Studies on chilling injury in plant cells II. Ultrastructural changes in cells rewarmed at 26?C after chilling treatment

Both the restoration and deterioration of ultrastructures wereobserved during therewarming of cultured cells of Cornus stoloniferain which chilling at 0?C had caused an apparent change in themorphology of the organelles. Complete restoration of the ultrastructures,moderately altered by the 12-hr chilling, took place within12 hr of wanning at 26?C. Even in cells chilled for 24 hr, severelyaltered ultrastructures were partially or completely repairedin more than fifty percent of the treated cells. Some cellschilled for 24 hr, however, displayed further deteriorationof their ultrastructures during rewarming. Restoration of therough endoplasmic reticulum and the development of polysomesin recovering cells were characteristic of the early stage ofrewarming. Rupture of the tonoplast was sometimes observed duringrewarming of cells chilled for 24 hr. A possible role for therough endoplasmic reticulum and for the integrity of the tonoplastin cell recovery during the chill-warm sequence is discussed. ¹Contribution No. 2026 from the Institute of Low TemperatureScience, Hokkaido University. ²This work was supported in part by Grant 248004 from the Ministryof Education. (Received November 6, 1978; )     

Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Despite their relevance for neuronal Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ dependencies, defined by low, moderate, or high maximal fractional open time (P_o), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR. Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor     

Detection of a Precursor Polypeptide of the Rapidly-Synthesized 32,000-Dalton Thylakoid Protein in Spinach Chloroplasts

Spinach chloroplast RNA was translated in a wheat germ cell-freesystem in the presence of [³⁵S]methionine or [³H]lysine, andthe products were analyzed by SDS polyacrylamide gel electrophoresisand fluorography. A polypeptide with molecular mass of 2,000-Dalarger than the 32,000-Da thylakoid protein was detected asa major product labeled by [³⁵S]methionine but not by [³H]lysine.Peptide mapping of this polypeptide showed a pattern very closeto that of the 32,000-Da protein synthesized in isolated chloroplasts.A better separation of this polypeptide from the 32,000-Da proteinwas observed in the electrophoresis on polyacrylamide gel includingurea at 8 M. Pulse-labeling of the isolated chloroplasts showedthe occurrence of the larger molecular weight form, which wasconverted to the mature size by a chasing incubation with coldmethionine. These results suggested that the 32,000-Da proteinof spinach is translated primarily as a high molecular weightprecursor in the chloroplasts, as has been reported for otherplant species. (Received March 30, 1985; Accepted April 23, 1985)     

Isolation of membranes containing protease from the cotyledons of germinating pea seeds

Membranes containing protease were isolated from the cotyledonsof germinating pea seeds by differential and two successivesucrose density gradient centrifugations. Membranes were recoveredin the microsomal fraction, but could be clearly separated frommembranes carrying antimycin A-insensitive NADH-cytochrome creductase EC 1.6.2.1 [EC] ), which seem to come from the endoplasmicreticulum. The density of protease-containing membranes was1.135. Membranes contained a lower amount of phospholipid ascompared with the endoplasmic reticulum. Electron microscopicobservations also showed that the membranes were different fromthe smooth-surfaced endoplasmic reticulum. ¹Present address: Department of Pathology, Aichi Medical University,Nagakute, Aichi, Japan. (Received September 12, 1973; )     

Size and Levels of mRNA for Acid Invertase in Ripe Tomato Fruit

Poly(A)⁺RNA was isolated from ripe tomato fruit and translatedin a wheat germ cell-free translation system. A 74-kDa polypeptidewas detected as a putative precursor of acid invertase by immunoprecipitationwith antiserum raised against SDS-treated acid invertase (denaturedform) from tomato fruit. The molecular mass of the mRNA foracid invertase was estimated to be about 8 ? 10⁵ Da (2.4 k nucleotides)by sucrose density gradient centrifugation. Mature, green tomatofruit contained very low levels of invertase mRNA. When mature,green tomato fruit were stored at 22?C, levels of invertasemRNA per gram fresh weight increased to a maximum after fourdays and then declined. (Received May 24, 1989; Accepted May 2, 1990)     

