Distribution of newly synthesized amylase in microsomal subfractions of guinea pigs pancreas

Amylase distribution was studied in guinea pig pancreas microsomes fractionated by centrifuging, for 2 hr at 57,000 g in a linear 10 to 30% sucrose gradient, a resuspended high speed pellet obtained after treating microsomes with 0.04% deoxycholate (DOC).¹ Amylase appeared in the following positions in the gradient: (a) a light region which contained ∼35% of total enzymic activity and which coincided with a monomeric ribosome peak; (b) a heavy region which contained ∼10% of enzymic activity in a sharp peak but which had very little accompanying OD₂₆₀ absorption; (c) a pellet at the bottom of the centrifuge tube which contained ∼20% of the enzymic activity. After 5 to 20 min'' in vivo labeling with leucine-1-C¹⁴, radioactive amylase was solubilized from these three fractions by a combined DOC-spermine treatment and purified by precipitation with glycogen, according to Loyter and Schramm. In all cases, the amylase found in the pellet had five to ten times the specific activity (CPM/enzymic activity) of the amylase found in the light or heavy regions of the gradient. The specific radioactivity (CPM/mg protein) of the proteins or peptides not extracted by DOC-spermine was similar for all three fractions. Hypotonic treatment of the fractions solubilized ∼80% of the total amylase in the fraction from the heavy region of the gradient, but only ∼20% of the amylase in the monomer or pellet fraction. Electron microscope observation indicates that the monomer region of the gradient contained only ribosomes, that the heavy region of the gradient contained small vesicles with relatively few attached ribosomes, and that the pellet was composed mostly of intact or ruptured microsomes with ribosomes still attached to their membranes. It is concluded from the above, and from other evidence, that most of the amylase activity in the monomer region is due to old, adsorbed enzyme; in the heavy region mostly to enzyme already inside microsomal vesicles; and in the pellet to a mixture of newly synthesized and old amylase still attached to ribosomes. Furthermore, the ribosomes with nascent, finished protein still bound to them are more firmly attached to the membranes than are ribosomes devoid of nascent protein.     

The intracellular distribution of calcium in the mucosa of the avian shell gland

The intracellular distribution of calcium has been studied in the mucosa of the avian shell gland, a tissue which transports large quantities of calcium during discrete time intervals. Ca⁴⁵ was administered to hens either in a single dose followed by sacrifice 5 min later or in repeated doses over an extended period followed by sacrifice 2 hr or 24 hr after the last injection. Subcellular fractions were isolated by differential centrifugation and analyzed for Ca⁴⁵. The Ca⁴⁵ was located principally in the particulate fractions; the concentration (CPM Ca⁴⁵/mg N) was highest in the mitochondrial fraction. Comparisons of (1) the Ca⁴⁵ distribution in shell gland cells with that of liver cells, (2) the alterations which occur due to the phase of the egg laying cycle, (3) the effects due to the time elapsed since the last injection of Ca⁴⁵, and (4) the Ca⁴⁵ distribution of the short term experiments with that of the long term experiments revealed that the mitochondrial fraction of the shell gland appeared to be active in the movement of calcium. The microsomal fraction showed increased values in CPM Ca⁴⁵/mg N when calcification was occurring, which may indicate that the subcellular components of this fraction have a role in calcium transport. The nuclear and supernatant fractions did not seem to be involved in the transport process. The implications of these results concerning the manner by which calcium may be controlled on a cellular level in this system are discussed.     

