the ATPase activity of these fractions were stimulated by cyclic AMP (10(-5)M) at an ATP concentration of 0.35 mM ATP. In the presence of higher concentrations of ATP (5 mM) cyclic AMP had no effect on the Ca-binding. The higher concentration of ATP markedly increased the cyclic AMP formation in relation to the activity found at the lower concentration of ATP. Isoprenaline (2 X 10(-6)M) stimulated the Ca-accumulation in the 35-45% fraction and increased the hydrolysis of ATP. These effects were absent in the fraction isolated at 35% sucrose. In the former fraction isoprenaline also stimulated the adenylate cyclase activity at 0.35 mM but not at 5 mM ATP. Both the effect of isoprenaline on the Ca-binding and the adenylate cyclase activity were inhibited by the adrenergic beta-receptor blocking agent sotalol. In the 35-45% fraction papaverine (1 X 10(-3)M) stimulated the Ca-accumulation and inhibited the phosphodiesterase activity. It is suggested that cyclic AMP and agents which influence the cyclic AMP metabolism in the microsomes may have a regulatory role on the Ca-binding of the microsomes.     

Evidence for an inherent,ATP-stimulated uptake of calcium into chromaffin granules

Summary Partially purified chromaffin granules (granular fraction), crude mitochondria (mitochondrial fraction) and a microsomal fraction were prepared from bovine adrenals by differential centrifugation and characterized by their catecholamine content, succinate dehydrogenase and glucose-6-phosphatase activity. During isotonic incubation with 0.1 mM ⁴⁵Ca²⁺ all fractions showed an uptake of ⁴⁵Ca²⁺, which was stimulated by ATP. In addition, after incubation the granular fraction was further fractionated by sucrose density gradient centrifugation (2.0–1.3 M sucrose; 170,000g·60 min). In some of these experiments the granular fraction was incubated simultaneously with ⁴⁵Ca²⁺ and [³H](-)noradrenaline to compare the uptake of both. The rate of uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction (characterized by the highest catecholamine content and the lowest succinate dehydrogenase activity) was doubled by ATP. The ATP-stimulated uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules was inhibited by N-ethylmaleimide (NEM) (0.1 mM), 2,4-dinitrophenol (DNP) (0.1 mM), azide (1 mM), carbonyl cyanide mchlorophenylhydrazone (CCCP) (20M), atractyloside (50 M), ruthenium red (40 M) and amobarbital (1 mM). This inhibition pattern was different from that of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the microsomal fraction, but it was similar to that of the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction. However, the following differences are incompatible with the view that a mitochondrial contamination, with a highly active uptake, is responsible for the ATP-stimulated uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules: a) The uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction was insensitive to 1 mM amobarbital, whereas this agent inhibited the uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules. b) Replacement of ATP by succinate stimulated the uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction only. c) The dependence of the ATP-stimulated uptake of ⁴⁵Ca²⁺ on the concentration of ATP (0.1–5 mM) was determined: while uptake into the 2.0 M sucrose fraction of chromaffin granules exhibited saturation kinetics, that into the mitochondrial fraction was linearly related to the concentration of ATP. Interestingly, uptake of ⁴⁵Ca²⁺ into those fractions of chromaffin granules that are known to be contaminated with mitochondria (1.6 M sucrose fraction) exhibited bot a saturable and a nonsaturable component. d) The uptake of ⁴⁵Ca²⁺ into the mitochondrial fraction was more sensitive to 0.1 mM DNP than the uptake of ⁴⁵Ca²⁺ into the 2.0 M sucrose fraction of chromaffin granules.The comparison of the ATP-stimulated uptake of ⁴⁵Ca²⁺ with that of [³H](-)noradrenaline into the 2.0 M sucrose fraction of chromaffin granules revealed that amobarbital, N,N-dicyclohexylcarbodiimide (DCCD), DNP and increasing concentrations of ATP had the same influence on both uptake processes. From our results we conclude that an inherent, ATP-stimulated uptake of ⁴⁵Ca²⁺ exists in chromaffin granules. The effects of agents on the uptake of ⁴⁵Ca²⁺ and/or [³H](-)noradrenaline into chromaffin granules are discussed with regard to the granular uptake mechanisms.This work was supported by the Deutsche ForschungsgemeinschaftSome results were reported at the 18th Spring Meeting 1977 of the Deutsche Pharmakologische Gesellschaft (Burger and Häusler 1977)     

