Protein and Lipid Compositions of Isolated Plasma Membranes from Orchard Grass (Dactylis glomerata L.) and Changes during Cold Acclimation

The chemical composition of plasma membrane fractions isolated from orchard grass seedlings (Dactylis glomerata L.) was analyzed and compared with endomembranes. The plasma membrane is characterized by an enrichment of sterols and a lower degree of unsaturation of phospholipids. Steryl glycosides were found to be one of the lipid components of the plasma membrane, but steryl esters and galactolipids were barely detectable. Diphosphatidyl glycerol was characteristically detected in the mitochondrial membrane, but not in the plasma membrane fraction. Plasma mambrane fraction was also characterized by its `lower fluidity' in comparison with the endomembranes. This may be due to the large amount of sterols and the lower degree of phospholipid unsaturation in plasma membranes.

Electrophoretic comparison of polypeptides was also made between different membranes. The distribution patterns of polypeptides revealed on one- and two-dimensional SDS-slab gels were characteristic for those membranes. The presence of glycopeptide complements was a useful criterion for distinguishing plasma membrane from other membranes. The plasma membrane and the ER + Golgi membranes were enriched in glycopeptides. However, a marked difference was revealed in the total number and the molecular weights of the peptides.

During cold acclimation of orchard grass seedlings, the degree of fatty acid unsaturation increased only slightly in plasma membrane, unlike in endomembranes. The change in sterols-to-phospholipids ratio in plasma membrane was also slight. On the other hand, the phospholipid-to-protein ratio increased significantly in the plasma membrane as cold hardiness increased. A significant change in the polypeptide complements of plasma membrane was also demonstrated during cold acclimation.

     

Phosphorylation of the adenosine triphosphatase in a deoxycholate-treated plasma membrane fraction from corn roots

The ATP phosphohydrolase (ATPase) activity of a corn (Zea mays L., WF9 × Mo17) root plasma membrane fraction was enriched almost 2-fold by selective extraction with 0.1% (w/v) deoxycholate. The detergent treatment solubilized about 30% of the total membrane protein and some ATP hydrolyzing activity that was not K⁺-stimulated, but the major portion of the ATPase activity could be pelleted with membranes. The properties of the ATPase associated with the detergent-extracted plasma membrane fraction were similar to those for the ATPase of the untreated plasma membrane fraction with respect to substrate specificity, pH optimum, kinetics with MgATP, ion stimulation, and inhibitor sensitivity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed only minor differences in protein composition resulting from the detergent treatment.

The plasma membrane fraction from corn roots contained an endogenous protein kinase activity. This was shown by the time course of phosphate incorporation and by the labeling of a number of protein bands on SDS-polyacrylamide gel electrophoresis. The deoxycholate treatment removed measurable protein kinase activity and allowed the demonstration of a rapidly turning over covalent phosphorylated intermediate associated with the detergent-extracted plasma membrane fraction. The phosphorylated intermediate was present as a 100,000 dalton polypeptide and may represent the catalytic subunit of the plasma membrane K⁺-ATPase.

     

The effects of salt stress on polypeptides in membrane fractions from barley roots

Cell fractions enriched in endoplasmic reticulum, tonoplast, plasma membrane, and cell walls were isolated from roots of barley (Hordeum vulgare L. cv CM 72) and the effect of NaCl on polypeptide levels was examined by two-dimensional (2D) polyacrylamide gel electrophoresis. The distribution of membranes on continuous sucrose gradients was not significantly affected by growing seedlings in the presence of NaCl; step gradients were used to isolate comparable membrane fractions from roots of control and salt-grown plants. The membrane and cell wall fractions each had distinctive polypeptide patterns on 2D gels. Silver-stained gels showed that salt stress caused increases or decreases in a number of polypeptides, but no unique polypeptides were induced by salt. The most striking change was an increase in protease resistant polypeptides with isoelectric points of 6.3 and 6.5 and molecular mass of 26 and 27 kilodaltons in the endoplasmic reticulum and tonoplast fractions. Fluorographs of 2D gels of the tonoplast, plasma membrane, and cell wall fractions isolated from roots of intact plants labeled with [³⁵S]methionine in vivo also showed that salt induced changes in the synthesis of a number of polypeptides. There was no obvious candidate for an integral membrane polypeptide that might correspond to a salt-induced sodium-proton anti-porter in the tonoplast membrane.     