Variability in transport rates of secretory glycoproteins through the endoplasmic reticulum and Golgi in human hepatoma cells

We have previously shown that newly synthesized liver secretory proteins are exported at three distinct characteristic rates, with intracellular retention half-times of 110-120 min (e.g. transferrin), 75-80 min (e.g. ceruloplasmin), and 30-40 min (e.g. alpha 1-protease inhibitor) (J. B. Parent, H. Bauer, and K. Olden (1985) Biochim. Biophys. Acta, in press). In the present study we have determined the average time required for specific glycoproteins to move through the various compartments of the intracellular transport pathway, consisting of endoplasmic reticulum and Golgi complex. Localization in particular compartments was monitored by the use of the following complementary approaches: (i) Percoll density gradient fractionation of the subcellular organelles, (ii) sensitivity of the glycan moiety of N-linked glycosylation to endo-beta-N-acetylglucosaminidase H, and (iii) by the lectin-binding characteristics. The cell fractionation studies revealed that alpha 1-protease inhibitor, ceruloplasmin, and transferrin were transported from the rough endoplasmic reticulum with a retention half-time of 10, 30, or 45 min, respectively. Measurements of the rate at which newly synthesized glycoprotein became endo H-resistant (an event localized near the medial region of Golgi) demonstrated that it took 60-70, 30, and 18 min for 50% of transferrin, ceruloplasmin, and alpha 1-protease inhibitor, respectively, to reach the medial Golgi. Consistent with this finding, maximal binding of transferrin to wheat germ agglutinin (also a medial Golgi event) and Ricinus communis agglutinin I (a trans Golgi event) required 75 and 90 min, respectively, and maximal binding of ceruloplasmin to both lectins occurred in approximately 30 min. Maximal binding of alpha 1-protease inhibitor to wheat germ agglutinin and Ricinus communis agglutinin I required 15 and 30 min, respectively. The results presented here clearly indicate that (i) the time required for protein secretion cannot be entirely accounted for by lag in transport from the rough endoplasmic reticulum to the Golgi since the glycoproteins examined are retained in the former organelle for no more than two-fifths of the total intracellular retention half-time, and (ii) the variability in rates of protein secretion is not due solely to differences in rates of transport from the rough endoplasmic reticulum to the Golgi as variability in retention within the Golgi is also demonstrated. The results are discussed in terms of their compatibility with receptor-mediated transport of glycoproteins in both the endoplasmic reticulum and Golgi.     

Isolation of Smooth Endoplasmic Reticulum and Tonoplast from Mung Bean Hypocotyls (Vigna radiata [L.] Wilczek) Using a Ficoli Gradient and Two-Polymer Phase Partition

A new method for the isolation of smooth endoplasmic reticulumand tonoplast from etiolated mung bean hypocotyls (Vigna radiata[L.] Wilczek) has been developed. After centrifugation in aFicoli density gradient [5.5% (w/w) in 15% (w/w) sucrose] ofa crude microsomal membrane fraction (10,000–156,000?gpellet) which had been prepared and resuspended in buffer systemsthat contained 0.25 M sorbitol, more than 80% of the total amountsof smooth endoplasmic reticulum and tonoplast were co-bandedat the interface between the sample load and the Ficoll layer,while most of the other cellular membranes, including plasmamembrane, Golgi membranes and yellow-colored membrane materials,which were presumably the etioplast envelopes, were sedimentedthrough the Ficoli layer. Smooth endoplasmic reticulum and tonoplastwere separated from each other to a high degree of enrichmentby a subsequent two-polymer phase partitioning. The separationis based on the principle that mung bean tonoplast has a highpartition coefficient for the polyethylene glycol-enriched upperphase and the smooth endoplasmic reticulum has a high partitioncoefficient for the Dextran-enriched lower phase. Assessed interms of degree of contamination by activities of membrane markerenzymes, the isolated smooth endoplasmic reticulum and tonoplastfractions were found to be highly purified. An ATPase sensitiveto neutral detergent and vanadate was found to be specificallyassociated with the smooth endoplasmic reticulum. ¹Contribution No. 3172 from the Institute of Low TemperatureScience (Received April 22, 1988; Accepted September 28, 1988)     

