scholarly journals Phytoferritin is synthesized in vitro as a high-molecular-weight precursor. Studies on the synthesis and the uptake in vitro of the precursors of ferritin and ferredoxin by intact chloroplasts

1983 ◽  
Vol 214 (3) ◽  
pp. 943-950 ◽  
Author(s):  
F van der Mark ◽  
W van den Briel ◽  
H G Huisman
Keyword(s):  
Gel Electrophoresis ◽  
Biosynthetic Pathway ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
French Bean ◽  
Polyacrylamide Gel ◽  
Cell Organelle ◽  
Bean Seed ◽  
Intact Chloroplasts

Evidence is presented that French-bean (Phaseolus vulgaris) seed ferritin is composed of one type of subunit with an apparent Mr of 26500. In normal and iron-loaded leaf tissues it is detected immunologically with an antiserum raised against purified bean seed ferritin and migrates in SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis with the same mobility as the bean seed ferritin subunit. The biosynthetic pathway of ferritin in normal and iron-loaded leaves was investigated. RNA was extracted, fractionated into polyadenylated RNA and translated in a cell-free rabbit reticulocyte lysate and a wheat-germ-extract system. The products were identified by SDS/polyacrylamide-gel electrophoresis after indirect immunoprecipitation. In all cases the ferritin product had an Mr 5000 higher than that of the native subunit. Uptake and processing of the precursor form of ferritin from iron-loaded leaves by intact chloroplasts was demonstrated. This indicates that, in iron-loaded leaves, ferritin acts as a chloroplast protein. We propose that the ferritin precursor in normal leaves follows the same biosynthetic pathway. This suggests that the iron-buffering function of ferritin in plants takes place in the chloroplast and that non-functional cellular iron will accumulate in this cell organelle.

scholarly journals Phosphorylation of synaptic-membrane proteins from ox cerebral cortex in vitro. Preparation of fractions enriched in phosphorylated proteins by using extraction with detergents and urea, and gel filtration

1977 ◽  
Vol 163 (2) ◽  
pp. 369-378 ◽  
Author(s):  
P R Dunkley ◽  
H Holmes ◽  
R Rodnight
Keyword(s):  
Cerebral Cortex ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Synaptic Membrane ◽  
Hydroxyl Groups ◽  
Phosphorylated Proteins

Synaptic-membrane fragments from ox cerebral cortex contain basal and cyclic AMP-stimulated protein kinase(s) that transfer 32P from [gamma-32P]ATP to hydroxyl groups of serine and threonine residues in membrane-protein substrates. In the present work, labelled membrane fragments were partitioned into soluble and insoluble fractions with Triton X-100, Nonidet P. 40, sodium deoxycholate and urea, and the distribution of 32P-labelled protein in the fractions was determined by polyacrylamide-gel electrophoresis and radioautography. A high percentage of phosphorylated protein sustrates remained insoluble, including those whose phosphorylation was most highly stimulated by cyclic AMP. Whole membrane fragments and samples prepared by detergent extraction were fractionated on Sepharose 6B columns in the presence of low concentrations of sodium dodecyl sulphate and pooled fractions were analysed by polyacrylamide-gel electrophoresis and radioautography. Phosphorylated proteins were fractionated on the basis of their molecular weight, but homogeneous protein was not obtained. The results are discussed in relation to the techniques used and the results obtained in other laboratories.

scholarly journals Purification and properties of β-mannanases I and II from the germinated seeds of Trifolium repens. Mode of galactomannan degradation in vitro

1978 ◽  
Vol 175 (3) ◽  
pp. 1079-1087 ◽  
Author(s):  
H Villarroya ◽  
J Williams ◽  
P Dey ◽  
S Villarroya ◽  
F Petek
Keyword(s):  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ph Optimum ◽  
Electrophoretic Method ◽  
Germinated Seeds

Two beta-mannanases (beta-mannosidases, EC 3.2.1.25) purified from the germinated seeds of Trifolium repens by a procedure that included chromatography on hydroxyapatite, gel filtration on acrylamide/agarose (Ultragel 5/4) and preparative polyacrylamide-gel-electrophoresis. The final purification step completely resolved two beta-mannanases with distinct specificities, which were termed beta-mannanase I and beta-mannanase II. beta-Mannanase I was purified 1400-fold and beta-mannanase II 1000-fold. The purified enzymes showed a single protein band when examined by polyacrylamide-gel disc electrophoresis. beta-Mannanase I, apparent mol.wt. 43 000, accounted for 49% of the total activity recovered from the final step of purification. beta-Mannanase II, apparent mol.wt. 38 000, accounted for the remaining 51% of activity. Molecular-weight determinations were by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and by the electrophoretic method of Hendrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. The substrate specificities of both enzymes were examined with the galactomannans of T. repens and of Medicago sativa, as well as with manno-oligosaccharides. The pH optimum was between pH 5.1 and 5.6 for both enzymes.

