Electronic structure and molecular design of simplified polyimide systems

The electronic structures of newly designed polyimide systems (ethenetetracarboxylic 1,2:1,2-dianhydride-diaminoethyne (PI-A) and ethenetetracarboxylic 1,1:2,2-dianhydride-diaminoethyne(PI-B)) are studied in detail with respect to their optimized geometries on the basis of the one-dimensional tight-binding self-consistent field crystal-orbital method. The computational results have revealed that PI-B shows intriguing properties such as a very small band gap and a wide bandwidth near the frontier level, compared with PI-A and other polyimides. Since PI-B would be a promising candidate for a new electric conducting material, a reaction diagram for this polymer is also proposed.Also affiliated to Central Research Laboratories, Matsushita Electric Industrial Co., Moriguchi 570, Japan.     

Transmembrane Ca2+ gradient is essential for high anion transport activity of human erythrocytes

The role of a transmembrane Ca²⁺ gradient in anion transport by Band 3 of human resealed erythrocyte ghosts has been studied. The results show that a transmembrane Ca²⁺ gradient is essential for the conformation of erythrocyte Band 3 with higher anion transport activity. The dissipation of the transmembrane Ca²⁺ gradient by the ionophore A23187 inhibits the anion transport activity. The extent of this inhibition approaches 90% as the Ca²⁺ concentration on both sides of the ghost membrane is increased to 1.0 mM and half-maximum inhibition is observed at 0.25 mM Ca²⁺. Addition of ATP (0.4 mM) to the resealing medium can partly reestablish the transmembrane Ca²⁺ gradient by activation of Ca²⁺-ATPase and alleviate the inhibition to some extent. N-ethylmaleimide, an inhibitor of erythrocyte Ca²⁺-ATPase, prevents such restoration. Electron micrographs reveal that numerous larger intramembranous particles can be observed on the P-faces of freeze-fractured resealed ghosts in the absence of a transmembrane Ca²⁺ gradient.Abbreviations DPA dipicolinic acid - EITC eosin 5-isothiocyanate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - TES N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid - PMSF phenylmethyl-sulfonylfluoride - NEM N-ethylamaleimide - BSA bovine serum albumin - EGTA ethyleneglycol-bis (aminoethylether)-tetra-acetic acid - EITC-Band 3 Band 3 labeled with EITC - Ca_i Ca²⁺ inside resealed ghosts - Ca_o Ca²⁺ outside resealed ghosts     

A new nucleosomal protein in normal liver related to the cytoplasmic polypeptide target of a carcinogen

Summary Normal adult rat liver contains a nucleosomal protein that is related to the principal target polypeptide of a carcinogen in cytoplasm. Normal rat liver was found previously to contain a 14 000-dalton polypeptide that is the principal. cytosolic target of the carcinogen, N-2-fluorenylacetamide (2-acetylaminofluorene; FAA), early during hepatocarcinogenesis. Elevated levels of immunohistochemically detectable target polypeptide in cytoplasm are associated with normal mitosis and carcinogen-induced hyperplasias in adult hepatocytes. A putatively related 17 500-dalton polypeptide was shown to be tightly bound to chromatin of normal liver nuclei. We report here that purified nucleosomes from normal rat liver contain the bound 17 500-dalton protein. Nuclei were digested with micrococcal nuclease, and the resultant nucleosomes were resolved into size classes by density gradient sedimentation. The monomers, dimers, and trimers of nucleosomes possessed bound 17 5019-dalton polypeptide, as determined by SDS gel electrophoresis followed by immunoelectroblot analyses. Alterations in the levels of the two polypeptides were shown previously to occur during liver carcinogenesis by FAA and 3-methyl-4-dimethylaminoazobenzene. The findings support the possibility that the 17 500-dalton polypeptide may function normally in a role related to the replication or expression of the hepatic genome, and may be connected with changes in hepatic genic activity brought about by the carcinogens.Abbreviations FAA N-2-fluorenylacetamide (2-acetylaminofluorene) - 3-Me-DAB 3-methyl-4-dimethylaminoazobenzene - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecyl sulfate     