Comparisons of plasma membrane polypeptides from soybean and alfalfa

Polypeptides were solubilized with sodium dodecyl sulfate from plasma membrane vesicles of eight varieties of soybean roots [Glycine max (L.) Merr.] and of cultured alfalfa cells (Medicago sativa L.). The solubilized polypeptides were analysed by 2D-polyacrylamide gel electrophoresis. Apparent isoelectric point and MW values were obtained for 80 soybean plasma membrane polypeptides and 44 alfalfa plasma membrane polypeptides. From these data composite distribution patterns were constructed, which are representative of the soybean or alfalfa 2D-gels, respectively. The results showed that the general polypeptide staining patterns were similar for all the soybean varieties, but some minor differences were evident. The alfalfa electrophoretograms differed markedly from the soybean electrophoretograms in specific details, though some general pattern similarities were noted. The data are discussed in terms of a physiological role for the integral plasma membrane polypeptides and in terms of the potential for distinguishing among soybean varieties and between species at the plasma membrane polypeptide level.     

NADH-Ferricyanide Reductase of Leaf Plasma Membranes : Partial Purification and Immunological Relation to Potato Tuber Microsomal NADH-Ferricyanide Reductase and Spinach Leaf NADH-Nitrate Reductase

Plasma membranes obtained by two-phase partitioning of microsomal fractions from spinach (Spinacea oleracea L. cv Medania) and sugar beet leaves (Beta vulgaris L.) contained relatively high NADH-ferricyanide reductase and NADH-nitrate reductase (NR; EC 1.6.6.1) activities. Both of these activities were latent. To investigate whether these activities were due to the same enzyme, plasma membrane polypeptides were separated with SDS-PAGE and analyzed with immunoblotting methods. Antibodies raised against microsomal NADH-ferricyanide reductase (tentatively identified as NADH-cytochrome b₅ reductase, EC 1.6.2.2), purified from potato (Solanum tuberosum L. cv Bintje) tuber microsomes, displayed one single band at 43 kilodaltons when reacted with spinach plasma membranes, whereas lgG produced against NR from spinach leaves gave a major band at 110 kilodaltons together with a few fainter bands of lower molecular mass. Immunoblotting analysis using inside-out and right-side-out plasma membrane vesicles strongly indicated that NR was not an integral protein but probably trapped inside the plasma membrane vesicles during homogenization. Proteins from spinach plasma membranes were solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio] 1-propane-sulfonate and separated on a Mono Q anion exchange column at pH 5.6 with fast protein liquid chromatography. One major peak of NADH-ferricyanide reductase activity was found after separation. The peak fraction was enriched about 70-fold in this activity compared to the plasma membrane. When the peak fractions were analyzed with SDS-PAGE the NADH-ferricyanide reductase activity strongly correlated with a 43 kilodalton polypeptide which reacted with the antibodies against potato microsomal NADH-ferricyanide reductase. Thus, our data indicate that most, if not all, of the truly membrane-bound NADH-ferricyanide reductase activity of leaf plasma membranes is due to an enzyme very similar to potato tuber microsomal NADH-ferricyanide reductase (NADH-cytochrome b₅ reductase).     

水稻根质膜蛋白质组双向电泳水化液的优化

以水稻(Oryza sativa L.)苗期幼嫩根尖作为材料,利用葡聚糖-聚乙二醇两相分配法纯化得到纯度达90%的质膜组分,使用4种不同的水化液溶解质膜蛋白,进行IEF/SDS-PAGE双向电泳和MALDI-TOF/TOF质谱分析.结果显示,4种水化液中,以7 mol/L Urea2、mol/L Thiourea、4%CHAPS、20 mmol/L DTE、1%ASB14的条件对膜蛋白的溶解效果和双向电泳分离效果最好;16个被鉴定蛋白中有9个为质膜相关蛋白,5个为未知蛋白,来自其它细胞器的蛋白仅有2个.研究表明,在常用水化液中添加磺基甘氨酸三甲内盐ASB14有利于植物细胞质膜蛋白质组的分析,并且该优化条件下的双向电泳适合分离水稻质膜中亲水性相对较高的膜附着蛋白.     

