Francis Kwok

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.

Porcine pyridoxal kinase: c-DNA cloning, expression and confirmation of its primary sequence

Porcine brain pyridoxal kinase has been cloned. A 1.2 kilo-based cDNA with a 966-base pair open reading frame was determined from a porcine brain cortex cDNA library using PCR technique. The DNA sequence was shown to encode a protein of 322 amino acid residues with a molecular mass of 35.4 kDa. The amino acid sequence deduced from the nucleotide sequence of the cDNA was shown to match the partial primary sequence of pyridoxal kinase. Expression of the cloned cDNA in E. coli has produced a protein which displays both pyridoxal kinase activity and immunoreactivity with monoclonal antibodies raised against natural enzyme from porcine brain. With respect to the physical properties, it is shown that the recombinant protein exhibits identical kinetic parameters with the pure enzyme from porcine brain. Although the primary sequence of porcine pyridoxal kinase has been shown to share 87% homology with the human enzyme, we have shown that the porcine enzyme carries an extra peptide of ten amino acid residues at the N-terminal domain.

Constitutive nitric oxide synthase (NOS) activities in big-head carp (Aristichthys nobilis)

Nitric oxide synthase (NOS) is an enzyme that catalyzes the formation of nitric oxide (NO), an important biological messenger from L-arginine. There are considerable evidence showing the expression of NOS in mammalian tissues. Information on distribution of NOS activities in various organs and tissues of fish is rare. Non-functional NOS activities were documented in fish semi-quantitatively either by an indirect nicotine-adenine-dinucleotide-phosphate diaphorase (NADPH-d) activity histochemical staining method or by an immunohistochemical method using a cross-reacting antibody to brain NOS. Report on the functional levels of NOS activities in fish is lacking. This report represent the first attempt to document the functional NOS levels in various fish organs and tissues. Constitutive NOS (cNOS) activities in various organs of big-head carp (Aristichthys nobilis) was measured by a chemiluminescence method with a detection limit as low as 10 ρmol of NO produced. It was found that constitutive NOS activity was highest in the brain, followed by the intestine, stomach, retina, olfactory lobe, swim bladder, skeletal muscle, heart, kidney, ovary and liver. NOS activity could not be detected in the gill filaments. Omission of NADPH in the reaction mixture caused a 57–100% decrease in cNOS activities. However, omission of arginine in the mixture only caused a 56–87% drop in cNOS activities. When compared with cNOS activities documented from other species, a similar pattern of cNOS activities in the various organs and tissues of big-head carp could be seen.

Development of a continuous coupled enzymatic assay for myo-inositol monophosphatase

Myo-inositol monophosphatase, an enzyme purified from brain tissues, catalyses the dephosphorylation of myo-inositol 1-phosphate. This enzyme has become the subject of intense research interest since myo-inositol is needed for the resynthesis of phosphatidylinositol in cell membranes. Since phosphate contamination has always been a problem for the assay of this enzyme activity, we have developed a coupled enzymatic assay for detecting the activity of the phosphatase with no interference by the presence of phosphate. The assay is based on the measurement of inositol release after dephosphorylation and subsequent conversion of inositol into scyllo-inosose by a second enzyme, inositol dehydrogenase from Enterobacter aerogenes. Since the second reaction requires the presence of beta-NAD+, the activity of the dephosphorylation reaction can be monitored continuously by the increase of absorbance at 340 nm spectrophotometrically.

Structure-function relationships of porcine pyridoxal kinase

Pyridoxal kinase (PK) catalyzes the formation of pyridoxal-5-phosphate (PLP) from pyridoxal (PL), ATP and a divalent cation (Zn2+). So far, there is no three-dimensional structure of PK available. Site-directed mutagenesis was carried out to study the importance of three conserved residues: Tyr137, Gly242 and G1y244. The mutants (Y137F, G242A and G244A) were constructed, expressed, purified and analyzed. The results demonstrated that the mutants had much less activity but with no dramatic variation in protein stability. Tyrl 37 residue was shown to be involved in PL binding but not ATP binding. For the G244A mutant, the absence of enzyme activity was probably due to the deficiency in PL binding rather than the lack of ATP binding. In the case of the G242A mutant, it did not bind to ATP or PL.

Three-Dimensional Model Of The ATP-Binding Domain Of Pyridoxal Kinase

With a combination of multiple sequence alignment, secondary structure prediction and fold recognition techniques, a molecular model of the ATP-binding domain of pyridoxal kinase was constructed. The overall fold of the model consists of a central β-sheet of five parallel strands sandwiched between six helices. It shows high resemblance with the typical Rossmann fold in nucleotide binding. The quality of this model was assessed with the program PROCHECK using conformation criteria. PROCHECK locates over 90% of the residues of the current model in the “most favored” and “additional allowed” regions in Ramachandran plot.

Partially Folded Conformations of Inositol Monophosphatase Endowed with Catalytic Activity

The stability of porcine brain inositol monophosphatase in the presence of increasing concentrations of urea was investigated at pH 7.5. Exposure of the enzyme to 8 M urea brings about the dissociation of the dimeric species of 58 kDa into monomeric forms as revealed by gel filtration chromatography. Unfolding of the protein by 8 M urea results in a decrease of the ellipticity at 220 nm (20%) together with a perturbation of the near-UV circular dichroism spectrum. Urea-treated inositol monophosphatase binds Co2+ ions with a dissociation constant of 3.3 μM. The enzyme is catalytically competent when assayed with 4-nitrophenyl-phosphate in the presence of the activating ion Co2+ at pH 7.5 in 8 M urea. The apparent activation constant for Co2+ is 2.5 mM. It is postulated that partially folded conformations of monomeric species preserve their catalytic function because the affinity of Co2+ ions for the metal coordination center of the protein is not perturbed by exposure to 8 M urea.