A rapid method for removal of [125I]iodide following iodination of protein solutions

Tuszynski, George P.; Knight, Linda C.; Piperno, Jeanette R.; Walsh, Peter N.

doi:10.1016/0003-2697(80)90126-8

Cited by 148 publications

(54 citation statements)

References 11 publications

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“…Iodination of streptavidin with Na'251I was carried out by the method of Fracker and Speck (11). Excess ['251jiodide was removed by gel filtration (42 Allium porrum plants were obtained from a local market. The section 7 to 10 cm above the meristem was used in all experiments.…”

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confidence: 99%

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Tissue Distribution of Acetyl-Coenzyme A Carboxylase in Leaves

Nikolau

Wurtele²,

Stumpf³

1984

Plant Physiol.

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Acetyl-CoA carboxylase [acetyl-CoA-carbon dioxide lipse (ADP forming), EC 6.4.1.21 is a biotin-containing enzyme catalyzing the formation of malonyl-CoA. The tissue distribution of this enzyme was determined for leaves of C3-and C4-plants. The mesophyll tissues of the C3-plants Pisum sativum and Aiium porrum contained 90% of the leaf acetyl-CoA carboxylase activity, with the epidermal tissues containing the remainder. Western blotting of proteins fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, using 1I-streptavidin as a probe, revealed biotinyl proteins ofmolecular weights 62,000,51,000, and 32,000 in P. sativum and 62,000, 34,000, and 32,000 in A. porrum.In the C4-plant sorghum, epidermal protoplasts, mesophyll protoplasts and strands of bundle sheath cells contained 35, 47, and 17%, respectively, of the total leaf acetyl-CoA carboxylase activity. In Zea mays leaves the respective figures were 10% for epidermal protoplasts, 56% for mesophyll protoplasts, and 32% for bundle sheath strands. Biotinyl proteins of molecular weights 62,000 and 51,000 were identified in leaves of sorghum and Z. mays.The results are discussed with respect to each tissue's requirements for malonyl-CoA for various metabolic pathways.

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“…The brei was filtered through a 20-,um mesh nylon net. An aliquot of the filtrate was immediately passed through a short column of Sephadex-G25 using a rapid centrifugation procedure (42). Columns were equilibrated with buffer A containing 0.01% Triton-X100.…”

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Tissue Distribution of Acetyl-Coenzyme A Carboxylase in Leaves

Nikolau

Wurtele²,

Stumpf³

1984

Plant Physiol.

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“…In studies of tissue distribution, the pellets enriched with either mesophyll or epidermal protoplasts were each washed with 10 ml of 20 mM TricineNaOH (pH 7.5) containing 0.5% (w/v) BSA and 0.5 M mannitol. Washed protoplasts were lysed in 1 to 2 ml of 50 mM containing 0.01% (w/v) Triton X-100 and immediately passed through a short column of Sephadex G-25 using a rapid centrifugation procedure (18).…”

Section: Methodsmentioning

confidence: 99%

Tissue Distribution and Subcellular Localization of Prephenate Aminotransferase in Leaves of Sorghum bicolor

Siehl¹,

Singh²,

Conn³

1986

Plant Physiol.

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The tissue and subcellular distribution of prephenate aminotransferase, an enzyme of the shikimate pathway, was investigated in protoplasts from leaves of Sorghum bicolor. Activity was detected in purified epidermal and mesophyll protoplasts, and in bundle sheath strands. EE Conn, unpublished data). In these examples, the plastid isozymes were regulated by shikimate pathway intermediates or end products, while the cytoplasmic isozymes, though quantitatively significant, were unregulated by the aromatic amino acids. In another study, minor, cytosolic forms ofEPSP synthase, DHQ dehydratase, and shikimate dehydrogenase were found in extracts from pea seedlings (13).Prephenate aminotransferase is required by all organisms which use the arogenate route for the biosynthesis of tyrosine and/or phenylalanine. In such organisms, production oftyrosine from prephenate proceeds by transamination, yielding arogenate, followed by dehydrogenation. All higher plants studied so far, including mung bean (15), corn (3), tobacco (7), and sorghum (4), were shown to contain arogenate dehydrogenase for tyrosine biosynthesis, implicating the presence of PAT in each of these species. PAT (2), and later in sorghum (16). The present study is the first attempt to characterize the distribution of PAT. MATERIALS AND METHODSPlant Material. Seeds (Sorghum bicolor x Sorghum sudanensis hybrid, WAC Forage 99, WAC Seed, Inc., Hereford, TX 79045) were soaked in aerated water for 24 h at room temperature, then grown in vermiculite under a 16:8 h (fluorescent light:dark) photoperiod. The temperature during light was 27°C and during dark, 22C.Preparation and Purification of Protoplasts. Protoplasts were obtained using a modification ofpreviously described procedures (11,14). Leaves from 6-d-old seedlings were abraded with carborundum, then incubated for 3 h at 30°C in a solution of 1.5% (w/v) Cellulysin (Calbiochem) in Mes-mannitol buffer (20 mM Mes [pH 5.6] and 0.6 M mannitol). The released protoplasts were filtered through a 44 gm mesh nylon net and pelleted by centrifugation at 100g for 10 min. The pellets were gently resuspended in 10 ml of 20% Ficoll in Mes-mannitol buffer. Five ml each of 10, 7.5, and 0% Ficoll were overlaid onto the 20% Ficoll mixture and the resulting gradient was centrifuged at 600g for 5 min. The protoplasts at the 0 to 7% interface (enriched with epidermal protoplasts) and at the 10 to 20% interface (enriched with mesophyll protoplasts) were collected using a wide mouthed pipette. The protoplast mixtures were diluted 5-to 10-fold with Mesmannitol buffer and collected by centrifugation at 300g for 3 min.Preparation of Protoplast Extracts. In studies of tissue distribution, the pellets enriched with either mesophyll or epidermal protoplasts were each washed with 10 ml of 20 mM TricineNaOH (pH 7.5) containing 0.5% (w/v) BSA and 0.5 M mannitol. Washed protoplasts were lysed in 1 to 2 ml of 50 mM containing 0.01% (w/v) Triton X-100 and immediately passed through a short column of Sephadex G-25 using a rapid centrifug...

