Microsomal Material Research Articles

Rat liver microsomal palmitoyl-coenzyme A synthetase. Structural properties.

The structural properties of purified palmitoyl-CoA synthetase from rat liver microsomal material were investigated. The active enzyme has a molecular weight of 168000 and contains about 8.2mol of phospholipid bound/mol of enzyme protein, as well as bound fatty acids. Association of the active enzyme was shown to occur, without impairment of catalytic activity. The lowest-molecular-weight species obtained under denaturing conditions was 27000 daltons.

Adrenal microsomal C19-steroid 5alpha-reductase activity in the Snell transplantable rat adrenocortical tumour 494 and the effect of oestradiol, testosterone propionate and adrenocorticotrophin in intact and gonadectomized rats.

The C(19)-steroid 5alpha-reductase activity in the microsomal fraction of rat adrenal tissue under various hormonal treatments was examined. In intact control rats the activity is similar in both males and females, and after gonadectomy it is markedly increased. Treatment with oestradiol (150mug/day per animal for 7 days) or testosterone propionate (2mg/day per animal for 7 days) lowered the activity of 5alpha-reductase in castrated animals to approximately the values for intact animals in both sexes, and in intact animals the activity was also decreased by these treatments. The enzyme activity was also decreased by adrenocorticotrophin treatment but to a lesser extent than by the steroid hormones. The activity of the 5alpha-reductase enzyme in the Snell adrenocortical tumour 494 is very low when incubated as a whole homogenate, but the activity in microsomal material of the tumour was measured and unexpectedly found to be similar to that in intact controls.

Isolation and characterization of a rough microsomal fraction from rat kidney that is enriched in lysosomal enzymes.

1. A special population of rough microsomal material (microsomes) rich in lysosomal acid hydrolases was separated by isopycnic centrifugation as a discrete fraction (RM(2)) from the bulk of rough microsomal material in rat kidney because of its greater density. 2. The specific activities of five acid hydrolases in the RM(2) fraction were approximately one-half those of a purified lysosomal (L) fraction and 10- to 30-fold greater than those of an ordinary rough microsomal (RM(1)) fraction. 3. These special rough microsomes have a distinctive ultrastructure and electron-cytochemical properties. Their cisternal content resembles the matrix of lysosomes in that it is electron-dense, osmiophilic and plumbophilic and gives a positive reaction for acid phosphatase activity. 4. Polyacrylamide-gel electrophoresis of soluble proteins from the L fraction resolved nine anionic glycoproteins, most of which exhibit acid hydrolase activities (Goldstone & Koenig, 1970, 1973; Goldstone et al., 1971a). The most anionic glycoprotein is the acidic lipoglycoprotein of the lysosomal matrix (Goldstone et al., 1970). 5. Polyacrylamide-gel electrophoresis of soluble proteins from the RM(2) fraction resolved two cationic glycoproteins with acid hydrolase activities (Goldstone & Koenig, 1973) and an anionic glycoprotein with the same electrophoretic mobility as the lysosomal lipoglycoprotein, but without its lipid constituents or capacity to bind the basic fluorochrome Acridine Orange. These constituents are considered to be the precursors of the lysosomal glycoproteins.

