Characterization of DNA-Binding Proteins Involved in the Assembly of Mitochondrial Nucleoids in the Yeast Saccharomyces cerevisiae

Mitochondrial nucleoids (mt-nucleoids) isolated from the yeastSaccharomyces cerevisiae were analyzed to identify the proteincomponents that are involved in the compact packaging of mtDNA.The isolated mt-nucleoids were disassembled by the additionof 2 M NaCl and the disassembled mt-nucleoids were reassembledonce again into compact structures by dialysis against a bufferthat contained NaCl at concentrations below 0.1 M, as monitoredby staining of the DNA with 4',6-diamidino-2-phenylindole. DNA-binding proteins with molecular masses of 67 kDa, 52 kDa,50 kDa, 38 kDa, 30 kDa and 20 kDa were separated from isolatedmt-nucleoids by column chromatography on DNA cellulose afterdigestion of mt-nucleoids by DNase I in the presence or absenceof 2 M NaCl. Purified mtDNA was compactly packaged into nucleoid-likestructures upon the addition of fractions that contained DNA-bindingproteins and subsequent dialysis to reduce the concentrationof NaCl. Five proteins, with molecular masses of 67 kDa, 52kDa, 50 kDa, 38 kDa and 30 kDa, respectively, had lower affinityfor double-stranded DNA than that of the 20-kDa protein. Thefraction that contained the five DNA-binding proteins otherthan the 20-kDa protein was also able to fold mtDNA compactlyinto nucleoid-like structures. By contrast, the combinationof the 20-kDa protein and mtDNA resulted in formation of lesstightly packed, string-of-bead structures. These results suggestthat at least six different DNA-binding proteins are involvedin the organization of the mt-nucleoids. (Received April 7, 1995; Accepted July 10, 1995)     

Changes in patterns of protein synthesis during haustorial development of Striga hermonthica (Del.) Benth. seedlings

This study focuses upon the developmental transition of theparasitic plant Striga hermonthica from its freeliving state(germinated seedling) to its parasitic state after developmentof an infection organ: the haustorium. A new method has beendeveloped that allows the production of gram quantities of germinatedand haustorially-induced Striga seedlings, thereby facilitatingbiochemical and molecular analysis of haustorial induction.Water-soluble proteins have been extracted from germinated seeds(stage A) and seedlings treated with 2,6-dimethoxy-p-benzoquinone(2,6-DMBQ) to induce haustorium (stage B). Samples were analysedby two-dimensional polyacrylamide gel electrophoresis and quantitativeas well as qualitative differences could be observed. In particulara group of four highly abundant acidic proteins (molecular weight39 kDa, pl 5.1, 5.3, 5.3, 5.6) and three other proteins (molecularweight 12 kDa, pl 6.9; 17 kDa, pl 4.4; 17 kDa, pl 4.45) wereseen in stage A while at least four proteins (molecular weight21.5 kDa, pl 6.4; 21.5 kDa, pl 6.3; 31 kDa, pl 5.1; 34 kDa,pl 6.2) were present in greater abundance in stage B. In orderto compare watersoluble protein with newly synthesized proteinpatterns, mRNAs from the two stages of development were isolatedand cell-free translation products analysed by 2-D PAGE. Two-Dgels of cell-free translation products showed the appearanceof six proteins in stage B (molecular weight ranging from 10to 35 kDa) and the presence of three acidic proteins in stageA with one protein (molecular weight 40 kDa) very similar insize to the triplet of proteins in the water-soluble protein2-D gels. Key words: Striga hermonthica (Del.) Benth., haustorium, 2-D PAGE, 2,6-DMBQ, translation in vitro     

Evidence from Crosslinking for a Close Association of the Extrinsic 33 kDa Protein with the 9.4 kDa Subunit of Cytochrome b 559 and the 4.8 kDa Product of the psb I Gene in Oxygen-Evolving Photosystem II Complexes from Spinach

