The relationship of hydrophobic tubulin with membranes in neural tissue

Brain membrane preparations contain tubulin that can be extracted with Triton X-114. After the extract is allowed to partition, 8% of the total brain tubulin is isolated as a hydrophobic compound in the detergent-rich phase. Cytosolic tubulin does not show this hydrophobic behaviour since it is recovered in the aqueous phase. Membrane tubulin can be released by 0.1 M Na₂CO₃ treatment at pH11.5 in such a way that the hydrophobic tubulin is converted into the hydrophilic form. These results suggest that tubulin exists associated with some membrane component that confers the hydrophobic behaviour to tubulin. If the tissue is homogenized in microtubule-stabilizing buffer containing Triton X-100, the hydrophobic tubulin is isolated from the microtubule fraction. This result indicates that the hydrophobic tubulin isolated from membrane preparations belongs to microtubules thatin vivo are associated to membranes. Therefore, hydrophobic tubulin (tubulin-membrane component complex) can be obtained from membranes or from microtubules depending on the conditions of brain homogenization.Abbreviations TBS Tris-buffered saline - Mes 2-(N-morpholine) ethane sulfonic acid     

Plasma membrane localization of palmitoylated tubulin

PC12 pheochromocytoma cells incorporate [(3)H]palmitic acid into tubulin in a time- and cell-density-dependent manner. The plasma membrane-enriched fraction contains most of the radioactivity of the membrane pellet. While palmitoylated tubulin is found in both the cytoplasm and particulate fraction, the bulk of [(3)H]palmitic acid bound to tubulin is present in the crude membrane pellet and the tubulin extracted from the plasma membrane is more heavily palmitoylated than that extracted from endoplasmic reticulum. Detergent-extracted tubulin from plasma membrane is, to a large extent, polymerization competent; a substantial fraction, increasing as a function of labeling time, is not hydroxylamine-labile. The requirement for detergent extraction, the accompanying changes in tubulin properties and the present findings of preferential incorporation of labeled tubulin into plasma membranes, make it clear that direct incorporation of tubulin into the plasma membrane can occur.     

Modulation of cytosolic and nuclear Ca2+ and Na+ transport by taurine in heart cells

Brain membranes contain tubulin that can be isolated as a hydrophobic compound by partitioning into Triton X-114. We have previously postulated: (a) that this kind of tubulin is a peripheral membrane protein that arises from microtubules that in vivo interact with membranes and (b) that the hydrophobic behaviour is due to the interaction of tubulin with a membrane component. Here we report the in vitro conversion of hydrophilic into hydrophobic tubulin by incubating microtubule associated proteins (MAPs) free taxol-stabilized microtubules with Triton X-100 solubilized membranes. After incubation, the microtubules were sedimented, depolymerized and subjected to partition into Triton X-114. Part of the tubulin was isolated in the detergent phase and contained, as observed in native membranes, a high proportion of the acetylated isotype. Because of the high proportion of acetylated tubulin the in vitro conversion resembles the in vivo interaction. Electrophoretic analysis of the detergent phase shows, besides tubulin, two major protein bands of 29 and 100 kDa molecular mass. The ability of the solubilized membranes to convert hydrophilic into hydrophobic tubulin is greatly diminished if the solubilized membrane preparation is preincubated in the presence of trypsin or heated at 90°C for 5 min, indicating that the membrane component that confers the hydrophobic behaviour to tubulin is of proteinaceous nature.     

Effects of microtubule agents on the spatial and electrical properties of the plasma membrane inChara corallina

The freshwater algaChara corallina Klein ex Willd., em. R.D.W. (=C. australis R.Br.) develops alternating outward (acid) and inward (alkaline) current areas on its surface when illuminated. Exposure of cells to vinblastine, colchicine, or oryzalin caused a reduction in and a shifting of this extracellular current pattern. Removal of these agents from the bathing media resulted in regeneration of the initial current profile. Because these agents all affect tubulin, microtubules may be responsible for orchestrating the transmembrane currents responsible for the acid and alkaline banding phenomenon. Analysis of the membrane potential showed a fast depolarization after vinblastine exposure; however, analysis of the current-voltage curve did not show a change in membrane conductance. A 30-min colchicine treatment decreased the conductance of the plasma membrane with either an hyperor a depolarization in the membrane potential. In contrast, although a 9-h exposure to oryzalin caused a major reduction in the extra-cellular current pattern, only minor changes were observed in the membrane potential and conductance. However, in the presence of oryzalin, the time constants in the light response of the membrane potential increased over this 9-h period. Collectively, these results implicate an involvement of microtubules in spatial control of plasma-membrane transport events inC. corallina. This research was supported by National Science Foundation grant DCB-88-16077.     

