Effect of sugars and amino acids on membrane potential in two clones of sugarcane

Sugarcane (Saccharum officinarum L.) leaf parenchyma cells bathed in 1X solution maintained an average membrane potential of −135 millivolts in the dark. No difference in membrane potential was found between clones 51 NG 97 and H50 7209. An electrogenic pump appears to contribute to membrane potential in these cells. Sugars (25 millimolar) added externally caused the following membrane potential depolarizations (in millivolts) in clone 51 NG 97: glucose, 18 ± 4; galactose, 24 ± 7; 3-O-methylglucose, 10 ± 4; sucrose, 22 ± 3; fructose, 21 ± 7; raffinose, 9 ± 3; mannitol, 0; lactose, 0; melibiose, 0; and 1-O-methyl-α-galactose, 0. Glycine (25 millimolar) and serine (10 millimolar) caused depolarizations of 47 ± 7 and 23 ± 2 millivolts, respectively. Depolarization shows saturation kinetics with respect to glucose concentration, with a K_m of 3 to 6 millimolar. The metabolic inhibitors KCN and salicyl hydroxamic acid together caused depolarization of the membrane potential and greatly inhibited depolarization by 25 millimolar glucose and 25 millimolar raffinose. In a series of substitution experiments, glucose (25 millimolar) caused almost total inhibition of depolarization by raffinose, sucrose, and 3-O-methylglucose (all 25 millimolar), but only partial inhibition of depolarization to 25 millimolar glycine. Glycine (25 millimolar), also, only partially inhibited depolarization by 25 millimolar glucose. Total depolarization to 25 millimolar glycine and 25 millimolar glucose was comparable to the amount of depolarization of membrane potential caused by 1 millimolar KCN plus 1 millimolar salicyl hydroxamic acid. The results are consistent with a co-transport mechanism of membrane transport, with sugars and amino acids being transported by separate carrier systems.     

Phosphate Transport across the Plasma Membrane of Wheat Leaf Protoplasts: Characteristics and Inhibitor Specificities

The kinetics and inhibitor specificities of phosphate transport across the plasma membrane of wheat leaf mesophyll protoplasts have been examined. Studies were also carried out on the effects of light and pH on phosphate transport and the plasma membrane electropotential. At pH 5.8 (30°C), protoplasts accumulated phosphate at the rate of 3.9 ± 0.2 nanomoles per milligram protein per hour. Phosphate uptake rates and inhibitor specificities for the leaf cell plasma membrane phosphate transporter were qualitatively similar to those observed with root protoplasts. Neither picrylsulfonic acid, or p-chloromercuribenzene sulfonate affected phosphate uptake significantly at 0.1 millimolar. Of all compounds tested, carbonyl cyanide-p-trifluoromethoxy phenylhydrazone was the most effective inhibitor of phosphate uptake (60% at 0.1 millimolar). Tribenzylphosphate inhibited uptake by 34% while dibenzylphosphate had no effect. The plasma membrane electropotential was found to be −37 ± 3 millivolts. Initiation of photosynthesis lowered the membrane potential to −39 ± 3 millivolts. Inhibition of phosphate uptake by 34% with the substrate analog tribenzylphosphate resulted in a measured membrane potential of −33 ± 3 millivolts. These changes in potential were not significant at the 5% probability level. Phosphate uptake rates remained constant under photosynthetic and nonphotosynthetic conditions. The utility of tribenzylphosphate as an inhibitor in plant systems is demonstrated.     

Active transport of charged substrates by a proton/sugar co-transport system. Amino-sugar uptake in the yeast Rhodotorula gracilis.

