Properties of Solubilized Microsomal Lipase from Germinating Brassica napus

Lipase (triacylglycerol acylhydrolase [EC 3.1.1.3.]) was extracted from the microsomal fraction of cotyledons of dark grown seedlings of Canola (Brassica napus L. cv Westar) by treatment with Triton X-100. The enzyme was partially purified by chromatography on Sephacryl S-300 and DEAE Bio-Gel and was stable when stored at −20°C in 50% (v/v) glycerol. The lipase aggregated readily but the distribution of species present in solution could be controlled by nonionic detergents. A species with an apparent M_r of about 250,000 was obtained by gel filtration chromatography in the presence of 1% (v/v) Triton X-100. Lipase activity was optimal near neutral pH, and the reaction approached maximum velocity at a concentration of 0.5 to 1 millimolar emulsified triolein. The reaction rate responded linearly to temperature up to about 40°C and the hydrolytic process had an activation energy of 18 kilocalories per mole. Microsomal lipase lost about 20% and 80% activity when heat-treated for 1 hour at 40°C and 60°C, respectively. At appropriate concentrations, the detergents Triton X-100, n-octyl-β-d-glucopyranoside, (3-[(3-cholamidopropyl-O-dimethylammonio]-1-propanesulfonate, cetyl trimethylammonium bromide, and sodium dodecyl sulfate all inhibited lipase activity. n-Octyl-β-d-glucopyranoside, however, was stimulatory in the 2 to 8 millimolar concentration range. The inhibitory effects of Triton X-100 were reversible.     

Partial Purification of an Ethylene-binding Component from Plant Tissue

An ethylene binding component(s) has been partially purified from mung bean sprouts. Tissue was homogenized in 0.3 molar sucrose and 0.2 molar potassium phosphate buffer (pH 7.0). The homogenate was centrifuged, and resuspended fractions were assayed by incorporating them onto cellulose fibers (0.7 grams per milliliter). These were exposed to [¹⁴C]ethylene (3.7 × 10⁻² microliters per liter of 120 millicurie per millimole) in the presence or absence of 1000 microliters per liter unlabeled ethylene. The cellulose was transferred to separate containers and the [¹⁴C]ethylene was absorbed in mercury perchlorate and counted. Distribution of ethylene binding to various fractions was: 0 to 3,000g, 3%; 3,000 to 12,000g; 4%; 12,000 to 100,000g, 69%; cellular debris, 24%; 100,000g supernatant, 0%. Adjustment of the pH to 4.0 precipitates the ethylene-binding component. Neutralization, addition of Triton X-100, and readjustment of the pH to 4.0 “solubilized” most of the binding component. Further purification was obtained by chromatography on CM-Sephadex in 10 millimolar potassium acetate buffer, (pH 5.0) containing 1% Triton X-100. Elution was with 200 millimolar potassium phosphate (pH 6.0) containing 1% Triton X-100. Upon treatment of the Triton “solubilized” component with cold acetone, over 90% of the binding capacity was lost. Extraction of the acetone-precipitated residue with 2% Triton X-100 restored some of the binding capacity which was found in the soluble fraction. The pH optimum for binding is 6.0. Passing the Triton X-100 extract of the acetone powder through Sepharose 6B provides considerable purification. The binding component moved ahead of most of the protein.     

Phosphatidylinositol synthesis in castor bean endosperm: cytidine diphosphate diglyceride:inositol transferase

CDP-diglyceride:inositol transferase in endoplasmic reticulum fractions from castor bean (Ricinus communis) endosperm was partially characterized. The enzyme had a pH optimum of 8.5 and required Mn²⁺ for activity. Maximal activity was at 1.5 millimolar MnCl₂. A K_m of 0.30 mM was calculated for myo-inositol and 1.35 millimolar was estimated for CDP-dipalmitoylglyceride. Concentrations of CDP-dipalmitoylglyceride above 1.2 millimolar inhibited the enzyme. A deoxycholate concentration of 0.1% (w/v) stimulated the reaction slightly while Triton X-100 inhibited at all concentrations tested. Some incorporation of myo-inositol into phosphatidylinositol occurred in the absence of CDP-diglyceride.     

