Sławomir Bartkowiak

The purification of aminoacyl-tRNA synthetases by affinity chromatography

Abstract The method of the purification of aminoacyl-tRNA synthetases by affinity chromatography is described on the basis of complex formation between the enzyme and aminoacyl-tRNA attached to bromoacetylamidobutyl-Sepharose. The purification of Escherichia coli isoleucyl-tRNA synthetase is given as an example.

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.

t-RNA-nucleotidyltransferase activity in Lupinus luteus seeds

Abstract t -RNA-nucleotidyltransferase activity was detected in Lupinus luteus seed.The enzyme was partly purified, and some of its properties are described.

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.