Effects of Cholinergic Drugs and Imidazole on Ca Release and Cyclic AMP Formation in Microsomal Fractions from Rabbit Colon

Abstract Following the addition of carbachol or acetylcholine to microsomal fractions isolated from rabbit colon which were preloaded with Ca, the ions were rapidly released. In the 35-45% fraction Ca was completely released within 10 min., but in the 35 % fraction only 30 % was released. Carbachol reduced the adenylate cyclase activity of the 35-45 % fraction. Both these effects were blocked by atropine. Exogenous cyclic AMP completely inhibited the Ca-releasing action of carbachol in the 35 % fraction and markedly reduced it in the 35-45 % fraction. Imidazole released Ca from the 35-45 % fraction and stimulated its phosphodiesterase activity. It is suggested that the microsomal fractions are parts of a Ca-sequestering system in smooth muscle which are able to bind Ca and which on the addition of some contracting drugs release the ions and thereby activate the contractile system. The release of Ca may partly at least be due to a reduction of the adenylate cyclase activity, although other mechanisms must also be considered.     

Effects of cholinergic drugs and imidazole on Ca release and cyclic AMP formation in microsomal fractions from rabbit colon.

Following the addition of carbachol or acetylcholine to microsomal fractions isolated from rabbit colon which were preloaded with Ca, the ions were rapidly released. In the 35-45% fraction Ca was completely released within 10 min., but in the 35% fraction only 30% was released. Carbachol reduced the adenylate cyclase activity of the 35-45% fraction. Both these effects were blocked by atropine. Exogenous cyclic AMP completely inhibited the Ca-releasing action of carbachol in the 35% fraction and markedly reduced it in the 35-45% fraction. Imidazole released Ca from the 35-45% fraction and stimulated its phosphodiesterase activity. It is suggested that the microsomal fractions are parts of a Ca-sequestering system in smooth muscle which are able to bind Ca and which on the addition of some contracting drugs release the ions and thereby activate the contractile system. The release of Ca may partly at least be due to a reduction of the adenylate cyclase activity, although other mechanisms must also be considered.     

Distribution and nature of epoxide hydrolase activity in subcellular organelles of mouse liver

Mouse liver light and heavy mitochondrial fractions contain significant epoxide hydrolase activity in addition to that present in the cytosol and microsomes. As the mitochondrial fraction itself contains a number of subfractions, experiments were designed to determine the localization of the epoxide hydrolase activity in these subfractions. Subcellular fractions were prepared using livers from 6- to 8-week-old Swiss-Webster male mice. Using trans-stilbene oxide (TSO) as substrate, the highest activity was localized in the cytosolic fraction, followed by the light mitochondrial fraction. Subfractionation of the light mitochondrial fraction by isopycnic sucrose density gradient resulted in the separation of mitochondria from peroxisomes as monitored by marker enzymes. The separation of these two subcellular organelles was also confirmed by the electron microscopic studies. Distribution of TSO-hydrolase activity in the sucrose density gradient fractions closely resembled the activity distribution of the peroxisomal markers catalase and urate oxidase, but significant activity was also found in mitochondria. Treatment of mice with clofibrate selectively induced TSO-hydrolase in the cytosol without affecting this enzyme activity in the peroxisomal fraction. There was no difference in the distribution pattern of TSO-hydrolase and marker enzymes in sucrose density gradients of mitochondrial fractions from clofibrate-treated and control mice. The epoxide hydrolase activity in the peroxisomes is immunologically similar to, and also has the same molecular weight as, the cytosolic epoxide hydrolase.     