Subfractionation of Rabbit Skeletal-Muscle Microsomal Fraction

In rabbit skeletal muscle a well-defined and highly organized system of internal membranes exists. These were first described by Retzius (1881) and Veratti (1902), subsequently overlooked and later rediscovered by Porter & Palade (1957) and Andersson-Cedergren (1959) and others. The skeletal-muscle cell is composed of three main structural components. These are, first, the myofibrils or contractile elements, the chief components of which are the thick and the thin filaments. The second group of structural components embraces the mitochondria, nuclei and ribosomes. Lysosomes have been reported in fractions prepared by the fractionation of skeletal muscle; however, their origin is obscure (Stagni & De Bernard, 1968). The internal membrane systems and the external membrane constitute the third group of structural components. The cell membrane or sarcolemma and the intracellular membrane systems together control the initiation of contraction and the subsequent relaxation of the muscle cell. Homogenization of skeletal muscle results in the formation of small rounded-off vesicles by an active pinching-off process of the various membrane systems (Palade & Siekevitz, 1956). These vesicles are to be found in the microsomal fraction (de Duve, 1964; Reid, 1967). The microsomal fraction would be expected to contain, in addition to the vesicles derived from the sarcoplasmic reticulum and from the transverse tubule system, elements of the outer cell membrane or sarcolemma and possibly vesicles arising by fragmentation of the mitochondria during the homogenization. Many enzymic activities have been demonstrated cytochemically to be associated with the internal membrane systems. Among these are ATPase* (EC 3.6.1.4) (Zebe, 1965) and acetylcholinesterase (EC 3.1.1.7) (Karnofsky, 1964). The ATPase activities associated with the internal membrane systems have been extensively studied since the discovery of the relaxing factor (Marsh, 1952). For a review of the various muscle ATPases see Duggan (1971). The ATP-dependent Ca2+-transporting system, which is responsible for the lowering of the sarcoplasmic Ca2+ concentration and thereby resulting in relaxation of the muscle, is present in the sarcoplasmic reticulum. Another microsomal ATPase activity has been found that is active in the absence of Ca2+ (Weber et al., 1966; Duggan, 1968b). This is the Ca2+-independent or basal ATPase. White skeletal muscle of the rabbit was homogenized in ice-cold 1 .OM-sucrose, forming a 25 % (w/v) homogenate. After a preliminary centrifugation for lOmin at 4°C at 4OOOg in an MSE High Speed 18 centrifuge (rotor no. 69179) the sediment was discarded. The supernatant, after filtration through several layers of cheesecloth, was diluted with half its volume of ice-cold distilled deionized water. This diluted supernatant was then centrifuged for 30min at 4°C at 15000g in the same centrifuge and rotor as used above. Again the sediment was discarded and the supernatant filtered through several layers of cheesecloth. This filtered supernatant served as the starting material for the preparation of concentrated microsomal suspensions. The preparation of concentrated microsomal suspensions was achieved by using the BXIV zonal rotor and a large sample volume (450ml). The microsomal material was sedimented from this sample into a lOOml layer of 1.0M-sucrose by centrifugation for 1 h at 4°C at 30000rev./min in an MSE Super Speed 50 centrifuge. After centrifugation the microsomal material was collected in four 25ml fractions. Analysis for the various enzymes and protein were carried out as described previously (Headon & Duggan, 1970). Fraction 2 was found to contain much of the total ATPase and Ca2+-uptake activities.

Isolation and characterization of membranes from normal and transformed tissue-culture cells

Homogenates of baby-hamster kidney cells and rat embryo fibroblasts prepared by nitrogen cavitation contain a small population of slowly sedimenting mitochondria or mitochondrial fragments, which contaminate the microsomal fraction. This appears to limit the resolution of surface membrane and endoplasmic reticulum on magnesium-containing dextran gradients. The microsomal material and mitochondria can, however, be completely separated on a 10-60% (w/w) sucrose zonal gradient containing a 30% sucrose plateau. On magnesium-containing dextran gradients this mitochondria-free microsomal material can be resolved into at least two surface membrane fractions and at least two endoplasmic reticulum fractions. Comparison of polyoma virus-transformed and normal baby-hamster kidney cells reveals some interesting differences in their microsomal fractionation patterns and the characteristics of the Na(+)/K(+)-Mg(2+) adenosine triphosphatase of their surface membranes, in particular a tenfold lower K(m) in the virus-transformed cells. The fractionation patterns of normal and spontaneously transformed rat embryo fibroblasts are also briefly discussed, particularly in relation to the significance of the observation that both the surface membrane and endoplasmic reticulum from these cells can be subfractionated.

Effects of safrole and isosafrole pretreatment on N- and ring-hydroxylation of 2-acetamidofluorene by the rat and hamster.

