Saul Zalik

Two dimensional acrylamide gel electrophoresis of wheat leaf cytoplasmic and chloroplast ribosomal proteins

Cytoplasmic and chloroplast ribosomes from the same plant species have been shown by acrylamide gel electrophoresis to differ greatly in protein composition [ l-61 . The limited resolving power of single-dimension electrophoresis did not, however, allow determination of the total number of proteins in each of the ribosome species. Two-dimensional acrylamide gel electrophoresis, which has been used to separate the ribosoma1 proteins of E. culi [7,8] , has recently been used to determine the approximate number of proteins in ribosomes of higher animals [9, lo]. So far similar analysis of plant ribosomal proteins have not been published. This paper reports the separation of the ribosomal proteins from cytoplasmic and chloroplast ribosomes of wheat leaf by two-dimensional acrylamide gel electrophoresis. The cytoplasmic and chloroplast rib@ some proteins were almost all different, with the cytoplasmic ribosomes containing more proteins. Both chloroplast 70 S and cytoplasmic 80 S wheat ribosomes contained more proteins than have been reported in E. coli 70 S ribosomes.

Gas chromatography of indole auxins

Abstract For effective separation of indole auxins by gas chromatography it was necessary to divide them into acidic and neutral indoles. The neutral indoles were volatile enough to be chromatographed directly, but the acidic indoles needed to be esterified in order to be chromatographed. Most indole acids were successfully esterified with diazomethane or by BF3 catalysis, but indolelactic acid could be esterified by diazomethane only. Esterification by diazomethane was preferred because this substance is a powerful esterifying reagent. Since the diazomethane was prepared as an ether solution, it had the additional advantage that after esterification the ether and excess diazomethane could be easily removed. The neutral indoles chromatographed well with a silicone substrate, SE-30 or SE-52, though some overlapping occurred with a 6 ft column. Excellent separations of indole esters were also obtained with the silicones, particularly SE-52, even on a 5 ft column. Other substrates which were effective to a certain extent included neopentyl glycol succinate and QF-1. Attempts to prepare acetal derivatives of indole aldehydes met with little success and it may be preferable to chromatograph indole aldehydes directly. The overlapping of neutral indole compounds was overcome by increasing the length of column. Higher efficiency was obtained and the longer column did not appear to destroy the compounds. Proper conditioning of the columns was necessary, especially for the more labile indoles, which could not be detected unless the column had been aged for a few days. Most efficient separation was obtained when the oven temperature was about 205°C, with the injector block at least 50° above that of the oven. Some prepurification of plant extracts was necessary before they could be applied to the gas chromatograph. However, sensitivity limitations made it difficult to detect the small amounts present in individual plants or plant parts. For larger quantities of material gas chromatography might be employed in the initial separation steps followed by spectrophotometric, or fluorometric analysis, or bioassay.

Magnesium replacement by polyamines in higher plant cell-free polyphenylalanine synthesis

Abstract The characteristics and specific requirements for the formation of polyphenylalanine from Phe-[ 14 C] in a barley ribosome cell-free incorporation system were detailed. The polyamines spermine, spermidine and putrescine, and the inorganic cations Ca 2+ , Ba 2+ and Mn 2+ demonstrated different capabilities for replacing the Mg 2+ requirement in the incorporation system. Spermine was extremely efficient in this respect, followed by spermidine; all of the cations tested showed discrete concentration ranges of effectiveness. The data supported the suggestion that, at least to a certain extent, the cation requirement for protein syntheis may be non-specific.

Physicochemical studies of bean and wheat chloroplast structural protein

Abstract Chloroplasts from wheat and bean seedlings were isolated, and after removing lipid and pigments the protein was solubilized. (NH4)2SO4 was used to precipitate the structural protein fraction. Sedimentation velocity studies showed homogeneous profiles in both species with s values lower than reported for spinach. The amino acid composition of this protein fraction from bean and wheat was similar. Optical rotatory dispersion measurements indicated the presence of α-helix in both. Gel electrophoresis showed that with respect to charge the structural protein fraction of wheat and bean chloroplasts was different and not homogeneous.

Chloroplast and cytoplasmic polysomes and ribosomal RNA from wheat

Abstract Ribosomal fractions from the chloroplasts and cytoplasm of Triticum durum and T. vulgare contained monomers with sedimentation coefficients of 66 and 80 S respectively. There were 4–5 other rapidly sedimenting particles which comprised more than 65 % of the material and were identified as polysomes. The differences in the S values of the monomers from the chloroplast and cytoplasm were reflected in their dimer, trimer, tetramer and pentamer series. The chloroplast and cytoplasmic ribosomal RNA of both species was made up of two components having sedimentation coefficients of 16 and 23 S and 17 and 25 S respectively. The faster sedimenting component of both types of ribosomal RNA had a molecular weight two times that of the slower sedimenting component.

Amino acid incorporation by cytoplasmic polysomes from wheat leaves

Abstract Sucrose gradient and analytical ultracentrifugation of cytoplasmic polysomes from 4-day-old seedlings revealed mono-, di-, tri-, tetra- and traces of pentasomes, whereas preparations from 7-day-old seedlings had only mono-, di- and traces of trisomes. Stability of the polysomes was related to Mg2+ concentration. In presence of cofactors, energy-donor and regenerating components, and supernatant factor, polysomes incorporated labeled phenylalanine. The incorporation was markedly decreased by the addition of pancreatic ribonuclease. Single ribosomes obtained by ribonuclease digestion of polysomes had a negligible activity which could be increased by poly U, but the activity was still less than for polysomes. In comparison with similar preparations from chloroplasts, the cytoplasmic polysomes showed much less amino acid incorporation even in the presence of poly U.

Ribulose-1,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase activity in barley and its virescens mutant

Abstract The most pronounced difference in carboxylase activities between Gateway barley and its virescens mutant occurred in seedlings grown in the light for 4 days. At this stage seedlings of Gateway had 11 times more ribulose-1,5-bisphosphate carboxylase and 18 times higher activity per gram fresh weight than the mutant. Although phosphoenolpyruvate carboxylase represented only 8% of the sum of the two carboxylase activities for the normal it accounted for 70% in the mutant. It represented 17% and 30% of the activity in 8-day dark-grown seedlings of the normal and mutant respectively but accounted for only 3% and 11% after 8 days in light. The high activity of phosphoenolpyruvate carboxylase in young seedlings of the mutant grown in light suggests an adaptation within the mutant during the period when the amount of RuBP-carboxylase is low.

Comparison of cytoplasmic ribosomal proteins of gateway barley and its mutant

Abstract The proteins of the cytoplasmic ribosomes isolated from dry embryos of Gateway barley and its virescens mutant were compared by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The monosomes of both the lines gave similar patterns with 60 basic proteins. Upon dissociation of the monosomes, for the mutant, the basic proteins of the large subunits migrated more slowly than those of the normal and lacked three proteins but had three additional spots. Also, the proteins of the small subunits differed. The mutant lacked three of the proteins present in the normal but had three additional spots. Therefore, the large and small subunits contained a total of 34 and 41 basic proteins, respectively, in both the lines. There were several spots with identical electrophoretic mobilities in the small and large subunits of these two lines.