Point Mutations in Dimerization Motifs of the Transmembrane Domain Stabilize Active or Inactive State of the EphA2 Receptor Tyrosine Kinase

The EphA2 receptor tyrosine kinase plays a central role in the regulation of cell adhesion and guidance in many human tissues. The activation of EphA2 occurs after proper dimerization/oligomerization in the plasma membrane, which occurs with the participation of extracellular and cytoplasmic domains. Our study revealed that the isolated transmembrane domain (TMD) of EphA2 embedded into the lipid bicelle dimerized via the heptad repeat motif L⁵³⁵X₃G⁵³⁹X₂A⁵⁴²X₃V⁵⁴⁶X₂L⁵⁴⁹ rather than through the alternative glycine zipper motif A⁵³⁶X₃G⁵⁴⁰X₃G⁵⁴⁴ (typical for TMD dimerization in many proteins). To evaluate the significance of TMD interactions for full-length EphA2, we substituted key residues in the heptad repeat motif (HR variant: G539I, A542I, G553I) or in the glycine zipper motif (GZ variant: G540I, G544I) and expressed YFP-tagged EphA2 (WT, HR, and GZ variants) in HEK293T cells. Confocal microscopy revealed a similar distribution of all EphA2-YFP variants in cells. The expression of EphA2-YFP variants and their kinase activity (phosphorylation of Tyr⁵⁸⁸ and/or Tyr⁵⁹⁴) and ephrin-A3 binding were analyzed with flow cytometry on a single cell basis. Activation of any EphA2 variant is found to occur even without ephrin stimulation when the EphA2 content in cells is sufficiently high. Ephrin-A3 binding is not affected in mutant variants. Mutations in the TMD have a significant effect on EphA2 activity. Both ligand-dependent and ligand-independent activities are enhanced for the HR variant and reduced for the GZ variant compared with the WT. These findings allow us to suggest TMD dimerization switching between the heptad repeat and glycine zipper motifs, corresponding to inactive and active receptor states, respectively, as a mechanism underlying EphA2 signal transduction.     

Ethylene Production and 1-Aminocyclopropane-1-Carboxylic Acid Conjugation in Thermoinhibited Cicer arietinum L. Seeds

The effect of supraoptimal temperatures (30°C, 35°C) on germination and ethylene production of Cicer arietinum (chick-pea) seeds was measured. Compared with a 25°C control, these temperatures inhibited both germination and ethylene production. The effect of supraoptimal temperatures could be alleviated by treating the seeds with ethylene. It was concluded that one effect of high temperature on germination was due to its negative effect on ethylene production. This inhibitory effect of high temperature was due to increased conjugation of 1-aminocyclopropane-1-carboxylic acid to 1-(malonylamino)cyclopropane-1-carboxylic acid and to an inhibition of ethylene-forming enzyme activity.     

Principles of environmental physics

Book Review

Principles of environmental physicsJ.L. Monteith and M.H. Unsworth Second edition. London: Edward Arnold, 1990. xii + 291 pages. £30.00 (hardback), £14.95 (paperback). ISBN 0-7131-2931-X     

Biochemical properties of 1-aminocyclopropane-1-carboxylate N-malonyltransferase activity from early growing embryonic axes of chick-pea (Cicer arietinum L.) seeds

In the present work, certain biochemical characteristics ofthe enzyme 1-aminocyclopropane-1-carboxylate N-malonyltransferase(ACC N-MTase) which is responsible for the malonylation of 1-aminocyclopropane-1-carboxylate(ACC) in chickpea (Cicer arietinum) are described. Phosphatebuffer was the most appropriate buffer with regard to enzymestability and, therefore, ACC N-MTase was extracted, assayedand purified in the presence of this buffer. ACC N-MTase waspartially purified approximately 900-fold from embryonic axesof chick-pea seeds using ammonium sulphate precipitation, hydrophobicinteraction and molecular filtration chromatography. By gelfiltration chromatography on Superose-12, the molecular massof the enzyme was estimated to be 54 4 kDa. ACC N-MTase hadan optimal pH and temperature of 7.5 and 40C, respectively,as well as a K_m for ACC and malonyl-CoA of 400 M and 90 M,respectively. D-Phenylalanine was a competitive inhibitor ofACC N-MTase with respect to ACC (K_i of 720 M), whereas co-enzymeA was a competitive product inhibitor with respect to malonyl-CoA(K_i of 300 M) and a non-competitive inhibitor with respectto ACC (K_i of 600 M). Under optimal assay conditions, ACC N-MTasewas strongly inhibited by (a)divalent [Zn²⁺>Mg²⁺>>Co²⁺>Co²⁺>(NH₄)²⁺>Fe²⁺]and monovalent metal cations (Li⁺>Na⁺>K⁺), without activitybeing detected in the presence of Hg²⁺, and (b) PCMB or mersalicacid, suggesting that sulphydryl group(s) are involved at theactive site of the enzyme. Key words: ACC-N-malonyltransferase, Cicer arietinum, embryonic axes, ethylene, germination, seeds