Reconstitution of ciliary membranes containing tubulin

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.     

The ultrastructure of plasma and thylakoid membrane vesicles from the fresh water cyanobacteriumAnacystis nidulans adapted to salinity

Summary Photoautotrophically growing cultures of the fresh water cyanobacteriumAnacystis nidulans adapted to the presence of 0.4–0.5 M NaCl (about sea water level) with a lag phase of two days after which time the growth rate reassumed 80–90% of the control. Plasma and thylakoid membranes were separated from cell-free extracts of French pressure cell treatedAnacystis nidulans by discontinuous sucrose density gradient centrifugation and purified by repeated recentrifugation on fresh gradients. Identity of the plasma and thylakoid membrane fractions was confirmed by labeling of intact cells with impermeant protein markers prior to breakage and membrane isolation. Electron microscopy revealed that each type of membrane was obtained in the form of closed and perfectly spherical vesicles. Major changes in structure and function of the plasma membranes (and, to a much lesser extent, of the thylakoid membranes) were found to accompany the adaptation process. On the average, diameters of plasma membrane vesicles from salt adapted cells were only one-third of the diameters of corresponding vesicles from control cells. By contrast, the diameters of thylakoid membrane vesicles were the same in both cases.Freeze-etching the cells and counting the number of membrane-intercalating particles on both protoplasmic and exoplasmic fracture faces of plasma and thylakoid membranes indicated a roughly 50% increase of the particle density in plasma membranes during the adaptation process while that in thylakoid membranes was unaffected. Comparison between particle densities on isolated membranes and those on corresponding whole cell membranes permitted an estimate as to the percentage of inside-out and right-side-out vesicles. Stereometric measurement of particle sizes suggested that two distinct sub-populations of the particles in the plasma membranes increased during the adaptation process, tentatively correlated to the cytochrome oxidase and sodium-proton antiporter, respectively. The effects of salt adaptation described in this paper were fully reversed upon withdrawal of the additional NaCl from the growth medium (deadaptation). Moreover, they were not observed when the NaCl was replaced by KCl.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - EF exoplasmic fracture face - PF protoplasmic fracture face - DABS diazobenzosulfonate; Hepes N-2-hydroxyethylpiperazine-N-2-ethane-sulfonate - PMSF phenylmethylsulfonylfluoride Dedicated to the memory of Professor Oswald Kiermayer     

Effectors of the mammalian plasma membrane NADH-oxidoreductase system. Short-chain ubiquinone analogues as potent stimulators

In the presence of effectors variations in the two recognized activities of the plasma membrane NADH-oxidoreductase system were studied in separate, specificin vitro assays. We report here that ubiquinone analogues that contain a short, less hydrophobic side chain than coenzyme Q-10 dramatically stimulate the NADH-oxidase activity of isolated rat liver plasma membranes whereas they show no effect on the reductase activity of isolated membranes. If measured in assays of the NADHferricyanide reductase of living cultured cells these compounds have only a limited effect; the oxidase activity of whole cells is not measurable in our hands. We have furthermore identified selective inhibitors of both enzyme activities. In particular, the NADH-oxidase activity can be significantly inhibited by structural analogues of ubiquinone, such as capsaicin and resiniferatoxin. The NADHferricyanide reductase, on the other hand, is particularly sensitive to pCMBS, indicating the presence of a sulfhydryl group or groups at its active site. The identification of these specific effectors of the different enzyme activities of the PMOR yields further insights into the function of this system.     

