Phenylalanine ammonia lyase and cinnamic acid hydroxylases as assembled consecutive enzymes on microsomal membranes of cucumber cotyledons: Cooperation and subcellular distribution

1. Cooperation between phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and cinnamic acid hydroxylases was investigated using microsomal fractions from cotyledons of cucumber (Cucumis sativus L.). The interpretations were based on experiments which demonstrate a limited exchange between the pool of cinnamic acid formed by the membrane-bound phenylalanine ammonia-lyase and the cinnamic acid pool external to the enzyme-membrane system. 2. The extent of cooperation between the microsomal enzymes was proved to be influenced by treatment of the cotyledons with light. On exposure to UV-light, which is known to enhance greatly the soluble phenylalanine ammonia-lyase activity in cell cultures, differential effects on the levels of microsomal and soluble phenylalanine ammonia-lyase, and of cinnamic acid hydroxylases, were observed. The time course of the enzyme activities and their cooperation in vitro after treatment of the cotyledons with light were studied. 3. The extent of cooperation in vitro was found to vary depending on the concentration of L-phenylalanine. 4. Homogenates obtained from etiolated cotyledons of Cucumis sativus in the absence of Mg²⁺ were fractionated by sucrose density gradient centrifugation and examined for phenylalanine ammonia-lyase, cinnamic acid o-hydroxylase, cinnamic acid o-hydroxylase, and several marker enzymes. Ammonia-lyase activity was highest in fractions with 25% sucrose, in which primarily smooth endoplasmic reticulum is localized. Hydroxylase activities co-occur with phenylalanine ammonia-lyase in these fractions (density=1.100 g/cm³), and also in fractions at higher densities (d=1.12–1.13 and 1.15 g/cm³).Abbreviations PAL L-phenylalanine ammonia-lyase - Tris tris-(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - ATPase ATP phosphohydrolase     

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.     

Colchicine-binding activity in particulate fractions of mouse brain

Both particulate and soluble fractions of brain homogenates bound [³H]colchicine. Approximately one-half of the total colchicine-binding activity in mouse brain was found in the particulate fraction. Of the particulate fractions, the microsomal and nerveending subfractions which sediment at the 1·0–1·2 m interface on sucrose gradients were richest in colchicine-binding activity. Intact microtubules were not found in these fractions, but colchicine-binding activity of these fractions may be related to the presence of microtubular protein.     

ISOLATION AND PROPERTIES OF SECRETORY GRANULES FROM RAT ISLETS OF LANGERHANS : I. Isolation of a Secretory Granule Fraction

A method has been devised for the isolation of a secretory granule fraction from isolated rat islets of Langerhans. The islets were homogenized in buffered sucrose, and the homogenate was separated into nuclear, mitochondrial, secretory granule, and microsomal fractions by differential centrifugation. The secretory granule fraction was purified by differential centrifugation in discontinuous sucrose density gradients. A greater degree of purification could be achieved by the use of two successive gradients of this type, although the final yield was greatly reduced. Biochemical and morphological characterization of the fractions was obtained; the secretory granule fraction contained both insulin and glucagon. The limiting membranes of the granules remained intact and the general appearance of the granules was similar to that seen within the whole islet cells.     

Development and intracellular localization of lipase activity in rapessed (Brassica napus L.) cotyledons

In homogenates of resting rapeseeds no lipase activity (glycerolester hydrolase, EC 3.1.1.3) could be detected using a titrimetric assay procedure. Following a 30-h lag-phase after imbibition, lipase activity increased sharply, reaching its maximum at day 4 after sowing. Simultaneously triglyceride content of the cotyledons decreased sharply. At any time during the 11-day period of seedling growth examined, only an alkaline lipase activity with a pH optimum around 9 was present. White light had essentially no effect on the development of lipase activity. However, the disappearance of lipase activity from the cotyledons after fat utilization was found to depend on nitrogen nutrition of the seedlings. The activities of the glyoxysomal enzymes catalase and malate synthetase showed the usual rise and fall patterns with peak activities at day 4 after sowing, independently of the mineral nutrition of the seedlings.About 90% of the lipase activity was associated with a microsomal membrane fraction. Resolution of this fraction by sucrose density gradient centrifugation (62,000 g for 14 h) yielded three distinct membrane fractions. Maximum activities of membrane marker enzymes were recovered from the gradients at following densities: The major portion of microsomal protein and lipase activity at 1.085 kg/l; microsomal malate synthetase and phosphorylcholineglyceride transferase at 1.116 kg/l; NADH-cytochrome c reductase and phosphorylcholinecytidyl transferase at 1.133 kg/l. Evidently in rapeseed cotyledons lipase activity is associated only with a discrete microsomal membrane fraction which sediments differently from membrane fractions of the endoplasmic reticulum.     

