A novel subtilase from common bean leaves

AbstractWe describe the isolation of a protease from common bean leaves grown in the field. On the basis of its biochemical properties it was classified as serine proteinase belonging to the subtilisin clan. Isoelectric focusing resulted in a single band at pH 4.6, and SDS–PAGE in a single band corresponding to Mr 72 kDa. The proteinase activity is maximal at pH 9.9 and shows high stability in the alkaline region. The relative activities of the proteinase for eight different synthetic substrates were determined. The requirement for Arg in the P1 position appeared obligatory. kcat/Km values indicate that, for highest catalytic efficiency, a basic amino acid is also required in the P2 position, presenting a motif typical of the cleavage site for the kexin family of subtilases. The sequence of the 17 N-terminal amino acids of this proteinase shows similarity to those of other plant subtilases, sharing the highest number of identical amino acids with proteinase C1 from soybean seedling cotyledons and a cucumisin-like proteinase from white gourd (Benincasa hispida)

Uber den Einfluss einiger aktiven Substanzen auf spezifische und unspezifische Cholinesterase

Es wurde ein Unterschied in der Wirkung von Atropin und von d-Tubocurarin auf die Hydrolyse von ACh und BCh durch die Serum-ChE festgestellt. Atropin inhibiert die Hydrolyse von ACh in allen Konzentrationen, die grosser als 1Q-4,5M sind, wahrend die Hydrolyse von BCh durch dieselbe Substanz im Konzentrationsbereiche von 10 - 4, 5 M bis 10- 2,5 M aktiviert wird. Durch d-Tubocurarin werden beide Hydrolysen zurilckgedrangt, doch die Hydrolyse von BCh weniger. Beide Aktivsubstanzen inhibieren viel weniger die ChE der Menschenerythrocyten als die Serum ChE

Purification and Characterization of Two Cysteine Proteinases from Potato Leaves and the Mode of Their Inhibition with Endogenous Inhibitors

Two cysteine proteinases, PLCP-1 and PLCP-2, were purified from potato leaves (Solanum tuberosum L.). SDS-PAGE of PLCP-2 gave a single band with Mr of 23400 and PLCP-1 gave a doublet within the same Mr range. Isoelectric focusing of PLCP-2 revealed two bands with pI = 4.6 and 4.9. Both enzymes demonstrate pH optima and maximum stability at slightly acidic pH, and strong inhibition by L-trans-epoxysuccionylleucylamido(4-guanidino)butane (E-64), cystatin C and stefin A, enabling them to be assigned to the papain family of cysteine proteinases. PLCP-1 and PLCP-2 were inhibited by Kunitz-type cysteine proteinase inhibitors (PCPIs) and multicystatin, all isolated from potato tubers. Among PCPIs, the strongest inhibitors were PCPI 9.4, with Ki in the 10–8 M range, and PCPI 8.3 in the 10–7 M range, while Kis for PCPI 6.6 and PCPI 5.4 were in the 10–6 M range. Multicystatin was the most potent inhibitor of both proteinases with Ki of about 0.5 nmol dm–3. The stoichiometry of inhibition of both proteinases with multicystatin was 1:4 (inhibitor : proteinase). The possible physiological significance of these endogenous inhibitors, also present in potato leaves, is discussed. PLCP-1 and PLCP-2 could not be differentiated in terms of their Kis