Functional evidence for distinct interaction of hydropholic arylisothiocyanates with the erythrocyte anion transport protein

Summary Human erythrocytes were treated with various hydrophobic arylisothiocyanates under conditions which favor modification of distinct proteinaceous nucleophiles. The morphological appearance of phenylisothiocyanate-treated cells was discoid and membrane-bound hydrolases (human acetylcholinesterase, sheep phospholipase A₂) were fully active following membrane modification. Noncharged hydrophobic arylisothiocyanates, including phenylisothiocyanate, -naphthylisothiocyanate and heterobifunctional azidoarylisothiocyanates inhibited [³⁵S]-sulfate efflux irreversibly. Protection against modification-induced inhibition of sulfate transport was attained by the simultaneous presence of the specific reversible anion transport inhibitor 4,4-dinitrostilbene-2,2-disulfonate. Selective protection of a functionally relevant domain of band 3 is concluded to occur based on the above-derived information.     

The composition of Erythrosins, Fluorescein, Phloxine and Rose Bengal: a study using thin-layer chromatography and solvent extraction

Synopsis Commercial samples of Erythrosin B (CI 45430), Erythrosin Y (CI 45425), Fluorescein (CI 45350), Phloxine (CI 45410) and Rose Bengal (CI 45440) have been analysed by thin-layer chromatography. The Erythrosins were found to be mixtures consisting in the main of 4-iodofluorescein, 4,5-di-iodofluorescein, 2,4,5-triiodofluorescein and 2,4,5,7-tetraiodofluorescein, in some instances together with 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein. Samples of Fluorescein were mixtures of the nominal dye usually with traces of several unidentified, fluorescent components. Those of Phloxine consisted mainly of mixtures of 4-bromo-4,5,6,7-tetrachlorofluorescein, 4,5-dibromo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tribromo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetrabromo-4,5,6,7-tetrachlorofluorescein, often with 4,5,6,7-tetrachlorofluorescein Samples of Rose Bengal were mixtures of 4-iodo-4,5,6,7-tetrachlorofluorescein, 4,5-di-iodo-4,5,6,7-tetrachlorofluorescein, 2,4,5-tri-iodo-4,5,6,7-tetrachlorofluorescein and 2,4,5,7-tetraiodo-4,5,6,7-tetrachlorofluorescein together with some unidentified components.Most of the commercial dye samples gave an insoluble residue when extracted with methanol. This residue was usually inorganic carbonate or halide. Some possible practical consequences of the various impurities are discussed.     

Photoreactive fatty acid analogues that bind to the rat liver fatty-acid binding protein: 11-(5′-azido-salicylamido)-undecanoic acid derivatives