1. In the yeast Rhodotorula gracilis several amino sugars were actively transported. Glucosamine, which is largely protonated at physiological pH (pK 7.75) was used as a model substrate. At pH 6.75 its half-saturation constant was 1 mM and the maximal velocity was 50 nmol/min per mg dry wt. 2. Amino sugars were taken up via the monosaccharide carrier. The transport of glucosamine was strongly restricted by monosaccharides. D-Xylose inhibited competitively the uptake of glucosamine. The inhibition constant was 1 mM. Cells preloaded with D-xylose showed exchange transport on subsequent addition of glucosamine. 3. Transport of glucosamine was energized by the membrane potential. Uncoupling agents such as carbonyl cyanide m-chlorophenyl-hydrazone and the lipophilic cation TPP+ (tetraphenylphosphonium ion) at concentrations that depolarized the membrane potential inhibited the uptake of glucosamine. Conversely the transport of glucosamine partly dissipated the membrane potential, which was monitored by radioactively labelled lipophilic cations. 4. The translocated charges were electrically compensated by the extrusion of protons and K+ (1 glucosamine molecule/0.85 H+ + 0.15 K+). 5. An increase of the pH in the range 4.75-8.75 lead to a decrease of the half-saturation constant from 5 mM to 1 mM and to an optimum of the maximal velocity at pH 6.75. We suggest that this fair constancy is due to the carrier not distinguishing between the protonated form of glucosamine (pH less than 7.75) and the deprotonated form (pH greater than 7.75). The increase of V(T) (maximal transport velocity) between pH 4.75 and 6.75 is due to the increase of the membrane potential: the decrease between pH 6.75 and 8.75 is due to the deprotonization of the carrier.     

Uptake of glutamine by the scutellum of germinating barley grain

Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up [¹⁴C]glutamine at an initial rate of about 10 micromoles·gram⁻¹·hour⁻¹ in the standard assay conditions (pH 5, 30°C, 1 millimolar glutamine). Inhibition by unlabeled glutamine and by dinitrophenol indicated that about 95% of the uptake was due to carrier-mediated active transport. The pH optimum of the uptake was 5, and after correction for a nonmediated component the uptake appeared to conform to Michaelis-Menten kinetics with an apparent K_m of about 2 millimolar and a V_max of about 25 micromoles·gram⁻¹·hour⁻¹.

The uptake of glutamine was inhibited by all of the 18 amino acids tested; the mode of inhibition was studied only with proline and was competitive. Eight of the ten amino acids tested at high concentrations appeared to be able to inhibit the mediated uptake of glutamine virtually completely. However, when the inhibitory effect of asparagine was extrapolated to an infinitely high concentration of asparagine, about 24% of the mediated uptake of glutamine remained uninhibited. These results suggest that glutamine is taken up by two (or more) rather unspecific amino acid uptake systems, the minor one having no affinity for asparagine.

Glutamine and alanine could completely inhibit the mediated uptake of 1 millimolar leucine, but about 12% of the mediated uptake appeared to be uninhibitable by asparagine. Furthermore, the ratio of the mediated uptake of glutamine to that of leucine changed from 0.9 to 1.7 between days 1 and 3 of germination. These results give further support for the presence of two unspecific amino acid uptake systems in barley scutella.

     

Uptake of proline by the scutellum of germinating barley grain

Scutella separated from germinating grains of barley (Hordeum vulgare L. cv Himalaya) took up 1 millimolar l-[¹⁴C]proline at an initial rate of about 6.5 micromoles gram⁻¹ fresh weight hour⁻¹ (pH 5, 30°C). The uptake had a pH optimum at 5. The bulk of the uptake (93%) was via carrier-mediated active transport. All of the 19 l-amino acids tested at 10 millimolar concentration inhibited the mediated uptake of 1 millimolar proline, the inhibitions varying from 18 to 76%. By studying how large a fraction of the mediated uptake was inhibitable by asparagine, alanine, glutamine, and leucine, the mediated uptake was shown to be due to three components. Two of these are most probably attributable to the two nonspecific uptake systems proposed earlier to act in the uptake of glutamine and leucine. The third component was not inhibited by glutamine, asparagine, or alanine, but was inhibited by unlabeled proline and leucine. The uptake by this system was apparently carrier-mediated active transport. d-Proline inhibited this system as strongly as l-proline. Nine of the 16 l-amino acids tested at 50 millimolar concentrations did not inhibit the uptake of 1 millimolar proline by this system. Valine, leucine, isoleucine, and the basic amino acids were inhibitory, but in spite of this, they did not appear to be taken up by this system. It seems therefore that in addition to two nonspecific amino acid uptake systems the scutella have an uptake system which is specific for proline. It is likely that this proline-specific system accounts for the bulk of proline uptake in a germinating grain.     

Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes

The effects of NO₃⁻ and assay temperature on proton translocating ATPases in membranes of barley (Hordeum vulgare L. cv California Mariout 72) roots were examined. The membranes were fractionated on continuous and discontinuous sucrose gradients and proton transport was assayed by monitoring the fluorescence of acridine orange. A peak of H⁺-ATPase at 1.11 grams per cubic centimeter was inhibited by 50 millimolar KNO₃ when assayed at 24°C or above and was tentatively identified as the tonoplast H⁺-ATPase. A smaller peak of H⁺-ATPase at 1.16 grams per cubic centimeter, which was not inhibited by KNO₃ and was partially inhibited by vanadate, was tentatively identified as the plasma membrane H⁺-ATPase. A step gradient gave three fractions enriched, respectively, in endoplasmic reticulum, tonoplast ATPase, and plasma membrane ATPase. There was a delay before 50 millimolar KNO₃ inhibited ATP hydrolysis by the tonoplast ATPase at 12°C and the initial rate of proton transport was stimulated by 50 millimolar KNO₃. The time course for fluorescence quench indicated that addition of ATP in the presence of KNO₃ caused a pH gradient to form that subsequently collapsed. This biphasic time course for proton transport in the presence of KNO₃ was explained by the temperature-dependent delay of the inhibition by KNO₃. The plasma membrane H⁺-ATPase maintained a pH gradient in the presence of KNO₃ for up to 30 minutes at 24°C.     

Electrical potentials in stomatal complexes

Guard cells of several species, but predominantly Commelina communis, were impaled by micropipette electrodes and potential differences measured that occurred between cell compartments and the flowing bathing medium. The wall developed a Donnan potential that was between −60 and −70 millivolt in 30 millimolar KCl at pH 7. The density of the fixed charges ranged from 0.3 to 0.5 molar; its dependence on pH was almost identical with the titration curve of authentic polygalacturonic acid. The vacuolar potential of guard cells of Commelina communis L., Zea mays L., Nicotiana glauca Graham, Allium cepa L., and Vicia faba L. was between −40 and −50 millivolt in 30 millimolar KCl when stomata were open and about −30 millivolt when stomata were closed. The vacuolar potential of guard cells of C. communis was almost linearly related to stomatal aperture and responded to changes in the ionic strength in the bathing medium in a Nernstian manner. No specificity for any alkali ion (except Li⁺), ammonium, or choline appeared. Lithium caused hyperpolarization. Calcium in concentrations between 1 and 100 millimolar in the medium led to stomatal closure, also caused hyperpolarization, and triggered transient oscillations in the intracellular potential. Gradients in the electrical potential existed across stomatal complexes with open pores. When stomata closed, these gradients almost disappeared or slightly reverted; all epidermal cells were then at potentials near −30 millivolt in 30 millimolar KCl.     

Ion Transport in Isolated Protoplasts from Tobacco Suspension Cells: III. Membrane Potential

The membrane electrical potential difference was measured in cultured cells and isolated protoplasts of tobacco (Nicotiana glutinosa L.) by inserting a microelectrode into cells held fast by a suction micropipette. The potential difference (± standard deviation) for unplasmolyzed tobacco cells was −52 ± 12 millivolts, for cells in 0.3 molar mannitol, −50 ± 11 millivolts; and for cells plasmolyzed in 0.7 molar mannitol, −49 ± 12 millivolts all inside negative. The potential difference for isolated protoplasts in 0.7 molar mannitol was −49 ± 16 millivolts, inside negative. In both cultured cells and protoplasts, the addition of 0.1 millimolar KCN caused a depolarization of the membrane potential. It was concluded that plasmolysis and enzymic release of the protoplast had no significant effect on the membrane potential of cultured tobacco cells.     