1. The effects of safrole and isosafrole pretreatment on both N- and ring-hydroxylation of 2-acetamidofluorene were studied in male rats and hamsters. 2. Isosafrole (100mg/day per kg body wt.) pretreatment of rats for 3 days did not have any effect on urinary excretion of hydroxy metabolites of 2-acetamidofluorene. However, similar pretreatment with safrole produced increased urinary excretion of N-, 3- and 5-hydroxy derivatives. 3. Similar treatment with these two chemicals for 3 days increased ring-hydroxylation activity by rat liver microsomal material. Increases in N-hydroxylation were much less than those in ring-hydroxylation. Isosafrole was twice as effective as safrole. 4. Increases in hydroxylating activity due to safrole or isosafrole treatment were inhibited by simultaneous administration of ethionine. Similarly, ethionine inhibition was almost completely reversed by the simultaneous administration of methionine. 5. Safrole or isosafrole (0.1mm and 1mm) inhibited 7-hydroxylation activity by liver microsomal material from control rats. At 1mm these two chemicals inhibited both 5- and 7-hydroxylation activity by liver microsomal material from 3-methylcholanthrene-pretreated rats. 3-Hydroxylation activity was not inhibited by 1mm concentrations of these two chemicals. 6. A single injection of safrole (50100 or 200mg/kg body wt.) 24h before assay had no appreciable effect on either N- or ring-hydroxylation activity by hamster liver microsomal material. However, isosafrole (200mg/kg body wt.) treatment inhibited N-, 3- and 5-hydroxylation activities by hamster liver microsomal material; it had no effect on 7-hydroxylation activity.

Preparation of plasma membrane from isolated neurons.

A bulk fraction enriched with respect to neuronal cell bodies was used as starting material for the isolation of neuronal plasma membrane The cells were gently homogenized in isotonic sucrose and a crude membrane containing fraction sedimented at 3000 g. Subsequently, the membrane fraction was purified on a discontinuous sucrose density gradient between 35% and 25 5% sucrose (w/w). Enzymatic analyses showed a 4-5-fold enrichment in plasma membrane markers, and a 10-15% contamination of mitochondrial and microsomal material. Electron micrographs of the membrane fraction confirmed the enzymatic data Fragmented membranes were found, mainly in vesicular form No ribosomes, but a few mitochondria and some multilamellar membranes were seen

Glycerolipid synthesis in milk: Evidence of glycerol kinase and other biosynthetic enzymes

1. 1. Freshly secreted bovine milk serum actively incorporated labelled glycerol into the various glycerolipid classes occurring in milk, particularly triglycerides, phosphatidylcholine, and phosphatidic acid. 2. 2. Addition of long-chain fatty acids and ATP significantly enhanced the amount of glycerol utilized. 3. 3. The evidence suggests that microsomal material containing the enzymes responsible for glycerolipid synthesis is secreted with other milk components.

Cation exchange binding of rubidium and cesium by rat liver cell microsomes.

AbstractThe rubidium and cesium binding characteristics of rat liver cell microsomes were studied by an equilibration, centrifugation and washing procedure. Concentration dependence experiments, in which microsomes were equilibrated in media containing 0 to 400 mM rubidium or cesium chloride at pH 6.9, yielded saturation type adsorption isotherms similar to those previously reported for sodium and potassium. Mass law analysis of the data yielded apparent dissociation constants of 21 × 10−3 eq/liter and 19 × 10−3 eq/liter for rubidium and cesium binding, respectively. The results indicate that cesium is bound slightly more strongly than rubidium, and that both these cations may be bound more strongly than sodium or potassium. The maximum binding capacity at pH 6.9 was approximately 1.3 meq rubidium or cesium/g nitrogen. Sodium, potassium, magnesium and calcium generally associated with the isolated microsomes decreased concomitantly with increasing bound rubidium or cesium, demonstrating the ion exchange nature of the binding. Results of pH‐dependence experiments showed that following equilibration of the microsomes in media containing approximately 96 mM rubidium or cesium at various pH values, bound rubidium or cesium was essentially zero at pH less than five, increased sharply between pH 5 and 7, and tended to level off at higher pH. The present results further characterize the cation binding properties of the microsomal material.

Protease and ribonuclease activities in bovine pituitary lobes.