Subcellular distribution of phospholipids during liver damage induced by rare earths

After intravenous injection of praseodymium nitrate, female Wistar rats develop fatty livers. In contrast to the marked increase of triglycerides, the phospholipid content was only increased by 50%. The subcellular distribution of phospholipids showed that major changes occur in the microsomal fraction within the first 24hrs. Among the individual phospholipids only phosphatidylcholine and phosphatidylethanolamine concentrations were elevated. Further subfractioning revealed that phospholipid concentration increased in the smooth endoplasmic reticulum, whereas it decreased in the rough endoplasmic reticulum. The individual phospholipids in the smooth endoplasmic reticulum increased to the same degree as did the total phospholipids. On the other hand, in the rough endoplasmic reticulum only the lecithin fraction decreased, while all other phospholipids remained unchanged. Cytochrome P₄₅₀, cytochrome b₅, and glucose 6-phosphatase activity were drastically reduced in the rough endoplasmic reticulum, while no changes could be observed in the smooth endoplasmic reticulum.In the serum, phospholipid concentration fell to half the normal value within the first 24 hrs after praseodymium intoxication.     

Preparation and characterization of subcellular fractions from the liver of the northern pike, Esox lucius

The present study was designed to prepare and characterize subcellular fractions from the liver of the Northern pike (Esox lucius), with special emphasis on the preparation of microsomal fractions suitable for studying xenobiotic metabolism. The purity of the different fractions obtained by differential centrifugation, as well as the recovery of different organelles, was determined using both enzyme markers and morphological examination with the electron microscope. Attempts were also made to increase the recovery of fragments of the endoplasmic reticulum in the microsomal fraction. Finally, the subcellular distribution of several drug-metabolizing enzymes (cytochrome P-450, benzpyrene monooxygenase, epoxide hydrolase and glutathione transferases) were determined. With the exception of the subcellular distribution of epoxide hydrolase, the results obtained here resemble closely those reported for rat liver and the microsomal fraction prepared is highly suitable for further studies of drug metabolism in pike liver.     

Comparison of the Effect of DDE on the Ca Metabolism of the Eggshell Gland and its Subcellular Fractions of the Duck and the Domestic Fowl

Abstract: In a strain of ducks sensitive to the eggshell-thinning effect of p-p'-DDE, administration of 40 mg/kg of the compound in the food for 45 days reduced the eggshell index (EI) by 13% and the content of calcium in the fluid of the shell gland forming an eggshell by 36%, and raised the calcium content of the shell gland mucosa by 19%, compared with the control values. DDE inhibited the translocation (secretion) of calcium between the gland mucosa and the uterine cavity. The ATP-dependent binding of Ca²⁺ to the subcellular fractions of the gland mucosa was reduced in DDE-treated ducks. The Ca²⁺ binding to a microsomal subfraction (FI) rich in fragments of the plasma membrane was reduced by 16%, whereas that to a subfraction Fill which bound Ca²⁺ at a very high rate was reduced by 36%. The latter may contain calcium-secreting granules of the gland. In the mitochondrial fraction the Ca²⁺ binding was reduced by 35%. In the domestic fowl DDE did not lower EI or interfered with the translocation of calcium between the shell gland mucosa and uterine cavity. DDE administration increased the Ca²⁺ binding to FI by 26%; the binding to other subfractions was not changed significantly. DDE may interfere with the stimulus-secretion mechanism of the eggshell gland in ducks through its effect on Ca²⁺ binding.     