Variation in microsomal subfractions obtained from normal animal tissues and transformed cells following cell disruption by nitrogen cavitation

Summary Microsomal membranes were obtained from MPC-11 cells, L-cells, Krebs II ascites cells and various normal animal tissues following cell disruption by nitrogen cavitation. Membrane preparations were applied to discontinuous sucrose gradients designed to separate three fractions — heavy rough (HR), light rough (LR) and smooth (S) microsomes. In each of the transformed cell lines all three fractions were found whilst in the normal tissues tested the HR fraction was absent. Of the normal tissues liver and pancreas were rich in both LR and S microsomes, the presence of large amounts of LR indicating a rich protein synthesizing activity on membrane-bound polysomes. Kidney also contained appreciable LR but much less than both liver and pancreas. Both heart and lung contained virtually only S microsomal material — a reflection of low protein synthetic activity on membrane-bound polysomes. Attempts to promote the appearance of the HR fraction in liver, kidney and pancreas by incubation in tissue culture medium, or, in the case of pancreas, by cholecystokinin/pancreozymin/secretin, stimulation bothin vivo andin vitro were unsuccessful.     

PREPARATION AND CHARACTERIZATION OF A PLASMA MEMBRANE FRACTION FROM ISOLATED FAT CELLS

A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25–30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.     

Regional and subcellular distribution of choline acetyltransferase in the brain of rats

—Homogenates of corpus striatum, cerebral cortex and hypothalamus excised from rat brain were fractionated on discontinuous Ficoll and sucrose density gradients, and the distribution of choline acetyltransferase (ChAc) in the mitochondrial and synaptosomal fractions was determined. In the hypothalamic and cortical regions the fractions enriched in synaptosomes showed much higher activity of ChAc than those containing mainly mitochondria. On the other hand, the corpus striatum showed an equal distribution of ChAc activity in those two fractions. The localization of ChAc was also studied in the postnuclear supernatants obtained from three brain regions, using continuous sucrose density gradients. The distribution of ChAc was compared to that of monoamine oxidase (MAO), potassium and protein. When the pellets obtained from the fractions collected from the gradient were suspended in sucrose, the peak of ChAc activity was close to that of MAO in all three brain regions. When 0.1 mm EDTA +1% butanol was used in order to liberate the occluded form of ChAc, the maximum liberation occurred in lighter fractions, resulting in a shift of the activity peak toward the top of the gradient. This was found with fractions from hypothalamic and cortical regions. In the striatum, the liberated ChAc remained in the same fractions as the occluded enzyme. The results indicate that ChAc is liberated only in those fractions where it is present in synaptosomes. In agreement with the results on the discontinuous gradients this occurs in particles of lower density than mitochondria in cortex and hypo-thalamus, but in particles of similar density to mitochondria in the corpus striatum, indicating regional differences in the distribution of ChAc in the brain. K+ containing particles centrifuged in less dense fractions than those containing ChAc, indicating that synaptosomes are heterogeneous with respect to these two marker substances.     

Sub-cellular fractionation of Trypanosoma brucei. Isolation and characterization of plasma membranes

A procedure is described for the isolation of sub-cellular fractions from bloodstream forms of Trypanosoma brucei. The method leaves intact most of the nuclei, mitochondria and microbodies. All the fractions have been chemically characterized and tested for 10 enzymatic markers. About 5% of total cell protein was isolated as a microsomal fraction containing mostly plasma membranes and endoplasmic reticulum vesicles. Plasma membranes were purified by high-speed centrifugation on magnesium-containing Dextran, and on linear sucrose-density gradients. The yield of membranes was approximately 0.3% of the total cell protein. The purified material had a sucrose density of 1.14 g/cm3 and consisted of smooth vesicles. Specific activity of the membrane markers Na+, K+, ouabain-sensitive ATPase and adenylate cyclase were 26- and 20-fold higher, respectively, than in total cells. Neither DNA nor RNA was detected. The sum of the cholesterol and phospholipid content was 0.99 mg/mg protein. The cholesterol/phospholipid molar ratio was 1:2.     

