No abstract

A casein kinase Il-type protein kinase has been purified from the cytosolic fraction of etiolated pea (Pisum sativum L.) plumules to about 90% purity as judged from Coomassie blue stained sodium dodecyl sulfate-polyacrylamide gels. This kinase has a tetrameric aa'a2 structure with a native molecular mass of 150 kD, and subunit molecular masses of 41 and 40 kD for the two catalytic subunits (a and a') and 35 kD for the putative regulatory subunit (8). Casein and phosvitin can be used as artificial substrates for this kinase.Both serine and threonine residues were phosphorylated when mixed casein, j3-casein, or phosvitin were used as the substrate, whereas only serine was phosphorylated if a-casein or histone 111-S was the substrate. l h e kinase activity was stimulated 130% by 0.5 mM spermine (the concentration required for 50% of maximal enzyme activity [ASO] = 0.1 mM) and 80% by 2.5 mM spermidine (As0 = 0.4 mM), whereas putrescine and cadaverine had no effect.l h e kinase was very sensitive to inhibition by heparin (concentration for 50% inhibition = 0.025 pg/mL). I n contrast to most other casein kinase 11-type protein kinases, this preparation was inhibited by K+ and Na+, with ls0 values of 75 and 65 mM, respectively. Pretreatment of the purified kinase preparation i n vitro with alkaline phosphatase caused a 5-fold decrease in its activity. Additionally, this kinase also lost its activity when its 8 subunit was autophosphorylated in the absence of substrate. These results suggest that the activity of this casein kinase II protein kinase may be regulated by the phosphorylation state of two different sites in its multimeric structure.

The changes in isozyme profiles of catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and 81utathione reductase (GR) during severe deactivation of total CAT activity by aminotriazole (AT) treatment were investigated in the leaves of Arabidopsis thaliana (Columbia ecotype) in relation to H202-mediated oxidative stress. In spite of striking deactivation of total CAT activity by 0.1 mM AT, there were no significant differences in H202 levels or total leaf soluble protein contents including a Rubisco in both the control and AT-treated leaves. On the other hand, one specific protein band (molecular mass, 66 kD) was observed on the SDS-gel from leaf soluble proteins whose staining intensity was strikingly enhanced by AT treatment for 6 h. However, this band disappeared at 12 h. In the native-gel assays of CAT, POD, APX and GR isozymes, AT remarkably inhibited the expression of the CAT1 isozyme with no effects on CAT2 and CAT3, and generally had no effect on POD isozyme profiles. However, AT stimulated the intensity of activities of pre-existing APX1 and GR1 isozymes. In particular, it induced a new synthesis of one GR isozyme. Therefore, these results collectively suggest that a striking deactivation of total CAT activity by AT in A. thaliana leaves largely results from the suppression of CAT1 isozyme, and that APXl, GR1, and a newly synthesized GR isozyme could complement the role of CAT1 to metabolize H202 into non-toxic water.

This study aimed to assess the skin-related anti-photoaging activities of the 2 epimeric forms of protopanaxadiol (PPD), 20(S)-PPD and 20(R)-PPD, in cultured human keratinocytes (HaCaT cells). The anti-photoaging activity was evaluated by analyzing the levels of reactive oxygen species (ROS) and matrix metalloproteinases (MMPs), as well as cell viability for HaCaT cells under UV-B irradiation. The activities for MMP-2 and -1 in conditioned medium were determined using gelatin zymography, and MMP-2 protein in the conditioned medium was detected using Western blot analysis. 20(S)-PPD, but not 20(R)-PPD, suppressed UV-B-induced ROS elevation. Neither of the epimers, at the concentrations used, exhibited cytotoxicity, irrespective of UV-B irradiation. 20(S)-PPD, but not 20(R)-PPD, exhibited an inhibitory effect on UV-B-induced MMP-2 activity and expression in HaCaT cells. In brief, only 20(S)-PPD, a major metabolic product of PPD-type ginsenosides, inhibits UV-B-induced ROS and MMP-2 elevation, implying its stereospecific anti-photoaging activity on the skin.