Summary Photoreactive probes for the hydrophobic pocket of the liver fatty acid-binding protein, 11-(5-azido-salicylamido)-undecanoic acid (5 ASU) and its acetyl ester (Ac5 ASU), were synthesized and their interaction with the protein was assessed. Fatty acid-binding proteins are closely related proteins which are abundantly expressed in tissues with active lipid metabolism. A simple model that assumes that the protein possesses a single kind of sites fitted the binding of radioiodinated 5 ASU to L-FABP satisfactorily. The apparent dissociation constant, 1.34×10^–7 M, evidenced a slightly higher affinity than that reported for C16–C20 fatty acids. Consistent with the binding curve, 5 ASU effectively competed with palmitic acid for the hydrophobic sites and the effect was nearly complete for concentrations of 1 gmM; oleic acid, in turn, displaced the radiolabelled probe. Irradiation at 366 nm of¹²⁵I-5 ASU bound to L-FABP caused the covalent cross-linking of the reagent. The amount of radioactivity covalently bound reached a maximum after 2 min thus agreeing with the photo-activation kinetics of the unlabelled compound that evidenced a t_1/2 of 31.1 sec. The yield with which probes bound to L-FABP became covalently linked to the protein, appraised after SDS-PAGE of irradiated samples, was estimated as 23 and 26 per cent for 5 ASU and Ac5 ASU respectively. In turn, irradiation of L-FABP incubated with 5ASU or Ac5 ASU resulted in the irreversible loss of about one fourth its ability to bind palmitic acid. Both results, taken together, suggested that the derivatives are linked to the protein through the sites for fatty acids. When cross-linking of¹²⁵I-5 ASU was performed after incubation with delipidated cytosol and products were analyzed by SDS-PAGE, a band was visualized in a position similar to that of purified L-FABP.Abbreviations FABP Fatty Acid-Binding Protein - L-FABP Hepatic FABP - I-FABP Intestinal FABP - C-FABP Cardiac FABP - 5 ASU-11 (5-azido-salicylamido)-undecanoic acid - Ac5 ASU-11 (O-acetyl-5-azido-salicylamido)-undecanoic acid     

Development of a selective pseudosubstrate-based peptide inhibitor of pp60c-src protein tyrosine kinase

Summary Using a combinatorial peptide library method, we identified YIYGSFK as an efficient and specific peptide substrate for pp60^c-src protein tyrosine kinase (PTK) [Lam et al., Int. J. Pept. Protein Res., 45 (1995) 587]. Employing YIYGSFK as a template, we synthesized and evaluated a series of pseudosubstrate-based inhibitors for pp60^c-src. We found that the efficiency of a given inhibitor was highly dependent on the specific tyrosine analog used at the phosphorylation site of the substrate. One of these pseudosubstrate inhibitors, YI(2-Nal)GSFK, selectively inhibited the kinase activity of pp60^c-src, with a K_i of 24 M. This peptide inhibitor exhibited selectivity for pp60^c-src as compared to other PTKs tested, such as c-Abl and Bcr-Abl. Our results suggest that selective inhibitors for a specific PTK can be developed when the structure of a specific and efficient small peptide substrate for this PTK can be used as a template for structure modification.Abbreviations 1-Nal l-1-naphthylalanine - 2-Nal l-2-naphthylalanine - BOP benzotriazolyl-N-oxy-tris(dimethylamino)-phosphonium hexafluorophosphate - BSA bovine serum albumin - cAPK cyclic AMP-dependent protein kinase - DIEA diisopropylethylamine - EGFR epidermal growth factor receptor - Fmoc fluorenylmethoxycarbonyl - HOBt 1-hydroxybenzotriazole - MES 2-[N-morpholino]ethanesulfonic acid - PBS phosphate-buffered salts - pCl l-p-chlorophenylalanine - pF l-p-fluorophenylalanine - PTK protein tyrosine kinase - TLC thin-layer chromatography     

Membrane proteins related to anion permeability of human red blood cells

Summary (³H)DIDS (4,4-diisothiocyano-2,2-ditritiostilbene-disulfonate) was used as a convalent label for membrane sites involved in anion permeability. The label binds to a small, superficially located population of sites, about 300,000 per cell, resulting in almost complete inhibition of anion exchange. The relationship of biding to inhibition is linear suggesting that binding renders each site nonfunctional. In the inhibitory range less than 1% of the label is associated with lipids but at higher concentrations of DIDS, the fraction may be as high as 4%. In ghosts, however, treatment with (³H)DIDS results in extensive labeling of lipids. In cells, a protein fraction that behavens on SDS acrylamide gels as thought its molecular weight is 95,000 daltons (95K) is predominatly labeled by (³H)DIDS. The only other labeled protein is the major sialoglycoprotein which contains less than, 5% of the total bound (³H)DIDS. Because of the linear relationship of binding to inhibition and the unique architecture of the site, it is suggested that the (³H)DIDS-binding site of the 95K protein is the substrate binding site of the anion transport system. The 95K protein is asymmetrically arranged in the membrane with the sites arranged on the outer face accessible to agent in the medium. In leaky ghost, only a few additional binding sites can be reached from the inside of the membrane in the 95K protein, in contrast to the extensive labeling of other membrane proteins in ghosts as compared to cells.Abbreviations DADS 4,4-Diamino-2,2-dihydrostilbene disulfonic acid - DIDS 4,4-Diisothiocyano-2,2-stilbene disulfonic acid - (³H)DADS 4,4-Diamino-2,2-ditritiostilbene disulfonic acid - (³H)DIDS 4,4-Diisothiocyano-2,2-ditritiostilbene disulfonic acid     