Heterogeneous distribution of glucose 6-phosphatase in rat liver microsomal fractions as shown by adaptation of a cytochemical technique

1. A novel technique for the subfractionation of rat liver smooth and rough microsomal fractions according to their content of glucose 6-phosphatase is described. This technique, based on the Gomori lead histochemical procedure, involves incubation of smooth and rough microsomal fractions with low concentrations of Pb(NO(3))(2) and glucose 6-phosphate. Control experiments, in which enzyme was assayed in the presence of various amounts of Pb(NO(3))(2) or in which microsomal fractions were reisolated after incubation with low concentrations of Pb(NO(3))(2) and glucose 6-phosphate, showed that lead does not interfere with glucose 6-phosphatase activity. 2. Discontinuous sucrose-density-gradient centrifugation of microsomal fractions which had previously been incubated with various amounts of Pb(NO(3))(2) and glucose 6-phosphate showed that it is possible to subfractionate both smooth- and rough-microsomal fractions into several bands, owing to a differential modification of the density of the microsomal vesicles by the trapping of lead phosphate within them. 3. When the material in the bands obtained by density-gradient centrifugation of incubated microsomal fractions was assayed for glucose 6-phosphatase activity, it was found that the modification of the density of the microsomal fractions was directly related to their relative enrichment in glucose 6-phosphatase activity. Control experiments, in which microsomal fractions were incubated with Pb(NO(3))(2) and glucose 6-phosphate and then treated with EDTA, showed that the subfractionation was not due to aggregation of microsomal vesicles, lead and glucose 6-phosphate. Thus the resolution of microsomal preparations into subfractions with different glucose 6-phosphatase activities is interpreted as indicating heterogeneity of glucose 6-phosphatase distribution in the microsomal vesicles. 4. Electron micrographs of both smooth- and rough-microsomal subfractions show deposits of lead phosphate within the microsomal vesicles. The frequency and extent of these deposits correlate with the different amounts of glucose 6-phosphatase activity measured biochemically. 5. The nature of the heterogeneous distribution of glucose 6-phosphatase is discussed and the more general applicability of the technique for studying membrane fractions containing a heterogeneous distribution of phosphatases is indicated.     

The subcellular localization of histamine and histamine methyltransferase in rat brain

Abstract— We have examined the subcellular localization of histamine and histamine methyl-transferase (S-adenosylmethionine: histamine 7V-methyltransferase; EC 2.1.1.8) in rat brain. The highest levels of histamine and histamine methyltransferase activity were found in the hypothalamus. A large proportion of hypothalamic histamine and histamine methyltransferase activity was found in particles with sedimentation properties in sucrose gradients similar to synaptosomes storing norepinephrine and serotonin. Histamine displayed a bimodal distribution in sucrose gradients. A substantial amount of a tracer dose of [³H]histamine added to hypothalamic homogenates at 4°C was bound to particulate fractions, suggesting that endogenous histamine may redistribute and bind to subcellular fractions during homogenization. The second, lighter peak of histamine in sucrose gradients was thought to be due to histamine that redistributed during homogenization.     

Thermal stability of ribosomal ribonucleic acid from baby hamster kidney cells

1. RNA has been prepared from baby hamster kidney cells by extraction with a phenol–EDTA mixture and further purified by passing through a column of Sephadex G-25 that had been equilibrated with water. 2. Aging of the total RNA extracts at 4° or heating at 95° followed by rapid cooling caused a conversion of 28s RNA into material sedimenting in sucrose gradients at approx. 18s. 3. When heated RNA was re-extracted with phenol the sedimentation profile was not returned to that of the unheated RNA. 4. The 28s and 18s RNA fractions were collected separately from sucrose gradients by precipitation with 2vol. of ethanol and passed through a Sephadex G-25 column equilibrated with water. 5. Heat treatment of purified 28s RNA at 95° caused the sedimentation coefficient to increase to approx. 40s, whereas similar treatment of 18s RNA caused no significant increase. If the RNA was heated before the Sephadex G-25 treatment the sedimentation coefficient of the 28s and 18s RNA decreased to approx. 12s and 8s. 6. Heating mixtures of purified 28s and 18s RNA at 95° caused some aggregation of 18s material with the 28s fraction.     