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“…Growth factors were iodinated by the solid phase lactoperoxidase method (Tower et al, 1977) using Enzymobeads (Bio-Rad Laboratories) and isolated by a microcolumn procedure (Tuszynski et al, 1980). Specific EGF binding was measured according to Carpenter (1985).…”

Section: Lodination Of Growth Factors and Binding Assaysmentioning

confidence: 99%

Long-term, heterologous down-regulation of the epidermal growth factor receptor in PC12 cells by nerve growth factor.

Lazarovici¹,

Dickens²,

Kuzuya³

et al. 1987

The Journal of Cell Biology

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Abstract. Cells of the rat pheochromocytoma clone PC12 possess receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), thus enabling the study of the interaction of these receptors in the regulation of proliferation and differentiation. Treatment of the cells with NGF induces a progressive and nearly total decrease in the specific binding of EGF beginning after 12 h and completed within 4 d. Three different measures of receptor show that the decreased binding capacity represents, in fact, a decreased amount of receptor: (a) affinity labeling of PC12 cell membranes by cross-linking of receptorbound ~25I-EGF showed a 60-90% decrease in the labeling of 170-and 150-kD receptor bands in cells treated with NGF for 1-4 d; (b) EGF-dependent phosphorylation of a src-related synthetic peptide or EGF receptor autophosphorylation with membranes from NGF-differentiated cells showed a decrease of 80 and 90% in the tyrosine kinase activity for the exogenous substrate and for receptor autophosphorylation, respectively; (c) analysis of 35S-labeled glycoproteins isolated by wheat germ agglutinin-Sepharose chromatography from detergent extracts of PC12 membranes showed a 70-90% decrease in the 170-kD band in NGF-differentiated cells. These findings permit the hypothesis that long-term heterologous down-regulation of EGF receptors by NGF in PC12 cells is mediated by an alteration in EGF receptor synthesis. It is suggested that this heterologous down-regulation is part of the mechanism by which differentiating cells become insensitive to mitogens.T HE rat pheochromocytoma clone PC12 has been used as a model of neuronal differentiation because the cells acquire neuronal properties and stop dividing in response to nerve growth factor (NGF) ~ (Greene and Tischler, 1976). The differentiation induced by NGF appears to be reversible, but, except for this, the changes that occur lead to cells with the phenotype of sympathetic neurons (Kimhi, 1981). The PC12 cells, of course, carry NGF receptors (Herrup and Thoenen, 1979;Landreth and Shooter, 1980) and the binding of NGF to these specific receptors triggers many and diverse arrays of cellular responses, including both shortterm, rapid membranal changes, and long-term alterations in cell properties associated with changes in transcription (Guroff, 1983). Among these responses, NGF-induced differentiation in PC12 involves a transition from a mitotic to a nonmitotic state.In this regard, it was of interest to observe that the PCI2 ceils also have receptors for epidermal growth factor (EGF) (Huff and Guroff, 1979;Huff et al., 1981;Boonstra et al., 1985). This peptide is a potent mitogen for many of the cells I. Abbreviations used in this paper: EGF, epidermal growth factor; NGF, nerve growth factor. with which it interacts, and is a mild mitogen for PC12 (Huff et al., 1981). Since the cells, then, have receptors for both a differentiating agent and a mitogen, experiments in this laboratory have been designed to explore what happens when the cells are treated with both ag...

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A rapid method for removal of [125I]iodide following iodination of protein solutions

Cited by 148 publications

References 11 publications

Tissue Distribution of Acetyl-Coenzyme A Carboxylase in Leaves

Tissue Distribution of Acetyl-Coenzyme A Carboxylase in Leaves

Tissue Distribution and Subcellular Localization of Prephenate Aminotransferase in Leaves of Sorghum bicolor

Long-term, heterologous down-regulation of the epidermal growth factor receptor in PC12 cells by nerve growth factor.

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