Characterization of a pathogenesis-related class 10 protein (PR-10) from Astragalus mongholicus with ribonuclease activity.
A pathogenesis-related (PR) class 10 protein (designated AmPR-10) was first isolated from the Chinese medicinal material Astragalus mongholicus using a combination of affinity chromatography on Zn-chelate Agarose 4B, ion exchange chromatography on QAE Sephadex A-25 and gel filtration on Sephadex G50. The purified AmPR-10 showed a single band with a molecular mass of 17.2kDa in SDS-PAGE. The molecular mass of intact AmPR-10 was determined to be 32.8kDa by gel filtration. Thus, AmPR-10 is a dimeric protein composed of two identical subunits. AmPR-10 was a glycoprotein detected by periodic acid-Schiff (PAS) staining and its neutral carbohydrate content was 13.7%. The carbohydrate was mainly composed of 73.0% (w/w) arabinose, 15.0% (w/w) glucose and 4.8% (w/w) fructose on the basis of high-performance anion exchange chromatography (HPAEC) analysis. Its N-terminal sequence of 15 amino acid residues was determined as GVISFNEETISTVAP, and showed significant sequence homology to some pathogenesis-related (PR) class 10 proteins. This sequence had 80% identity with the PR-10 protein LlPR10.1C from Lupinus luteus (yellow lupine) followed by 73.3% identity with the PR-10 protein PR10.2 from Medicago sativa (alfalfa), suggesting it is a new member of PR-10 proteins. AmPR-10 exhibited ribonuclease (RNase) activity as do some other PR-10 proteins. The optimal pH and temperature for RNase activity were pH 6.0 and 60 degrees C, respectively. The RNase activity was stable within pH 5.0-11.0. It was stable up to 60 degrees C at pH 6.0. The purification and characterization of AmPR-10 in this investigation furnish additional data to the relatively scanty literature pertaining to Astragali radix proteins.
Yan
Qiaojuan
Q
Bioresource Utilization Laboratory, College of Engineering, China Agricultural University, Beijing, China. yanqj@cau.edu.cn
Qi
Xiaowei
X
Jiang
Zhengqiang
Z
Yang
Shaoqing
S
Han
Lujia
L
D000595:Amino Acid Sequence; D027883:Astragalus Plant / Q000737:chemistry* / Q000235:genetics; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000737:chemistry / Q000235:genetics; D006023:Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D031297:Lupinus / Q000737:chemistry / Q000235:genetics; D000455:Medicago sativa / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid
D002241:Carbohydrates; D006023:Glycoproteins; D010940:Plant Proteins; C053376:pathogenesis-related proteins, plant; D012260:Ribonucleases