Comparison of chemical properties of purified plasma membranes and secretory vesicle membranes from the bovine adrenal medulla

Summary A highly enriched fraction of plasma membranes from the bovine adrenal medulla has been isolated by differential and sucrose gradient centrifugation. The membranes were found to occur as 0.1–0.5 diameter vesicles and to equilibrate at a density of 1.13–1.14 g/ml. This fraction was characterized by 4-fold elevated levels of adenylate cyclase and 20-fold elevated levels of 5-nucleotidase. Secretory vesicle membranes, isolated by repeated hypotonie and hypertonic shocks of whole vesicles, were found to equilibrate between d = 1.08 and d = 1.12 on a sucrose density step gradient. These membranes were highly enriched in cytochrome b₅₆₂ and dopamine--hydroxylase. Proteins in the two membranes were compared by SDS gel electrophoresis. All protein size classes found in the vesicle membrane fraction were also represented in the plasma membrane fraction, though in different proportions on the basis of staining intensity. The plasma membrane fraction contained prominent bands co-migrating with the - and -bands of tubulin, as well as a component co-migrating with actin. These bands were absent from the vesicle membranes. Fingerprint analysis of stained bands from the membrane fraction demonstrated that the components were indeed tubulin and actin. The plasma membranes contained twice as much sialic acid residues as did the chromaffin granule membranes, but had only half the cholesterol content on a weight basis. The cholesterolphospholipid ratio in the plasma membranes was 0.63, while in the secretory vesicle membranes it was 1.04. These results show that plasma membranes and secretory vesicle membranes are functionally and structurally different.Supported, in part, by a stipend to O.Z. from The Grant Foundation, New York     

Lateral diffusion in the plasma membrane of maize protoplasts with implications for cell culture

Plasma-membrane dynamics in live protoplasts from maize (Zea mays L.) roots were characterized and examined for relationships as to the ability of the protoplasts to synthesize new cell walls and develop to cells capable of division. The lateral diffusion-coefficients and mobile fractions of fluorescence-labeled plasma-membrane proteins and lipids were measured by fluorescence photobleaching recovery. Small but significant effects on the diffusion of membrane proteins were observed after treatments with oryzalin or amiprophosmethyl, microtubule-disrupting drugs that increased the mobile fraction, and after treatments with cytochalasins B or D, microfilament-disrupting drugs that decreased the diffusion coefficient. A number of parameters were tested for correlative effects on membrane dynamics and protoplast performance in culture. Protoplasts isolated with a cellulase preparation from Trichoderma viride showed faster membrane-protein diffusion and a lower frequency of development to cells capable of division than did protoplasts isolated with a cellulase preparation from T. reesei. Membrane proteins in maize A632, a line less capable of plant regeneration from callus, diffused with a smaller diffusion coefficient but a greater mobile fraction than did membrane proteins in maize A634, a line with greater regeneration capacity. The plasma membranes of A632 and A634 protoplasts also differed with regard to lateral-diffusion characteristics of phospholipid and sterol probes, although the presence of both rapidly and slowly diffusing lipid components indicated the apparent existence of lipid domains in both A632 and A634. The protoplasts of the two lines did not differ significantly, however, in either wall regeneration or frequency of development to cells capable of division.Abbreviations and symbols D lateral diffusion coefficient - FITC fluorescein-5-isothiocyanate - FPR fluorescence photobleaching recovery - LY Lucifer yellow - LY-Chol dilithium 4-amino-N-[(-(carbo(5-cholesten-3-yl)oxy)hydrazinocarbonyl)aminol]-1,8-naphthalimide-3,6-disulfonate - LY-DC_16:0PE dilithium 4-amino-N-[3-(-(dipalmitoyl-sn-glycero-3-phosphoethanol-amino)ethylsulfonyl)phenyl]-1,8-naphthalimide-3,6-disulfonate     

Impact of salt adaptation on esterified fatty acids and cytochrome oxidase in plasma and thylakoid membranes from the cyanobacterium Anacystis nidulans