Enzymatic esterification of vitamin a in the pigment epithelium of bovine retina

The kinetic properties and subcellular distribution of an esterifying enzyme in the pigment epithelium of bovine retina have been studied using both [1-³H]retinol and [³H]retinol bound to cellular retinol-binding protein as substrates. The most active esterifying fraction in pigment epithelial cell preparations was the microsomes, but the lysosome plus mitochondria fraction also showed some activity, probably due to endoplasmic reticulum present as an impurity. The microsomal enzyme showed optimum activity at pH 7.5, and the reaction was linear up to 30 μg protein and for the first 10–15 min. The apparent K_m values were 16.6 · 10^?6 and 5.5 · 10^?6 M for [³H]retinol and bound [³H]retinol, respectively. This is the first time that retinol bound to cellular retinol-binding protein has been shown to undergo metabolic stransformation. The microsomal esterifying activity was destroyed by boiling for 1 min, or after freezing for 2 months. No clear requirement for ATP, CoA or fatty acid could be demonstrated.Of all the other tissues examined under the same experimental conditions as those used for the pigment epithelium, onlt intestine showed measurable activity. With larger amounts of tissue protein and longer incubation periods, activity was also detectable in microsomes of liver, testis and retina     

THE NUCLEAR ANNULI AS PATHWAYS FOR NUCLEOCYTOPLASMIC EXCHANGES

Colloidal gold particles, 25 to 55 A in diameter, which had been coated with polyvinylpyrrolidone, were microinjected into the ground cytoplasm of amebas (Chaos chaos). At time intervals of 1 minute, 2 minutes, 10 minutes, and 24 hours after injection the cells were fixed for electron microscopy. After 24 hours, gold particles were found in both the nuclei and the ground cytoplasm, the concentration being higher in the nuclei. Colloidal particles were also present in the nuclei after 10 minutes, but at this time interval the concentration did not appear to be greater than that in the ground cytoplasm. One and 2 minutes after injection, the gold particles were located almost exclusively in the ground cytoplasm; however, individual particles were often found within the annuli of the nuclear envelope, and were located specifically in the centers of these structures. The results suggest that at least some of the gold particles which enter the nuclei pass through the annuli, and that passage through these structures may be restricted to a central channel.     

Comparison of 45Ca2+ uptake activity by microsomes from control and stimulated mouse pancreatic acini

The ability of microsomal preparations to transport ⁴⁵Ca²⁺ was studied in preparations of control and secretagogue-stimulated pancreatic acini. ATP-dependent ⁴⁵Ca²⁺ uptake activity was present in the pancreatic post-mitochondrial supernatant and microsomes but little activity was present in the postmicrosomal supernatant. Treatment of acini with the secretagogues cholecystokinin (CCK) and carbamylcholine (CCh) prior to cell fractionation increased the subsequently measured microsomal ⁴⁵Ca²⁺ uptake. The effect of CCK was maximal after 10 min stimulation and at a not. The effect of CCK was maximal after 10 min stimulation and at a concentration of 1 nM; these conditions are comparable to the effects of CCK on ⁴⁵Ca²⁺ fluxes in intact acini. The increased microsomal ⁴⁵Ca²⁺ uptake induced by CCK was due to an increase in the maximal rate of ⁴⁵Ca²⁺ uptake as there was no effect on the K_m for Ca²⁺ (1 μM). It is concluded that secretagogues increase the ATP-dependent uptake of ⁴⁵Ca²⁺ by an isolated pancreatic microsomal component under the same conditions that also stimulate both digestive enzyme secretion and bi-directional Ca²⁺ movements.     

Intracellular transport of mouse kidney β-glucuronidase induced by gonadotrophin

1. The response of renal beta-glucuronidase with time to the injection of gonadotrophin was investigated in each submicrosomal fraction of rough and smooth microsomal fractions of mouse kidney homogenate. 2. The increase in beta-glucuronidase activity appeared initially in membranes of the rough microsomal fraction, 24h after injection. 3. Afterwards the newly synthesized enzyme appeared in the contents of the rough microsomal fraction and was subsequently found in the smooth microsomal fraction, reaching a maximum concentration in this fraction at 72h. 4. At this juncture, a decrease in the enzyme activity was observed in rough microsomal contents whereas the lysosomal fraction had reached its maximum value. 5. The time-course of the appearance of beta-glucuronidase in the submicrosomal fractions after the gonadotrophin stimulation suggests that the newly synthesized enzyme at the site of membrane-bound ribosomes is transferred across the membrane into cisternae of the rough endoplasmic reticulum, and then is transported into lysosomes via the smooth endoplasmic reticulum. 6. The properties of microsomal and lysosomal beta-glucuronidases were compared.     