1. Acid and alkaline protease activities in bovine anterior and posterior pituitary lobes were reinvestigated by measurement of u.v. and Folin-Ciocalteu colour values of trichloroacetic acid-soluble digestion products of denatured haemoglobin. 2. Both lobes of the pituitary gland contain a cathepsin with a pH optimum at 3.8. 3. When release of u.v.-absorbing material was used as the assay there was also an optimum at pH8.3-9.7, but this proved to be due to the release of nucleosides from an endogenous substrate. 4. The presence of a ;cyclizing' ribonuclease active at alkaline pH on endogenous RNA was confirmed by the inhibitory effects of phosphate, arsenate and bentonite. The activity was unaffected by heat, EDTA or metal ions. The enzyme also acted on exogenous RNA. 5. A purified preparation of neurosecretory granules from fresh bovine posterior pituitary lobes was free from alkaline ribonuclease activity. Most of the activity present in the tissue was recovered in the supernatant plus microsomal material. 6. The distribution of RNA did not follow that of the alkaline ribonuclease.

Ion association reactions with biological membranes, studied with the fluorescent dye 1-anilino-8-naphthalenesulfonate

(1) When salts are added to buffered suspensions of membrane fragments containing the fluorochrome 1-anilino-8-naphthalenesulfonate (ANS), there is an increased fluorescence. This is caused by increased binding of the fluorochrome; the intrinsic fluorescence characteristics of the bound dye remain unaltered. These properties make ANS a sensitive and versatile indicator of ion association equilibria with membranes. (2) Alkali metal and alkylammonium cations bind to membranes in a unique manner. Cs(+) binds most strongly to rat brain microsomal material, with the other alkali metals in the order Cs(+)>Rb(+)>K(+)>Na(+)>Li(+). The reaction is endothermic and entropy driven. Monovalent cations are displaced by other monovalent cations. Divalent cations and some drugs (e. g., cocaine) displace monovalent cations more strongly. (3) Divalent cations bind to membranes (and to lecithin micelles) at four distinct sites, having apparent association constants between 50 and 0.2MM (-1). The characteristics of the titration suggest that only one species of binding site is present at any one time, and open the possibility that structural transitions of the unassociated coordination sites may be induced by divalent cation binding. Divalent cation binding at the weakest site (like monovalent cation binding) is endothermic and entropy driven. At the next stronger site, the reaction is exothermic. Monovalent cations affect divalent cation binding by reducing the activity coefficient: they do not appear to displace divalent cations from their binding sites.

New observations on β-glucuronidase in human cervical cancer

β-glucuronidase activity was measured in cytoplasmic fractions prepared from malignant and non-malignant human cervix uteri. Enzyme activity in the supernatant fraction from malignant tissue was very much greater than that from non-malignant specimens, relative to protein content and tissue weight. No significant difference was found in β-glucuronidase activity/mg protein of the mitochondrial and microsomal material from malignant and non-malignant tissues. The cancer specimens with very high β-glucuronidase activities showed a different cytoplasmic distribution of the enzyme from those with lower β-glucuronidase activities. It appeared that the higher enzyme activities were due to increases in the free soluble cytoplasmic enzyme and not particle-bound enzyme.

Investigation of mixed function oxidation by means of chemiluminescence

The chemiluminescent oxidation of luminol in the peroxidase-O 2 system and the microsomal NADPH oxidase system has been analyzed. 1. 1. It is concluded that the chemiluminescence is probably mainly due to the formation of H 2O 2 which is utilized for catalytic peroxidation of luminol, and not directly due to the action of free radicals. On the other hand, the formation of H 2O 2 may involve the free, ionized perhydroxyl radical as an intermediate. 2. 2. By quantitative determination of the amounts of dihydroxyfumaric acid consumed by radiolytic oxidation in aqueous solution, it was demonstrated that free perhydroxyl radicals readily attack the redogenic donor dihydroxyfumaric acid. 3. 3. The NADPH-dependent oxidation of luminol which is catalyzed by liver microsomes is greatly stimulated by menadione, a reaction which is accompanied by an increased rate of NADPH oxidation. The reaction is inhibited by p-chloromercuribenzoate, diphenylphenylenediamine, cytochrome c and substrates susceptible to peroxidation like ascorbate and pyrogallol. The reactions did not involve cytochrome P-450 but required the participation of NADPH-cytochrome c reductase. The menadione-independent system was found to be verty labile upon storage, and only the menadione-independent activity could be solubilized. 4. 4. Under certain conditions, the catalase test for involvement of H 2O 2 may lead to erraneous conclusions. Due to the very high affinity of microsomal material for H 2O 2, only very high concentrations of externally added catalase will affect microsomal reactions involving small amounts of H 2O 2.