ISOLATION AND CHARACTERIZATION OF PLASMA MEMBRANE FRACTIONS FROM GARFISH LEPISOSTEUS OSSEUS OLFACTORY NERVE

Garfish Lepisosteus osseus olfactory nerve, because of its large size and the unusually high concentration of axonal membrane, is an excellent source of axonal membrane. A procedure is described for the isolation of two types of plasma membranes from the nerve which are obtained in yields of about 20 mg (fraction I) and 1.5 mg (fraction II) per g of wet nerve. Both membrane fractions consist mostly of rounded membrane vesicles, with a unit membrane thickness of ~7.5 nm. The two membrane fractions are different in their lipid to protein ratios, Na-K ATPase activities, polypeptide patterns on sodium dodecyl sulfate (SDS) gel electrophoresis, and fatty acid compositions. They have similar phospholipid composition. On the basis of the relative concentration of axonal and Schwann cell plasma membranes in the nerve, the Na-K ATPase activities of the two membrane fractions and a comparison of the properties of the membrane fractions to those of squid and lobster nerve membrane preparations, fraction I seems to be the axonal membrane and fraction II the Schwann cell plasma membrane. Fraction I has a low protein to lipid ratio. Its polypeptide pattern on SDS gel appears to be much more complex as compared to that of fraction II membrane.     

Lipid Characterization of an Enriched Plasma Membrane Fraction of Dunaliella salina Grown in Media of Varying Salinity

We have developed a rapid procedure for isolating a fraction enriched in plasma membrane from Dunaliella salina using an aqueous two-phase system (dextran/polyethylene glycol, 6.7%/6.7%). An enriched plasma membrane fraction, free of chloroplast and mitochondrial contamination, could be obtained in 2.5 hours. Plasma membrane proteins, which accounted for approximately 1% of the total membrane protein, contained a number of unique proteins compared with the other cell fractions, as shown by gel electrophoresis. The lipids of the plasma membrane fraction from 1.7 molar NaCl-grown cells were extracted and characterized. Phosphatidylethanolamine and phosphatidylcholine were the two most prevalent phospholipids, at 20.6% and 6.0% of the total lipid, respectively. In addition, inositol phospholipids were a significant component of the D. salina plasma membrane fraction. Phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate accounted for 5.2% and 1.5% of the plasma membrane phospholipid, respectively. Diacylglyceryltrimethylhomoserine accounted for 7.9% of the plasma membrane total lipid. Free sterols were the major component of the plasma membrane fraction, at 55% of the total lipid, and consisted of ergosterol and 7-dehydroporiferasterol. Sterol peroxides were not present in the plasma membrane fraction. The lipid composition of enriched plasma membrane fractions from cells grown at 0.85 molar NaCl and 3.4 molar NaCl were compared with those grown at 1.7 molar NaCl. The concentration of diacylglyceryltrimethylhomoserine and the degree of plasma membrane fatty acid saturation increased in 3.4 molar plasma membranes. The relative concentration of sterols in the plasma membrane fraction was similar in all three NaCl concentrations tested.     

Nitrate-induced polypeptides in membranes from corn seedling roots

The polypeptide composition of the membranes from corn (Zeamays L.) seedling roots upon nitrate induction was determinedby two-dimensional gel electrophoresis and silver-staining.The synthesis of five polypeptides (49, 48, 35, 33, and 32 kDa)in the tono-plast fraction and four polypeptides (50, 49, 38,and 33 kDa) in the plasma membrane fraction was induced by both2.5 mM Ca(NO₃)₂ and 5 mM KNO₃. Extensive washing of the membraneswith salt and NaOH demonstrated that three induced polypeptides(49, 48, and 35 kDa) in the tonoplast fraction and two inducedpolypeptides (49 and 33 kDa) in the plasma membrane fractionwere integral proteins. After incubation of seedlings in N-freemedium for 4 d, the 49 and 32 kDa polypeptides in the tonoplastfraction had disappeared. By the sixth day in N-free medium,the 35 kDa polypeptide had disappeared from the tonoplast fraction.The 50 kDa polypeptide of the plasma membrane fraction was nolonger detectable in seedlings incubated for 6 d in N-free medium.The size of the spots corresponding to the 33 kDa polypeptidesof both membrane fractions and to the 49 kDa polypeptide ofthe plasma membrane fraction was reduced following incubationof seedlings in N-free medium. The changes in nitrate-inducedpolypeptides in both membrane fractions following transfer toN-free medium correlated with a reduced capacity to take upnitrate in the treated seedlings. The results support the conclusionthat the nitrate-induced polypeptides may be involved in nitratetransport across the tonoplast and plasma membrane. Key words: Nitrate transport, induction, membrane peptides     