CHARACTERISTICS OF THE ISOPRENALINE STIMULATED PROLIFERATIVE RESPONSE OF RAT SUBMAXILLARY GLAND

A simple stochastic model has been developed to determine the cell cycle kinetics of the isoprenaline stimulated proliferative response in rat acinar cells. The response was measured experimentally, using ³H-TdR labelling of interphase cells and cumulative collections of mitotic cells with vincristine. The rise and fall of the fraction of labelled interphase cells and of metaphase cells is expressed by the product of the proliferative fraction and a difference of probability distributions. The probability statements of the model were formulated and then compared by an iterative fitting procedure to experimental data to obtain estimates of the model parameters. The model when fitted to the combined fraction labelled interphase (FLIW) and fraction metaphase (FMW,) waves gave a mean G_is transit time of 21-2 hr, mean G_is+ S transit time of 270 hr, and mean G_is+ S + G₂ transit time of 35-8 hr for a single injection of isoprenaline, where G_is is the initiation to S phase time. When successive injections of isoprenaline were given at intervals of 24 and 28 hr the corresponding values after the third injection were 12-4 hr, 20-8 hr and 25-7 hr respectively. The variance of the G_is phase dropped from 18-1 to 1–3 while the other variances remained unchanged. The estimated proliferative fraction was 0–24 after a single injection of isoprenaline, and 0–31 after three injections of the drug. Independently determined values of the proliferative fraction, obtained from repeated ³H-TdR injections, were 0–21 and 0–36 respectively.     

Lung surfactant synthesis: a Ca++-dependent microsomal phospholipase A2 in the lung.

We present the first direct evidence for a highly active, Ca⁺⁺-dependent phospholipase A₂ in the microsomal fraction of rat lung homogenate. Several previously reported studies from other laboratories strongly implicate this enzyme as a key metabolic step in the biosynthesis of dipalmitoyl lecithin, the primary component of pulmonary surfactant. In the present study, stoichiometric amounts of [³H]lysophosphatidylethanolamine and [¹⁴C]fatty acid were released during incubation of 1-[9, 10-³H]palmitoyl-2-sn-[1′-¹⁴C]linoleoyl phosphatidylethanolamine with the lung microsomal fraction. Marker enzyme measurements showed that the microsomal activity cannot be due to contamination with mitochondria, which also show phospholipase A₂ in both lung and liver. In contrast, liver microsomes show predominantly a phospholipase A₁ activity.     

Dynamics of the distribution of labeled etimizol in brain cell structures

The time course of 14C-etimizol distribution in cell structures of the rat cerebral cortex was studied. Two minutes after intraperitoneal injection etimizol penetrates brain cells. At this time the greater portion of the drug is found in cytosol. In five minutes the maximal part of the label gets bound with cell nuclei and microsomal protein fraction extracted with 0.14 M NaCl. These two cell fractions show the highest radioactivity throughout the entire observation period (up to 7 days). Since these fractions are reported to be capable of calcium accumulation, a suggestion is made that metabolic effects of etimizol are likely to be related to calcium metabolism.     

Solubilization and characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: A novel,participate glutathione S-transferase