The influence of increasing chlorine content on the accumulation and metabolism of polychlorinated biphenyls (PCBs) by Paul's Scarlet Rose cells

Four radiolabled congeners of biphenyls with increasing chlorine content (biphenyl; 1-monochlorobiphenyl; 2,2,4,4-tetrachlorobiphenyl; and 2,2,4,4,5,5-hexachlorobiphenyl) were provided to suspension cultures of rose (Rosa sp. cv. Paul's Scarlet) for 4 days. Both the kinetics of ¹⁴C exchange between the cells and medium, and the metabolism of the parent compounds depended on the chlorine content of the congeners. Analysis of both the cells and their medium showed that of the recovered radioactivity 88%, 86%, and 3% of the biphenyl, 1-PCB, and 2,2,4,4-PCB were metabolized respectively to polar and insoluble residue products. The 2,2,4,4,5,5-PCB did not appear to be metabolized.     

Photoaffinity labeling and partial purification of the putative plant receptor for the fungal wilt-inducing toxin,fusicoccin

The high-affinity fusicoccin-binding protein (FCBP) was solubilized from plasma-membrane vesicles prepared from leaves of Vicia faba L. by aqueous two-phase partitioning. Conditions for the solubilization of intact FCBP-radioligand complexes were worked out. About 60–70% of the complexes can be solubilized with 50–60 mM nonanoyl-N-methylglucamide in the presence of 1 mg· ml^-1 soybean phosphatidylcholine, type IV S, and 20% (v/v) glycerol at pH 5.5. The slow dissociation of the radioligand, 9-nor-fusicoccin-8-alcohol-[³H] from the FCBP at low temperatures permits the purification of FCBP-radioligand complexes at 4–10° C by fast protein liquid chromatography on anion-exchange and gel permeation columns. The FCBP, extracted from plasma membranes with cholate and chromatographed in the presence of this detergent, gave an apparent molecular mass (M_r) of 80±20 kDa on gel permeation columns under the conditions used. By comparison of the elution profiles of the fraction most enriched in FCBP-radioligand complexes with polypeptide patterns obtained on sodium dodecyl sulfate-polyacrylamide gels, a polypeptide with an M_r of approx. 34kDa co-separated with the radioactivity profile. A second, faint band of approx. 31 kDa was sometimes also observed co-electrophoresing. Photoaffinity labeling of plasma-membrane vesicles with the new compound 9-nor-8[(3,5-[³H]-4-azidobenzoy)ethylenediamine]-fusicoccin ([³H]ABE-FC) and subsequent separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis labeled a single band with an M_r of 35±1 kDa. Labeling in this band was strongly reduced when the membranes were incubated with [³H]ABE-FC in the presence of 0.1–1 M fusicoccin. From our data, we conclude (i) that the 34-35-kDa polypeptide represents the FCBP and (ii) that in detergent extracts of plasma membranes this polypeptide is probably present as a di- or trimeric structure.Abbreviations ABE-FC [(4-azidobenzoyl)-ethylenediamine]-fusicoccin - ABE-NHS (4-azidobenzoyl)-N-hydroxysuccinimide ester - FC fusicoccin - FCBP fusicoccin-binding protein - FCol 9-norfusicoccin-8-alcohol - MAB monoclonal antibody - Mega-9(10) nonanoyl(decanoyl)-N-methylglucamide - M_r apparent molecular mass - PMSF phenylmethyl-sulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TCA trichloroacetic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Inhibition of anion transport associated with chymotryptic cleavages of red blood cell band 3 protein