Oxygen-evolving photosystem II complexes from spinach, whichlack light-harvesting chlorophyll a/b proteins, were treatedwith a bifunctional crosslinking reagent, hexamethylene-diisocyanate.Identification of crosslinked proteins with antisera raisedagainst various constituent proteins of the oxygen-evolvingPS II complex showed that the extrinsic 33 kDa protein is locatedless than 11 Å from the 9.4 kDa subunit of cytochromeb 559 and the 4.8 kDa product of psb I gene. (Received October 14, 1991; Accepted February 6, 1992)     

Light-dependent GTP-binding proteins in squid photoreceptors.

Previous biochemical and electrophysiological evidence suggests that in invertebrate photoreceptors, a GTP-binding protein (G-protein) mediates the actions of photoactivated rhodopsin in the initial stages of transduction. We find that squid photoreceptors contain more than one protein (molecular masses 38, 42 and 46 kDa) whose ADP-ribosylation by bacterial exotoxins is light-sensitive. Several lines of evidence suggest that these proteins represent distinct alpha subunits of G-proteins. (1) Pertussis toxin and cholera toxin react with distinct subsets of these polypeptides. (2) Only the 42 kDa protein immunoreacts with the monoclonal antibody 4A, raised against the alpha subunit of the G-protein of vertebrate rods [Hamm & Bownds (1984) J. Gen. Physiol. 84. 265-280]. (3) In terms of ADP-ribosylation, the 42 kDa protein is the least labile to freezing. (4) Of the 38 kDa and 42 kDa proteins, the former is preferentially extracted with hypo-osmotic solutions, as demonstrated by the solubility of its ADP-ribosylated state and by the solubility of the light-dependent binding of guanosine 5'-[gamma-thio]triphosphate. The specific target enzymes for the observed G-proteins have not been established.     

Functional Structure of the Oxygen-Evolving Unit of Photosystem II as Determined by Radiation Inactivation

The size of the complex that is essential for the electron-transferactivity from the oxygen-evolving center to the secondary electronacceptor, Q_B, is about 250 kDa, as determined by target-sizeanalysis after the radiation inactivation of functions of photosystemII (PS II). Inter-Chl tranfer of excitation energy was insensitiveto the radiation inactivation indicating that the masses ofCP47, CP43, and light-harvesting Chi a/b proteins are not includedin the functional size of the oxygen-evolving PS II complex.The transfer of electrons from the secondary electron donor,Z, to Q_B was catalyzed by a unit of only 65 kDa. The sizes ofthe complexes involved in these light-induced functions of PSII were dependent on the intensity of actinic light. Under saturatingintensities of light, the functional size of the complex fortransfer of electrons from Z to Q_B was 38 kDa, with a correspondingdecrease in the size of the oxygen-evolving PS II from 250 kDato 125 kDa [Takahashi, Mano and Asada (1985) Plant Cell Physiol.26: 383]. The protein of about 30 kDa functions in the photoreductionof the pheophytin molecule, as well as in the electron transferfrom Z to Q_A. Under low-intensity light, complexes having thesame sizes as those of the basal functional complexes undersaturating-intensity light are further required, probably tostabilize separated charges in the PS II reaction center andthe oxygen-evolving center. (Received June 20, 1990; Accepted September 18, 1990)     

Detection of Polypeptides Involved in Early Stages of Nodulation in Soybean Roots