Isolation of amyloplasts from developing maize endosperm

Methods for the formation of protoplasts from developing maize endosperm and for the aqueous isolation of intact amyloplasts from such protoplasts are described. Protoplasts were obtained after incubating endosperm slices in a medium containing cellulase and pectolyase for 5 days at 4°C or 5 hours at 30°C. After purification in a Ficoll density gradient, the protoplasts were reptured by forcing the suspension through a Nitex mesh (20 micrometer) positioned at the lower end of a modified disposable syringe. The resulting filtrate was layered on a discontinuous Ficoll density gradient of 30, 15, and 10%. Each Ficoll solution contained 0.7 molar sucrose, 10 millimolar arginine, 10 millimolar dl-dithiothreitol, 50 millimolar 2-(N-morpholino)ethanesulfonic acid (pH 5.6), and 2 millimolar CaCl₂. After 3 hours in the cold, an amyloplast fraction 50 to 93% intact and free from cytoplasmic, mitochondrial, and glyoxysomal contamination was recovered in the 15% Ficoll layer. Amyloplast intactness was estimated by fluorescent microscopy and activity of certain amyloplast marker enzymes before and after rupture of the amyloplast membrane. Starch branching enzyme, ADPG-pyrophosphorylase, and nitrite reductase were used as amyloplast marker enzymes.     

Sugar transport in isolated corn root protoplasts

Isolated corn (Zea mays L.) root protoplasts were used to study sucrose and hexose uptake. It is found that glucose was preferentially taken up by the protoplasts over sucrose and other hexoses. Glucose uptake showed a biphasic dependence on external glucose concentration with saturable (K_m of 7 millimolar) and linear components. In contrast, sucrose uptake only showed a linear kinetic curve. Sucrose and glucose uptake were linear over a minimum of 1 hour at pH 6.0 and 1 millimolar exogenous sugar concentration. Glucose uptake showed a sharp 42°C temperature optimum, while sucrose uptake showed a lower temperature sensitivity which did not reach a maximum below 50°C. Uptake of both sugars was sensitive to several metabolic inhibitors and external pH. Differences between sucrose and glucose uptake in two different sink tissue (i.e. protoplasts from corn roots and soybean cotyledons) are discussed.     

Transport of Glycerate across the Envelope Membrane of Isolated Spinach Chloroplasts

Uptake of d, l-glycerate into the chloroplast stroma has been studied using the technique of silicone oil filtering centrifugation. Glycerate uptake was 3 to 5 times higher in the light than in darkness, the stimulation by light being abolished by the proton ionophore carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. The pH optimum for uptake was 7.0 at 2°C and 8.5 at 20°C, but at all pH values the rate of uptake was higher at 20°C than at 2°C. Uptake was concentration dependent, saturating above 8 millimolar glycerate. At 2°C, the K_m was 0.3 millimolar and the V_max was 13 micromoles per milligram of chlorophyll per hour. At 20°C initial rates of glycerate uptake were higher than 40 micromoles per milligram of chlorophyll per hour.     

Transport of NAD in Percoll-Purified Potato Tuber Mitochondria: Inhibition of NAD Influx and Efflux by N-4-Azido-2-nitrophenyl-4-aminobutyryl-3'-NAD

A mechanism by which intact potato (Solanum tuberosum) mitochondria may regulate the matrix NAD content was studied in vitro. If mitochondria were incubated with NAD⁺ at 25°C in 0.3 molar mannitol, 10 millimolar phosphate buffer (pH 7.4), 5 millimolar MgCl₂, and 5 millimolar α-ketoglutarate, the NAD pool size increased with time. In the presence of uncouplers, net uptake was not only inhibited, but NAD⁺ efflux was observed instead. Furthermore, the rate of NAD⁺ accumulation in the matrix space was strongly inhibited by the analog N-4-azido-2-nitrophenyl-4-aminobutyryl-3′-NAD⁺. When suspended in a medium that avoided rupture of the outer membrane, intact purified mitochondria progressively lost their NAD⁺ content. This led to a slow decrease of NAD⁺-linked substrates oxidation by isolated mitochondria The rate of NAD⁺ efflux from the matrix space was strongly temperature dependent and was inhibited by the analog inhibitor of NAD⁺ transport indicating that a carrier was required for net flux in either direction. It is proposed that uptake and efflux operate to regulate the total matrix NAD pool size.     

