Resolution Two-dimensional Polyacrylamide Gel Electrophoresis Research Articles

Aldosterone-induced proteins in toad urinary bladders. Identification and characterization using two-dimensional polyacrylamide gel electrophoresis.

Aldosterone-stimulated Na+ transport is mediated by new protein synthesis, but the identification of specific aldosterone-induced proteins (AIPs) has proven difficult and the cellular function of such proteins is unknown. Using high resolution two-dimensional polyacrylamide gel electrophoresis and autoradiography we have identified AIPs of similar isoelectric points (5.8 to 6.4) and molecular weights (70,000 to 80,000) in membrane-rich and cytosolic subcellular fractions of epithelial cells derived from single toad urinary bladders. The ability of actinomycin D to inhibit both AIP synthesis and aldosterone-induced Na+ transport is consistent with a role for these proteins in the natriferic action of aldosterone. In addition, since non-natriferic concentrations of cortisol did not induce similar proteins, AIP synthesis appears to be mineralocorticoid-specific. The relationship of AIP synthesis to Na+ transport was also studied. Since amiloride, which blocks Na+ transport in high resistance epithelia, did not affect the synthesis of these proteins, Na+ transport is not required for their synthesis. In addition, similar proteins were not induced when Na+ transport was stimulated by antidiuretic hormone and theophylline. Consequently, AIP synthesis is not merely a nonspecific consequence of the cellular metabolic changes associated with Na+ transport.

Protein Patterns of Early Mouse Embryos During Development

Using high resolution two-dimensional polyacrylamide gel electrophoresis, the major protein species (labeled in vitro with 35S-methionine) of mouse embryos were examined starting with the unfertilized egg up to the blastocyst stage (fourth day of development). The analysis was then continued using in vitro culture techniques up to the 10th equivalent gestation day. At all periods of development distinct protein changes could be seen. However, major alterations in the protein synthesis pattern were noted between the 2nd and 3rd day in vivo and around the 8th equivalent gestation day in vitro. A complete series of gels is presented such that proteins can be easily identified in terms of their molecular weights and isoelectric points.

Additional platelet membrane glycoprotein abnormalities in glanzmann's thrombasthenia: A comparison with normals by high resolution two-dimensional polyacrylamide gel electrophoresis

The platelets from sixteen patients with Glanzmann's thrombasthenia have been analysed by isoelectric focusing and gradient sodium dodecyl sulphate-discontinuous polyacrylamide gel electrophoresis in a two-dimensional technique and compared with normals. As well as the previously described absence or deficiency of glycoproteins IIb and IIIa and a marked reduction in fibrinogen levels there were changes in the isoelectric point of glycoproteins Ib and IIIb and increased periodic acid-Schiff's reagent staining of some other glycoproteins. These differences imply that in Glanzmann's thrombasthenia there is more general perturbation of platelet membrane glycoproteins than previously suspected.

Basic Mechanisms of Ovarian Function: Germ Cells

Recent work is reviewed on several molecular aspects of gene expression during oocyte maturation and early development. Several analyses have been carried out on protein synthesis patterns using the high resolution two-dimensional polyacrylamide gel electrophoresis technique. The protein pattern of some 400–500 spots in sea urchin oocytes before and just after fertilization are quite similar. By gastrula, major stage specific protein changes have been noted. In a similar study major protein changes are noted during the meiotic maturation process in mammals. With the use of the single-copy DNA hybridization technique, the quantitative levels of rare mRNA sequence expression have been determined during oogenesis and early development in the sea urchin model. The mature oocyte sequence set of some 37 × 106 nucleotides of information (or potentially 18,500 different proteins) is synthesized during oogenesis and slowly utilized during development. The sea urchin gastrula mRNA is predominantly synthesized by the embryo genome; however, essentially all those gastrula mRNA sequences can also be found in the mature oocyte (maternal) set. It is proposed by Hough-Evans et al. that this maternal sequence set, which is made during oogenesis and both utilized (translated in 10,000–20,000 proteins), and in part, continually synthesized by all stages of the embryo, plays a critical role in the morphogenic formation of a sea urchin embryo.

Basic mechanisms of ovarian function: germ cells

Recent work is reviewed on several molecular aspects of gene expression during oocyte maturation and early development. Several analyses have been carried out on protein synthesis patterns using the high resolution two-dimensional polyacrylamide gel electrophoresis technique. The protein pattern of some 400-500 spots in sea urchin oocytes before and just after fertilization are quite similar. By gastrula, major stage specific protein changes have been noted. In a similar study major protein changes are noted during the meiotic maturation process in mammals. With the use of the single-copy DNA hybridization technique, the quantitative levels of rare mRNA sequence expression have been determined during oogenesis and early development in the sea urchin model. The mature oocyte sequence set of some 37 x 10(6) nucleotides of information (or potentially 18,500 different proteins) is synthesized during oogenesis and slowly utilized during development. The sea urchin gastrula mRNA is predominantly synthesized by the embryo genome; however, essentially all those gastrula mRNA sequences can also be found in the mature oocyte (maternal) set. It is proposed by Hough-Evans et al. that this maternal sequence set, which is made during oogenesis and both utilized (translated in 10,000-20,000 proteins), and in part, continually synthesized by all stages of the embryo, plays a critical role in the morphogenic formation of a sea urchin embryo.

Two-dimensional gel patterns of protein synthesis before and after fertilization of sea urchin eggs

Eggs and embryos of the sea urchin Lytechinus pictus were labeled with [ 35S]methionine. Aqueous extracts of protein were prepared and analyzed by a high resolution two-dimensional polyacrylamide gel electrophoresis system described recently by O'Farrell utilizing isoelectric focusing and sodium dodecyl sulfate electrophoresis. Out of about 400 distinctly resolved newly synthesized proteins, all but a few detectable in the zygote were being synthesized in the egg. Thus, the activation of translation of stored maternal messenger RNA following fertilization is due to a quantitative rather than qualitative change in the population of messenger RNA available for translation. The patterns of protein synthesis change only slightly during cleavage, but major differences appear by the beginning of gastrulation.

Proteins of Friend leukemia cells. Comparison of hemoglobin-synthesizing and noninduced populations.

Proteins of Friend leukemia cells induced to form large amounts of hemoglobin by dimethylsulfoxide treatment were compared with proteins from noninduced cells by high resolution two-dimensional polyacrylamide gel electrophoresis. Approximately 98% of more than 500 proteins separated by this technique were qualitatively and quantitatively the same in both cell populations. Changes representing more than 50% of the control cell amount were detected in six non-histone chromosomal proteins, two nucleoplasmic proteins, and three cytoplasmic proteins. It is concluded that dimethylsulfoxide induces an extremely specific pattern of erythroid differentiation in these cells, which should be susceptible to detailed analysis. Comparison of protein patterns from Friend leukemia cells and HeLa cells revealed electrophoretic identity of approximately 20% of cytoplasmic proteins and 50% of non-histone chromosomal proteins.