Isolation and characterization of carboxypeptidase Ⅲ from germinating triticale grains  

作　　者：Adam Drzymala Wieslaw Bielawski 

机构地区：[1]Department of Biochemistry, Warsaw University of Life Sciences-SGGW, Nowoursynowska 159, 02-776 Warsaw, Poland

出　　处：《Acta Biochimica et Biophysica Sinica》2009年第1期69-78,共10页生物化学与生物物理学报（英文版）

摘　　要：Carboxypeptidase Ⅲ from germinating triticale grains was purified 434.2-fold with a six-step procedure including： homogenization, ammonium sulfate precipitation, cation-exchange chromatography on CM-cellulose, gel filtration chromatography on Sephadex G-150, cation-exchange chromatography on SP8HR column （HPLC）, and affinity chromatography on CABSSepharose 4B. Triticale carboxypeptidase Ⅲ is a monomer with a molecular weight of 45 kDa, which optimally hydrolyzes peptides at temperature 30-50℃ and pH 4.6. N-CBZ-Ala-Phe, N-CBZ-Ala-Leu, and N-CBZ-AIa-Met are hydrolyzed at the highest rates. Amino acids with aromatic or large aliphatic side chains are preferred in position P1＇, whereas the presence of these types of groups in position P1 of the substrate results in a lower rate of hydrolysis. Peptides containing glutamic acid in positions P1 are poor substrates for the enzyme. This phenomenon suggests the hydrophobic substrate-binding sites S1 and S1＇. The active site contains serine since diisopropylfluorophosphate and phenylmethanesulfonyl fluoride reduce the activity by 89.9% and 81.5%, respectively. Moreover, the activity of triticale carboxypeptidase Ⅲ is reduced by mercury ions and organomercurial compounds, which suggests the presence of a sulfhydryl group adjacent to the active site of the enzyme. Identification of purified enzyme by mass spectrometry method demonstrated that the enzyme is a homolog of barley carboxypeptidaseⅢ.Carboxypeptidase Ⅲ from germinating triticale grains was purified 434.2-fold with a six-step procedure including： homogenization, ammonium sulfate precipitation, cation-exchange chromatography on CM-cellulose, gel filtration chromatography on Sephadex G-150, cation-exchange chromatography on SP8HR column （HPLC）, and affinity chromatography on CABSSepharose 4B. Triticale carboxypeptidase Ⅲ is a monomer with a molecular weight of 45 kDa, which optimally hydrolyzes peptides at temperature 30-50℃ and pH 4.6. N-CBZ-Ala-Phe, N-CBZ-Ala-Leu, and N-CBZ-AIa-Met are hydrolyzed at the highest rates. Amino acids with aromatic or large aliphatic side chains are preferred in position P1＇, whereas the presence of these types of groups in position P1 of the substrate results in a lower rate of hydrolysis. Peptides containing glutamic acid in positions P1 are poor substrates for the enzyme. This phenomenon suggests the hydrophobic substrate-binding sites S1 and S1＇. The active site contains serine since diisopropylfluorophosphate and phenylmethanesulfonyl fluoride reduce the activity by 89.9% and 81.5%, respectively. Moreover, the activity of triticale carboxypeptidase Ⅲ is reduced by mercury ions and organomercurial compounds, which suggests the presence of a sulfhydryl group adjacent to the active site of the enzyme. Identification of purified enzyme by mass spectrometry method demonstrated that the enzyme is a homolog of barley carboxypeptidaseⅢ.

关 键 词：carboxypeptidase Ⅲ enzymatic properties GERMINATION TRITICALE 

分 类 号：Q51[生物学—生物化学]