Evidence for the presence of two different types of protein bodies in wheat endosperm

Storage proteins of wheat grains (Triticum L. em Thell) are deposited in protein bodies inside vacuoles. However, the subcellular sites and mechanisms of their aggregation into protein bodies are not clear. In the present report, we provide evidence for two different types of protein bodies, low- and high-density types that accumulate concurrently and independently in developing wheat endosperm cells. Gliadins were present in both types of protein bodies, whereas the high molecular weight glutenins were localized mainly in the dense ones. Pulse-chase experiments verified that the dense protein bodies were not formed by a gradual increase in density but, presumably, by a distinct, quick process of storage protein aggregation. Subcellular fractionation and electron microscopy studies revealed that the wheat homolog of immunoglobulin heavy-chain-binding protein, an endoplasmic reticulum-resident protein, was present within the dense protein bodies, implying that these were formed by aggregation of storage proteins within the endoplasmic reticulum. The present results suggest that a large part of wheat storage proteins aggregate into protein bodies within the rough endoplasmic reticulum. Because these protein bodies are too large to enter the Golgi, they are likely to be transported directly to vacuoles. This route may operate in concert with the known Golgi-mediated transport to vacuoles in which the storage proteins apparently condense into protein bodies at a postendoplasmic reticulum location. Our results further suggest that although gliadins are transported by either one of these routes, the high molecular weight glutenins use only the Golgi bypass route.     

Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum

Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.     

Studies on chilling injury in plant cells I. Ultrastructural changes associated with chilling injury in callus tissues of Cornus stolonifera

This study was designed to elucidate the primary ultrastructuralchanges associated with chilling injury of cultured cells ofCornus stolonifera (TK-1). The cultured cells suffered seriousinjury after exposure to 0?C for longer than 24 hr, while noinjury was observed with less than 12 hr of treatment. Earlyultrastructural responses to chilling treatment were detectedin proplastids and the rough endoplasmic reticulum within 12hr of treatment. A remarkable ultrastructural change in thetonoplast, i.e., invagination, infolding and partial disruption,became apparent in the 24-hr stage of chilling treatment. Novisible change, however, was observed in mitochondria, nucleiand plasma membranes within 24 hr. Upon exposure to 0?C for48 hr, an abrupt degradation proceeded in the cytoplasm. Thesequential ultrastructural changes observed in cell organdies,especially proplastids, rough endoplasmic reticulum and thetonoplast, were closely related to the degradation of the cellscaused by chilling treatment. ¹ Contribution No. 1840 from the Institute of Low TemperatureScience, Hokkaido University. ² This work was supported in part by Grant 248004 from the Ministryof Education. (Received September 8, 1977; )     

β-N-acetylglucosaminidase and β-galactosidase from aleurone layers of resting wheat grains

We have examined the characteristics of binding to wheat germ agglutinin-Sepharose of β-N-acetylglucosaminidase and β-galactosidase from aleurone layers of resting wheat grains. Although the enzymes interacting with wheat germ agglutinin-Sepharose could be extracted by a procedure which did not involve any solubilizing treatments, the highest activity of these enzymes was obtained by extracting and sonicating the tissues in the presence of 0.5% Triton X-100. The pH optimum and time-course of binding as well as the effect of some divalent ions on the binding were studied. The largest part of the bound enzymes was eluted at low concentration of N-acetyl-D-glucosamine (0.05 M), although smaller amounts were still eluted at higher molarities (0.1 and 0.2 M). D-Mannose, D-glucose and L-fucose failed to replace N-acetyl-D-glucosamine in eluting the enzymes bound to wheat germ agglutinin-Sepharose, whereas N-acetyl-D-galactosamine was much less effective than N-acetyl-D-glucosamine. The catalytic properties of the enzymes remained unchanged after the binding to wheat germ agglutinin-Sepharose, although the K_m values of the free and lectin-bound enzymes were slightly different. A rapid and easy three-step procedure of purification, mainly based on affinity chromatography on wheat germ agglutinin-Sepharose, is described. It allows purification of β-galactosidase and β-N-acetylglucosaminidase over 200-fold. β-N-Acetylglucosaminidase has been further purified to electrophoretic homogeneity and also characterized.     