Rat basophil leukemia cell homogenates effectively catalyze the conversion of leukotriene A₄ to a mixture of leukotrienes C₄ and D₄ in the presence of glutathione. These homogenates also catalyze the formation of adducts of halogenated nitrobenzene with glutathione, as determined spectrophotometrically. While all the classical glutathione S-transferase activity resides in the soluble fraction of the homogenates, the thiol ether leukotriene-generating activity is found in the particulate fraction. This “leukotriene C synthetase” activity has been solubilized from a crude high-speed particulate fraction by means of the nonionic detergent, Triton X-100. The solubilized enzyme is incapable of converting 2,4-dinitrochlorobenzene to a colored product in the presence of glutathione. Nor will it react with 3,4-dichloronitrobenzene. On the other hand, under optimal conditions, this enzyme preparation is capable of generating about 0.1 nmol leukotriene C mg protein^?1 min^?1 in a reaction which continues in linear fashion for at least 10 min. This dissociation in substrate specificity, as well as differences in the inhibition profile, distinguish the enzyme activity in the particulate fraction from rat basophil leukemia cell homogenates from the microsomal glutathione S-transferase which has been described in rat liver homogenates, suggesting that this “leukotriene C synthetase” is a new and unique enzyme.     

Intra-amniotic prostaglandin F2α and urea for abortion

Prostaglandin F_2α (PGF_2α) 20 mg combined with urea 80 g was injected intra-amniotically in 20 patients to induce mid-trimester abortion. Abortion resulted in all subjects within 24 hours in a mean time of 12 hours 38 minutes (range 5 hours 50 minutes to 20 hours 45 minutes).Plasma sex steroids were evaluated before and hourly for 5 hours after the injection. A progressive decline in levels occurred with time. Decreases in plasma progesterone, estrone, estradiol and estriol were significant as soon as one hour after injection.Gastrointestinal side effects occurred with a greater frequency than when a comparable dose of PGF_2α is given alone and 2 patients had small cervical lacerations requiring suture. Further studies are indicated to establish whether a lower dose of PGF_2α will be associated with fewer side effects and be as effective.     

On the synthesis and degradation of the multiple forms of catalase in mouse liver

The incorporation of ⁵⁵Fe-labeled ferrous sulfate and ³H-labeled γ-aminolaevulinic acid into the catalase of mouse liver was measured at intervals up to 96 hr after intraperitoneal injection, and the intracellular location of radioactive catalase followed, as well as the distribution of radiolabel between the multiple forms of this enzyme. At 10 min, catalase radioactivity was present in all the cellular fractions studied, but after this time, label began to disappear from the microsomal fraction and from the peroxisomal detergent extract. By comparison, catalase incorporation reached a peak at about 6 hr in the peroxisomal aqueous extract, and rose to a broad peak after about 30 hr in the cytosol fraction. On resolving the multiple forms of catalase in the supernatant fraction by electrophoresis, it was found that label first appeared in the fastest moving heteromorph, and appeared sequentially in the other multiple forms over a period of 96 hr.The sequence of degradation of catalase was also studied by examination of residual catalase activity subsequent to the injection of allyl-isopropyl acetamide, a heme synthesis antagonist which blocks catalase synthesis. Blood catalase levels did not seem to be significantly affected by this treatment, but in the liver, the decay rates of catalase activity were appreciable, and varied significantly between the intracellular pools. The rate of decrease was greatest in the peroxisomal detergent extract, and least in the supernatant fraction.These findings have been discussed in relation to current understanding of the subcellular disposition, multiplicity, and turnover of hepatic catalase.     

Incorporation of [14C]glucosamine and [14C]leucine into mouse kidney β-glucuronidase induced by gonadotrophin

Male BALB/C mice were injected intraperitoneally with 2.5 i.u. of gonadotrophin. After the injection, increase of β-glucuronidase activity was first observed in the microsomal fraction. By 36h 45–50% of the total homogenate activity was found in the microsomal fraction compared with 20–25% in the control microsomal fraction. From 36 to 80h not only microsomal β-glucuronidase but also lysosomal β-glucuronidase increased progressively. After 69h stimulation with 2.5 i.u. of gonadotrophin, d-[1-¹⁴C]glucosamine or l-[U-¹⁴C]leucine was injected intraperitoneally. After a further 3h the kidneys were homogenized and five particulate fractions were prepared by differential centrifugation. The β-glucuronidase in the microsomal and lysosomal fractions was released respectively by ultrasonication and by freezing and thawing treatment. The enzyme was purified by organic-solvent precipitation and by sucrose-density-gradient centrifugation. The results demonstrated the incorporation of these two labels into the mouse renal β-glucuronidase. The microsomal β-glucuronidase was much more radioactive than the lysosomal enzyme and approx. 80% of the newly synthesized enzyme appeared in microsomes and approx. 20% of that was found in lysosomes at this period. These results suggest that the mouse renal β-glucuronidase is a glycoprotein and that the newly synthesized enzyme is transported from endoplasmic reticulum to lysosomes.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase. A comparison of the modulation in vitro by phosphorylation and dephosphorylation to modulation of enzyme activity by feeding cholesterol- or cholestyramine-supplemented diets