Polyamine Uptake, Kinetics, and Competition among Polyamines and between Polyamines and Inorganic Cations

Polyamine uptake, the kinetics of this uptake, and the competition among polyamines and between polyamines and inorganic cations were studied in petals of Saintpaulia ionantha Wendl. Uptake experiments using ¹⁴C-labeled polyamines were carried out on single petals, at room temperaure (20°C) and in the light. The results show that putrescine, spermidine, and spermine uptake was dependent on the external pH and occurred up to high external polyamine concentrations with K_m values of 8.6, 1.2, and 2.1 millimolar, respectively, with spermidine being the most absorbed at low concentration (17 micromolar). Putrescine and spermidine did not seem to compete for the same site of absorption. Furthermore, putrescine and spermidine uptake was not inhibited by Ca²⁺, Mg²⁺, and K⁺ at the same concentrations (17 micromolar), whereas 1.7 millimolar Ca²⁺ inhibited and K⁺ enhanced spermidine uptake. The intracellular localization of the absorbed putrescine was determined using two different methods. Very little label was found in the apoplast, while most of it was localized in the 98,500g supernatant. According to our data the vacuole, which represents a substantial part of Saintpaulia parenchyma cells, could be a site of putrescine accumulation. 2,4-Dinitrophenol and diethylstilbestrol did not inhibit uptake; however, at 0°C there was a 35% inhibition of spermidine uptake, compared with the controls kept at 20°C as well as a 68% inhibition with 20 millimolar NaSCN.     

Purification and properties of two endochitosanases from Mucor rouxii implicated in its cell wall degradation

Abstract From autolysed cultures of Mucor rouxii , two chitosanases, A and B, were purified to electrophoretic homogeneity. Apparent M _r values of 76 000 and 58 000 and p I values of 4.9 and 4.7 were determined for A and B, respectively. Both chitosanases showed a high specificity for chitosan and chitosan derivatives. They had optimum activities at pH 5.0 and at temperatures of 55°C and 50°C for A and B, respectively. Enzyme A was inhibited by acetate ions and enzyme B by high substrate concentration. Both enzymes showed an endo-splitting type of activity, and the end product of chitosan degradation contained a mixture of dimer, trimer and higher molecular mass oligomers of glucosamine. Glucosamine oligosaccharides were poorly hydrolysed by these enzymes. Both enzymes extensively degraded the chitosan extracted from M. rouxii cell walls.     

Effects of calcium on chlorophyll synthesis and stability in the early phase of greening in cucumber cotyledons

The effects of calcium on chlorophyll accumulation and its stability in the early phase of greening in cucumber cotyledons were investigated. Chlorophyll accumulation was hardly affected by dark preincubation of cotyledons with 10 millimolar calcium solution, but was inhibited almost completely when 50 or 100 millimolar solution was used. On the other hand, 50 millimolar calcium inhibited δ-aminolevulinic acid formation in the light by only 75%. Calcium had little effect on the lag for initiation of protochlorophyllide₆₅₀ regeneration, but slowed down the rate of accumulation of protochlorophyllide₆₅₀. In calcium-treated cotyledons, the chlorophyll formed by primary photoconversion was quickly decomposed in the dark. The present results show that calcium inhibited chlorophyll accumulation by inhibiting δ-aminolevulinic acid formation in the light and by stimulating the decomposition of newly formed chlorophyll, both effects being completely prevented by potassium.     

Effect of vanadate, molybdate, and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots

The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 × MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase.

Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K⁺. K⁺-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K⁺ transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate.

     

Glucosamine prevents <Emphasis Type="Italic">in vitro</Emphasis> collagen degradation in chondrocytes by inhibiting advanced lipoxidation reactions and protein oxidation

Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction. Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.     

