RESUMO
LUTI (Linum usitatissimum trypsin inhibitor), a member of the potato inhibitor I family, has been isolated from seeds of flax by ethanol fractionation, ion exchange chromatography on CM-Sephadex C-25, affinity purification on immobilized methylchymotrypsin (alpha-chymotrypsin in which His57 has been converted to 3-methylhistidine) in the presence of 5M NaCl, and finally by reversed-phase HPLC. The 7655 Da inhibitor consists of a single polypeptide chain of 69 residues with one disulfide bridge. The molecule is acetylated at the N terminus. Its primary structure has been determined after limited proteolysis of the native molecule with trypsin at the reactive site, cleavage with cyanogen bromide or arginyl endopeptidase (Arg-gingipain), and alcoholytic deacetylation of the N-terminally blocked serine. The association constants (K(a)) of LUTI with bovine beta-trypsin and alpha-chymotrypsin are 3.58x10(10) M(-1) and 5.02x10(5) M(-1), respectively. High NaCl concentration (3M) increased the association constant of LUTI with alpha-chymotrypsin to 6.64x10(7) M(-1). To our knowledge, LUTI is the first serine-proteinase-type inhibitor isolated from a plant of the Linaceae family.
Assuntos
Linho/química , Proteínas de Plantas/química , Inibidores de Serina Proteinase/química , Sequência de Aminoácidos , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Quimotripsina/antagonistas & inibidores , Quimotripsina/química , Eletroforese em Gel de Poliacrilamida , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas de Plantas/isolamento & purificação , Sementes/química , Inibidores de Serina Proteinase/isolamento & purificação , Inibidores da Tripsina/química , Inibidores da Tripsina/isolamento & purificaçãoAssuntos
Entorpecentes/intoxicação , Transtornos Relacionados ao Uso de Opioides/enzimologia , Síndrome de Abstinência a Substâncias/enzimologia , Fosfatase Ácida/sangue , Doença Aguda , Adolescente , Adulto , Butirilcolinesterase/sangue , Humanos , Masculino , Entorpecentes/administração & dosagem , Transtornos Relacionados ao Uso de Opioides/terapia , alfa 1-Antitripsina/sangueRESUMO
The main polymorphic form of acid phosphatase was isolated from Poa pratensis seeds by chromatography on DEAE- and CM-cellulose and gel filtration on Bio-Gel P-100. The enzyme migrated as a single band in disc electrophoresis at pH 4.5 and 8.4. The purified enzyme is a glycoprotein of molecular weight about 33 000. Carbohydrate content accounts for 40% of the total weight. The optimum pH is at 5.2 and the apparent Km for p-nitrophenylphosphate, 0.55 mM. Fluoride ions are non-competitive and zinc ions-uncompetitive inhibitors, with the apparent Km values of 0.55 and 0.28 mM, respectively.