The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase) was considerably inhibited during incubation with ATP+Mg²⁺. The inactivated enzyme was reactivated on further incubation with partially purified cytosolic phosphoprotein phosphatase. The inactivation was associated with a decrease in the apparent K_m of the reductase for hydroxymethylglutaryl-CoA, and this was reversed on reactivation. The slight increase in activity observed during incubation of microsomal fraction without ATP was not associated with a change in apparent K_m and, unlike the effect of the phosphatase, was not inhibited by NaF. Liver microsomal fraction from rats given cholesterol exhibited a low activity of hydroxymethylglutaryl-CoA reductase with a low apparent K_m for hydroxymethylglutaryl-CoA. Mícrosomal fraction from rats fed cholestyramine exhibited a high activity with a high K_m. To discover whether these changes had resulted from phosphorylation and dephosphorylation of the reductase, microsomal fraction from rats fed the supplemented diets and the standard diet were inactivated with ATP and reactivated with phosphoprotein phosphatase. Inactivation reduced the maximal activity of the reductase in each microsomal preparation and also reduced the apparent K_m for hydroxymethylglutaryl-CoA. There was no difference between the preparations in the degree of inactivation produced by ATP. Treatment with phosphatase restored both the maximal activity and the apparent K_m of each preparation, but never significantly increased the activity above that observed with untreated microsomal fraction. It is concluded that hydroxymethylglutaryl-CoA reductase in microsomal fraction prepared by standard procedures is almost entirely in the dephosphorylated form, and that the difference in kinetic properties in untreated microsomal fraction from rats fed the three diets cannot be explained by differences in the degree of phosphorylation of the enzyme.     

Antimutagenic properties of a polyphenol-enriched extract derived from sesame-seed perisperm

A polyphenolic mixture derived from sesame-seed perisperm (SSP) strongly reduced the mutagenicity of hydrogen peroxide (H₂O₂), sodium azide (NaN₃), and benzo[a]pyrene (BaP) in strains TA100 and/or TA98 of Salmonella typhimurium. It exhibited desmutagenic activity against H₂O₂, BaP in TA98 and/or TA100 and biomutagenic activity (apparently by affecting the DNA-repair system) against NaN₃ in strain TA100. According to in vitro experiments the polyphenolic mixture inhibited the activity of the CYP1A1 (EROD) enzyme responsible for the activation of BaP in the Ames’ test, as well as that of the cytosolic enzyme GST.A cytosolic fraction from liver of male Wistar rats treated with either 20% SSP in the food, or 3 mg or 6 mg of polyphenolic mixture/20 g food/day for a time period of 8 weeks reduced the mutagenic potential of BaP in strains TA100 and TA98, with the cytosolic fraction from rats treated with SSP causing the strongest reduction. Furthermore, a microsomal fraction from the 20% SSP-treated rats inhibited the mutagenicity of BaP in strains TA100 (26.3%) and TA98 (23%). In contrast, a microsomal fraction from rats treated with 3 mg of polyphenolic mixture stimulated the mutagenicity of BaP in TA100 but reduced it in TA98, while for the microsomal fraction from rats treated with 6 mg of polyphenolic mixture, these effects on TA100 and TA98 were reversed.     