Inhibition of Enzyme Activities by Free Fatty Acids

The inhibition of several enzyme activities by free fatty acids has been examined. The inhibition of rat liver microsomal linolenate-activating enzyme activity by linolenate was found to be due to the inactivation of enzyme by substrate fatty acid. Stearoyl coenzyme A was also found to inhibit linolenate-activating enzyme. Likewise, microsomal glucose 6-phosphatase was inhibited by oleate; the extent of inhibition by oleate was related to the relative concentration of microsomal material. Citrate synthase from pig heart was inhibited by linoleate and glucose 6-phosphate dehydrogenase of yeast was inhibited by oleate. The inhibition of glucose 6-phosphate dehydrogenase was time-dependent and the presence of glucose 6-phosphate gave considerable protection against inactivation. The marked similarity in the observed inhibition of diverse enzymes by long chain unsaturated fatty acids to that of reported inhibitions by fatty acyl-CoA esters suggests that inhibitory effects are due to detergent properties of these inhibitors. In view of these and other considerations it is suggested that the extrapolation of observed inhibitions in vitro of enzyme activities by free fatty acids to regulation of metabolism in vivo must be made with caution.

Fatty acid biosynthesis. IV Properties of acetyl-CoA carboxylase in lactating-rabbit mammary gland

1. 1. The distribution of acetyl-CoA carboxylase between the microsomal and particle-free supernatant fractions of lactating-rabbit mammary gland has been investigated. Conditions are described which radically alter the distribution of the enzyme between the two fractions. The presence of acetyl-CoA carboxylase in the microsomal fraction is not an artefact of preparation. 2. 2. Repeated washing of the microsomal fraction with 0.3 M sucrose caused a preferential release of acetyl-CoA carboxylase of high specific activity. Addition of KCl to the sucrose did not increase the release. Attempts to release the particulate enzyme by sonication or detergents were unsuccessful. 3. 3. Sucrose density gradient studies showed a single acetyl-CoA carboxylase (which could be completely sedimented) in the microsomal plus particle-free supernatant fraction. This enzyme had a density lower than that of the majority of microsomal protein, but higher than that of the majority of soluble protein present. The enzyme in the microsomal fraction behaved as did the enzyme in the microsomal plus particle-free supernatant fraction. The enzyme recovered in the particle-free supernatant fraction ( i.e., after separation from microsomal material) had changed its molecular characteristics into those of a soluble enzyme. 4. 4. Acetyl-CoA carboxylase in the particle-free supernatant fraction of lactatingrabbit mammary gland was unusual in binding onto microsomal material from a number of tissues including lactating-rabbit mammary gland. In contrast, the microsomal fraction from lactating-rabbit mammary gland could bind much more soluble enzyme from this tissue than from the other tissues investigated.

Distribution of Nephric Antigens in Australian Vertebrates

NOTABLE improvements in immunohistological techniques and the availability of a new fauna prompted us to apply anti-kidney sera previously described1 to further studies of the species distribution of kidney-specific antigens. We used two sera prepared by immunizing rabbits with microsomal material from human and hamster kidneys respectively, and examined their cross-reactivity with the kidneys of other species by immunofluorescence2. Contrary to the earlier reports that cross-reactions are limited to mammals, birds and fish, we found that both sera cross-reacted with all kidneys examined from vertebrates of every class. There was also some cross-reaction with seminal vesicle epithelium in the few species studied.

Species distribution of gastrointestinal antigens.

IMMUNOFLUORESCENCE provides a simple means of studying the species distribution of antigenic components of animal tissues1. The availability of gastrointestinal-specific antisera as immunohistological reagents prompted us to explore the distribution of gastrointestinal antigens in mammalian and other vertebrate fauna of Australia. Two types of sera were used. One, prepared in rabbits by immunization with microsomal material from fresh surgical specimens of human colonic mucosa, had been previously shown to react with an acid mucopolysaccharide component of human and some other mammalian gastrointestinal mucosal cells and with no other tissue in the body2. The other, obtained from patients with pernicious anaemia, contains naturally occurring autoantibody to human gastric parietal cells; this antibody has also been shown not to be species-specific3.