Characterization of a nuclear phosphatidylinositol 4-kinase in carrot suspension culture cells

We have shown previously that a nuclear phosphatidylinositol (PI) 4-kinase activity was present in intact nuclei isolated from carrot suspension culture cells (Daucus carota L.). Here, we further characterized the enzyme activity of the nuclear enzyme. We found that the pH optimum of the nuclear-associated PI kinase varied with assay conditions. The enzyme had a broad pH optimum between 6.5–7.5 in the presence of endogenous substrate. When the substrate was added in the form of phosphatidylinositol/phosphatidylserine (PI/PS) mixed micelles (1 mM PI and 400 μM PS), the enzyme had an optimum of pH 6.5. In comparison, the pH optimum was 7.0 when PI/Triton X-100 mixed micelles (1 mM PI in 0.025 %, v/v final concentration of Triton X-100) were used. The nuclear-associated PI kinase activity increased 5-fold in the presence of low concentrations of Triton X-100 (0.05 to 0.3 %, v/v); however, the activity decreased by 30 % at Triton X-100 concentrations greater than 0.3 % (v/v). Calcium at 10 μM inhibited 100 % of the nuclear-associated enzyme activity. The K_m for ATP was estimated to be between 36 and 40 μM. The nuclear-associated PI kinase activity was inhibited by both 50 μM ADP and 10 μM adenosine. Treatment of intact nuclei with DNase, RNase, phospholipase A₂ and Triton X-100 did not solubilize the enzyme activity. Based on sensitivity to calcium, ADP, detergent, pH optimum and the product analysis, the nuclear-associated PI 4-kinase was compared with previously reported PI kinases from plants, animals and yeast.     

Partial Purification and Characterization of Uracil-DNA Glycosylase Activity from Chloroplasts of Zea mays Seedlings

A uracil-DNA glycosylase activity has been purified about 750-fold from the chloroplasts of light-grown Zea mays seedlings. This report represents the first direct demonstration of a DNA-glycosylase repair activity in chloroplasts. The activity, in part, was associated with a chloroplast Triton X-100 sensitive membrane. Its apparent K_m was 1.0 micromolar for a poly(dA-dT/U) substrate, and its molecular weight, as determined by gel filtration, was 18,000. The enzyme exhibited optimal activity at pH 7.0 with an atypically narrow pH tolerance. Activity was inhibited greater than 60% by 10 millimolar NaCl, 5 millimolar MgCl₂, or 5 millimolar EDTA. Enzyme activity was inhibited 80% by 10 millimolar N-ethylmaleimide, a sulfhydryl group-blocking agent. The activity removed uracil more rapidly from single-stranded DNA than from double-stranded DNA. With this report, uracil-DNA glycosylase activity has now been attributed to all three DNA-containing organelles of eucaryotic cells.     

Intracellular Localization of GDP-Fucose: Polysaccharide Fucosyl Transferase in Corn Roots (Zea mays L.)

The intracellular site of synthesis of the fucose-rich polysaccharide slime secreted by corn roots was localized by monitoring the distribution of GDP-fucose:polysaccharide fucosyl transferase activity in subcellular fractions of corn roots. Root tip sections were chopped in the presence of 0.56 molar sucrose and 100 millimolar Tris (pH 7.0). After a brief centrifugation, the homogenate was applied to a Sepharose 4B column (1.5 × 30 cm). The turbid, particulate portion of the supernatant fraction eluted at the void volume. Ninety per cent of the enzyme activity was found in the pooled particulate fractions. The particulate fraction was purified on linear sucrose gradients. Gradient fractions were characterized by buoyant density, 280 nanometer absorbance, electron microscope observation, and distributions of NADH-cytochrome c oxidoreductase and fucosyl transferase activities.     