THE LYSOSOME PERIPHERY: BIOCHEMICAL AND ELECTROKINETIC PROPERTIES OF THE TRITOSOME SURFACE

Normal rat liver lysosomes were isolated by the technique of loading with Triton WR-1339. Purity of the preparation was monitored with marker enzymes; a high enrichment in acid hydrolases was obtained in the tritosome fraction. In 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO₃, pH 7.2 at 25°C the tritosomes had an electrophoretic mobility of -1.77 ± 0.02 µm/s/V/cm, a zeta potential of 23.2 mV, a surface charge of 1970 esu/cm², and 33,000 electrons per particle surface assuming a tritosome diameter of 5 x 10^-7 m. Treatment of the tritosomes with 50 µg neuraminidase/mg tritosome protein lowered the electrophoretic mobility of the tritosome to -1.23 ± 0.02 µm/s/V/cm under the same conditions and caused the release of 2.01 µg sialic acid/mg tritosome protein. Treatment of the tritosomes with hyaluronidase did not affect their electrophoretic mobility, while trypsin treatment elevated the net negative electrophoretic mobility of the tritosomes. Tritosome electrophoretic mobilities indicated a homogeneous tritosome population and varied greatly with ionic strength of the suspending media. pH vs. electrophoretic mobility curves indicated the tritosome periphery to contain an acid-dissociable group which likely represents the carboxyl group of N-acetylneuraminic acid; this was not conclusively proven, however, since the tritosomes lysed below a pH of 4 in the present system. Total tritosome carbohydrate (anthrone-positive material as glucose equivalents) was 0.19 mg/mg tritosome protein while total sialic acid was 3.8 µg (11.4 nmol)/mg tritosome protein. A tritosome "membrane" fraction was prepared by osmotic shock, homogenization, and sedimentation. Approximately 25% of the total tritosome protein was present in this fraction. Analysis by gas-liquid chromatography and amino acid analyzer showed the following carbohydrate composition of the tritosome membrane fraction (in microgram per milligram tritosome membrane protein): N-acetylneuraminic acid, 14.8 ± 3; glucosamine, 24 ± 3; galactosamine, 10 ± 2; glucose, 21 ± 2; galactose, 26 ± 2; mannose, 31 ± 5; fucose, 7 ± 1; xylose, 0; and arabinose, 0. The results indicate that the tritosome periphery is characterized by external terminal sialic acid residues and an extensive complement of glycoconjugates. Essentially all the tritosome N-acetylneuraminic acid is located in the membrane and about 53% of it is neuraminidase susceptible.     

Phospholipid composition of a plasma membrane-enriched fraction from developing soybean roots

Phospholipid polar head group and fatty acid composition were determined for plasma membrane enriched fractions from developing soybean root (Glycine max [L.] Merr. cult. Wells II). Plasma membrane vesicles were isolated from meristematic and mature sections of four-day-old dark grown soybean roots at pH 7.8 and in the presence of 5 millimolar ethylenediaminetetraacetate, 5 millimolar ethyleneglycol-bis (β-aminoethyl ether)N,N tetraacetic acid, and 10 millimolar NaF. Lipid extracts analyzed for phospholipid composition revealed two major phospholipid components: phosphatidylcholine and phosphatidylethanolmine. Minor phospholipid components identified were phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, and diphosphatidylglycerol. Lipid degradation by endogenous phospholipase D during membrane isolation at pH 6.5 and in the absence of chelating agents and NaF resulted in the recovery of large amounts of phosphatidic acid. Phosphatidylcholine was the principal substrate for phospholipase D.     

Purification and degradation of ribulose bisphosphate carboxylase from soybean leaves

A rapid method is described for the preparation of up to 500 milligrams of pure ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase) from 250 grams of field-grown soybean leaves. Leaves were extracted in 20 millimolar phosphate (pH 6.9) at 4°C, containing 4% (w/v) polyvinylpolypyrrolidone, 10 micromolar leupeptin, 1 millimolar phenylmethyl sulfonylfluoride, 1 millimolar diethyldithiocarbamate, 5 millimolar MgCl₂, 1 millimolar dithiothreitol, 0.2 millimolar ethylene-diaminetetraacetic acid, 50 millimolar 2-mercaptoethanol. The extract was incubated in the presence of 5 millimolar ATP at 58°C for 9 minutes, then centrifuged and concentrated. Sucrose gradient centrifugation into 8 to 28% (w/v) sucrose on a vertical rotor for 2.5 hours yielded pure enzyme with a specific activity of 1.1 to 1.3 micromoles per minute per milligram protein at pH 8.0, 25°C. Soybean plants of the same line grown (at 400 microeinsteins per square meter per second) in growth chambers yielded enzyme with a specific activity of 0.6 to 0.7 micromoles per minute per milligram protein. During prolonged purification procedures a proteolytic degradation of RuBP carboxylase caused complete loss of catalytic activity. Without destroying the quaternary structure of the enzyme, a 3 kilodalton peptide was removed from all large subunits before further breakdown (removal of a 5 kilodalton peptide) occurred. Catalytic competence of the enzyme was abolished with the loss of the first (3 kilodalton) peptide.