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PURIFICATION AND CHARACTERIZATION OF TWO DOUBLE‐HEADED TRITICALE ISOINHIBITORS OF ENDOGENOUS ALPHA‐AMYLASE AND SUBTITLISIN 1
Author(s) -
ZAWISTOWSKA URSZULA,
LANGSTAFF JOHN,
FRIESEN ALBERT D.
Publication year - 1989
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.1989.tb00395.x
Subject(s) - subtilisin , isoelectric focusing , chemistry , chromatography , triticale , fast protein liquid chromatography , isoelectric point , amylase , alpha amylase , biochemistry , enzyme , high performance liquid chromatography , biology , botany
Two double‐headed isoinhibitors of endogenous alpha‐amylase and subtilisin have been purified from triticale by immobilized copper affinity chromatography followed by S‐Sepharose Fast Flow ion exchange chromatography and preparative isoelectric focusing on a sucrose density gradient ampholyte column. The inhibitors had isoelectric points at pH 7.25 and 6.95, and were present in triticale grains in approximately equal amounts. Both proteins, designated inhibitor 7.25 and inhibitor 6.95, have been purified to homogeneity as assessed by nondenaturing polyacrylamide gel electrophoresis (PAGE) at pH 8.3. They also showed approximately 90% purity as determined by size exclusion limit FPLC. Their properties were similar to the endogenous alpha‐amylase/subtilisin inhibitors found in wheat and barley, since they were active against cereal alpha‐amylases and subtilisin and they were inactive against salivary and bacterial alpha‐amylases as well as against trypsin. Apparent MW of both inhibitors was 20 kilodaltons as estimated from sodium dodecyl sulfate‐PAGE under reducing conditions. No carbohydrate was detected in isoinhibitor preparations. The triticale alpha‐amylase‐triticale inhibitor systems studied revealed a mixed type of inhibition with apparent dissociation constant (Ki) values of 6.2 and 5.8 nM for the inhibitor 7.25 and inhibitor 6.95, respectively.