Biosynthesis of Cytidine 5'-Diphosphate-diacylglycerol in Endoplasmic Reticulum and Mitochondria of Castor Bean Endosperm

Cytidine 5′-triphosphate (CTP):phosphatidate cytidyltransferase from the endoplasmic reticulum and mitochondria of Ricinus communis L. var Hale was characterized. The endoplasmic reticulum enzyme has a pH optimum of 6.5 and a divalent cation is required, Mn²⁺ being preferred and giving maximum activity at 2.5 millimolar. The estimated K_m for CTP is 16.7 micromolar, but that for phosphatidate could not be determined accurately. The activity was inhibited by both deoxycholate and Triton X-100 at concentrations as low as 0.01% (w/w).

The mitochondrial enzyme has a pH optimum of 6.0 and a divalent cation requirement similar to that of the endoplasmic reticulum. Maximum stimulation of the reaction by substrates occurred with 1.5 millimolar phosphatidate (from egg phosphatidylcholine) and about 400 micromolar CTP. The apparent K_m for phosphatidate could not be estimated accurately since activity was obtained in the absence of added lipid, apparently utilizing endogenous substrate. The K_m estimated for CTP was altered by the presence of the detergent Triton X-100; in its absence the value was 33.3 micromolar, but in its presence the value was 66.7 micromolar. Inclusion of 0.6% (w/w) Triton X-100 in the assay mixture stimulated the activity about 2.5-fold.

     

Phospholipase A2 activity towards phosphatidylcholine in mixed micelles: surface dilution kinetics and the effect of thermotropic phase transitions

Phospholipase A₂ will act on dipalmitoyl phosphatidylcholine as substrate when the phospholipid is part of a mixed micelle with Triton X-100 at a molar ratio of Triton to phospholipid of 2:1 or greater. Kinetic studies at high molar ratios of Triton X-100 to phospholipid are reported and show that the binding of phospholipase A₂ to substrate depends on the total concentration of Triton X-100 and phospholipid, but that the rate of enzymatic catalysis decreases proportionally to the Triton X-100 concentration. These results are interpreted in terms of a model involving surface dilution kinetics. The relationship of this model to that of competitive inhibition is discussed. In addition, the activity of phospholipase A₂ towards dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine at different temperatures is reported, and the results show a direct effect of the thermotropic phase transition of dipalmitoyl phosphatidylcholine on enzymatic activity.     

GM1-ganglioside-triton X-100 mixed micelles: Changes of micellar properties studied by laser-light scattering and enzymatic methods

The micellar properties of mixtures of GM1 ganglioside and the non-ionic amphiphile Triton X-100 in 25 mM Na phosphate-5 mM di Na EDTA buffer (pH = 7.0) were investigated by quasielastic light scattering in a wide range of Triton/GM1 molar ratios and in the temperature range 15–37°C. These measurements: (a) provided evidence for the formation of mixed micelles; (b) allowed the determination of such parameters as the molecular weight and the hydrodynamic radius of the mixed micelles; (c) showed the occurrence of statistical aggregates of micelles with increasing temperature and micelle concentration. Galactose oxidase was chosen for studying the relation between enzyme activity and micellar properties. The action of the enzyme on GM1 was found to be strongly dependent on the micellar structure. In particular: (a) galactose oxidase acted very poorly on homogeneous GM1 micelles, while affecting mixed GM1/Triton X-100 micelles; (b) at fixed GM1 concentration the oxidation rate increased by enhancing Triton X-100 concentration and followed a biphasic kinetics with a break at a certain Triton X-100 concentration; (c) the formation of statistical micelle aggregates was followed by inhibition of the enzyme activity.     

Formation and characterization of mixed micelles of the nonionic surfactant Triton X-100 with egg, dipalmitoyl, and dimyristoyl phosphatidylcholines