Electron-Microscopic Study of Osmium Ferricyanide Staining in Meristematic, Extending and Differentiated Mesophyll Cells of Winter Rye Seedlings

The osmium ferricyanide (OsFeCN) cytochemical technique wasused in the electron microscopic study of organelles in cellsof functionally different leaf zones of winter rye seedlings.In meristematic cells, the endoplasmic reticulum and nuclearenvelope were the main structures reacting with OsFeCN; in cellsof the extension zone and in differentiated mesophyll cellsit was the peripheral reticulum and the intermembrane spaceof the chloroplast envelope. The change in OsFeCN staining character of organelles betweenthe various zones of leaves is considered to be due to cellexit from the meristem. The participation of the peripheralreticulum of chloroplasts in the sequestration of Ca²⁺ ionsis proposed. Secale cereale L., leaf zones, OsFeCN-staining, Ca²⁺-sequestring organelles     

Gonadotropin and prostaglandins binding sites in rough endoplasmic reticulum and Golgi fractions of bovine corpora lutea.

Highly purified rough endoplasmic reticulum and three subfractions of golgi were prepared from 105,000g pellet of the homogenate by centrifugation in floatation and sedimentation discontinuous sucrose gradients. Highly purified plasma membranes were also prepared from 9,000g pellet of the same homogenates for assessment under the same experimental conditions. Although 5′-nucleotidase, a marker for plasma membranes, was markedly enriched in plasma membranes, very little or none of this enzyme activity was found in other fractions. Very little or no NADH cytochrome c reductase activity, a marker for rough endoplasmic reticulum, was found in fractions other than rough endoplasmic reticulum. Galactosyl transferase, a marker for golgi, was found and enriched in all the fractions; however, enrichment in golgi fractions was higher than in other fractions. Very little or no lysosomal marker activity, i.e., acid phosphatase, was found in rough endoplasmic reticulum or golgi fractions as compared to lysosomes. These marker enzyme data suggested that rough endoplasmic reticulum and golgi fractions were relatively pure with little or no cross contamination with other organelles. The [¹²⁵I]human choriogonadotropin ([¹²⁵I]hCG), [³H]prostaglandin (PG)E₁, and [³H]PGF_2a specifically bound to rough endoplasmic reticulum and golgi fractions in addition to plasma membranes. The enrichments of binding in the former two fractions, in some cases, were as high as plasma membranes itself. The specific binding of some of the ligands was found to be partially latent in rough endoplasmic reticulum and golgi fractions but not in plasma membranes. Marker enzyme data, ratio between bindings and marker enzyme activities (an index of organelle contamination), and partial latency of binding suggest that rough endoplasmic reticulum and golgi fractions intrinsically contain gonadotropin and PGs binding sites.     

Characterization of cell membrane and sarcoplasmic reticulum from bovine uterine smooth muscle

Subcellular fractions, enriched in sarcoplasmic reticulum or in cell membrane, were separated from one another. Starting material was a microsomal pellet (15–40 × 1000g) obtained by differential centrifugation from the uteri of close-to-term pregnant cows. A microsomal fraction enriched in ATP-dependent calcium accumulation was shown to contain sarcoplasmic reticulum and cell membrane. Only 8% or less of the protein in this fraction could be recovered, using affinity chromatography on Sepharose 6MB wheat germ agglutinin. The small yields did not allow extensive characterization. A method was developed to separate sarcoplasmic reticulum from cell membrane using discontinuous sucrose density gradient centrifugation. Protein was collected at the 24–28, the 28–33, and the 33–45% sucrose interfaces. Characterization was by enzyme assays and by specific receptor assay. ATP-dependent calcium accumulation was fourfold greater in the 24–28% sucrose layer than in the 33–45% layer. In contrast, 5′-nucleotidase was more than threefold as high in the 33–45% sucrose layer as in the 24–28% layer. Ouabain-inhibited p-nitrophenylphosphatase doubled and ouabain-inhibited Na,K-ATPase tripled in the 28–33% layer, compared with the 24–28% layer, specific ouabain binding was also doubled in the 28–33% sucrose layer. ¹²⁵I-Labeled wheat germ agglutinin binding was greatest in the 33–45% sucrose layer. It is concluded that the 24–28% layer consists primarily of sarcoplasmic reticulum, whereas the 28–33 and the 33–45% layers are concentrated in the cell membrane. Specific prostaglandin (PGE₂) binding was found to be a property of the cell membrane.     