Uptake and intracellular transport in rat liver of formaldehyde-treated bovine serum albumin labelled with 125I-tyramine-cellobiose

1. Endocytosis of formaldehyde-treated bovine serum albumin by rat liver sinusoidal cells has been followed by injecting rats with the protein labelled with 125I-tyramine cellobiose (125I-TCfBSA). 125I-TCfBSA is quickly taken up by the liver; the radioactivity present in the organ reaches a plateau 5-10 min after injection and is maintained for up to at least 180 min. During the first 5 min most of radioactivity remains acid-precipitable. After which, labelled acid-soluble components are produced at a constant rate for up to 30-40 min. 2. Differential centrifugation shows that radioactivity is first recovered mainly in the microsomal fraction. Within a few minutes it exhibits a distribution pattern similar to that of lysosomal enzymes, being chiefly located in the mitochondrial fractions. 3. Isopycnic centrifugation in a sucrose gradient of the microsomal fraction isolated 1 min after injection indicates a similar distribution for radioactivity and alkaline phosphodiesterase. Later, the microsomal radioactivity distribution curve is shifted towards higher densities and becomes distinct from that of the plasma-membrane enzyme. After isopycnic centrifugation in a sucrose gradient of the total mitochondrial fraction a considerable overlapping of acid-precipitable and acid-soluble radioactivity distributions is observed without significant changes with time. The same is observed in a Percoll gradient except that after a relatively long time (greater than 30 min) of injection a marked shift of radioactivity distribution towards higher densities occurs. 4. A pretreatment of rats with Triton WR 1339, a density perturbant of liver lysosomes, causes a striking shift of acid-soluble radioactivity distribution in a sucrose gradient towards lower densities while having markedly less influence on the acid-precipitable distribution. As a result, a distinction between the distribution of both kinds of radioactivity becomes clearly apparent. A preinjection of yeast invertase, modifies the acid-soluble distribution without having a significant effect on the acid-precipitable distribution up to 30 min after 125I-TCfBSA injection. 5. Glycyl-1-phenylalanine-2-naphthylamide largely releases acid-soluble radioactivity associated with the mitochondrial fraction, whatever the time after 125I-TCfBSA injection. On the other hand the proportion of acid-precipitable radioactivity present in the fraction that can be released is almost zero at 10 min after injection, and it later increases. 6. The results presented here are best explained by supposing that, after being trapped in small pinocytic vesicles, 125I-TCfBSA is quickly delivered to the endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of the System for the Mixed Function Oxidation of Kaurene and Kaurene Derivatives in Microsomes of the Immature Seed of Marah macrocarpus: Electron Transfer Components

Cytochrome P-450 and cytochrome b₅ at levels of approximately 0.10 and 0.60 nanomole per milligram of microsomal protein were detected by spectral measurements in microsomes prepared from endosperm tissue of immature Marah macrocarpus seeds. TPNH-cytochrome c reductase, DPNH-cytochrome c reductase, andDPNH-cytochrome b₅ reductase activities were also present in these microsomes at levels of approximately 0.060, 0.22, and 0.52 unit per milligram of microsomal protein, respectively. (One unit of reductase is the amount of enzyme catalyzing the reduction of 1 micromole of electron acceptor per minute.) Treatments of microsomes with steapsin or trypsin were not effective in solubilizing any of these electron transport components in detectable form. However, treatment of a microsomal suspension in 25% glycerol with 1% sodium deoxycholate led to the release of about 60% of the protein and each of the above hemoproteins and electron transfer activities to the fraction which was not pelleted after centrifugation for 2 hours at 105,000g. Some ent-kaur-16-ene oxidase activity could be detected in the solubilized fraction after removal of the detergent. Cytochrome b₅ and DPNH-cytochrome b₅ reductase activity were largely separated from one another and from an overlapping mixture of TPNH-cytochrome c reductase and DPNH-cytochrome c reductase when the sodium deoxycholate-solubilized fraction was chromatographed on a DEAE-cellulose column. No cytochrome P-450 or cytochrome P-420 was detected in the column fractions and no ent-kaur-16-ene oxidase activity was detected when the column fractions were tested singly or in combination.     