During adaptation of photoautotrophically growing fresh water cyanobacterium Anacystis nidulans to high salinity the cells showed a pronounced increase of proton-sodium antiporter activity, and of cytochrome c oxidase in isolated and purified plasma membrane. At the same time the concentrations of plasma membrane-bound EDTA-resistant copper and iron (determined by inductively coupled plasma atomic emission spectrometry) rose proportionately, accompanied by an increase in whole cell respiration. In plasma membranes from salt adapted cells lipid/protein ratios were markedly higher than in control cells, levels of esterified saturated and long-chain fatty acids being significantly higher than the respective levels of unsaturated and short-chain fatty acids which explains the higher lipid-phase transition temperatures derived from Arrhenius plots. Immunoblotting of the membrane proteins with antisera raised against the cytochrome c oxidases from Paracoccus denitrificans and A. nidulans gave two cross-reacting bands with apparent molecular weights around 50000 and 30000 (subunits I and II, respectively) which were more pronounced in plasma membranes from salt adapted cells when compared to control cells. The protein pattern of plasma membranes from salt adapted cells also showed the appearance of bands at apparent molecular weights of 44000–48000 and 54000–56000 which might stem from the proton/sodium-antiporter in this membrane.Abbreviations CM cytoplasmic or plasma membrane - ICM intracytoplasmic or thylakoid membrane - cyt cytochrome - DCCD N,N-dicyclohexylcarbodiimide - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - ICP-AES inductively coupled plasma atomic emission spectrometry - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - EPR electron paramagnetic resonance spectrometry     

Activation of PMCA by calmodulin or ethanol in plasma membrane vesicles from rat brain involves dissociation of the acetylated tubulin/PMCA complex

We have recently shown that acetylated tubulin interacts with plasma membrane Na(+),K(+)-ATPase and inhibits its enzyme activity in several types of cells. H(+)-ATPase of Saccharomyces cerevisiae is similarly inhibited by interaction with acetylated tubulin. The activities of both these ATPases are restored upon dissociation of the acetylated tubulin/ATPase complex. Here, we report that in plasma membrane vesicles isolated from brain synaptosomes, another P-type ATPase, plasma membrane Ca(2+)-ATPase (PMCA), undergoes enzyme activity regulation by its association/dissociation with acetylated tubulin. The presence of acetylated tubulin/PMCA complex in membrane vesicles was demonstrated by analyzing the behavior of acetylated tubulin in a detergent partition, and by immunoprecipitation experiments. PMCA is known to be stimulated by ethanol and calmodulin at physiological concentrations. We found that treatment of plasma membrane vesicles with these reagents induced dissociation of the complex, with a concomitant restoration of enzyme activity. Conversely, incubation of vesicles with exogenous tubulin induced the association of acetylated tubulin with PMCA, and the inhibition of enzyme activity. These findings indicate that activation of synaptosomal PMCA by ethanol and calmodulin involves dissociation of the acetylated tubulin/PMCA complex. This regulatory mechanism was shown to also operate in living cells.     

Iron limitation and its effect on membrane proteins and siderophore transport inNeurospora crassa

Summary Cells of the fungusNeurospora crassa were grown under iron-deficient and iron-sufficient conditions and their plasma membrane proteins were compared. Three strains were studied:N. crassa 74A (wild type), a siderophore-free mutantN. crassa (arg-5 ota aga) as well as a slime variant ofN. crassa which lacks a cell wall. Plasma membranes were purified, solubilized and analyzed by one-dimensional SDS/polyacrylamide gel electrophoresis yielding approximately 50 distinct protein bands with molecular masses in the range 14–160 kDa. Iron-sufficient and iron-deficient growth resulted in nearly identical plasma membrane protein profiles in all strains. Although minor alterations in the proportion of certain proteins could be detected, significant overproduction of certain membrane proteins during iron limitation could not be observed. Transport of⁵⁵ Fe-labeled siderophores seems to be correlated to the degree of iron limitation. For example, transport rates were enhanced five-fold after 16 h of growth in iron-deficient medium compared to growth in iron-sufficient medium. Extraction and HPLC measurement of siderophores from conidiospores yielded approximately 10^–15 mol/spore, indicating that germination tubes and young cells used for transport measurements are not iron-deficient. It is suggested that the putative transport systems for siderophores in fungal plasma membranes are constitutively expressed and enhanced uptake of siderophores during iron limitation is rather the result of cellular transport regulation mechanisms.     