In Vitro Localization of Hydroxyproline O-Glycosyl Transferases in Chlamydomonas reinhardii

The intracellular location of the two major O-glycosylatingenzymes (hydroxyproline-arabinosyl and -galactosyl transferases)involved in the synthesis of the cell wall glycoproteins ofChlamydomonas reinhardii was determined by isopycnic sucrosedensity gradient centrifugation. A comparison of gradients preparedunder low and high Mg²⁺-conditions has enabled us to clearlyallocate the galactosyl transferase to membranes of the Golgiapparatus. In contrast, the membranes which bear the arabinosyltransferase respond to a change in Mg²⁺-concentration in justthe same way as the endoplasmic reticulum does. Analysis ofthe product formed in vitro from UDP-[¹⁴C]arabinose and microsomalmembranes has confirmed the synthesis of an arabinose-containinghydroxyproline-rich glycoprotein. Our results indicate thatwhilst the Golgi apparatus is responsible for some of the glycosylationreactions in hydroxyproline-rich glycoprotein biosynthesis anappreciable portion of the arabinosylation is accomplished whilethe polypeptide is still in the lumen of the endoplasmic reticulum. ³This paper is dedicated to Professor Lothar Jaenicke on theoccasion of his 65th birthday. (Received July 2, 1988; Accepted March 8, 1989)     

Isolation and properties of gamma protein,the major transmembrane sialoglycoprotein of the HeLa cell

The surface of the HeLa cell is composed of a heterogeneous population of sialogly coproteins which undergo lectin-mediated endocytosis (Kramer and Canellakis, Biochim Biophys Acta 551:328, 1979). One such sialoglyco-protein, gamma protein, is the major periodate-Schiff-reactive and [³H]-glucosamine-labeled component of the plasma membrane; it has an apparent molecular weight of 165,000. Gamma protein is also the major [¹²⁵I]-wheat germ agglutinin-binding component in sodium dodecyl sulfate gels. Neuraminidase digestion of HeLa cells abolishes binding of [¹²⁵I]-wheat germ agglutinin to gamma protein, and pretreatment of cells with wheat germ agglutinin protects gamma protein from desialation by neuraminidase. suggesting that wheat germ agglutinin binds to the sialic acid residues of gamma protein at the cell surface. Gamma protein can be extracted with various detergents but not with high-salt, chelating, or chaotropic agents. Intact inside-out plasma membrane vesicles have been prepared from HeLa cells that had phagocytosed latex particles. Treatment of these isolated vesicles with trypsin reduces the molecular weight of gamma protein. These results suggest that gamma protein is an integral membrane protein that spans the plasma membrane. Gamma protein can be purified to homogeneity by sequential lithium diiodosalicylate-phenol extraction, wheat germ agglutinin-agarose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Heterogeneity in wheat germ agglutinin binding by mouse peritoneal macrophages

The binding of the lectin wheat germ agglutinin (WGA) to the cell surface of monocytes and macrophages obtained from the stimulated peritoneal cavity of mice was investigated electron microscopically, using ovomucoid-gold as an indirect marker. Resident (tissue) macrophages, identified by the presence of PO activity in the rough endoplasmic reticulum as well as in the nuclear envelope, showed low WGA binding, whereas monocytes and monocyte-derived macrophages with PO activity in the granules showed high WGA binding. Since cells devoid of PO activity showed variable WGA binding, the value of this gold-WGA-binding technique for discrimination on a quantitative basis between resident macrophages and monocytes or monocyte-derived macrophages, is discussed.     

