(13) Assay Card

General Information
Summary Kalata B1 and B2 were found to have no trypsin or chymotrypsin inhibitory activity.
Condition Tris¬HCl, pH 8.0,¬was used for bovine trypsin and chymotrypsin. Enzyme and various amounts of kalata B1, kalata B2, or Nicotiana alata proteinase inhibitor were mixed and preincubated in a 60-¬µl volume for 30¬min at 30¬C before the addition of substrate (40¬¬µl). The reactions were continued for 30¬min at 30¬C before release of p-nitroanilide was recorded at 405¬nm on a SpectraMax 250¬microtiter plate reader (Molecular Devices). Trypsin activity was determined by using 4¬¬µg (1.67¬¬µM) bovine trypsin [Type XIII 1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK) treated, Sigma] or 3.2¬¬µg of gut protein. Chymotrypsin assays used 0.1¬¬µg (0.04¬¬µM) of bovine chymotrypsin (Type VII TLCK treated, Sigma) or 2.6¬¬µg of gut protein. Amylase assays were performed at 30¬C in 200 ¬µl of reaction mixture containing 26 ¬µg of gut protein and 0.5% (wt/vol) starch (Sigma) in CAPS buffer. Samples (20 ¬µl) were removed at 10, 20, and 30 min for reducing sugar assay by using dinitrosalicyclic acid reagent. Potential inhibitory activity of kalata B1 and B2 was measured by mixing 20 ¬µg with gut protein (26 ¬µg) and preincubating at 30¬C for 1 h in CAPS buffer before the enzyme assay was initiated by the addition of an equal volume of 1% (wt/vol) starch in the same buffer. The -amylase inhibitor from wheat seeds (Sigma A1520) was used as a positive control.
Result Inhibition of endogenous digestive proteinases is one mechanism by which small disulfide-rich defense molecules exert their effect, as exemplified by the proteinase inhibitors from N.¬alata. This possibility was tested for kalata by using a series of enzyme inhibition assays. Kalata B1 and B2 had no effect on bovine or H.¬punctigera trypsins and chymotrypsins, whereas the N.¬alata proteinase inhibitors abolished all trypsin and most chymotrypsin activity under the same conditions. Similarly, the kalata peptides failed to inhibit -amylases from Helicoverpa gut.
AssayType Protease inhibition

Jennings C, West J, Waine C, Craik D, Anderson M (2001) Biosynthesis and insecticidal properties of plant cyclotides: the cyclic knotted proteins from Oldenlandia affinis. Proc Natl Acad Sci U S A 98:10614-9

Proteins Assayed kalata B2
kalata B1