Feasibility of Sijunzi Tang (Chinese medicine) to enhance protein disulfide isomerase activities for reactivating malate dehydrogenase deactivated by polycyclic aromatic hydrocarbons.

The objective of this research is to investigate the enzymatic activities between protein disulfide isomerase (PDI) found in animals and plants and the properties found in a commonly used Chinese medicine called Sijunzi Tang. During the investigation, PDI, which is a monomer with a molecular mass of 57.0 kDa, was used to reactivate malate dehydrogenase (MDH). However, with the interference of polycyclic aromatic hydrocarbons (PAHs), evidence indicates that such chemicals are carcinogenic, mutagenic, and toxic to humans. The enzymatic activity of PDI found in animal's liver and plant was 1657 folds of purification; 0.284 unit/mg of enzyme activity, and 5694.4 folds of purification; 1.00 unit/mg of enzyme activity, respectively. PDI extracted in treated animal and plant tissue revealed 2.40% and 80.44% of regaining MDH enzymatic activity, respectively. Although in its initial phase of investigation, it is assumed that the properties found in Sijunzi Tang can help regain enzymatic activity in those affected by xenobiotic substances, thus, making it a potential ingredient in assisting with PDI functions.

Immunohistochemical studies of brain pyridoxine-5'-phosphate oxidase.

A total of six hybridoma cell lines, which produce monoclonal antibodies (mAbs) against the sheep brain pyridoxine-5'-phosphate oxidase (PNP oxidase), were established. Isotype analysis revealed that all antibodies corresponded to the IgG 2B kappa subclass. Immunoblotting with various tissue homogenates indicated that all the mAbs specifically recognize a single protein band of 30 kDa. They also appear to be extensively cross-reactive among different mammalian and avian sources. These results demonstrated that only one type of immunologically similar PNP oxidase is present in all of the mammalian tissues tested. When the purified PNP oxidase was incubated with the mAbs, the enzyme activity was inhibited up to a maximum of 81%. Furthermore, these antibodies were successfully applied in immunohistochemistry in order to detect PNP oxidase in various regions of rat brain tissues. The immunoreactive neurons in PNP oxidase were found in cerebellar cortex, hippocampus, amygdala, paraventricular nucleus, cerebral cortex and ependyma. This result suggests that PNP oxidase may play an important role in the neuronal metabolism.

Identification of the heterogeneous nuclear ribonucleoprotein A2/B1 as the antigen for the gastrointestinal cancer specific monoclonal antibody MG7.

MG7 is an early gastrointestinal cancer specific monoclonal antibody. It can detect gastric cancer with high sensitivity and specificity. However, the target antigen for MG7 has not been identified. Western blot analysis revealed that the MG7 antibody reproducibly recognized two approximately 35 kDa proteins in the total cell lysates of human gastric carcinoma cell lines KATO III and MKN-45. Using a proteomic approach, we identified these MG7 immunoreactive proteins as the human heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1). Western blot analysis of nuclear and cytosolic fraction of KATO III cells using either MG7 or hnRNP A2/B1 antibodies confirmed that the target antigen is located exclusively in the nucleus. With the use of archival samples, we also found that the level of hnRNP A2/B1 protein was increased in gastric cancer tissues (4 out of 5 patients), when compared to their corresponding matching normal stomach tissue.

Cloning and characterization of Arabidopsis thaliana pyridoxal kinase.

Pyridoxal kinase (PK; EC 2.7.1.35), a key enzyme in vitamin B(6) metabolism, was cloned from Arabidopsis thaliana (L.) Heynh. and characterized. The amino acid sequence of the A. thaliana PK was found to be similar to the mammalian enzyme, with a homology of more than 40%. Characterization studies showed that the kinase is a dimeric molecule consisting of two identical subunits, each subunit having a molecular mass of approximately 35 kDa. The enzyme exhibited maximal activity at pH 6.0. Similar to the mammalian enzyme, the enzyme from A. thaliana preferred Zn(2+) instead of the commonly used Mg(2+) as the divalent cation for catalysis. Under optimal conditions, the V(max) of the enzyme was 604 nmol pyridoxal 5'-phosphate (PLP) mg(-1) min(-1), and the K(m) values for pyridoxal and ATP were 688 micro M and 98 micro M, respectively. Examination of levels of enzyme expression showed that leaves, stems, roots and flowers can generate PLP independently at similar levels. Furthermore, expression of the PK gene in A. thaliana seeds was found to start 60 h after imbibition. Results from the present study suggest that plant tissues depend on PK for the production of PLP.