Hepatic subcellular distribution of [H]T-2 toxin

and . Hepatic subcellular distribution of [³H]T-2 toxin. Toxicon 27, 1307–1311, 1989.—The subcellular distribution of T-2 mycotoxin and its metabolites was studied in isolated rat livers perfused with [³H]T-2 toxin. After a 120-min perfusion, the distribution of radiolabel was to bile 53%, perfusate 38% and liver 7%. Livers were fractionated into mitochondria, endoplasmic reticulum (smooth and rough), plasma membrane and nuclei. Plasma membrane fractions contained 38% of the radiolabel within 5 min, decreasing to < 1% at the end of the 120-min perfusion. Smooth endoplasmic reticulum contained 27% of the radiolabel by 5 min and increased to 43% over the 120-min perfusion. The mitochondrial fraction contained 3% of the radiolabel by 30 min and increased to 10% after 120-min perfusion. Label in the nuclear fraction remained constant at 7% from 30 to 120 min. By 15 min, only the parent toxin was detected in the mitochondrial fraction. In the other fractions, radiolabel was associated with HT-2, 4-deacetylneosolaniol, T-2 tetraol, and glucuronide conjugates. Glucuronide conjugates accounted for radiolabel eliminated via the bile. The time course for distribution of radiolabel in liver suggested an immediate association of [³H]T-2 with plasma membranes and a subsequent association of toxin and metabolites with endoplasmic reticulum, mitochondria and nuclei, the known sites of action of this toxin.     

Comparison between procaine and isocarboxazid metabolism in vitro by a liver microsomal amidase-esterase.

Characteristics of a liver microsomal amidase with isocarboxazid (ISOC) as substrate were compared to characteristics of a liver microsomal esterase with procaine (PROC) as substrate. Both amidase and esterase activities were mainly localized in the microsomal fraction with low or null activities in other fractions. Higher specific activities were found in smooth endoplasmic reticulum (s-ER) compared with rough endoplasmic reticulum (r-ER). The microsomal amidase-esterase has a pH optimum of 8.5 to 9.0 and a K_m for ISOC of 0.19 mM and for PROC of 0.53 mM. On the basis of sensitivity to certain esterase inhibitors, this amidase-esterase was considered to be a carboxylesterase type; however, no divalent cations were found to activate the amidase-esterase. Since there were no significant differences between ISOC and PROC in their enzymic properties, i.e. subcellular localization, stability, pH optimum and susceptibility to inhibition, it is possible to speculate that this amidase-esterase is responsible for the metabolism of various drugs possessing amido or ester linkage.     

Effects of quinidine on calcium transport activities of the rabbit heart mitochondria and sarcotubular vesicles.

The effects of quinidine on the calcium-transporting properties of sarcotubular (heavy microsomal) and mitochondrial fractions of the rabbit heart were investigated and compared with those of procaine amide and lidocaine. High concentrations of quinidine (10^?4-10^?3 M) markedly decreased microsomal and mitochondrial calcium uptake; these inhibitory actions were observed at all concentrations of ATP but not when low concentrations of Mg²⁺ were used in the incubation medium. Calcium binding by mitochondrial fraction, unlike microsomal fraction, was also decreased by high concentrations of quinidine. Procainc amide (10^?3M) had no effect on microsomal or mitochondrial calcium uptake whereas lidocaine (10^?3 M) showed a stimulatory action only on the microsomal calcium uptake at initial intervals of incubation. The ATPase activities of both microsomal and mitochondrial fractions were inhibited by quinidine but not by procaine amide or lidocaine. Quinidine, unlike procaine amide and lidocaine. was also found to release about 15% of the calcium bound by microsomes and mitochondria. The results presented in this study suggest that quinidine impairs microsomal and mitochondrial calcium transport; however, other antiarrhythmic agents such as procaine amide and lidocaine, which in high concentrations also depress myocardial contractility, do not decrease calcium transport by heart subcellular particles.     