Mixed micelles of the nonionic surfactant Triton X-100 and egg phosphatidylcholine were isolated by column chromatography on 6% agarose and by centrifugation at 35,000g. It was found that egg phosphatidylcholine bilayers are able to incorporate Triton X-100 at molar ratios of Triton to phospholipid below about 1:1, whereas above a molar ratio of about 2:1 Triton/phospholipid all of the phospholipid is converted into mixed micelles. Mixed micelles at a molar ratio of about 10:1 Triton/phospholipid were found to be in the same size range as pure micelles of Triton X-100. The formation of mixed micelles with dipalmitoyl phosphatidylcholine at room temperature, when the phospholipid is below its thermotropic phase transition, is shown to require relatively high concentrations of Triton X-100. The point at which dimyristoyl phosphatidylcholine bilayers are converted to mixed micelles was found to be less clear cut than with egg phosphatidylcholine, but above a molar ratio of about 2:1 Triton/phospholipid, all of this phospholipid is also in mixed micelles. The relevance of these results to the solubilization of membrane-bound proteins with Triton X-100 and the action of phospholipase A₂, which hydrolyzes phosphatidylcholine when it is in mixed micelles with Triton X-100, is discussed.     

Ubiquinol-cytochrome c reductase (EC 1.10.2.2). Isolation in Triton X-100 by hydroxyapatite and gel chromatography. Structural and functional properties

1. A method for the isolation of a monodisperse ubiquinol-cytochrome c reductase (complex III) from beef heart mitochondria has been developed. The procedure consists of an enzyme solubilization in Triton X-100 followed by hydroxyapatite and gel chromatography.2. The minimum unit of the isolated complex is composed of 9 polypeptide subunits with M_r of 49000, 47000, 30000, 25000, 12000, 11000 and 6000. It contains 8 μmol of cytochrome b, 4 μmol of cytochrome c₁ 7–8 μmol of nonheme iron, corresponding to 3.5–4 μmol of the Rieske iron-sulfur protein, less than 1.0 μmol of ubiquinone and about 60 μmol of phospholipids, per g of protein. The specific detergent binding amounts to 0.2 g of Triton X-100 per g protein.3. Cytochrome b exhibits an α-absorbance maximum at 562 nm. In redox titrations it reveals two half-reduction potentials, i.e. ?10 and +100 mV, at pH 7.0. The absorbance maximum of cytochrome c₁ lies at 553 nm and its half-reduction potential amounts to +250 mV.4. The reductase reveals electron-transferring activity with ubiquinol-1, -2, -3, and -9 as donor and cytochrome c as acceptor. The activity with ubiquinol-9 was analyzed according to the surface dilution scheme developed for the action of phospholipases. The molecular activity amounts to 75 mol of cytochrome c reduced per s at 20°C.5. A dissociation constant K′_s of 5.5 mM has been determined for the Triton-solubilized enzyme: ubiquinol-containing micelle association. In this case the total concentration of ubiquinol plus Triton X-100 has been substituted for the concentration of binding areas on the ubiquinol-containing micelles. This substitution makes the reasonable assumption that the sum of ubiquinol concentration plus Triton X-100 is proportional to the number of available binding areas.6. A K′_m value of 0.025 was found for ubiquinol-9. This is an analog to the Michaelis constant and is expressed as mol fraction of ubiquinol in the ubiquinol-Triton micelle.     

Purification and Properties of UDP-GlcNAc:Dolichyl-Pyrophosphoryl-GlcNAc GlcNAc Transferase from Mung Bean Seedling

The N-acetylglucosamine (GlcNAc) transferase that catalyzes the formation of dolichyl-pyrophosphoryl-GlcNAc-GlcNAc from UDP-GlcNAc and dolichyl-pyrophosphoryl-GlcNAc was solubilized from the microsomal enzyme fraction of mung beans with 1.5% Triton X-100, and was purified 140-fold on columns of DE-52 and hydroxylapatite. The partially purified enzyme preparation was quite stable when stored in 20% glycerol and 0.5 millimolar dithiothreitol, and was free of GlcNAc-1-P transferase and mannosyl transferases. The GlcNAc transferase had a sharp pH optimum of 7.4 to 7.6 and the K_m for dolichyl-pyrophosphoryl-GlcNAc was 2.2 micromolar and that for UDP-GlcNAc, 0.25 micromolar. The enzyme showed a strong requirement for the detergent Triton X-100 and was stimulated somewhat by the divalent cation Mg²⁺. Uridine nucleotides, especially UDP and UDP-glucose inhibited the enzyme as did the antibiotic, diumycin. However, a variety of other antibiotics including tunicamycin were without effect. The product of the reaction was characterized as dolichyl-pyrophosphoryl-GlcNAc-GlcNAc.     

