Marek Radłowski

Maize pollen enzymes after two-dimensional polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate.

Twelve enzymes from mature pollen grains of maize were separated by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). The separation in the second dimension was both in the presence and absence of sodium dodecyl sulfate (SDS). Ten of the investigated enzymes lost activity after separation in the presence of SDS, but those of esterases and acid phosphatase could be recovered. On the other hand, 2‐D electrophoresis without SDS is suitable for the analysis of maize pollen pectinesterase, malate dehydrogenase, glutamic‐oxalacetic transaminase, diaphorase, superoxide dismutase, and phosphoglucose isomerase. 1‐D PAGE and isoelectric focusing (IEF) are sufficient to analyze glucose‐6‐phosphate dehydrogenase, alcohol dehydrogenase, shikimic dehydrogenase, and glutamate dehydrogenase. The possibility of applying 2‐D electrophoresis for the analysis of enzymes from single stigma and stigma exudate is dicussed.

Effects of interaction between pollen coat eluates and pistil at the molecular level in self-compatible and self-incompatible plants ofLolium multiflorum Lam.

Two-dimensional electrophoresis (2-DE) of soluble proteins and enzymes was performed and specific activities of 5 enzymes (esterase, pectinesterase, acid phosphatase, protease and diaphorase) were determined in stigmas ofLolium multiflorum (Italian ryegrass) treated with self or foreign pollen coat eluates (pc). Also, a low-molecular-weight fraction of the treated self-compatible (SC) and self-incompatible (SI) stigmas was analyzed by high-pressure liquid chromatography (HPLC). The treatment of stigmas with foreign pollen induced the loss of 42% of the control sample proteins in SC plants but only of 5.5% in SI plants. In contrast, the treatment of stigmas with foreign pollen induced the loss of 15% proteins in SC plants and of 29% in SI plants. Specific activities of esterase, pectinesterase and diaphorase were higher in SC than in SI stigmas. The 2-DE enzyme patterns indicated qualitative relationships between the presence of some isoforms of acid phosphatase or protease and the treatment with self or foreign pc in SC and SI stigmas. No changes were observed in HPLC profiles of the low-molecular-weight fraction from SC and SI stigmas treated or not with pc. The presented results revealed different reactions of SC and SI stigmas to the treatment with self or foreign pc. Further investigations may explain if any of the observed reactions represent specific reorientations in the style, facilitating cross- or self-pollination.

Protease activity from maize pollen

Abstract Protease activity from maize pollen has been purified by chromatography on DEAE-Sepharose CL 6B, Sephadex G-100 columns and an HPLC procedure. The final purification factor of the protease was ca 52. The single band after SDS-PAGE showed its M , to be 21 000. The enzyme works at the optimum pH of 4.8 and in the temperature range 45–50°. The isolated protease appears to have a serine group at the active site. No loss in the activity of the enzyme was observed after storage for several months at −18°.

A factor affecting stimulation of aminocylation in plants

The presence of a factor stimulating the reaction of aminoacylation tRNAs was found in the seeds of lupin, with a molecular weight of 950 as estimated by gel filtration. The influence of this factor on the kinetics of the aminoacylation reactions of lupin, Escherichia coli, Bakers yeast and heterogeneous systems was investigated. This factor inhibits the esterification reaction of aminoacyl-tRNA synthetases from bacteria and yeast. Its influence on the optimum pH activity of isoleucyl-tRNA synthetase from lupin was determined.

Differential influence of stimulating factors on cytoplasmic and chloroplastic aminoacyl-tRNA synthetase activity

Abstract The effect of stimulating factors on the activity of aminoacyl- t RNA synthetases from bean leaves and Euglena gracilis cytoplasm and chloroplasts in the reaction of aminoacylation was investigated. Leucyl- t RNA synthetase was chosen as a model system. Stimulating factors isolated from bean, lupin and maize seeds have different effects on the activity of leucyl- t RNA synthetase. In all cases the stimulating factor from maize appeared to be active. The stimulating factors I and II from bean in a homologous system stimulate only the cytoplasmic enzyme. The most interesting systems were taken for further examination of the 32 PP i -ATP exchange reaction, where a weaker effect was found.

Detection of Three DNA‐Binding Proteins from Lupine Mitochondria1

To investigate the mechanism of transcription in plant mitochondria, proteins displaying binding activity to mitochondrial plasmid promoters were studied. We have identified a conserved nonanucleotide motif at the 5′ end of a Lupinus albus mitochondrial plasmid transcript. Three proteins with molecular masses of ca. 25, 30, and 48 kD interacted with ds oligonucleotides, representing different parts of the plasmid promoter. Proteins with molecular masses 25 and 30 kD seem to represent two components of a heterodimer. The DNA‐binding activities of these proteins have been confirmed by electrophoretical mobility shift assay (EMSA), SDS‐PAGE, Southwestern, and UV cross‐linking assays. The specificity of DNA‐binding reaction was examined by competition experiments.