Highly enriched,minimally disrupted plasma membrane vesicles from aortic myocytes grown in primary culture

A plasma membrane-enriched fraction (fraction 1B) has been obtained from rat aortic myocytes grown in primary culture. Plasma membrane markers, 5′-nucleotidase and ouabain-sensitive (Na⁺ + K⁺)-ATPase, are enriched 4.1- and 8.7-fold, respectively, in this fraction. Although endoplasmic reticulum marker NADPH-cytochrome c reductase is the most enriched in mitochondrial and heavy sucrose density gradient fractions, substantial enrichment of this marker is also observed in membrane fraction 1. This membrane preparation therefore contains a certain quantity of endoplasmic reticulum. Cytochrome c oxidase is de-enriched by a factor of 0.04 in fraction 1, indicating that it is essentially clear of mitochondrial contamination. Homogenization of aortic media-intima layers using a whole-tissue technique induces greater disruption of mitochondria and subsequent contamination of membrane fractions than does the procedure for cell disruption. Analysis of electrophoretic gels, vesicle density distribution and electron micrographs of enriched membrane fractions provide evidence that plasma membrane enriched from cultured myocytes is less traumatized than comparable fractions obtained from intact tissue. The potential value of such a highly enriched, minimally disrupted plasma membrane preparation is discussed.     

Photoaffinity labeling of a synaptic vesicle specific nucleotide transport system from Torpedo marmorata

We have employed azido derivatives of ATP and AMP to identify the ATP translocase of synaptic vesicles. Azido-AMP inhibits transport of both ATP and AMP in vitro. The affinity of the translocase for the azido derivatives is similar to that of the native ligands. Upon UV irradiation of vesicles incubated with radiolabeled azido-AMP or -ATP, a molecular weight (Mr) 34000 polypeptide is selectively modified. On two-dimensional gel electrophoresis, the single radiolabeled polypeptide has a pI of approximately 7.7. Analysis of the fractions obtained when vesicles were purified on linear sucrose density gradients reveals that the Mr 34000 polypeptide is highly enriched in the vesicle-containing fractions. The findings support the notion that this polypeptide is identical with a previously described vesicle-specific component of the same molecular size [Stadler, H., & Tashiro, T. (1979) Eur. J. Biochem. 101, 171-178], and we conclude on the basis of uptake inhibition and photoaffinity labeling results that this protein is directly involved in ATP translocation of synaptic vesicles.     

Protein heterogeneity of lipoprotein particles containing apolipoprotein A-I without apolipoprotein A-II and apolipoprotein A-I with apolipoprotein A-II isolated from human plasma

The protein heterogeneity of fractions isolated by immunoaffinity chromatography on anti-apolipoprotein A-I and anti-apolipoprotein A-II affinity columns was analyzed by high resolution two-dimensional gel electrophoresis. The two-dimensional gel electrophoresis profiles of the fractions were analyzed and automatically compared by the computer system MELANIE. Fractions containing apolipoproteins A-I + A-II and only A-I as the major protein components have been isolated from plasma and from high density lipoproteins prepared by ultracentrifugation. Similarities between the profiles of the fractions, as indicated by two-dimensional gel electrophoresis, suggested that those derived from plasma were equivalent to those from high density lipoproteins (HDL), which are particulate in nature. The established apolipoproteins (A-I, A-II, A-IV, C, D, and E) were visible and enriched in fractions from both plasma and HDL. However, plasma-derived fractions showed a much greater degree of protein heterogeneity due largely to enrichment in bands corresponding to six additional proteins. They were present in trace amounts in fractions isolated from HDL and certain of the proteins were visible in two-dimensional gel electrophoresis profiles of the plasma. These proteins are considered to be specifically associated with the immunoaffinity-isolated particles. They have been characterized in terms of Mr and pI. Computer-assisted measurements of protein spot-staining intensities suggest an asymmetric distribution of the proteins (as well as the established apolipoproteins), with four showing greater prominence in particles containing apolipoprotein A-I but no apolipoprotein A-II.     