Analysis of lectin-specific cell surface glycoprotein on neuroblastoma cells

The binding sites for the lectins wheat germ agglutinin, Ricinus communis agglutinin and concanavalin A on mouse neuroblastoma cell membranes were identified using SDS-gel electrophoresis in combination with fluorescent lectins. Ricinus communis agglutinin and wheat germ agglutinin were found to bind almost exclusively to a single polypeptide with an apparent molecular weight of 30 000. Concanavalin A labeled over 20 different polypeptides, most with molecular weights greater than 50 000. However, when the neuroblastoma cells were treated with concanavalin A so as to internalize all the concanavalin A binding sites visible at the level of the fluorescent microscope and the purified plasma membranes analyzed for their concanavalin A binding polypeptides, only four of the 20 glycopolypeptides were missing or significantly reduced in amount. Thus, these four high molecular weight concanavalin A-binding polypeptides appear to be the major cell surface receptors for concanavalin A. Binding studies with iodinated concanavalin A indicated that these polypeptides represented the high affinity concanavalin A binding sites $\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 2 · 10}{}^{\mathrm{?7}}\text{M}$). Low affinity concanavalin A binding sites were present on the cell surface after internalization of high affinity concanavalin A binding sites.     

ATP-dependent CA2+ transport in endoplasmic reticulum isolated from roots ofLepidium sativum L.

Endoplasmic reticulum membranes were isolated from roots of garden cress (Lepidium sativum L. cv Krause) using differential and discontinuous sucrose gradient centrifugation. The endoplasmic reticulum fraction was 80% rough endoplasmic reticulum oriented with the cytoplasmic surface directed outward and contaminated with 12% unidentified smooth membranes and 8% mitochondria. Marker enzyme analysis showed that the activity for endoplasmic reticulum was enriched 2.4-fold over total membrane activity while no other organelle activity showed an enrichment. All evidence indicated that the fraction was composed of highly enriched endoplasmic reticulum membranes. Ca²⁺ uptake activity was measured using the filter technique described by Gross and Marmé (1978). The results of these experiments showed an ATP-dependent, oxalate-stimulated Ca²⁺ uptake into vesicles of the endoplasmic reticulum fraction. The majority of the transport activity was microsomal since specific inhibitors of mitochondrial Ca²⁺ transport (ruthenium red, LaCl₃ and oligomycin) inhibited the activity by only 25%. Sodium azide showed no inhibition. The transport was likely directly coupled to ATP hydrolysis since there was no inhibition with carbonylcyanidem-chlorophenylhydrazone. The transport activity was specific for ATP showing only 36% and 29% of the activity with inosine diphosphate and guanosine 5′-triphosphate, respectively. The results indicate a Ca²⁺ transport function located on the endoplasmic reciculum of garden cress roots.     

Transfer of phospholipids from microsomes to mitochondria in germinating castor bean endosperm

¹⁴C-labeled microsomes were prepared by feeding [1-¹⁴ C]acetateto endosperm tissues from 4-day-old seedlings of castor beanseeds and incubated with unlabeled mitochondria from the sametissues. The loss of ¹⁴C-lipids from the microsomes was accompaniedby an increase of ¹⁴C-lipids in the mitochondria. The additionof 105,000?g supernatant and also pH 5.1-treated supernatant,both of which had been prepared from castor bean endospermsat the same stage, markedly enhanced the lipid transfer frommicrosomes to mitochondria. The activity in this fraction wasprecipitated by ammonium sulfate and lost with trypsin or heattreatment. The transfer of lipids was limited to phospholipids.Thus, it is concluded that in castor bean endosperms, phospholipidsare transferred from the endoplasmic reticulum to the mitochondriaby a phospholipid-exchange protein contained in the cytosol. (Received August 8, 1977; )