Right-side-out vesicles derived from red blood cells treated with chymotrypsin retain specific anion transport function (defined as transport sensitive to the specific inhibitor, 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS)), even though the transport protein, band 3, is cleaved into two segments of 60 and 35 kdaltons. In contrast, vesicles derived from alkali-stripped ghosts treated with relatively high concentrations of chymotrypsin retain almost no specific anion function. The loss of function appears to be related to additional cleavages of band 3 protein that occur in treated ghosts, the 60-kdalton segment being reduced first to a 17- and then to a 15-kdalton segment and the 35-kdalton segment being reduced to a 9-kdalton segment plus a carbohydrate containing fragment. The chymotryptic cleavages of band 3 protein of ghosts are preferentially inhibited by high ionic strength, the production of the 9-kdalton segment being somewhat slower than that of the 15-kdalton segment. Vesicles derived from ghosts treated with chymotrypsin at different ionic strengths show a graded reduction in specific anion transport activity, but it was not possible to determine, definitively, which of the additional cleavages was inhibitory. In the light of these data and other information, the functional role of the segments of band 3 is discussed.     

Full 1H NMR assignment of a 24-nucleotide RNA hairpin: Application of the 1H 3D-NOE/2QC experiment

The subject RNA models the binding site for the coat protein of the R17 virus, as well as the ribosome recognition sequence for the R17 replicase gene. With an RNA of this size, overlaps among the sugar protons complicate assignments of the ¹H NMR spectrum. The cross peaks that overlap significantly in 2D-NOE spectra can frequently be resolved by introducing a third, in our approach the double-quantum, frequency axis. In particular the planes in a 3D-NOE/2QC spectrum perpendicular to the 2Q axis are extremely useful, showing a highly informative repeating NOE-2Q pattern. In this experiment substantial J-coupling confers special advantages. This always occurs for geminal pairs (H5/H5 for RNA plus H2/H2 for DNA), as well as for H5/H6, for H3/H4 in sugars with substantial populations of the N-pucker, for H1/H2 for S-puckered sugars, and usually for H2/H3. For the 24-mer RNA hairpin the additional information from the 3D-NOE/2QC spectrum allowed assignment of all of the non-exchangeable protons, eliminating the need for stable-isotope labeling.     

Role of cyclic adenosine-3′,5′-monophosphate in olfactory reception

The action of cyclic adenosine-3,5-monophosphate (3,5-AMP) and of substances modifying the rate of its breakdown (inhibitors and activators of phosphodiesterase) on the olfactory epithelium was investigated in frogs. The slow electrical response of the olfactory epithelium to stimulation by solutions of various substances was recorded. Cyclic 3,5-AMP and its dibutyryl derivative were found to excite the olfactory receptors effectively. Responses to these substances developed after an appreciably longer delay than responses to stimulation by solutions of odiferous substances. It is postulated that the depolarizing action of 3,5-AMP and dibutyryl 3,5-AMP is manifested only after they have penetrated inside the receptor cell through its membrane. Both 5-AMP and cyclic 2,3-AMP were ineffective. In the next series of experiments the integral receptor potential was recorded in response to short stimulation by the vapor of an odiferous substance. The duration of this potential was increased after treatment of the olfactory epithelium with phosphodiesterase inhibitors: methylxanthines or papaverine. Conversely, the negative wave of the integral receptor potential was shortened under the influence of the phosphodiesterase activator imidazole. Cyclic 3,5-AMP is considered to play the role of mediator in the mechanism of excitation of the olfactory receptor; during interaction between an odiferous substance and the receptor, adenyl cyclase is activated and the concentration of 3,5-AMP increases; this, in turn, causes depolarization of the receptor cell membrane.Institute of Chemical Physics, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 5, No. 4, pp. 415–422, July–August, 1973.     