Conformational changes in the reaction of pyridoxal kinase.

To understand the processes involved in the catalytic mechanism of pyridoxal kinase (PLK),1 we determined the crystal structures of PLK.AMP-PCP-pyridoxamine, PLK.ADP.PLP, and PLK.ADP complexes. Comparisons of these structures have revealed that PLK exhibits different conformations during its catalytic process. After the binding of AMP-PCP (an analogue that replaced ATP) and pyridoxamine to PLK, this enzyme retains a conformation similar to that of the PLK.ATP complex. The distance between the reacting groups of the two substrates is 5.8 A apart, indicating that the position of ATP is not favorable to spontaneous transfer of its phosphate group. However, the structure of PLK.ADP.PLP complex exhibited significant changes in both the conformation of the enzyme and the location of the ligands at the active site. Therefore, it appears that after binding of both substrates, the enzyme-substrate complex requires changes in the protein structure to enable the transfer of the phosphate group from ATP to vitamin B(6). Furthermore, a conformation of the enzyme-substrate complex before the transition state of the enzymatic reaction was also hypothesized.

Crystal structure of brain pyridoxal kinase, a novel member of the ribokinase superfamily.

The three-dimensional structures of brain pyridoxal kinase and its complex with the nucleotide ATP have been elucidated in the dimeric form at 2.1 and 2.6 A, respectively. Results have shown that pyridoxal kinase, as an enzyme obeying random sequential kinetics in catalysis, does not possess a lid shape structure common to all kinases in the ribokinase superfamily. This finding has been shown to be in line with the condition that pyridoxal kinase binds substrates with variable sizes of chemical groups at position 4 of vitamin B(6) and its derivatives. In addition, the enzyme contains a 12-residue peptide loop in the active site for the prevention of premature hydrolysis of ATP. Conserved amino acid residues Asp(118) and Tyr(127) in the peptide loop could be moved to a position covering the nucleotide after its binding so that its chance to hydrolyze in the aqueous environment of the active site was reduced. With respect to the evolutionary trend of kinase enzymes, the existence of this loop in pyridoxal kinase could be classified as an independent category in the ribokinase superfamily according to the structural feature found and mechanism followed in catalysis.

Proteome of Oriental ginseng Panax ginseng C. A. Meyer and the potential to use it as an identification tool.

Oriental ginseng (Panax ginseng C. A. Meyer) and American ginseng (Panax quinquefolius) are two widely used valuable traditional Chinese medicines (TCM). Previously, the identification of ginseng was mainly performed by analyzing the ginsengnosides using high performance liquid chromatography and amplification of polymorphic DNA using polymerase chain reaction. However, these methods cannot be used to distinguish TCM samples which are from different parts (main root, lateral roots, rhizome head and skin) of ginseng and ginseng culture cells from wild-grown ginseng. The present study aimed to identify different species of ginseng, different parts of the same ginseng and cultured cells of ginseng using a proteomic approach. Two-dimensional electrophoresis (2-DE) maps were established from the American ginseng main root, different parts (main root, lateral roots, rhizome head and skins) of Oriental ginseng and Oriental ginseng culture cells. Our results show that the 2-DE maps of different ginseng samples contain sufficient differences to permit easy discrimination. We have also identified common and specific protein spots in the 2-DE maps of different ginseng samples. The use of these "marker proteins" may help to speed up the identification process.

Crystallization and preliminary crystallographic studies of pyridoxal kinase from sheep brain.

Pyridoxal kinase (ATP:pyridoxal 5'-phosphotransferase; EC 2.7.1.35) is a key enzyme in the transformation of vitamin B(6) to pyridoxal-5'-phosphate. Pyridoxal-5'-phosphate is the crucial cofactor required by numerous enzymes involved in the metabolism of amino acids and the synthesis of many neurotransmitters. Pyridoxal kinase from sheep brain was crystallized in an orthorhombic form using the hanging-drop vapour-diffusion method with sodium citrate as the precipitant. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 59.8, b = 94.4, c = 128.2 A, and diffract to a resolution of 2.1 A. Crystals were transferred into a soaking liquid without citrate and two heavy-atom derivatives were prepared.