Abstract
Two new double-headed protease inhibitors have been isolated from black-eyed peas. The isoinhibitors can be purified to homogeneity with greater than 90% recovery in a four-step procedure by means of sequential affinity chromatography on trypsin-Sepharose and chymotrypsin-Sepharose affinity columns. The isoinhibitors both have molecular weights near 8,000 and both have the same NH1-terminal residue serine. Black-eyed pea chymotrypsin and trypsin inhibitor (BEPCI) has an isoelectric point of 5.1 and inhibits trypsin and chymotrypsin simultaneously. Black-eyed pea trypsin inhibitor (BEPTI) has an isoelectric point of 6.5 and inhibits 2 molecules of trypsin simultaneously. BEPTI binds to chymotrypsin-Sepharose above pH 6 but does not inhibit chymotrypsin in the standard inhibitor assay with 10-3 M substrate. These new inhibitors are distinct from the Ventura inhibitor isolated from Serido black-eyed peas. An endogenous seed protease has been isolated from black-eyed peas by affinity chromatography on soybean inhibitor-carboxymethylcellulose affinity columns. A protease-BEPCI complex has been isolated by ion exchange chromatography. A dual physiological function of inhibition and protection of the seed protease is suggested as a plausible role of seed protease inhibitors.
Highlights
Indicated that there is no inhibitor which bound to chymotrypsin but not to trypsin. This suggests that BEPCI is a single isoinhibitor rather than a mixture
Reduced, carbamidomethylated samples of BEPCI and BEPTI, denatured in 1% sodium dodecyl sulfate for 10 min, 60”, plus standing overnight at room temperature, produced single bands at about 10,000 and 9,000, respectively
The same spot, which co-chromatographed with dansyl serine, was found for both BEPCI and BEPTI
Summary
Two new double-headed protease inhibitors have been isolated from black-eyed peas. Sepharose above pH 6 but does not inhibit chymotrypsin in the standard inhibitor assay with 10e3 M substrate These new inhibitors are distinct from the Ventura inhibitor isolated from Serido black-eyed peas. An endogenous seed protease has been isolated from black-eyed peas by affinity chromatography on soybean inhibitor-carboxymethylcellulose affinity columns. Serido black-eyed peas [5, 6], two new protease isoinhibitors have been isolated Both are double-headed; one (BEPCI’) inhibits trypsin and chymotrypsin simultaneously, the other (BEPTI) inhibits 2 molecules of trypsin simultaneously. This paper describes the development of a simple method of isoinhibitor purification by sequent,ial affinity chromatography of crude black-eyed pea extracts on trypsin-Sepharose and chymotrypsin-Sepharose affinity columns. Preliminary investigations of an endogenous seed protease and its interactions with BEPCI are described in an attempt to examine the physiological role of protease inhibitors in uiuo
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