Soluble proteins extracted from the roots of nodulating soybean[Glycine max (L.) Merr. cv. T202] and from roots of the non-nodulatingisoline rj₁ of cv. T202 (cv. T201), which had been inoculatedwith Bradyrhizobium japonicum, were analyzed by two-dimensionalpolyacrylamide gel electrophoresis and silver staining, in anattempt to identify polypeptides involved in early stages ofnodulation. Almost identical patterns of polypeptides were generatedby extracts of 3-day-old roots of uninoculated T201 and T202and of inoculated T201 and T202, but a unique spot, correspondingto a polypeptide of 38 kDa was detected in the case of inoculatedroots of T202. Western blotting analysis using "inoculated-T202-rootspecific" antiserum, prepared by titration of antiserum againstproteins from inoculated T202 roots with proteins from inoculatedT201 roots, revealed spots corresponding to polypeptides of26,27, and 33 kDa that were detectable only in the extractsof roots of inoculated T202. However, no unique polypeptidespots were detected in the case of roots of inoculated T201and T202, as compared with those from uninoculated T201 andT202 roots by Western blotting analysis using "inoculated-T201-rootspecific" antiserum prepared by titration of antiserum againstproteins from inoculated T201 roots with proteins from uninoculatedT201 roots. (Received May 27, 1991; Accepted September 30, 1991)     

Specific Secretion of Proline-Rich Proteins by Salt-Adapted Winged Bean Cells

Six proteins, designated SAP1 through SAP6, were secreted specificallyby salt-adapted cells of winged bean (Psophocarpus tetragonolobus)in suspension cultures. The amino-terminal amino acid sequencesof SAP2 (57 kDa), SAP4 (21 kDa), SAP5 (19 kDa) and SAP6 (17kDa) were homologous to the sequences of proline-rich proteins,indicating that proline-rich proteins are secreted specificallyby these salt-adapted cells. In addition, the amino-terminalamino acid sequence of SAP2 was identical to that of SAP4, andthe amino-terminal sequence of SAP5 was identical to that ofSAP6. Secretion of SAP2 was significantly enhanced by additionof AlCl₃ but not of KCl, LiCl, CaCl₂, MgCl₂, mannnitol or sucroseto suspension cultures. Furthermore, secretion of SAP4, SAP5and SAP6 was stimulated by addition of abscisic acid to cultures,suggesting that these proteins might be secreted in responseto salt or osmotic stress. (Received September 12, 1994; Accepted January 20, 1995)     

Cry1A毒素与二化螟中肠刷状缘膜囊泡受体蛋白配基结合分析

分离和鉴定二化螟Chilo suppresalis幼虫中肠刷状缘膜囊泡（BBMV）中Cry1A毒素的受体蛋白,对于阐明Cry1A毒素作用机理和二化螟抗性机理具有十分重要的意义。为此,本文就Cry1A毒素对二化螟杀虫活性及Cry1Ac与二化螟中肠受体的配基结合进行了研究。结果表明: Cry1Ab对二化螟室内品系(CN)的毒力高于Cry1Ac,而Cry1Ac高于Cry1Aa。配基结合分析表明二化螟CN品系幼虫中肠BBMV中有6个Cry1Ac结合蛋白(分子量分别为50,70,90,120,160和180 kDa), 其中180,160和90 kDa结合蛋白的条带颜色明显深于其他结合蛋白的条带,表明这3个受体蛋白具有较高的结合浓度。同源竞争结合研究表明,180和90 kDa结合蛋白为Cry1Ac的低亲合性结合蛋白,其他4个为高亲合性结合蛋白。为了研究Cry1Ac和Cry1Ab受体结合部位的相互作用,进行了异源竞争结合研究。Cry1Ab可以与Cry1Ac所有的6个结合蛋白进行竞争性结合,与180,120,70和50 kDa结合蛋白具有高亲合性,而与160和90 kDa结合蛋白具有低亲合性。结果显示,Cry1Ac与Cry1Ab在二化螟幼虫中肠BBMV上拥有多个共享的结合位点,但对每个结合位点的亲合性有差异。基于毒素结合部位的相似性,Cry1Ac和Cry1Ab不宜同时用于转基因Bt水稻来控制二化螟。     

Genetic Variability in Recovery Growth and Synthesis of Stress Proteins in Response to Polyethylene Glycol and Salt Stress in Finger Millet