Isolation of Cell Membranes from Saccharomyces cerevisiae for Evaluation of Their Protein Composition

We describe a simple method for the isolation of membrane fractions from Saccharomyces cerevisiae yeasts containing a complex of plasma membranes and cell walls. The method is based on cell disruption on an INBI flow disintegrator. This device spares subcellular structures, which simplifies the isolation of cell membranes. The membrane fraction obtained by this method was suitable for studies of the protein composition of these structures by means of two-dimensional gel electrophoresis.     

Surface properties of right side-out plasma membrane vesicles isolated from barley roots and leaves

Highly purified plasma membrane vesicles were obtained from roots and leaves of 7-day-old light-grown barley (Hordeum vulgare L. cv Kristina) seedlings by partitioning of crude microsomal fractions in a dextran-polyethylene glycol two-phase system. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed the polypeptide composition of plasma membranes from the two organs to be qualitatively similar, but with different relative amounts of some of the polypeptides. Between 80 and 100% of the K⁺,Mg²⁺-ATPase activity was latent indicating that the vesicles were sealed and right side-out. The isoelectric points of the outer surface of root and leaf plasma membranes as determined by cross-partitioning were similar and quite acidic—about pH 3.6. In contrast, the net negative surface charge density at pH 7.0 as measured by 9-aminoacridine fluorescence differed significantly, being −29 mC·m⁻² for the leaf plasma membrane and only −19 mC·m⁻² for the root plasma membrane. As isolated, both types of plasma membrane vesicles had Ca²⁺ and Mg²⁺ bound to the outer surface as shown by the combined use of chelators and 9-aminoacridine fluorescence; however, the leaf plasma membrane had a relatively higher proportion of Ca²⁺ bound (0.57) than did the root plasma membrane (0.45). This difference probably reflects differences in the in vivo conditions as no chelator was present during the isolation procedure. Also Ni²⁺ could bind to the root vesicles as indicated by the effect of Ni²⁺ on 9-aminoacridine fluorescence, and by the binding of ⁶³Ni²⁺ (44 nanomoles bound per milligram protein) at 100 micromolar NiCl₂.     

Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis

A plasma membrane-enriched fraction prepared from barley roots was analyzed by two-dimensional gel electrophoresis. Four methods of sample solubilization were assessed on silver stained gels. When membranes were solubilized with 2% sodium dodecyl sulfate followed by addition of Nonidet P-40, gels had high background staining and few proteins because of incomplete solubilization. Gels of membranes solubilized in urea and Nonidet P-40 had a greater number of proteins but proteins with molecular weights greater than 85,000 were absent and proteins with low molecular weights were diffuse. High molecular weight proteins were present in gels of membranes solubilized in 4% sodium dodecyl sulfate followed by acetone precipitation but background staining and streaking remained a problem. Gels of the best quality were obtained when membrane proteins were extracted with phenol and precipitated with ammonium acetate in methanol; background staining and streaking were diminished and proteins were clearly resolved. This method makes possible the resolution required for meaningful qualitative and quantitative comparisons of protein patterns on two-dimensional gels of plant membrane proteins.     

Isolation and characterization of the plasma membrane from the yeast Pichia pastoris

Despite similarities of cellular membranes in all eukaryotes, every compartment displays characteristic and often unique features which are important for the functions of the specific organelles. In the present study, we biochemically characterized the plasma membrane of the methylotrophic yeast Pichia pastoris with emphasis on the lipids which form the matrix of this compartment. Prerequisite for this effort was the design of a standardized and reliable isolation protocol of the plasma membrane at high purity. Analysis of isolated plasma membrane samples from P. pastoris revealed an increase of phosphatidylserine and a decrease of phosphatidylcholine compared to bulk membranes. The amount of saturated fatty acids in the plasma membrane was higher than in total cell extracts. Ergosterol, the final product of the yeast sterol biosynthetic pathway, was found to be enriched in plasma membrane fractions, although markedly lower than in Saccharomyces cerevisiae. A further characteristic feature of the plasma membrane from P. pastoris was the enrichment of inositol phosphorylceramides over neutral sphingolipids, which accumulated in internal membranes. The detailed analysis of the P. pastoris plasma membrane is discussed in the light of cell biological features of this microorganism especially as a microbial cell factory for heterologous protein production.     

