Kunitz-type protease inhibitors group B from Solanum palustre.
Five Kunitz protease inhibitor group B genes were isolated from the genome of the diploid non-tuber-forming potato species Solanum palustre. Three of five new genes share 99% identity to the published KPI-B genes from various cultivated potato accessions, while others exhibit 96% identity. Spls-KPI-B2 and Spls-KPI-B4 proteins contain unique substitutions of the most conserved residues usually involved to trypsin and chymotrypsin-specific binding sites of Kunitz-type protease inhibitor (KPI)-B, respectively. To test the inhibition of trypsin and chymotrypsin by Spls-KPI proteins, five of them were produced in E. coli purified using a Ni-sepharose resin and ion-exchange chromatography. All recombinant Spls-KPI-B inhibited trypsin; K(i) values ranged from 84.8 (Spls-KPI-B4), 345.5 (Spls-KPI-B1), and 1310.6 nM (Spls-KPI-B2) to 3883.5 (Spls-KPI-B5) and 8370 nM (Spls-KPI-B3). In addition, Spls-KPI-B1 and Spls-KPI-B4 inhibited chymotrypsin. These data suggest that regardless of substitutions of key active-center residues both Spls-KPI-B4 and Spls-KPI-B1 are functional trypsin-chymotrypsin inhibitors.
Speransky
Anna S
AS
Faculty of Biology, M. V. Lomonosov Moscow State University, Vorobjevy Gory, Moscow, Russia.
Cimaglia
Fabio
F
Krinitsina
Anastasya A
AA
Poltronieri
Palmiro
P
Fasano
Pasqua
P
Bogacheva
Anna M
AM
Valueva
Tatiana A
TA
Halterman
Dennis
D
Shevelev
Alexei B
AB
Santino
Angelo
A
D000595:Amino Acid Sequence; D007611:Aprotinin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D032322:Solanum / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology
D010940:Plant Proteins; D011994:Recombinant Proteins; D014361:Trypsin Inhibitors; C098973:recombinant Kunitz protease inhibitor; D007611:Aprotinin; D002918:Chymotrypsin; D014357:Trypsin