Purification and characterization of two cysteine proteases from germinated barley seeds

Different stages during barley [Hordium vulgare cv. Giza 126] gennination showed endoproteolytic activity. The changes of endoproteolytic activity and protein were detected with different stages during germination. In presence of gibberellic acid [GA 3], no changes could be detected in the profile of protease activity except on day 4 where the pinteolytic activity was increased to a 1.25 fold over its value in absence of GA3. During purification of H. vulgare protease, ion exchange chromatography on DEAE-cellulose led to five separate forms [from P1 to P5]. Proteases P3 and PS with the highest specific activities were pure after chromatography on Sephacryl 5-200. The molecular weights of P3 and PS were 25,000 and 30,000, respectively. By SDS-PAGE, the P3 and PS were composed of a single band of molecular weights of 24,000 and 30,000, respectively, indicating that the two proteases are brobably monomers. Barley P3 and P5 exhibited pH optima at 3.5 and 4.0, respectively. Km values for barley P3 and P5 were estimated to be 5.4 and 4.2 mg azocaseine/ml, respectively. Varying protease activity was detected for P3 and P5 when supplied with various proteins as substrates. P3 and P5 were found to have temperature optima at 30 and 40°C, respectively. P3 and P5 were stable up to 40°C and retained 45% and 35% of their activities at 60°C, respectively. The stability of P5 toward metal ions inactivation was considerably higher than the stability of P3. Only inhibitors of cysteine proteases significantly inhibited P3 and P5, while DTT as a reducing agent enhanced P3 and P5 activities. These results indicated unequivocally that P3 and P5 are cysteine proteases. With the purification of barley P3 and PS, the physiological roles of these specific proteases in germinating barley and the regulation of their expression by GA 3 can be addressed

Cloning and sequencing of the nitrogenase structural genes nifHDK of Herbaspirillum seropedicae

The nitrogenase structural genes (nifHDK) of the endophytic diazotroph Herbaspirillum seropedicae were isolated from a genomic bank by plate hybridization. Sequence analysis of the DNA showed a consensus promoter region upstream from the nifH gene containing a -24/-12 type promoter together with NifA- and integration host factor (IHF)- binding sites. The derived protein sequences of NifH, NifD and NifK contained conserved cysteine residues for binding iron-sulfur clusters and the iron-molybdenum cofactor. These protein sequences showed the strongest similarities to the nifHDK gene products of the symbiotic diazotroph Bradyrhizobium japonicum (93.5 per cent, 91.3 per cent and 83.3 per cent, respectively), the plant-associated diazotrophAzospirillum brasilense (90.0 per cent, 83.7 per cent and 75.1 per cent, respectively) and to Thiobacillus ferrooxidans (91.0 per cent, 83.4 per cent and 81.1 per cent, respectively) of the same phylogenetic group of the protobacteria.