Activation of the plasma membrane H-ATPase of Saccharomyces cerevisiae by glucose is mediated by dissociation of the H(+)-ATPase-acetylated tubulin complex

In the yeast Saccharomyces cerevisiae, plasma membrane H(+)-ATPase is activated by d-glucose. We found that in the absence of glucose, this enzyme forms a complex with acetylated tubulin. Acetylated tubulin usually displays hydrophilic properties, but behaves as a hydrophobic compound when complexed with H(+)-ATPase, and therefore partitions into a detergent phase. When cells were treated with glucose, the H(+)-ATPase-tubulin complex was disrupted, with two consequences, namely (a) the level of acetylated tubulin in the plasma membrane decreased as a function of glucose concentration and (b) the H(+)-ATPase activity increased as a function of glucose concentration, as measured by both ATP-hydrolyzing capacity and H(+)-pumping activity. The addition of 2-deoxy-d-glucose inhibited the above glucose-induced phenomena, suggesting the involvement of glucose transporters. Whereas total tubulin is distributed uniformly throughout the cell, acetylated tubulin is concentrated near the plasma membrane. Results from immunoprecipitation experiments using anti-(acetylated tubulin) and anti-(H(+)-ATPase) immunoglobulins indicated a physical interaction between H(+)-ATPase and acetylated tubulin in the membranes of glucose-starved cells. When cells were pretreated with 1 mm glucose, this interaction was disrupted. Double immunofluorescence, observed by confocal microscopy, indicated that H(+)-ATPase and acetylated tubulin partially colocalize at the periphery of glucose-starved cells, with predominance at the outer and inner sides of the membrane, respectively. Colocalization was not observed when cells were pretreated with 1 mm glucose, reinforcing the idea that glucose treatment produces dissociation of the H(+)-ATPase-tubulin complex. Biochemical experiments using isolated membranes from yeast and purified tubulin from rat brain demonstrated inhibition of H(+)-ATPase activity by acetylated tubulin and concomitant increase of the H(+)-ATP ase-tubulin complex.     

Specific binding of a hypersensitive lignification elicitor from Puccinia graminis f. sp. tritici to the plasma membrane from wheat (Triticum aestivum L.)

We have recently reported the isolation and characterization of a glycoprotein (M_r 67 000) from germ-tube walls of Puccinia graminis f. sp. tritici which elicits the cellular hypersensitive lignification response in wheat (G. Kogel et al., 1988, Physiol. Mol. Plant Pathol. 33, 173–185). The present study uses this glycoprotein, referred to as Pgt elicitor, to identify putative elicitor targets in wheat cell membranes. In enzyme-linked immunosorbent assays using anti-Pgt elicitor antibodies, specific binding sites for Pgt elicitor were detected in highly purified plasma-membrane vesicles of wheat (Triticum aestivum L.) primary leaf cells. Binding proved to be independent of the presence or absence in wheat of the Sr5 gene for rust resistance, and also occurred on barley (Hordeum vulgare L.) plasma membrane. The binding sites have an Mr of 30 000 and 33 000, respectively, and binding activity was not lost in the presence of sodium dodecyl sulfate. [¹⁴C]imido-Pgt elicitor was used to determine the apparent K _d value for specific binding, found to be 2.0 M, and the maximum content of binding sites, found to be 250 pmol per mg of plasma-membrane protein. The relevance of the elicitor binding for the outcome of the interaction of P. graminis and wheat is discussed.Abbreviations BSA bovine serum albumin - ELISA enzyme linked immunosorbent assay - IDPase inosine 5-diphosphatase - MPLC medium-pressure liquid chromatography - MF microsomal fraction - Pgt elicitor elicitor of Puccinia graminis f. sp. tritici - SDS sodium dodecyl sulfate - Pre U₃, Pre U₁ pure plasma membrane from wheat cultivar Prelude and plasma membrane contaminated by intracellular membrane, respectively This work was supported by the Deutsche Forschungsgemeinschaft. We wish to thank C. Larsson, Lund, Sweden for his kind support in the preparation of plasma membrane.     