Localization of Galactolipid Biosynthesis in Etioplasts Isolated from Dark-Grown Wheat (Triticum aestivum L.)

Etioplasts were isolated from leaves of dark-grown wheat (Triticum aestivum L. var Starke II). Galactolipid biosynthesis was assayed in an envelope-rich fraction and in the fraction containing the rest of the etioplast membranes by measuring incorporation of ¹⁴C from uridine-diphospho[¹⁴C]galactose into monogalactosyl diacylglycerol and digalactosyl diacylglycerol. More than half of the galactolipid biosynthetic capability was found in the fraction of inner etioplast membranes. This fraction was subfractioned into fractions enriched in prolamellar bodies and membrane vesicles (prothylakoids), respectively. All membrane fractions obtained from etioplasts were able to carry out galactolipid biosynthesis, although the activity was very low in prolamellar body-enriched fractions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed markedly different polypeptide patterns between the different fractions. It is concluded that the capability of galactolipid biosynthesis of etioplasts probably is not restricted to the envelope, but is also present in the inner membranes of this plastid.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with ¹²⁵I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 × g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with a apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similary, the free receptor also showed higher sedimentation profile with a apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of ¹²⁵I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI.U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the performed ¹²⁵I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Fractionation of renal brush border membrane proteins with Triton X-114 phase partitioning.

Analysis of brush border membrane proteins by gel electrophoresis has revealed a complex polypeptide composition. We have investigated the use of Triton X-114 phase partitioning to fractionate such proteins on the basis of their degree of hydrophobicity. Each of the fractions was composed of a complex but distinct set of proteins. Most proteins were solubilized by Triton X-114 and partitioned into the detergent-poor fraction. Trehalase, gamma-glutamyl transpeptidase, and leucine aminopeptidase were well solubilized (greater than 80%) and enriched 5.1-, 3.9-, and 2.5-fold in the detergent-rich fraction. In contrast, alkaline phosphatase and 5'-nucleotidase were poorly solubilized. The specific activities of these enzymes were increased 2.7- and 2.3-fold in the insoluble protein fraction. Maltase was almost completely solubilized and partitioned into the detergent-poor fraction with a small enrichment factor (1.3). These results suggest that Triton X-114 phase partitioning could be useful as a first step in the purification of many brush border membrane proteins.     

Nitrate-induced changes in protein synthesis and translation of RNA in maize roots

Nitrate regulation of protein synthesis and RNA translation in maize (Zea mays L. var B73) roots was examined, using in vivo labeling with [³⁵S]methionine and in vitro translation. Nitrate enhanced the synthesis of a 31 kilodalton membrane polypeptide which was localized in a fraction enriched in tonoplast and/or endoplasmic reticulum membrane vesicles. The nitrate-enhanced synthesis was correlated with an acceleration of net nitrate uptake by seedlings during initial exposure to nitrate. Nitrate did not consistently enhance protein synthesis in other membrane fractions. Synthesis of up to four soluble polypeptides (21, 40, 90, and 168 kilodaltons) was also enhanced by nitrate. The most consistent enhancement was that of the 40 kilodalton polypeptide. No consistent nitrate-induced changes were noted in the organellar fraction (14,000g pellet of root homogenates). When roots were treated with nitrate, the amount of [³⁵S]methionine increased in six in vitro translation products (21, 24, 41, 56, 66, and 90 kilodaltons). Nitrate treatment did not enhance accumulation of label in translation products with a molecular weight of 31,000 (corresponding to the identified nitrate-inducible membrane polypeptide). Incubation of in vitro translation products with root membranes caused changes in the SDS-PAGE profiles in the vicinity of 31 kilodaltons. The results suggest that the nitrate-inducible, 31 kilodalton polypeptide from a fraction enriched in tonoplast and/or endoplasmic reticulum may be involved in regulating nitrate accumulation by maize roots.