Carotenoid pigments of Stigmatella aurantiaca (Myxobacterales)

Summary In this first article on the carotenoids of Myxobacterales we report on the minor carotenoids of Stigmatella aurantiaca: phytoene, phytofluene, lycopene, -carotene, 4-keto--carotene, 1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy--carotene, 4-keto-1,2-dihydro-1-hydroxy-torulene, and 1,2,1,2-tetrahydro-1,1-dihydroxy-lycopene. These pigments account for about 10% of total carotenoids.     

Localization of adenosine 5′-phosphosulfate sulfotransferase in spinach leaves

Roots of spinach (Spinacia oleracea L.) seedlings contained only a very low activity of adenosine 5-phosphosulfate sulfotransferase compared to the cotyledons. Adenosine 5-phosphosulfate sulfotransferase activity increased about tenfold in cotyledons during greening. Preparation of organelle fractions from spinach leaves by a combination of differential and isopycnic density gradient centrifugation showed that adenosine 5-phosphosulfate sulfotransferase banded with NADP-glyceraldehyde-3-phosphate dehydrogenase, a marker enzyme for intact chloroplasts. In the fractions of peroxisomes, mitochondria and broken chloroplasts virtually no adenosine 5-phosphosulfate sulfotransferase activity was measured. Comparison with the chloroplast enzyme NADP-glyceraldehyde-3-phosphate dehydrogenase indicates that in spinach, adenosine 5-phosphosulfate sulfotransferase is localized almost exclusively in the chloroplasts.Abbreviations APS Adenosine 5-phosphosulfate - APSSTase Adenosine 5-phosphosulfate sulfotransferase - BSA Bovine serum albumin - BRIJ58 Polyethylene glycolmonostearylether - DTE Dithioerythritol - DTT Dithiothreitol - EDTA Ethylenediaminetetraacetic acid - ME 2-Mercaptoethanol - NADP-GPD NADP-linked glyceraldehyde-3-phosphate dehydrogenase - PAPS Adenosine 3-phosphate 5-phosphate 5-phosphosulfate - POPOP 1,4 Di [2-(5-phenyloxazolyl)]-benzene - PPO 2,5-Diphenyloxazol The results presented in this paper are taken from the Ph. D. thesis of H.F.     

Direct Measurement of Nitrite Transport Across Erythrocyte Membrane Vesicles Using the Fluorescent Probe, 6-Methoxy-N-(3-sulfopropyl) quinolinium

Nitrite was shown to quench the fluorescence of 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) almost twofold more than chloride. SPQ loaded inside vesicles prepared from asolectin and isolated erythrocyte ghosts allowed for the direct measurement of nitrite movement across these membranes. Movement of nitrite across asolectin occurred by diffusion as HNO₂ in a pH-dependent manner. By contrast, erythrocyte ghosts had very low diffusion rates for nitrous acid. Erythrocyte ghosts preloaded with 50 mM nitrite to quench SPQ fluorescence were utilized to study heteroexchange with externally added anions. SPQ fluorescence increases (becomes unquenched) with added bicarbonate and nitrate, indicating that nitrite is moving out of the preloaded vesicles. The pH optimum for this exchange was approximately 7.6 and exchange was inhibited by N-ethylmaleimide (NEM) and dihydro-4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS). These data indicate that nitrite moves across erythrocyte plasma membranes as NO₂- by a heteroexchange mechanism with other monovalent anions.     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

The sulfhydryl groups of the 35,000-dalton C-terminal segment of band 3 are located in a 9000-dalton fragment produced by chymotrypsin treatment of red cell ghosts