Marked differences were found among 28 finger millet genotypes(Eleusine coracana Gaertn.) in acquired tolerance to osmoticstress as assessed by the recovery of root growth from severestress [-1·2 MPa polyethylene glycol, (PEG) or 400 mMNaCl]. However, these differences in tolerance were observedonly when the seedlings were subjected to a preceding mild inductionstress (-0·6 MPa PEG or 200 mM NaCl). In two contrastinggenotypes, synthesis of stress-induced proteins was studied.Proteins with apparent molecular weight of 70-72, 52, 37, 34and 23 kDa were synthesized in the highly responsive genotype(GE 415) and poorly responsive (VL 481) genotype following amild induction stress (200 mM NaCl). However, GE-415 synthesizeda 54 kDa protein that was not observed in VL-481. Addition ofabscisic acid (ABA) to the induction medium containing 200 mMNaCl enhanced the acquired tolerance of finger millet seedlingsover those without ABA in association with the appearance ofseveral ABA-responsive proteins. GE-415 required much less ABAthan VL-481 to obtain the same response. With 10 µM ABA+ 200 mM, A NaCl induction stress, GE-415 had significantlyhigher endogenous ABA. In association with higher levels ofABA, GE-415 had greater recovery root growth following severestress from 600 mM NaCl. Pretreatment with 10 µM ABA +200 mM NaCl induced several proteins with apparent molecularweights of 70-72, 54, 45, 36, 29 and 21 kDa in both genotypes.Qualitatively, GE-415 synthesized a unique 23-24 kDa proteinand quantitatively there was significantly more of the 21 kDaprotein in GE-415 compared to VL-481. The results indicate thatthe synthesis of stress proteins is correlated with the observedvariation in acquired tolerance of the two genotypes.Copyright1995, 1999 Academic Press Eleusine coracana Gaertn., salinity, polyethylene glycol, stress proteins, ABA, ABA-responsive proteins, finger millet seedlings     

The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells

Cell suspension cultures of Beta vulgaris L., treated with calciumchelators or untreated, were used to characterize pyndine nucleotide-dependentdiaphorases of microsomes. The microsomal activity of NADH-dependentduroquinone reductase from cultures treated with 10 mM Na₂EGTAfor 24 h increased by a factor of 1.8 with respect to controlmicrosomes, and was mainly associated with particles of d=1.17gml^–1. NADPH-duroquinone reductase and NADH-ferricyanidereductase activities showed smaller increases. Bacterial protein-lipopolysaccharidecomplexes (prLPS) also promoted the increase of microsomal diaphorases;CaEGTA was Ineffective. EGTA effects on enzymes of supernatantand mitochondria were negligible, although Na₂EGTA treatmentinduced cell aggregation and strong acidification of the medium. When microsomes from control cultures were solubilized with1% LPC and fractionated in high-efficiency gel permeation columns(FPLC) the diaphorase activities were found associated to threemajor proteins: (i) NADH-specific quinone reductase (NADH-QR)of 340 kDa; (ii) pyndine nucleotide-nonspecific quinone reductase(NAD(P)H-QR) of 85 kDa also having ferricyanide reductase activity;(iii) NADH-specific ferricyanide reductase (NADH-FCR) of 38kDa. The microsomes from EGTA-treated cells also showed a highlyactive NADH-QR having a larger molecular mass (440 kDa) thanin control cells. NAD(P)H-QR also showed increased activity.We conclude that external Ca²⁺ chelation induces changes indehydrogenase components in microsomes. Furthermore, prLPS probablyexert part of their effect on plants through Ca²⁺ chelation. Key words: Beta vulgaris, cell cultures, calcium chelators, diaphorase, NAD(P)H-dehydrogenase, lipopolysaccharide, EGTA, quinone reductase     

Identification of Chitinase and Osmotin-Like Protein as Actin-Binding Proteins in Suspension-Cultured Potato Cells