Carbohydrate antigens and lectin receptors of the plasma membrane of carrot cells

Summary A monoclonal antibody (JIM 1) has been derived, subsequent to immunization of rats with carrot protoplasts and a hybridoma screen of protoplast immunoagglutination, that recognizes a determinant at the outer face of the plasma membrane of carrot cells. The binding of JIM 1 is readily inhibitable by -D-galactosyl residues. Although weakly cross-reacting with an extracellular arabinogalactan protein, isolated from the conditioned medium of suspension-cultured carrot cells, JIM 1 does not recognize arabinogalactan proteins associated with the plasma membrane. The plasma membrane antigen recognized by JIM 1 was of low molecular weight and was sensitive to both periodate treatment and a protease. JIM 1 therefore defines a new class of galactosyl-residue containing plant cell surface antigen, distinct from the arabinogalactan proteins. However, the extracellular arabinogalactan protein and related plasma membrane-associated glycoproteins are demonstrated to bind the anti-galactose plant lectin peanut agglutinin.Abbrevations AGP arabinogalactan protein - McAb monoclonal antibody - PNA peanut agglutinin     

Effect of Anti-Microtubular Agents on Respiration and Ultrastructural Organization of Wheat Leaf Cells

The effect of anti-microtubular agents oryzalin (15 M) and colchicine (1 mM) on respiration and fine cellular organization of leaves was studied in wheat (Triticum aestivum L.) seedlings. Unlike oryzalin, colchicine considerably suppressed total respiration and the activities of cytochrome and cyanide-resistant pathways of electron transport, probably due to its rotenone-like effect since the latter was negated by vicasol that shunts the first complex of respiratory chain. Oryzalin-induced changes in the ultrastructure of mitochondria and polysomes corresponding to a decrease in their functional activity were detected. The changes in the shape of mitochondria were observed in the companion cells, whereas the mesophyll organelles did not respond to oryzalin. Disintegration of polysomes induced by oryzalin was detected in the cells of both tissues. Association of microtubules with mitochondria, polysomes, and plasma membrane was detected in situ. These data suggest that the intact cytoskeleton may participate in the spatial organization of energy transformation and protein synthesis machinery of the cells; the fact that the observed changes were tissue-specific points at the functional role of microtubules and/or to the number of targets for the inhibitor.     

Inhibition of the NADH oxidase activity of plasma membranes isolated from xenografts and cell lines by the antitumor sulfonylurea,N-(4-methylphenylsulfonyl)-N′-(4-chlorophenyl)urea (LY 181984) correlates with drug susceptibility of growth

Summary Inhibition of NADH oxidase activity of plasma membranes isolated from a series of human xenografts and cell lines by the antitumor sulfonylurea, N-(4-methylphenylsulfonyl)-N-(4-chlorophenyl) urea (LY 181984), correlated with the ability of the sulfonylurea to inhibit cell growth. Growth of rat kidney cells either untransformed or transformed with Kirsten-ras (K-ras) were unaffected by the sulfonylurea. Similarly, the NADH oxidase activity of isolated plasma membranes from K-ras transformed cells was unaffected by LY 181984. In contrast, when transformed with Harvey-ras (H-ras), both growth and NADH oxidase activity were inhibited. With the inactive but structurally related LY 181985 (N-4-methylphenyl-sulfonyl)-N-(phenyl)urea), neither growth nor plasma membrane NADH oxidase activity of either sulfonylurea-susceptible or -resistant tissues or cell lines was inhibited. Both sulfonylureas were inactive with rat liver plasma membranes but NADH oxidase activity of plasma membranes and growth with HeLa cells was inhibited by the active (LY 181984) but not by the inactive (LY 181985) sulfonylurea. The findings suggest a possible correlation between inhibition of plasma membrane NADH oxidase activity by the antitumor sulfonylureas and their oncolytic action.     

A possible plasma membrane particle containing malic enzyme activity

A definite membrane fraction from Cucurbita hypocotyls, maize coleoptiles, and other plant tissues contains a NADP-dependent malic enzyme activity, up to 10% of overall tissue activity, and probably other soluble proteins. This malic enzyme particle is identified as plasmalemma on the basis of sedimentation behavior, density distribution in sucrose gradients, in comparison with enzyme markers, and sluggish penetration by the sugar Metrizamide. Enzyme binding to the plasma membrane is stable and scarcely sensitive to salts and EDTA, although all activity is released to the supernatant in the presence of Triton-X-100 or under hypotonic conditions. The properties of bound enzyme are similar to those of free enzyme in cell extracts. It is proposed that osmotically sensitive plasma membrane vesicles, containing cytoplasm fragments, are formed during homogenization. Low malic enzyme activities are also associated with Cucurbita proplastids.Abbreviations Tris tris(hydroxymethyl)amino methane - NPA naphthylphtalamic acid - malic enzyme L-malate - NAD(P) oxidoreductase, decarboxylating (EC 1.1.1.40) - ER endoplasmic reticulum     