Five sulfhydryl groups of band 3, the anion-transport protein of the red blood cell membrane, can be labeled byN-ethylmaleimide (NEM). Two of these are located in a 35,000-dalton, C-terminal segment produced by chymotrypsin treatment of cells. Extensive treatment of unsealed ghosts with chymotrypsin results in the disappearance of the 35,000-dalton segment, but its two NEM-binding sites are preserved in a 9000-dalton peptide. The latter must therefore be a proteolytic product of the larger segment. Labeling of sulfhydryl groups of band 3 by an impermeant analog of NEM occurs in inside-out, but not in right-side-out vesicles derived from red cell ghosts, supporting the conclusion that NEM-reactive sulfhydryl groups, including those in the 35,000- and 9000-dalton segments, are exposed at the cytoplasmic face of the membrane. These findings support the conclusion that the 35,000-dalton segment crosses the bilayer, and suggest that the 9000-dalton segment may be a membrane-crossing portion of the 35,000-dalton segment.     

Cytoskeleton components of inside-out and right-side-out plasma membrane vesicles from plants

Summary Isolated plasma membrane vesicles purified by aqueous polymer two-phase partitioning were used as a model system for studies on the membrane-associated (cortical) cytoskeleton in plants. Actin, as identified by immunoblotting, was found to be specifically attached to plasma membrane vesicles from cauliflower (Brassica oleracea L.). The actin was not washed off as the vesicles were turned inside-out, indicative of a fairly strong attachment. Triton X-100 extraction of plasma membrane vesicles resulted in an insoluble and hence pelletable fraction where actin could be found together with several other proteins. Our results show that the cortical cytoskeleton is to some extent co-purified with the plasma membrane, and we believe that well defined, inside-out and right-side-out plasma membrane vesicles can be used to study the structure and dynamics of the plant cortical cytoskeleton.Abbreviations ATP adenosine 5-triphosphate - BCIP 5-bromo-4-chloro-3-indolyl phosphate - BSA bovine serum albumin - CCD counter-current distribution - DTT dithiothreitol - EDTA ethylene-diamine-tetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether) - GSII 1,3--glucan synthase - HEPES N-[2-hydroxyethyl]-piperazine-N-[2-ethane sulfonic acid] - MES 2-(N-morpholino)ethane sulfonic acid - NBT p-nitro blue tetrazolium chloride - IDP inosine 5-diphosphate - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethane-sulfonic acid) - PPB potassium phosphate buffer - PM plasma membrane - PMSF phenylmethylsulfonyl fluoride - PVDF polyvinylidene difluoride - PVPP polyvinylpolypyrrolidone - SDS sodium dodecyl sulfate - TBS Tris-buffered saline - TTBS Tris-buffered saline with Tween 20 - Tris tris(hydroxymethyl) aminomethane     

Optimization of the synthesis of porcine somatotropin in Escherichia coli

We report on the influence of choice of promoter and RNA polymerase, 5-untranslated regions and ribosome binding sites, codon usage, leader peptide coding sequences and poly A tail in the 3-untranslated region on the synthesis of porcine somatotropin (PST) in Escherichia coli. A total of 12 different constructs were tested in this study for the production of porcine somatotropin (PST) in E. coli. Several factors have significant effects on PST synthesis. In the presence of a strong promoter and a strong ribosome binding site, the next most important factor seems to be the combination of sequences at the 5-end of the mRNA including both the 5-untranslated region and the start of the coding sequence. Codon usage in the 5-coding sequence per se is not important in determining the level of PST synthesis where high level expression is achieved from a strong ribosome binding site. However, where low level synthesis of recombinant PST (rPST) is achieved, codon usage in the 5-coding sequence is important in determining the level of PST synthesis. Leader sequences dramatically reduce the level of PST synthesis. The presence of a poly A tail in the 3-untranslated region has no significant effect on PST synthesis.