Further Evidence for an Intrinsic Neuraminidase in CNS Myelin

An intrinsic neuraminidase activity in rat brain CNS myelin has been demonstrated and compared with the neuraminidase activity in rat brain microsomes. With use of ganglioside GM3 as a substrate, the myelin-associated neuraminidase exhibited a shallow pH curve with an optimum at pH 4.8 whereas the microsomal activity had a marked optimum at pH 4-4.3. Neuraminidase activity in both fractions was optimized in 0.3% Triton CF-54 but activation was much greater in the microsomes. When the neuraminidase activities were examined at 60 degrees C, the myelin neuraminidase activity was more than sevenfold of that observed at 37 degrees C and was linear for at least 2 h; the microsomal activity increased only fivefold initially and exhibited a continual loss in activity. Addition of excess microsomes to the total homogenate prior to myelin isolation resulted in no change in myelin neuraminidase activity. When the two membrane fractions were examined at equivalent protein concentrations in the presence of additional cations or EDTA (1 mM), similar but not identical effects on neuraminidase activity were seen. The microsomal neuraminidase was considerably more susceptible to inhibition by divalent copper ion. Activity in both fractions was markedly inhibited by Hg2+ and Ag+ whereas EDTA had no effect on either activity. The myelin-associated neuraminidase activity was the highest in cerebral hemispheres, followed by brainstem, cerebellum, and spinal cord and was extremely low in sciatic nerve. In fact, the myelin neuraminidase activity was higher than the microsomal enzyme activity in the cerebral hemispheres.(ABSTRACT TRUNCATED AT 250 WORDS)     

The subcellular fractionation of the bovine caudate nucleus

Two synaptosomal fractions could be obtained from bovine caudate nucleus on sucrose density gradients one of which had a much greater capacity for high affinity choline uptake than the other but comparable amounts of CAT and choline kinase activity. Specific binding of QNB was widely distributed among all the subcellular fractions except the mitochondrial fraction and in quantitative terms by far the greatest amount was in the microsomal fraction. Only the microsomal fraction contained measurable amounts of glycerophosphocholine phosphodiesterase.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

Cytoplasmic and outer membranes separation in Rhodopseudomonas sphaeroides

A cell envelope fraction has been prepared after mechanical disruption of lysozyme-EDTA spheroplasts from depigmented Rhodopseudomonas sphaeroides (aerobically grown in the light). On linear sucrose gradients this fraction can be separated in a cytoplasmic membrane fraction and an outer membrane fraction. The cytoplasmic fraction (buoyant density: 1.18 g/cm³) has been characterized by its succinic dehydrogenase activity and by its composition. The outer membrane fraction (buoyant density: 1.21 g/cm³) does not contain any respiratory activity nor hemoproteins. The same fractionation has been done on cells repigmented in the dark by lowering the O₂ pressure. In that case the same two fractions have been detected in addition to the chromatophore fraction (buoyant density: 1.14 g/cm³). However both, and specially the outer membrane fraction, were contaminated by chromatophore material.     

The submicrosomal localization of acyl-coenzyme A–cholesterol acyltransferase and its substrate, and of cholesteryl esters in rat liver

To determine the submicrosomal distribution of acyl-CoA–cholesterol acyltransferase and of cholesteryl esters, the microsomal fraction and the digitonin-treated microsomal preparation of rat liver were subjected to analytical centrifugation on sucrose density gradients. With untreated microsomal fractions the distribution profile and the median density of acyl-CoA–cholesterol acyltransferase were very similar to those of RNA. This is in contrast with hydroxymethylglutaryl-CoA reductase and cholesterol 7α-hydroxylase, which are confined to endoplasmic reticulum membranes with low ribosomal coating. In digitonin-treated microsomal preparations activity of acyl-CoA–cholesterol acyltransferase was not detectable. The labelling of untreated microsomal fractions with trace amounts of [¹⁴C]cholesterol followed by subfractionation of the labelled microsomal fraction showed that the specific radioactivity of cholesteryl esters obtained in vitro by the various subfractions was similar with all subfractions but different from the specific radioactivity of the 7α-hydroxycholesterol obtained in vitro by the same subfraction. These results demonstrate the existence of two pools of cholesterol confined to membranes from the endoplasmic reticulum, one acting as substrate for cholesterol 7α-hydroxylase and the other acting as substrate for acyl-CoA–cholesterol acyltransferase. The major part of cholesteryl esters present in both untreated and digitonin-treated microsomal fractions was distributed at densities similar to those of membranes from the smooth endoplasmic reticulum and at densities lower than those of smooth membranes from Golgi apparatus. The ratio of the concentrations of non-esterified to esterified cholesterol in the subfractions from both untreated and digitonin-treated microsomal fractions was highest at the maximum distribution of plasma membranes.     