Haloperidol-sensitive (+)[3H]SKF-10,047 binding sites (σ sites) exhibit a unique distribution in rat brain subcellular fractions

The distribution of haloperidol-sensitive (+)[³H]N-allylnormetazocine ((+)[³H]SKF-10,047) binding sites (σ sites) in subcellular fractions of rat brain homogenates was extensively characterized. In synaptosomal fractions, enriched in choline acetyltransferase activity, σ sites were present in lower concentrations than in whole brain homogenates. On the other hand, microsomal and myelin fractions were found to be enriched in σ sites. A similar pattern of enrichment was seen for 5′-nucleotidase activity, a general plasma membrane marker. However, subsequent experiments in which microsomes were subfractionated on linear sucrose gradients led to the recovery of σ sites over a significantly lower density range than 5′-nucleotidase activity or ATP-stimulated [³H]ouabain binding, an additional plasma membrane marker. In addition, previously reported distributions of a number of other subcellular markers, including those for endoplasmic reticulum, were found to contrast with the observed distribution of σ sites. It is concluded that rat brain σ sites are not concentrated at synaptic regions of plasma membrane. However, the possibility that σ sites are localized to specialized areas of nonsynaptic plasma membrane cannot be excluded.     

Spiperone: a ligand of choice for neuroleptic receptors. 3. Subcellular distribution of neuroleptic drugs and their receptors in various rat brain areas.

Tissue fractionation was used as an analytical tool to study in various rat brain regions the subcellular distribution of the neuroleptic receptor and labeled neuroleptics previously injected into the animals. In order to assess the composition of the different subcellular fractions, the distribution pattern of various marker enzymes was also determined.After differential centrifugation of rat striatum homogenate, [³H]spiperone binding receptors together with 5′ nucleotidase were found to be mainly enriched in the microsomal fraction. Similarly, after injection of [³H]spiperone or [³H]pimozide into rats, the radioactivity was specifically recovered in the microsomal fraction in the striatum, the olfactory tubercle and nucleus accumbens and the frontal cortex but not in the cerebellum.After equilibration through a sucrose density gradient, the distribution pattern of [³H]spiperone revealed a main peak in a gradient region of low density very close to that of 5′ nucleotidase. The present results indicate that the receptor sites of neuroleptic drugs in the brain dopaminergic areas is associated with membrane-like structures but not with mitochondria or nerve terminals containing dopamine.     

Decreased incorporation of14C-leucine in different liver nuclear protein fractions at early stages of carbon tetrachloride poisoning in the rat

Administration of CCl₄ to 12–14 h starved Sprague Dawley male rats (220–240 g) at a dose of 5 ml of a 20% (v/v) olive oil solution/kg i.p. decreased the incorporation of¹⁴C-leucine into microsomal and nuclear proteins at 1 or 2 h after the hepatotoxin. Decreased amino acid incorporation capacity in nuclear but not in microsomal proteins returned to control values at 4 h after poisoning. Cycloheximide given i.p. at a dose of 1 mg/kg in saline either alone or before CCl₄ also decreased to a similar or greater extent the incorporation of¹⁴C-leucine into both, microsomal and nuclear proteins at 1 h. The CCl₄-induced decrease in amino acid incorporation was observable and significant even 1 h after CCl₄ and involved histone and different non-histone nuclear protein fractions. Results suggest that decreased protein synthesis at the endoplasmic reticulum occurring in the very early stages of CCl₄ poisoning might provoke temporary deficiencies in protein supplies to nuclei with as yet unknown consequences. However, some of the proteins involved have major regulatory properties in DNA expression.     

Effects of glucagon on 45Ca outflow exchange in the isolated perfused rat heart

The effects of glucagon on cellular calcium distribution in cardiac muscle were studied using an isolated, spontaneously-beating, perfused rat heart preparation and a ⁴⁵Ca outflow exchange technique. The rate of loss of ⁴⁵Ca from the cellular space (intracellular calcium and calcium bound to extracellular sites) was determined, using [¹⁴C]sucrose to estimate the rate of loss of ⁴⁵Ca from the extracellular space. The data were analysed by an iterative non-linear, least squares curve-fitting procedure. The simplest system which was found to be consistent with ⁴⁵Ca outflow exchange data obtained for control and glucagon-treated hearts is one in which three kineticaliy-distinct compartments of exchangeable calcium are associated with the cellular space. For control hearts, the fractional transfer rates (rate constants) for the outflow of calcium from compartments 1–3 were 0.61, 0.14 and 0.022min^?1, respectively. Measurement of the amounts of ⁴⁵Ca present in subcellular fractions of perfused hearts indicated that compartment 3 represents calcium present in intracellular stores, including mitochondria and possibly the sarcoplasmic reticulum. The predominant effects of glucagon were to increase the fractional transfer rate for calcium outflow from compartment 1, and the quantity of exchangeable calcium in compartment 3.     

