ABSTRACT
Rice (Oryza sativa) has two betaine aldehyde dehydrogenase homologs, BAD1 and BAD2, encoded on chromosome four and chromosome eight respectively. BAD2 is responsible for the characteristic aroma of fragrant rice. Complementary DNA clones of both BAD1 and BAD2 were isolated and expressed in E. coli. BAD2 had optimum activity at pH 10, little to no affinity towards N-acetyl-gamma-aminobutyraldehyde (NAGABald) with a Km of approximately 10 mM and moderate affinity towards gamma-guanidinobutyraldehyde (GGBald) and betaine aldehyde (bet-ald) with Km values of approximately 260 microM and 63 microM respectively. A lower Km of approximately 9 microM was observed with gamma-aminobutyraldehyde (GABald), suggesting BAD2 has a higher affinity towards this substate in vivo. The enzyme encoded on chromosome four, BAD1, had optimum activity at pH 9.5, showed little to no affinity towards bet-ald with a Km of 3 mM and had moderate affinity towards GGBald, NAGABald and GABald with Km values of approximately 545, 420 and 497 microM respectively. BAD1 had a half life roughly double that of BAD2. We discuss the implications of these findings on the pathway of fragrance generation in Basmati and Jasmine rice and the potential of rice to accumulate the osmoprotectant glycine betaine.
Subject(s)
Betaine-Aldehyde Dehydrogenase/metabolism , Oryza/enzymology , Oryza/physiology , Betaine-Aldehyde Dehydrogenase/isolation & purification , Enzyme Activation , Escherichia coli , Half-Life , Hydrogen-Ion Concentration , Kinetics , Plant Proteins/isolation & purification , Plant Proteins/metabolism , Proline/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Substrate Specificity , TemperatureABSTRACT
Tinospora smilacina Benth. has been used in Australian indigenous medicine for the treatment of headache, rheumatoid arthritis and other inflammatory disorders. As part of an investigation into the anti-inflammatory potential of plants using an ethnopharmacological approach, the present study sought to evaluate the efficacy and safety of Tinospora smilacina. An ethanol extract of this plant was evaluated in vitro for anti-inflammatory activities on cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LO) and phospholipase A(2) (PA(2)). The ethanol extract of Tinospora smilacina showed inhibitory activities on COX-1, COX-2, 5-LO and PA(2) with the IC(50) values of 63.5, 81.2, 92.1 and 30.5 micro g/mL respectively. Cytotoxic effect of the extracts of Tinospora smilacina was investigated in vitro using ATP-based luminescence assay and the results showed no cytotoxic effect on cell lines of skin fibroblasts (1BR3), human Caucasian hepatocyte carcinoma (Hep G2) and human Caucasian promyelocytic leukaemia (HL-60). This paper also describes the results of fractionations and bioassay guided chemical studies, suggesting that the anti-inflammatory activity is due to triterpene-fatty acid esters and free fatty acids.