Removal of bound Triton X-100 from purified bovine heart cytochrome bc1

Cytochrome bc₁ isolated from Triton X-100-solubilized mitochondrial membranes contains up to 120 nmol of Triton X-100 bound per nanomole of the enzyme. Purified cytochrome bc₁ is fully active; however, protein-bound Triton X-100 significantly interferes with structural studies of the enzyme. Removal of Triton X-100 bound to bovine cytochrome bc₁ was accomplished by incubation with Bio-Beads SM-2 in the presence of sodium cholate. Sodium cholate is critical because it does not interfere with the adsorption of protein on the hydrophobic surface of the beads. The resulting Triton X-100-free cytochrome bc₁ retained nearly full activity, absorption spectra, subunit, and phospholipid composition.     

Studies on the Secretion of Maize Root Cap Slime: II. Localization of Slime Production

The distribution of fucose-containing polysaccharides in apical 1-cm sections of corn (Zea mays cv. SX-17) root tips was analyzed. Fucose-containing polysaccharides were localized predominantly in the apical 1 mm of the root, i.e., in the apical initials and root cap. An analysis of the distribution of incorporated radioactive label from l-fucose[(3)H] gave similar results. After a 2-hr incubation with fucose[(3)H], label was found principally in two components, namely a water-soluble slime fraction and hemicellulose. The incorporation of fucose into the water-soluble, ethanol-insoluble fraction was primarily in the apical 1 mm of the root, whereas incorporation into a water-insoluble, potassium hydroxide-soluble fraction was in the region 2 to 5 mm behind the root cap. Addition of sucrose to the incubation medium during fucose[(3)H] incorporation reduces label uptake but increases the amount of label in the fucose-rich secreted polysaccharide. The utility of fucose as a marker for the secreted polysaccharide was confirmed by demonstrating that no appreciable metabolism of this sugar occurs.     

Partial Purification, Photoaffinity Labeling, and Properties of Mung Bean UDP-Glucose:Dolicholphosphate Glucosyltransferase

UDP-glucose:dolichylphosphate glucosyltransferase has been purified 734-fold from Triton X-100 solubilized mung bean (Phaseolus aureus) microsomes. The partially purified enzyme has broad pH optima of activity from 6.0 to 7.0 and is maximally stimulated with 10 millimolar MgCl₂. The K_m for UDP-glucose was determined as 27 micromolar, and the K_m for dolichol-P was 2 micromolar. Using the UDP-glucose photoaffinity analog, 5-azido-UDP-glucose, a polypeptide of 39 kilodaltons on sodium dodecyl sulfate-polyacrylamide gels was identified as the catalytic subunit of the enzyme. Photoinsertion into this 39-kilodalton polypeptide with [³²P]5-azido-UDP-glucose was saturable, and was maximally protected with the native substrate UDP-glucose. 5-Azido-UDP-glucose behaves competitively with UDP-glucose in enzyme assays, and upon photolysis inhibits activity in proportion to its concentration. This study represents the first subunit identification of a plant glycosyltransferase involved in the biosynthesis of the lipid-linked oligosaccharides that are precursors of N-linked glycoproteins.     

Characterization of Nitrate Reductase from Corn Leaves (Zea mays cv W64A x W182E) : Two Molecular Forms of the Enzyme

