Production of recombinant bovine lactoferrin N-lobe in insect cells and its antimicrobial activity.

Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.

Antimicrobial peptide of korean native goat lactoferrin and identification of the part essential for this activity.

The antimicrobial activity of lactoferrin isolated from Korean native goat (KN goat) milk was studied and its antimicrobial domain was identified using synthetic peptides. Antimicrobial activity was assayed by a micro-method using 96-well microplates and a microplate reader. The amino acid sequence of the antimicrobial domain was suggested to be YQWQRRMRKLGAPSIT and this sequence corresponds to amino acid residues 20 to 35 of KN goat lactoferrin. Five peptides with certain amino acid residues deleted were synthesized in an effort to identify the residues essential for antimicrobial activity and it was found that the part with the sequence RRMRK (24-28) is the region most important for this activity. On the other hand, the conformation of the peptides did not influence the antimicrobial activity.

Blue light activates the plasma membrane H(+)-ATPase by phosphorylation of the C-terminus in stomatal guard cells.

The opening of stomata, which is driven by the accumulation of K(+) salt in guard cells, is induced by blue light (BL). The BL activates the H(+) pump; however, the mechanism by which the perception of BL is transduced into the pump activation remains unknown. We present evidence that the pump is the plasma membrane H(+)-ATPase and that BL activates the H(+)-ATPase via phosphorylation. A pulse of BL (30 s, 100 micromol/m(2)/s) increased ATP hydrolysis by the plasma membrane H(+)-ATPase and H(+) pumping in Vicia guard cell protoplasts with a similar time course. The H(+)-ATPase was phosphorylated reversibly by BL, and the phosphorylation levels paralleled the ATP hydrolytic activity. The phosphorylation occurred exclusively in the C-termini of H(+)-ATPases on both serine and threonine residues in two isoproteins of H(+)-ATPase in guard cells. An endogenous 14-3-3 protein was co-precipitated with H(+)-ATPase, and the recombinant 14-3-3 protein bound to the phosphorylated C-termini of H(+)-ATPases. These findings demonstrate that BL activates the plasma membrane H(+)-ATPase via phosphorylation of the C-terminus by a serine/threonine protein kinase, and that the 14-3-3 protein has a key role in the activation.