Electrophoretic determination of sulfhydryl groups and its application to complex protein samples, in vitro protein synthesis mixtures, and cross-linked proteins

1986 ◽  
Vol 64 (2) ◽  
pp. 154-160 ◽  
Author(s):  
Helga Stan-Lotter ◽  
Philip D. Bragg
Keyword(s):  
Protein Synthesis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sulfhydryl Groups ◽  
Additional Parameter ◽  
In Vitro Protein Synthesis ◽  
Molecular Weights ◽  
Complex Protein ◽  
Vitro Protein

Without prior fractionation, the number of sulfhydryl groups of individual polypeptides in a protein mixture can be determined, provided their molecular weights and approximate isoelectric points are known. Urea-denatured protein samples are reacted with iodoacetamide and iodoacetate in a modified version of Creighton's procedure. After separation by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and isoelectric focusing, the number of sulfhydryl groups is determined by counting the protein bands which have additional negative charges. This method requires little material and provides an additional parameter, besides the molecular weight and isoelectric point, for the identification and characterization of a protein. The sensitivity may be enhanced for nonradioactive proteins by using 14C-labeled iodoacetamide and iodoacetate. The procedure has been applied to prokaryotic in vitro protein synthesis mixtures, bacterial membrane protein, and trypsin-cleaved or chemically cross-linked subunits of the F1 ATPase from Escherichia coli.

The effect of synthetic lysine and methionine on in vitro protein synthesis of weaning pigs

1995 ◽  
Vol 15 (3) ◽  
pp. 429-437 ◽  
Author(s):  
Y.M. Kwon ◽  
K.N. Heo ◽  
Y.J. Choi ◽  
I.K. Han ◽  
J.H. Woo
Keyword(s):  
Protein Synthesis ◽  
In Vitro Protein Synthesis ◽  
Weaning Pigs ◽  
Vitro Protein

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

[37] In vitro protein synthesis

1991 ◽  
pp. 536-545 ◽  
Author(s):  
Michael J. Leibowitz ◽  
Francis P. Barbone ◽  
Denise E. Georgopoulos
Keyword(s):  
Protein Synthesis ◽  
In Vitro Protein Synthesis ◽  
Vitro Protein

Effects of oestradiol, the oestrous cycle and pregnancy on weight, metabolism and cytosol receptors in the uterus of the brush-tail possum (Trichosurus vulpecula)

1986 ◽  
Vol 108 (2) ◽  
pp. 201-210 ◽  
Author(s):  
J. D. Curlewis ◽  
G. M. Stone
Keyword(s):  
Protein Synthesis ◽  
Protein Content ◽  
Corpus Luteum ◽  
Metabolic Activity ◽  
Weight Ratio ◽  
Oestrous Cycle ◽  
Uterine Weight ◽  
In Vitro Protein Synthesis ◽  
Vitro Protein

ABSTRACT Uterine weight, RNA, DNA, protein content, in-vitro rate of protein synthesis, cytosol oestrogen and progesterone receptors were examined after administration of oestradiol to ovariectomized animals and on days 0, 5, 9 and 13 of the non-pregnant cycle and day 13 of pregnancy. In ovariectomized animals, oestradiol increased uterine weight, RNA: DNA and protein: DNA ratios and the concentration of cytosol receptors for oestradiol and progesterone. During the oestrous cycle there was a linear increase in uterine weight and a significant effect of the corpus luteum on the weight of the ipsilateral uterus. Changes in RNA, DNA and protein content between days 0 and 5 were not observed, but between days 5 and 13 RNA: DNA and protein: DNA ratios increased and the DNA: tissue weight ratio decreased. Thus, cellular hypertrophy and/or increased metabolic activity rather than hyperplasia occur over this period, which is coincident with the known rise in plasma progesterone levels. The rate of in-vitro protein synthesis (per unit tissue protein) during the non-pregnant cycle was greatest at day 0. These changes in uterine metabolic activity were associated with alterations in cytosol receptor concentrations for both steroids. Cytosol progesterone receptor concentrations were highest at day 0 after which they declined to a minimum at day 13. Cytosol oestradiol receptor concentrations, however, rose between days 0 and 5 and then declined. Although lutectomy on day 8 of the cycle does not interfere with the development of a histologically normal luteal phase, high peripheral progesterone levels which occur after day 8 in intact animals are associated with major increases in uterine metabolic activity. The unilateral effect of the corpus luteum on uterine weight was associated with a decrease in DNA: g tissue ratio and an increase in rate of in-vitro protein synthesis indicating hypertrophy and/or extracellular accumulation of secreted material as well as enhanced metabolic activity. There was a significant effect of pregnancy on uterine weight at day 13 and this was associated with an increase in DNA content of both uteri. There was a unilateral effect of pregnancy on RNA: DNA ratio and in-vitro rate of protein synthesis, but not on uterine weight. J. Endocr. (1986) 108, 201–210

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