Subcellular localization of human placental aryl hydrocarbon hydroxylase

Homogenates of human placentas were fractionated by a variety of differential centrifugation techniques. Prepared subfractions were assayed by ultrastructure analysis and marker enzyme quantitation. The lowest ratios of cytochrome oxidase/aryl hydrocarbon hydroxylase activities were observed in a 6.24 × 10⁶g-min sediment fraction following sequential precentrifugations at 1.5 × 10⁴, 9.75 × 10⁴, 2.18 × 10⁵, 3.03 × 10⁵ and 4.64 × 10⁵g-min. Investigations of subfractions with respect to acid phosphatase, glucose-6-phosphatase, nucleoside disphosphatase, glucose-6-phosphate dehydrogenase, and aryl sulfatases A, B, and C also tended to support a concept of localization of the mixed-function oxidase system in the endoplasmic reticulum of human placental cells. Consistent with these chemical observations was the enrichment of membranous components seen in electron micrographs of the respective homogenate subfractions.     

Ca2+-uptake into noradrenaline-storing granules of bovine splenic nerves

Summary Noradrenaline-storing granules, a mitochondrial fraction and a microsomal fraction of bovine splenic nerve trunks were prepared by differential centrifugation. These particulate fractions were characterized by their noradrenaline content, succinate dehydrogenase and glucose-6-phosphatase activity. In the presence of ATP-Mg²⁺ all three fractions accumulated ⁴⁵Ca²⁺ during incubation with 0.1 mM ⁴⁵CaCl₂, buffered with potassium phosphate or glycylglycine (pH 7.5; 28°C). The accumulated ⁴⁵Ca²⁺ was not removable by EGTA, and the uptake was absent at 0°C or after destruction of the particles by sonication. The behaviour of the ⁴⁵Ca²⁺-uptake into all three fractions against varying ATP-concentrations, metabolic inhibitors (pentachlorophenol, desaspidine, 2,4-dinitrophenol, N-ethylmaleimide, p-chloromercuribenzoate, sodium azide, amobarbital) and drugs (phenoxybenzamine, verapamil, prenylamine, reserpine, bretylium, phentolamine) was studied. Under nearly all conditions there were differences between the ⁴⁵Ca²⁺-uptake into mitochondria and that into microsomes, which suggests two distinct uptake processes. The ⁴⁵Ca²⁺-uptake into the granule fraction behaved intermediate between the two other fractions under many conditions, but not under all. Therefore, it is not possible to explain the ⁴⁵Ca²⁺-uptake into the granule fraction as being due to contamination with mitochondria and microsomes; an inherent ATP-Mg²⁺-dependent ⁴⁵Ca²⁺-uptake into the nerve granules must be postulated, which is not directly coupled with the noradrenaline transport into these particles.A particulate fraction (14000–1000000 g), containing noradrenaline granules, was prepared from the vas deferens of the rat. Incubation with 5×10^–6 M (-)-noradrenaline and 0.1 mM ⁴⁵Ca²⁺ showed that the particles of this fraction take up noradrenaline and ⁴⁵Ca²⁺. The uptake of both was dependent on ATP-Mg²⁺. The ATP-Mg²⁺-dependent uptake of both noradrenaline and ⁴⁵Ca²⁺ was substantially reduced in the corresponding tissue fraction prepared from denervated vasa deferentia.This work was supported by the Deutsche ForschungsgemeinschaftPreliminary accounts were presented at the 12th spring meeting 1971 of the Deutsche Pharmakologische Gesellschaft (Burger and Philippu, 1971)