Cytoplasmic aggregation is an early resistance-associated eventthat is observed in potato tissues either after penetrationof an incompatible race of Phytophthora infestans, the potatolate blight fungus, or after treatment with hyphal wall components(HWC) prepared from P. infestans. In potato cells in suspensionculture, the number of cells with cytoplasmic aggregation increasedupon treatment with HWC, but such an increase was suppressedby treatment with cytochalasin D prior to treatment with HWC.This result suggested that cytoplasmic aggregation in culturedpotato cells might be connected with the association of actinfilaments. To identify the molecular basis of cytoplasmic aggregation,we purified actin and actin-related proteins by affinity chromatographyon a column of immobilized DNase I from cultured potato cellsand isolated proteins of 43 kDa, 32 kDa and 22 kDa. Analysisof the amino-terminal amino acid sequences indicated that the43 kDa, 32 kDa and 22 kDa proteins were potato actin, basicchitinase and osmotin-like protein, respectively. This conclusionwas supported by the results of Western blotting analysis ofthe 43 kDa and 32 kDa proteins with antibodies against actinand basic chitinase. Binding analysis with actin coupled toactin-specific antibodies and biotinylated actin suggested thatthe 32 kDa and 22 kDa proteins had actin-binding activity. Inaddition, examination of biomolecular interactions using anoptical biosensor confirmed the binding of chitinase to actin.These results imply the possibility that basic chitinase andosmotin-like protein might be involved in cytoplasmic aggregation,hereby participating in the potato cell's defense against attackby pathogen. (Received June 11, 1996; Accepted January 27, 1997)     

Immunological Quantification of Proteins Related to Light-Harvesting Chlorophyll a/b Protein Complexes of the Two Photosystems in Rice Mutants Totally and Partially Deficient in Chlorophyll b

Ten rice chlorina mutants of Type I, which totally lack chlorophyllb and hence are unable to synthesize light-harvesting chlorophylla/b protein complexes of photosystem II (LHC-II), containedmRNA for proteins related to LHC-II. Immunoblotting with anantiserum, which had been raised against the 24 and 25 kDa apoproteinsof LHC-II and found to cross-react with the 26 kDa protein ofLHC-II and the 20 and 21 kDa apoproteins of light-harvestingchlorophyll a/b protein complexes of photosystem I (LHC-I),revealed that all the five proteins related to LHC-Iand LHC-IIwere present in reduced amounts in the Type I mutants. ThreeType HA mutants, which have a chlorophyll a/b ratio of 10, weremore abundant in the apoproteins, while three Type IIB mutantswith the ratio of 15 were heterogeneous in terms of the apoproteincontent. All the chlorina mutants contained less P700 comparedwith the wild type rice, but were relatively more abundant inthe LHC-I proteins than the LHC-II proteins. The results showthat all the rice chlorina strains are mutants of chlorophyllb synthesis and the deficiency of chlorophyll b differentlyaffects accumulation of the apoproteins of LHC-I and LHC-II.To balance light absorption between the two photosystem, lossof LHC-II is partly counter-balanced by a decrease in the numberof PSI complexes in the mutants. (Received January 21, 1988; Accepted April 28, 1988)     

Purification and characterization of napin-like proteins from radish

Nine 2S albumin proteins from garden and sea radish seeds (Raphanussativus and Raphanus raphanistrum, respectively) have been purified.Their molecular weights and amino acid compositions have beendetermined. Most of these proteins exhibit a molecular massof 14.5 kDa, but one component of each species shows a slightlyhigher value (15.1 kDa). The amino acid compositions obtainedare typical of napin-like proteins, although a different contentof Tyr and basic residues was observed. The isolated proteinspresent circular dichroism and fluorescence spectra nearly identicalto each other, but different from those of rapeseed or mustardnapins. The 2S albumins from radish exhibit about 53% -helix,14% ß-bend, 16% ß-turn and 17% aperiodicconformation. The microheterogeneity of these proteins has beenanalysed by high pressure liquid chromatography of the reducedmolecules, and a high degree of polymorphism has been observedin most of the obtained fractions. On the basis of the aminoacid contents of the individual chains, as well as the carboxypeptidaseY digestion data, new sites have been suggested for the processingof their polypeptide precursors to render the mature proteins. Key words: Storage proteins, Brassicaceae, spectroscopy, processing     

Immunopurification and structural analysis of a putative epithelial Cl- channel protein isolated from bovine trachea.