Phosphorylation of prolactin and growth hormone

1992 ◽  
Vol 8 (3) ◽  
pp. 183-191 ◽  
Author(s):  
C. Arámburo ◽  
J. L. Montiel ◽  
J. A. Proudman ◽  
L. R. Berghman ◽  
C. G. Scanes
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Pituitary Cells ◽  
Charge Heterogeneity ◽  
Kinase A

ABSTRACT To determine whether GH and prolactin could be phosphorylated, turkey GH, chicken GH, chicken prolactin and turkey prolactin were incubated in vitro with the catalytic subunit of protein kinase A and [γ-32P]ATP. Phosphorylation was assessed after sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Western blotting and autoradiography. Polyacrylamide electrophoresis showed that both purified native chicken GH and turkey GH were phosphorylated under the conditions employed. However, the glycosylated variant of chicken GH did not appear to be labelled. Chicken prolactin, turkey prolactin and the glycosylated variant of turkey prolactin were all intensely phosphorylated by protein kinase A. Ovine and rat prolactins could also be phosphorylated by protein kinase A. The phosphate content of different native prolactin (turkey, ovine and rat) and GH (ovine and chicken) preparations was also determined and found to be significant. Chicken pituitary cells in primary culture incorporated P in GH- and prolactin-like bands isolated by non-denaturing polyacrylamide gel electrophoresis, and this was stimulated by phorbol myristate acetate. Phosphorylation of GH and prolactin may thus explain some of the charge heterogeneity of these hormones.

scholarly journals Initiation and processing in vitro of the primary translation products of guinea-pig caseins

1979 ◽  
Vol 184 (2) ◽  
pp. 261-267 ◽  
Author(s):  
R K Craig ◽  
P A J Perera ◽  
A Mellor ◽  
A E Smith
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Secretory Proteins ◽  
Post Translational Modifications ◽  
Microsomal Membranes

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal ‘signal’-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.

scholarly journals KONSENTRASI PROTEIN DAN PENENTUAN BERAT MOLEKUL EKSKRETORI/SEKRETORI L3 Ascaridia galli

2016 ◽  
Vol 3 (1) ◽  
Author(s):  
Darmawi D ◽  
Ummu Balqis ◽  
Risa Tiuria ◽  
Retno D Soejoedono ◽  
Fachriyan H Pasaribu
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Ascaridia Galli ◽  
Sds Page

Penelitian ini bertujuan menentukan konsentrasi dan berat molekul protein  ekskretori/sekretori larva (L3) Ascaridia galli (A. galli). Larva L3 diperoleh dari usus halus 100  ayam tujuh hari setelah pemberian dosis 6000 L2 melalui esofagus ayam. Sebanyak 5–10  L3 dikultur secara in vitro  dalam setiap ml medium Rosswell Park Memorial Institute (RPMI 1640), pH 6,8, tanpa merah fenol dalam inkubator pada temperatur 37 0C dan 5% CO2 selama 3 hari. Ke dalam medium ditambahkan 100 unit ml-1 penisilin G, 100 µg ml-1 streptomisin, 5 µg ml-1 gentamisin dan 0,25 µg ml-1 kanamisin. Ekskretori/sekretori dipreparasi dari produk metabolisme L3 yang dilepaskan ke dalam medium kultur. Untuk mendapatkan protein ekskretori/sekretori, medium kultur dipekatkan dengan vivaspin 30.000 MWCO, dan kuantitas protein dihitung dengan metode Bradford. Berat molekul protein ekskretori/sekretori divisualisasikan dengan sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE). Hasil penelitian  menunjukkan bahwa konsentrasi protein ekskretori/sekretori adalah 0,595 mg/ml dengan berat molekul 28 kDa.

scholarly journals α-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro

1978 ◽  
Vol 175 (3) ◽  
pp. 1069-1077 ◽  
Author(s):  
J Williams ◽  
H Villarroya ◽  
F Petek
Keyword(s):  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ph Optimum

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).

scholarly journals mraY Is an Essential Gene for Cell Growth in Escherichia coli

1998 ◽  
Vol 180 (23) ◽  
pp. 6429-6432 ◽  
Author(s):  
David S. Boyle ◽  
William D. Donachie
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Essential Gene ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Dodecyl Sulfate

ABSTRACT The synthesis of the murein precursor lipid I is performed by MraY. We have shown that mraY is an essential gene for cell growth. Cells depleted of MraY first swell and then lyse. The expression of mraY DNA in vitro produces a 40-kDa polypeptide detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

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