SODIUM-POTASSIUM-ACTIVATED ATPASE AND POTASSIUM-ACTIVATED P-NITROPHENYLPHOSPHATASE: A COMPARISON OF THEIR SUBCELLULAR LOCALIZATIONS IN RAT BRAIN.

The association of a Na+-K+-activated ATPase (ATP phosphohydrolase, EC 3.6.1.4.) with the active transport of these cations is supported by substantial evidence.l-4 An effect of sodiuml ions on a brain ATPase was first reported by Utter.5 Further work has led to the concept that the Na+-K+-ATPase activity may be a resultant of transphosphorylations within the cell membrane which could couple phosphate bond energy directly to cation transport.6-s Consequently, the enzyme system would be an integral component of specialized membranes. Several workers have suggested that the final step in the ATPase reaction sequence may involve a K+-activated phosphatase6 9 and in particular, a phosphoprotein phosphatase.6 Since phenyl phosphate serves as substrate for some phosphoprotein phosphatasesl? and a K+-activated p-nitrophenylphosphatase occurs in brain,ll the structural association of a K+-activated phosphatase with the Na+-K+-ATPase in well-defined subcellular units would support their possible functional relationship. However, the operationally defined subcellular fractions of brain as prepared in various laboratories have produced discordant accounts of ATPase localizations: Deul and McIlwainl2 found Na+-activated ATPase mainly in cerebral microsomes; Schwartz et al.13 found 75 per cent of the Na+-activated ATPase associated with microsomal material other than ribosomes; JirnefeltL4 studied brain homogenates in 0.25 Mf sucrose in which the major ATPase activity was found in a heavy microsomal fraction recovered after centrifuging at 25,000 g X 45 min. In a more recent paper by Hayashi et al.,15 a large percentage of activity was found in a nuclear fraction containing cell debris and intact cells and also in the mitochondrial and microsomlal fractions.

CHANGES IN THE COMPOSITION OF RABBIT LIVER AND OF LIVER MICROSOME FRACTIONS FOLLOWING BIRTH.

The conditions for maximal recovery of the microsome fraction from rabbit liver homogenates have been established. Glucose‐6‐phosphatase is a poor index of the recovery of microsomal material from a cell sap microsome fraction mixture, as it is more rapidly sedimented than RNA and total protein. The sedimentation of glucose‐6‐phosphatase parallels that of microsomal phospholipid.The changes in composition of neonatal rabbit livers and liver microsome fractions were determined. The concentrations of dry matter, phospholipid and RNA are relatively constant during neonatal development, but total lipid, which is about 15–20 per cent of wet weight at birth, drops to adult levels in about 10 days. Total nitrogen increases from about 65 per cent of adult values at birth to adult values during the month after birth. The number of nuclei and the liver DNA content per unit wet weight of liver drops by about two‐thirds during the first week after birth. This is probably due to the loss of erythropoietic cells. The mean DNA per liver nucleus alters little in the first 2 weeks after birth and is then about 20 per cent below the adult value.The microsomal RNA and nitrogen concentrations are relatively constant during neonatal development. The main microsomal change is an increase of phospholipid, adult concentrations being reached 2–4 weeks after birth.

Gangliosides, phospholipids, protein and ribonucleic acid in subfractions of cerebral microsomal material.

Research Article| August 01 1962 Gangliosides, phospholipids, protein and ribonucleic acid in subfractions of cerebral microsomal material JR WHERRETT; JR WHERRETT Search for other works by this author on: This Site PubMed Google Scholar H McILWAIN H McILWAIN Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1962) 84 (2): 232–237. https://doi.org/10.1042/bj0840232 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation JR WHERRETT, H McILWAIN; Gangliosides, phospholipids, protein and ribonucleic acid in subfractions of cerebral microsomal material. Biochem J 1 August 1962; 84 (2): 232–237. doi: https://doi.org/10.1042/bj0840232 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1962 The Biochemical Society1962 Article PDF first page preview Close Modal You do not currently have access to this content.