Oryzalin,a dinitroaniline herbicide,binds to plant tubulin and inhibits microtubule polymerization in vitro

The effects of oryzalin, a dinitroaniline herbicide, on chromosome behavior and on cellular microtubules (MTs) were examined by light microscopy and immunogold staining, respectively, in endosperm cells from Haemanthus katherinae Bak. Brief treatments with 1.0·10^-8 M oryzalin reduced markedly the migration rate of anaphase chromosomes and 1.0·10^-7 M oryzalin stopped migration abruptly. Oryzalin (1.0·10^-7 M) depolymerized MTs and prevented the polymerization of new MTs at all stages of the mitotic cycle. The chromosome condensation cycle was unaffected by oryzalin. Endothelial cells from the heart of Xenopus leavis showed no chromosomal or microtubular rearrangements after oryzalin treatment. The inhibition by oryzalin of the polymerization of tubulin isolated from cultured cells of Rosa sp. cv. Paul's scarlet was examined in vitro by turbidimetry, electron microscopy and polymer sedimentation analysis. Oryzalin inhibited the rapid phase of taxol-induced polymerization of rose MTs at 24°C with an apparent inhibition constant (K _i ) of 2.59·10⁶ M. Shorter and fewer MTs were formed with increasing oryzalin concentrations, and maximum inhibition of taxol-induced polymerization occurred at approx. 1:1 molar ratios of oryzalin and tubulin. Oryzalin partially depolymerized taxol-stabilized rose MTs. Ligand-binding experiments with [¹⁴C]oryzalin demonstrated the formation of a tubulin-oryzalin complex that was time- and pH-dependent. The tubulin-oryzalin interaction (24°C, pH 7.1) had an apparent affinity constant (K _app) of 1.19·10⁵ M^-1. Oryzalin did not inhibit taxol-induced polymerization of bovinebrain MTs and no appreciable binding of oryzalin to brain tubulin or other proteins was detected. The results demonstrate pharmacological differences between plant and animal tubulins and indicate that the most sensitive mode of action of the dinitroaniline herbicides is the direct poisoning of MT dynamics in cells of higher plants.Abbreviations MT microtubule - SIB sucrose isolation buffer - TO tubulin-oryzalin complex     

Tyrosinated,detyrosinated and acetylated tubulin isotypes in rat brain membranes. Their proportions in comparison with those in cytosol

The heterogeneity of -tubulin and the relative proportions of the tubulin isotypes were investigated in brain membranes of rats of 1, 25 and 180 days of age by using four anti--tubulin antibodies: a) the monoclonal DM1A antibody, specific for -tubulin; b) the monoclonal 6-11B-1 antibody, specific for acetylated tubulin; c) a polyclonal antibody (Glu antibody), specific for detyrosinated tubulin; and d) a polyclonal antibody (Tyr antibody), specific for tyrosinated tubulin. We found that rat brain membranes contain the three tubulin isotypes mentioned above. The proportions of tyrosinated and detyrosinated tubulin relative to total -tubulin were somewhat lower in membrane than in cytosol in animals of 25 and 180 days of age. At day one of development, the proportions in membrane were similar to those found in cytosol. With respect to the acetylated form, it was about 20 times higher in membrane than in cytosol at the three ages studied. The proportion of acetylated tubulin was determined in different subcellular fractions: myelin, synaptic vesicles, mitochondria, microsomes, and plasma membrane. While the amount of total tubulin differed between the different subcellular fractions, the proportion of acetylated tubulin relative to total -tubulin was constant and similar to that found in total membranes. The proportion of acetylated tubulin was also investigated in non-neural tissues (kidney, liver and lung). Although values for cytosol were about 10-fold higher than that found in brain cytosol, no detectable values for membranes could be obtained in these organs.