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Summary A short distance migrating cationic peroxidase from Korean radish seeds (Raphanus sativus) was detected. Cationic peroxidase C s was purified to apparent homogeneity and characterized. The molecular mass of the purified cationic peroxidase C s was estimated to be about 44 kDa on SDS-PAGE. After reconstitution of apoperoxidase C s with protohemin, the absorption spectra revealed a new peak in the Soret region around 400 nm, which is typical in a classical type III peroxidase family. The optimum pH of peroxidase activity for o-dianisidine oxidation was observed at pH 7.0. Kinetic studies revealed that the reconstituted cationic peroxidase C s has K m values of 1.18 mM and of 1.27 mM for o-dianisidine and H2O2, respectively. The cationic peroxidase C s showed the peroxidase activities for native substrates, such as coumaric acid, ferulic acid, and scopoletin. This result suggested that cationic peroxidase Cs plays an important role in plant cell wall formation during seed germination.

Prothrombin, a protein involved in blood coagulation, is a plasma glycoprotein composed of the Gla domain, two adjacent kringle domains, and a serine protease domain. Kringles are triple-disulfide-loop folding domains, which are found in several other blood proteins. In this study, we showed that recombinant human prothrombin kringle-1, -2. and -1-2 (rk-1, -2, -1-2) all have potent anti-angiogenic activities, which inhibit Lewis lung carcinoma (LLC) tumor growth and metastases. Recombinant human prothrombin kringles were expressed by an E. coli expression system and purified to apparent homogeneity from crude E. coli extracts. Purified rk-1, -2, -1-2 migrated with a molecular mass of 14, 19, and 31 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. rk-1, -2, -1-2 exhibited potent inhibitory effects on bFGF-stimulated bovine capillary endothelial cell growth with half-maximal concentrations (ED50) of approximately 41, 55, and 156 nM, respectively. All of the recombinant human prothrombin kringles also inhibited angiogenesis in the chorioallantoic membrane (CAM) of chick embryos at a dose of 20 microg. Systemic administration of rk-1, -2, -1-2 at a dose of 0.5 mg/kg/day suppressed the growth of primary LLC and at dose of 0.5 and 1.0 mg/kg/day inhibited LLC metastases in C57BL6/J mice lungs through their anti-angiogenic effects.

The Korean radish cationic peroxidase promoter (KRPP) and the β -glucuronidase (GUS) gene were fused on the genome of tobacco BY-2 cells via Agrobacterium -mediated transformation method. A fragment of the KRPP comprising nucleotides −471 to +704 relative to the transcriptional initiation site confers constituent expression of GUS in transgenic tobacco BY-2 cells (BYK12). The expressed GUS activity was increased by as much as 40% at 577 nM gibberellic acid (GA 3 ). This increase was time and dose dependent. The abscisic acid (ABA) from 10 to 100 μM slowly decreased the GUS activity and at 100 μM reduced completely the inductive effect of GA 3 on GUS expression. Thus, GA 3 and ABA have antagonistic effects on the GUS expression mediated by the Korean radish cationic peroxidase promoter in transgenic BYK12 cells. Exogenous GA 3 activated the activity of the Korean radish isoperoxidases and also increased the intensity of two bands of Korean radish cationic isoperoxidases, when hypocotyl portions of the Korean radish seedlings grown in a green house for 10 days were examined using starch gel electrophoresis. However, ABA decreased the intensity of the Korean radish cationic isoperoxidase bands and also reduced the inductive effect of GA 3 in seedlings.

The carbohydrate moieties of two anionic peroxidases, termed A1 and A2, and one cationic peroxidase, named C3, from Korean radish (Raphanus sativus) were studied. For profiling of N-glycans, each peroxidase was treated with peptidyl N-glycosidase F and hydrazine. These peroxidases were more susceptible to hydrazine than to peptidyl N-glycosidase F. When these three peroxidases were subjected to trifluoroacetic acid treatment, mannose, fucose and N-acetylglucosamine were released. Two major N-glycans of peroxidase C3 were isolated and treated with several glycohydrolases. Analysis of digested products of the two major N-glycans on polyacrylamide gel suggested that core-fucosylated trimannosylchitobiose may contain a different linkage from the typical α-1,6 of native N-linked oligosaccharide.

The valine sensitive acetolactate synthase (ALS) isozyme from Serratia marcescens ATCC 25419 was purified to homogeneity. Analysis of the native molecular weight of the purified enzyme by the native pore gradient polyacrylamide gel electrophoresis indicated the molecular weight of about 178,000 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the enzyme to be composed of two different types of subunits with molecular weights of 62,000 and 35,000. The molar ratio of the two polypeptides was estimated to be 1, suggesting that native enzyme is composed of two large subunits and two small subunits. The enzyme exhibits homotropic allosterism with pyruvate unlike other enteric ALS isozymes. The specificity ratio R (V[acetohydroxybutyrate]/V[acetolactate] = R.[alpha-ketobutyrate]/pyruvate]), of the enzyme was found to be 0 suggesting that the Serratia ALS has very high specificity for pyruvate. The pH optimum was around 7.5, and the enzyme was stable at 50 degrees C for 30 min. The pI value for the purified enzyme was 5.2. The concentration of branched chain amino acids for 50% inhibition of the enzyme was 0.1 mM for valine, and 1 mM for leucine and isoleucine, respectively.