Atomic force microscope visualization of lipid bilayer degradation due to action of phospholipase A2 and Humicola lanuginosa lipase

An important application of liquid cell Atomic Force Microscopy (AFM) is the study of enzyme structure and behaviour in organized molecular media that mimic in-vivo systems. In this study we demonstrate the use of AFM as a tool to study the kinetics of lipolytic enzyme reactions occurring at the surface of a supported lipid bilayer. In particular, the time course of the degradation of lipid bilayers by Phospholipase A₂ (PLA₂) and Humicola Lanuginosa Lipase (HLL) has been investigated. Contact mode imaging allows visualization of enzyme activity on the substrate with high lateral resolution. Lipid bilayers were prepared by the Langmuir-Blodgett technique and transferred to an AFM liquid cell. Following injection of the enzyme into the liquid cell, a sequence of images was acquired at regular time intervals to allow the identification of substrate structure, preferred sites of enzyme activation, and enzyme reaction rates.     

A comparative study of the effects of cadmium and nickel on liver microsomal drug metabolizing enzymes of guinea-pig in vitro

1. In vitro addition of cadmium chloride (CdCl₂) or nickel chloride (NiCl₂) to an incubation mixture produced a concentration-dependent inhibition of liver microsomal aniline 4-hydroxylase activity of male guinea-pig. The inhibitory effect of CdCl₂ on the enzyme activity was stronger than that of NiCl₂.2. While CdCl₂ also caused a concentration-dependent inhibition of liver microsomal ethylmorphine N-demethylase activity, NiCl₂ increased the enzyme activity between the concentrations 10⁻⁵ and 10⁻³ M and caused a rather abrupt decline at higher concentrations.3. When the liver 10,000 g supematants were preincubated in the presence of metals, metal-induced inhibitions increased as the time of preincubation progressed and attained their maximal rates at about 5 and 15 min for microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase activities, respectively. However, no change was noted by NiCl₂ on liver microsomal ethylmorphine N-demethylase activity as the time of preincubation progressed.4. After preincubations, the concentration-dependent inhibitions produced by metals on liver microsomal drug metabolizing enzyme activities were found to be stronger and in favour of CdCl₂.     

Characterization of cell membrane and sarcoplasmic reticulum from bovine uterine smooth muscle

Subcellular fractions, enriched in sarcoplasmic reticulum or in cell membrane, were separated from one another. Starting material was a microsomal pellet (15–40 × 1000g) obtained by differential centrifugation from the uteri of close-to-term pregnant cows. A microsomal fraction enriched in ATP-dependent calcium accumulation was shown to contain sarcoplasmic reticulum and cell membrane. Only 8% or less of the protein in this fraction could be recovered, using affinity chromatography on Sepharose 6MB wheat germ agglutinin. The small yields did not allow extensive characterization. A method was developed to separate sarcoplasmic reticulum from cell membrane using discontinuous sucrose density gradient centrifugation. Protein was collected at the 24–28, the 28–33, and the 33–45% sucrose interfaces. Characterization was by enzyme assays and by specific receptor assay. ATP-dependent calcium accumulation was fourfold greater in the 24–28% sucrose layer than in the 33–45% layer. In contrast, 5′-nucleotidase was more than threefold as high in the 33–45% sucrose layer as in the 24–28% layer. Ouabain-inhibited p-nitrophenylphosphatase doubled and ouabain-inhibited Na,K-ATPase tripled in the 28–33% layer, compared with the 24–28% layer, specific ouabain binding was also doubled in the 28–33% sucrose layer. ¹²⁵I-Labeled wheat germ agglutinin binding was greatest in the 33–45% sucrose layer. It is concluded that the 24–28% layer consists primarily of sarcoplasmic reticulum, whereas the 28–33 and the 33–45% layers are concentrated in the cell membrane. Specific prostaglandin (PGE₂) binding was found to be a property of the cell membrane.