We have purified to homogeneity a 38-kDa protein (called p38) from bovine tracheal epithelium. This protein, when reconstituted into liposomes, mediates stilbene disulfonate-sensitive 125I- conductive uptake. On nonreduced or partially reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this protein associates into a doublet of 62-64 kDa. In some experiments a multimer of 141 kDa was also observed. Rabbit polyclonal anti-P38 antibodies have been produced and used to immunopurify the native transporter. Upon reconstitution of the immunoaffinity-purified protein into liposomes, a 260-fold enhancement of 4,4'-bis(isothiocyano)-2,2'-stilbenedisulfonate and valinomycin-sensitive 125I- uptake was observed as compared to proteoliposomes containing unseparated material. On Western blots of total solubilized tracheal membrane proteins or semipurified fractions, the antibody recognized the 62-64-kDa doublet much better than the original 38-kDa antigen. Similar protein bands were detected in T84 and CFPAC cells as well. However, if apical membrane proteins were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, the antibody recognized major bands at 140 and approximately 240 kDa. Upon partial reduction, immunolabeling of these proteins diminished with the concomitant appearance of the 62-64-kDa doublet. Upon complete reduction, the appearance of 32- and 38-kDa proteins was evident with the disappearance of the 62-64-kDa doublet. We hypothesize that the native Cl-channel is a heteromer containing at least four subunits connected by S-S bridges.     

Purification and Further Characterization of Pyrenoid Proteins and Ribulose-1,5-bisphosphate Carboxylase-Oxygenase from the Green Alga Bryopsis maxima

Pyrenoid proteins and ribulose-1,5-bisphosphate carboxylase-oxygenase(RuBisCO) in the green alga Bryopsis maxima were purified tohigh degrees and their peptide compositions were studied bySDS-polyacrylamide gel electrophoresis. RuBisCO had a largesubunit of 50 kDa and a small one of 16 kDa. The apparent molecularweight of the purified RuBisCO was estimated as 460 kDa by gelfiltration. Pyrenoid proteins had two major polypeptides: 52kDa and 17 kDa. The peptide map of the 52 kDa pyrenoid polypeptidecoincided well with that of the large subunit of RuBisCO, stronglysuggesting that the major component of the pyrenoid of thisalga was RuBisCO. We attempted to survey the distribution ofRuBisCO in the chloroplasts. The results suggested that muchof the RuBisCO of Bryopsis maxima was localized in the pyrenoid.The pyrenoid also contained more than 10 minor polypeptidesnot found in the RuBisCO fraction. The minor polypeptides comprisedabout 15% of the total pyrenoid protein and differed from thepolypeptides of the thylakoid membranes and from those foundin the starch grains surrounding the pyrenoid. (Received February 3, 1984; Accepted July 21, 1984)     

Evidence from Crosslinking for Nearest-Neighbor Relationships among the Three Extrinsic Proteins of Spinach Photosystem II Complexes that are Associated with Oxygen Evolution

Treatment of oxygen-evolving Photosystem II complexes, whichlack light-harvesting chlorophyll a/b proteins, with a seriesof disuccinimidyl esters with different chain lengths yieldeda crosslinked product which consisted of one molecule each ofthe extrinsic 33 kDa and 23 kDa proteins. In addition, crosslinkingbetween the 33 kDa protein and the chlorophyll-carrying 47 kDaprotein and between the 23 kDa and 17 kDa proteins was confirmed.Thus, the three extrinsic proteins are closely associated witheach other to form a complex which is attached to the PS IIreaction center complexes. (Received December 1, 1989; Accepted May 2, 1990)     