Pro-inflammatory mediators, such as nitric oxide (NO), prostaglandin E{sub 2} (PGE{sub 2}), and several cytokines (tumor necrosis factor (TNF)-{alpha}, interleukin (IL)-1{beta}, and IL-6) are responsible for central nervous system (CNS) injuries that include ischemia, Alzheimer's disease, and neural death. Inhibition of these pro-inflammatory mediators would be an effective therapy to reduce the progression of neurodegenerative diseases. In this study, we examined the anti-inflammatory effects of a human prothrombin fragment-2-derived peptide, NSA9 (NSAVQLVEN), on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-activated brain microglia. NSA9 significantly inhibited the release of NO, PGE{sub 2}, and pro-inflammatory cytokines in a dose-dependent manner. Furthermore, NSA9 reduced the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 mRNA and protein, which control the production of NO and PGE{sub 2}, respectively. Moreover, NSA9 suppressed the LPS-induced nuclear translocation and activation of nuclear factor-{kappa}B (NF-{kappa}B). These results suggest that NSA9 strongly inhibits the pro-inflammatory responses of microglia through the modulation of NF-{kappa}B activity.

Prothrombin is a plasma glycoprotein involved in blood coagulation and, as we have previously reported, prothrombin kringles inhibit BCE (bovine capillary endothelial) cell proliferation. To reveal the mechanism, we investigated the influence of rk-2 (recombinant human prothrombin kringle-2) on the BCE cell cycle progression and ROS (reactive oxygen species) generation using FACS (fluorescence-activated cell sorter) analysis. Cell cycle analysis showed a decrease of G(1) phase cells in cells treated with bFGF (basic fibroblast growth factor) and an increase in cells treated with rk-2, as compared with the control cells. But, the portion of the S phase was reversed. In Western blot analysis, bFGF induced cytoplasmic translocation of p21(Waf1/Cip1) and p27(Kip1) and phosphorylation of p27(Kip1) but rk-2 treatment inhibited translocation of p21(Waf1/Cip1) and p27(Kip1) from nucleus to cytoplasm and phosphorylation of p27(Kip1). Also, rk-2 induced up-regulation of p53 and nuclear p21(Waf1/Cip1) and inhibited the cyclin D1/CDK4 (cyclin-dependent kinase 4) complex. The ROS level of rk-2-treated BCE cells was increased 2-fold when compared with the control, but treatment with NAC (N-Acetyl-L: -cysteine), an anti-oxidant, decreased ROS generation about 55% as compared with the rk-2 treatment. NAC treatment also restored cell cycle progression inhibited by rk-2 and down-regulated p53 and nuclear p21(Waf1/Cip1) expression induced by rk-2.These data suggest that rk-2 induces the BCE cell cycle arrest at G(0)-G(1) phase through inhibition of the cyclin D1/CDK4 complex caused by increase of ROS generation and nuclear cyclin-dependent kinase inhibitors.

Studies of the regulation of the activity of the Korean radish cationic peroxidase ( KRCP ) promoter during dedifferentiation and redifferentiation are reported here. Histochemical staining with 5-bromo-4-chloro-indolyl glucuronide (X-gluc) showed that only dedifferentiated marginal cells of leaf discs of the transgenic plants, but not of the interior region, were stained blue, as leaf discs were incubated on dedifferentiation-inducing medium from 5 days after callus induction (DACI). The levels of cationic peroxidase activity and of KRCP transcripts in Korean radish seedlings ( Raphanus sativus L. F1 Handsome Fall) were also upregulated by a low ratio of cytokinin to auxin, but not by high concentrations of cytokinin. To identify important cis -regulatory regions controlling callus-specific expression, a series of 5′ promoter deletions was carried out with KRCP::GUS gene fusion systems. The data suggest that at least two positively regulatory regions are involved in the KRCP::GUS expression during dedifferentiation induced by a low ratio of cytokinin to auxin: one from –471 to –242 and another from –241 to +196. GUS expression, however, was quickly decreased to a basal level during regeneration of root and shoot. Thus, the downstream region between +197 and +698 seems to be enough to suppress GUS expression of all constructs during regeneration. We further show that the 142-bp fragment (–471 to –328) has at least one cis -element to bind to the nuclear proteins from Korean radish seedlings induced by dedifferentiation.

Two cationic isoperoxidases (designated C1 and C3) and four anionic isoperoxidases (designated A1, A2, A3n and A3) from Korean radish (Raphanus sativus L.) root have been purified to apparent homogeneity, and some of their enzymatic properties were characterized. All six isoperoxidases are glycoproteins composed of a single polypeptide chain. The Mrs Of C1, C3, A1 and A2 were ca 44 000, while anionic isoperoxidase A3n and A3 have Mrs of 31 000 and 50 000, respectively. Deglycosylated A2 and C3 by trifluoromethanesulphonic acid treatment showed Mrs of 37 000 and 40 000, respectively, suggesting that the carbohydrate contents for these isoenzymes are 14 and 9%, respectively. Relative amino acid compositions of four anionic isoperoxidases (designated A1, A2, A3n and A3) and one cationic isoperoxidase C3 were determined. N-Terminal amino acid sequences were determined for A1, A3n and C3, while A2 was found to have a blocked amino terminal residue. Kinetic studies with respect to various synthetic and naturally occurring substrates were also investigated.