Subcellular Localization of Inorganic Pyrophosphatases in Rabbit Skeletal Muscle and Some Properties of a Microsomal Acid Pyrophosphatase

Subcellular localization of muscle inorganic pyrophosphatase was examined using rabbit skeletal muscle homogenates. The pyrophosphatases were found to be contained in the microsomal, mitochondrial, and cytosol fractions. The microsomal and mitochondrial pyrophosphatases were most likely bound to the respective subcellular fractions. The pyrophosphatases associated with microsome and mitochondria showed their optimal activities at about pH 5.5 and 7, respectively. They were not dissociated from the particles by washing with salt solution or by ten times freezing-thawing. The activity of microsomal acid pyrophosphatase was not affected by Mg,²⁺ Ca,²⁺ or EDTA, but that of the mitochondrial neutral pyrophosphatase was enhanced by the addition of Mg.²⁺ The microsomal acid pyrophosphatase was stable between pH values of 5.5 and 8.5 during storage at 4°. The activity was inhibited by p-chloromercuribenzoate. The activity was irreversibly inhibited by sodium dodecyl sulfate, but reversibly inhibited by neutral salts and membrane solubilizing detergents such as Triton X-100, octaethylene glycol mono-n-dodecylether, and sodium cholate.     

QUAKING MOUSE MYELIN: BIOCHEMICAL CHARACTERIZATION OF ZONAL GRADIENT SUBFRACTIONS

The brains of Quaking and littermate control mice were fractionated by differential and density gradient centrifugation into soluble, microsomal, myelin and related (SN 4) fractions. There were no apparent differences in protein composition between any Quaking and control fraction with the exception of myelin and SN 4. Analysis of CNP activity indicated that in Quaking animals a high proportion of the total activity was localized in microsomal fractions, while in controls a large percentage of activity was found in myelin and SN4; in contrast, there were no marhcd differences in the distribution of AChE activity between Quaking and control fractions. The yield of myelin isolated from Quaking animals was 3.6%, of that from controls by electron microscopy myelin fractions from both Quaking and controls consisted of compact myelin whorls. Zonal centrifugation on continuous sucrose gradients demonstrated that both control and Quaking myelin was distributed in a bell-shaped mode with peak densities at 0.66 0.68 and 0.71-0.75 M-sucrose, respectively. The specific activity of CNP was generally lower in mutant subfractions than in controls. Protein analysis revealed that there were similar qualitative trends between light and heay myelin subfractions from both mutant and control animals, although the levels of proteolipid and small basic proteins were substantially lower in all Quaking fractions. These results indicate that. although all mutant myelin subfractions are compositionally abnormal, the type of particle heterogeneity in Quaking myelin is similar to that observed in controls.     

MYELIN SUBFRACTIONS IN DEVELOPING RAT BRAIN: CHARACTERIZATION AND SULPHATIDE METABOLISM

Centrifugation of isolated myelin on discontinuous sucrose gradients resulted in a separation into three bands and a pellet. The three bands were morphologically identical to myelin, whereas the pellet consisted primarily of vesicular membranes. These four fractions differed from one another in their lipid-to-protein ratios and in molar ratios of cholesterol:phospholipid:galactolipid. All of the fractions contained proteins typical of myelin, although the proportions of the proteins varied, with the pellet being the lowest in basic protein and proteolipid protein. High activity of 2′,3′-cyclic nucleotidase and low activity of cerebroside sulphotransferase further distinguished these fractions from the microsomal fraction. Distribution of radioactive sulphatide in the subfractions at 15 min after intracranial injection of radioactive sulphate indicated that newly-labelled sulphatide first appeared in the lipid-poor fractions, followed by the lipid-rich fractions; results of pulse-chase experiments also suggested this relationship. Several days or weeks after the injection of radioactive sulphate, most of the radioactive sulphatide was in the lipid-rich fractions.     

The markers of pig heart mitochondrial sub-fractions: II. — On the association of malate dehydrogenase with inner membrane

Evidence is presented about the dual location of NADPH-cytochrome c reductase in mitochondrial outer membranes as well as in microsomes, from pig heart.A high specific activity, was found in both fractions, even after their purification by washing, digitonin treatments, or passages on sucrose gradients. A large fraction of the total activity was associated with both mitochondria and microsomes.Mitochondrial outer membrane differs from microsomes by a low choline phosphotransferase activity and the absence of cytochrome P-450.The properties of mitochondrial and microsomal rotenone-insensitive NADH- and NADPH-cytochrome c reductases were studied. In microsomes, both activities have the same optimum pH (8.5) ; in contrast, in mitochondria they have a different one. The K_m-NADPH were always much higher than those for NADH. In mitochondria the K_m for NAD(P)H were dependent on cytochrome c concentration.The results show that the rotenone-insensitive NADH- and NADPH-cytochrome c reductases of mitochondria and microsomes have quite different behavior and do not appear to be supported by the same enzyme.