Determination of molecular mass, stokes' radius, frictional coefficient and isomer‐type of non‐denatured proteins by time‐dependent pore gradient gel electrophoresis

Abstract

Molecular mass, Stoke's radius, frictional coefficient and isomer-type of non-denatured proteins can be obtained by time-dependent gradient gel electrophoresis by evaluating the resulting data using a two-step mathematical procedure. Provided a histochemical staining procedure is available to locate the position of an enzyme in the gel, crude cell extracts can be used for estimating their molecular size properties. The computation of molecular properties of non-denatured proteins is demonstrated for isozymes of aspartate aminotransferase (EC 2.6.1.1), peroxidase (EC 1.11.1.42) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) from current-year needles of spruce. The resulting data as well as those which were calculated for esterase (EC 3.1.1.1), glutamate dehydrogenase (EC 1.4.1.4), isocitrate dehydrogenase (EC 1.4.1.42), and shikimate dehydrogenase (EC 1.1.1.25) are in accordance with those reported in the literature. The method described may be applied to various scientific areas such as genetics or environmental pollution. It could be shown here that current-year needles of injured spruce (damage class 3) contained two more peroxidase isozymes and one more glucose-6-phosphate dehydrogenase isozyme than those from non-injured trees. These differences may mark two genotypes of spruce of different susceptibilities towards present-day air and soil pollutants.

Changes in shikimate dehydrogenase and the end products of the shikimate pathway, chlorogenic acid and lignins, during the early development of seedlings of Capsicum annuum

“…Staining of shikimate dehydrogenase (Diaz, Bernal & Merino de Caceres, 1994) was by a modification of the method described by Rothe (1988). A solution (30 ml) containing 5-2 mg of shikimic acid, 5 mg of NADP, 10 mg of MTT [3-(4,5-dimethyl-2-il)-2,5-diphenyltetrazolium bromide)], and 1 mg of PMS (phenazine methosulphate), in 0-1 M Tris-HCl buffer, pH 9-0, was used.…”

“…Polyacrylamide gel electrophoresis was performed in 7-7 % (w/v) polyacrylamide slab gels using 0-2 M Tris, 2 mM EDTA and 0-15 M boric acid, pH 8-5, as electrode buffer (Guries & Ledig, 1978). Staining of shikimate dehydrogenase (Diaz, Bernal & Merino de Caceres, 1994) was by a modification of the method described by Rothe (1988). A solution (30 ml) containing 5-2 mg of shikimic acid, 5 mg of NADP, 10 mg of MTT [3-(4,5-dimethyl-2-il)-2,5diphenyltetrazolium bromide)], and 1 mg of PMS (phenazine methosulphate), in 0-1 M Tris-HCl buffer, pH 9-0, was used.…”

Kinetic and electrophoretic characterization of NADP dependent dehydrogenases from root tissues of Norway spruce [Picea abies (L.) Karst.] employing a rapid one-step extraction procedure

“…The number of multiple forms which can be separated by electrophoresis of extracts from spruce needles are reported to be 1-3 for G6PDH (Rothe 1988(Rothe , 1989), 3-6 for 6PGDH (Rothe 1989), and 1-3 for ICADH (Muona et al 1987). The number of multiple forms is influenced by the genotype (Muona et al 1987;Rothe 1989) and by the assay conditions, especially the extraction procedure (H. Thelen and M. Guttenberger, unpublished data).…”

Shikimate dehydrogenase from pepper (Capsicum annuum) seedlings. Purification and properties

“…On the other hand, the inhibition by 2,4-D is particularly interesting. This compound was only tested previously by Rothe (1974), but was not found to have an inhibitory effect on purified pea SKDH. 2,4-D is Physiol.…”

“…The electrode buffer consisted of 0.2 M Tris, 2 mM EDTA and 0.15 M boric acid, pH 8.5 (Guries and Ledig 1978). Gels were stained for SKDH with a modified form of the method of Rothe (1988), using a staining solution consisting of 20 mg of NADP, 21 mg of shikimic acid, 20 mg of MTT and 1.5 mg of PMS in 60 ml of 0.1 M Tris-HCl buffer pH 9.0.…”