Heterogeneity of Odorant-binding Proteins in the Antennae of Bombyx mori

Different odorant-binding proteins (OBPs) were isolated fromtotal antennal homogenates of male and female Bombyx mori. Proteinswere separated according to their isoelectric point by usingpreparative fast-flow isoelectrofocusing. Odorant-binding proteinswere identified in immunoblots by antisera raised against thepheromone-binding protein (anti-PBP) and the general odorant-bindingprotein (anti-GOBP2) of Antheraea polyphemus. Four proteinscross-reacting with anti-PBP were detected in males and twoin females, while three proteins cross-reacting with anti-GOBP2were found in males and five in females. Both anti-PBP and anti-GOBP2cross-reacting proteins had an apparent molecular weight of15–16 kDa. In parallel, the same two antisera were usedin immunocytochemical studies in order to determine the distributionof these proteins within the various subtypes of olfactory sensilla.The presence of multiple odorant-binding proteins within onemoth species as well as their complex distribution pattern supportthe suggestion that soluble OBPs might have a function in odorantdiscrimination. Chem. Senses 22: 503–515, 1997.     

Agonist-enhanced palmitoylation of platelet proteins

When washed human platelets were incubated with [3H]palmitic acid, radioactivity was incorporated into a major 38 kDa doublet and several minor proteins that were resolved on polyacrylamide gels. The radioactivity associated with the proteins remained after extractions with organic solvents, but it was lost after hydroxylamine treatment or mild alkali methanolysis. The products of these reactions were analyzed by thin-layer chromatography and HPLC. They were identified as palmitohydroxamate and methyl palmitate, respectively, indicating that the palmitic acid was covalently linked to the proteins via oxygenester or thioester bonds. In resting platelets, radioactivity was detected in the 38 kDa proteins 2 min after the addition of [3H]palmitic acid. A plateau was reached between 5 and 11 min, at which time radioactivity was also detected in a 23 kDa protein. Thrombin elicited faster and greater incorporation of label into both proteins. Phorbol 12-myristate 13-acetate (PMA) led to a similar, but slower increase of radioactivity in the 38 kDa proteins, while collagen and A23187 were less effective. Enhanced palmitoylation may be closely linked to platelet activation, as suggested by the following observations: (1) in thrombin- or PMA-activated platelets, the time-course of aggregation correlated with the time-course of enhanced palmitoylation of the 38 kDa proteins; (2) in platelets activated by various concentrations of thrombin with or without prostacyclin, aggregation was correlated with the enhanced incorporation of radioactivity into the 38 kDa proteins.     

Evidence for Shared Receptor Proteins for Human Interleukin-3 and Granulocyte-Macrophage Colony-Stimulating Factor in the Human M-07 Cell Line

Abstract

The biologic response of the human leukemia cell line M-07 to granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin 3 (IL-3) and interleukin 4 (IL-4) is mediated by a low number of high affinity receptors. Cross-competition studies revealed that IL-3 and GM-CSF partially inhibited the specific binding of the heterologous radiolabeled ligand, whereas IL-4 binding was not affected by these cytokines. The molecular mechanism of cross-competition was investigated by chemical crosslinking and immuno-precipitation. Trimolecular receptor complexes consisting of a major 73kDa and two minor 120 and 128kDa membrane proteins for IL-3, and a major 84kDa and two minor 120 and 130 kDa proteins for GM-CSF were found on M-07 cells. The 73 and 84kDa proteins represent distinct and non-linked membrane proteins and are identical with the cloned, low affinity IL-3 and GM-CSF receptor proteins (Gearing et al, 1989, Hayashida et al, 1990). The higher molecular weight proteins share common binding sites as evidenced by immunoprecipitation of double-crosslinked membranes. The 120/128kDa proteins are most likely identical with the recently cloned and shared β-subunit of the IL-3 and GM-CSF receptor (Kitamura et al, 1991) containing a single or two IL-3 and/or GM-CSF molecules.