Cytokinin Dehydrogenase activity in primary roots and characterization of primary metabolites from leaves and rootlets of Ricinus Communis

The aim of this study was to determine the activity of cytokinin dehydrogenase (CKX) and to measure other biochemical components in the primary leaves and radicles of castor seedlings (BRS Energia) in the initial phase of growth. The crude protein extract obtained after a 1-h extraction from the root tissues of seedlings showed no detectable CKX enzymatic activity when incubated with the substrate isopentenyl adenine for 1 h. However, after precipitation with ammonium sulfate at 70% saturation, the pellet showed CKX activity. The peroxidase enzyme activity was higher in the leaves than in the radicles. The total and reducing sugar content was 1.5 times higher in the leaves than in the radicles. The amino acid and protein contents were 6.4 and 9.2 times higher in the leaves than in the radicles, respectively.

Purification and characterization of a mannose/N-acetyl-D-glucosamine-specific lectin from the seeds of Platymiscium floribundum Vogel.

Platymiscium floribundum lectin (PFL), a mannose/N-acetyl-D-glucosamine-specific lectin, was isolated from P. floribundum seeds using Sepharose-mannose affinity media chromatography. PFL is a glycoprotein that is a potent agglutinin for rabbit erythrocytes. In addition, PFL is highly stable because it is able to maintain its hemagglutinating activity after exposure to temperatures of up to 60 °C for 1 h and exposure to a wide pH range. The PFL purification process was monitored using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the results showed that the purified lectin consists of a single band with a molecular mass of approximately 29 kDa in either the presence or the absence of a reducing agent. The analysis of purified PFL by electrospray ionization-mass spectrometry showed that most ions had a molecular weight of 27,053 ± 2 Da, and other less abundant ions had similar molecular weights. Gel filtration shows that the lectin exists as a dimer in solution with mass at approximately 65 kDa. Sixteen peptides were sequenced, and as a result, a total of 130 amino acids were identified and resulted in a coverage of approximately 65% of the PFL sequence. The partial sequence of PFL was aligned with sequences of other lectins from evolutionarily related species, and PFL showed considerable similarity to the other lectins.

Native crystal structure of a nitric oxide-releasing lectin from the seeds of Canavalia maritima.

Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide.

Crystallization and preliminary X-ray diffraction analysis of a lectin from Canavalia maritima seeds.

A lectin from Canavalia maritima seeds (ConM) was purified and submitted to crystallization experiments. The best crystals were obtained using the vapour-diffusion method at a constant temperature of 293 K and grew in 7 d. A complete structural data set was collected to 2.1 A resolution using a synchrotron-radiation source. The ConM crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 67.15, b = 70.90, c = 97.37 A. A molecular-replacement search found a solution with a correlation coefficient of 69.2% and an R factor of 42.5%. Crystallographic refinement is under way.