The primary leaves from corn seedlings grown for 6 days were harvested, frozen with liquid N₂ and extracted in a Tris buffer (pH 8.5, 250 millimolar) containing 1 millimolar dithiothreitol, 10 millimolar cysteine, 1 millimolar EDTA, 20 micromolar flavin adenine dinucleotide and 10% (v/v) glycerol. Nitrate reductase (NR) in the crude extract was stable for several days at 0°C and for several months at −80°C. The enzyme was purified using (NH₄)₂SO₄ fractionation, brushite-hydroxyl-apatite chromatography and blue-sepharose affinity chromatography. The enzyme was eluted from the blue-sepharose column with a linear gradient of NADH (0-100 micromolar) or with 0.3 molar KNO₃. About 10% of the original activity was recovered with NADH (NADH-NR). It had a specific activity of about 60 to 70 units (micromoles NO₂⁻ per minute per milligram protein). A sequential elution with NADH followed by KNO₃ (0.3 molar) or KCl (0.3 molar) yielded 2 peaks. Rechromatography of each peak gave two peaks again. These results indicate that we are dealing with two forms of the same enzyme rather than two different NR proteins. The two NRs had different molecular weights as judged by chromatography on Toyopearl. The NADH-NR was more sensitive than the NO₃⁻-NR to antibody prepared against barley leaf NR. In Ouchterlony assays a single precipitin line, with completely fused boundaries, was observed.     

Purification and characterization of membrane-bound aldehyde dehydrogenase from Acetobacter polyoxogenes sp. nov.

Summary Membrane-bound aldehyde dehydrogenase (ALDH) was purified from the membrane fraction of an industrial-vinegar-producing strain, Acetobacter polyoxogenes sp. nov. NBI1028 by solubilization with Triton X-100 and sodium N-lauroyl sarcosinate and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneos on polyacrylamide disc gel electrophoresis. Upon sodium dodecyl sulphate-polyacrylamide gelelectrophoresis, the enzyme showed the presence of two subunits with a molecular mass of 75 000 daltons and 19 000 daltons, respectively. From the absorption and fluorescence spectra, the absence of cytochrome c and the presence of pyrroloquinoline quinone in the purified enzyme were demonstrated. The ALDH preferentially oxidized aliphatic aldehyde with a straight carbon chain except for formaldehyde. The apparent K _m for acetaldehyde was 12 mM. The optimum pH and temperature were 7.0 and 50°–60°C, respectively. The enzyme remained active after storage at 4°C for 20 days. p-Chloromercuribenzoic acid and heavy metal salts such as CuSO₄ were inhibitory to the enzyme. Ferricyanide was effective as an electron acceptor.Offprint requests to: M. Fukaya     

PARTICULATE AND SOLUBILIZED FUCOSYL TRANSFERASES FROM MOUSE BRAIN

The transfer of [¹⁴C]fucose from GDP-[U-¹⁴C]fucose to endogenous and exogenous acceptors by particulate and solubilized preparations from mouse brain is described. Suspensions of brain microsomes incorporated [¹⁴C]fucose into a heterogenous group of glycoprotein products, which have a distribution on gel electrophoresis similar to those synthesized in vivo. Fucosyl transferase, extracted from brain microsomes by Triton X-100, transferred [¹⁴C]fucose from GDP-[U-¹⁴C]fucose to terminal galactose residues exposed by mild acid hydrolysis of porcine plasma glycoprotein. Comparison of the specific activities of the solubilized fucosyl transferase from a number of organs showed that, in the presence of the exogenous acceptor which was used, the transferase of brain was more active than the transferases from all other organs tested, with the exception of kidney. Examination of subcellular fractions of brain, with endogenous and exogenous acceptors, showed that activity was limited to fractions containing microsomal membranes, whereas synaptosomal and other fractions were virtually inactive.     

Characteristics of phospholipase D in reverse micelles of Triton X-100 and phosphatidylcholine in diethyl ether

Summary Phospholipase D, from cabbage, is active in reverse micelles formed from its substrate phosphatidylcholine and Triton X-100 in diethyl ether. The activity is optimum at w₀=12.5. The increase of the molar ratio of Triton X-100/substrate from 1:4 to 2:1 results in an activity decrease by 25 %. At 136 mM Triton X-100 the K_M value in reverse micelles is 136 mM, whereas it is 0.40 mM in the aqueous system containing SDS.     

Relationship between Cottonseed Malate Synthase Aggregation Behavior and Suborganellar Location in Glyoxysomes and Endoplasmic Reticulum

Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocyto-chemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCl or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supernatants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20-40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes.