[Isolation of trypsin PC from the Kamchatka crab Paralithodes camtschatica and its properties].
Trypsin PC from the hepatopancreas of the king crab Paralithodes camtschatica was isolated and purified to apparent homogeneity by ion-exchange chromatography on Aminosilochrom and DEAE-Sephadex and affinity chromatography on arginine-agarose. The yield of the enzyme was 37.7%, and the purification degree was 21. Trypsin PC has a molecular mass of 29 kDa and pI < 2.5. It hydrolysis N-benzoyl-L-arginine p-nitroanilide at the optimum pH of 7.5-8.0 and at the temperature optimum of 55 degrees C (K(m) = 0.05 mM). Trypsin PC retained its activity within the pH range of 5.8-9.0 in the presence of Ca2+. The enzyme was inhibited by the specific inhibitors of serine proteases diisopropyl fluorophoshate and phenylmethylsulfonyl fluoride, by the trypsin inhibitor N-tosyl-L-lysylchloromethylketone, and by the trypsin inhibitors from soybean and potato. Trypsin PC was found to hydrolyze amide bonds formed by carboxylic groups of lysine and arginine in peptide substrates. The N-terminal sequence of this enzyme is IVGGTEVTPG.
Rudenskaia
G N
GN
Department of Chemistry, Moscow State University, Russia. Rudenskaya@biorg.chem.msu.su
Isaev
V A
VA
Kalebina
T S
TS
Stepanov
V M
VM
Mal'tsev
K V
KV
Shvets
S V
SV
Luk'ianova
N A
NA
Kislitsin
Iu A
IuA
Miroshnikov
A I
AI
D000595:Amino Acid Sequence; D000818:Animals; D003386:Brachyura / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D005340:Fibrinogen / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014357:Trypsin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014361:Trypsin Inhibitors / Q000494:pharmacology
D014361:Trypsin Inhibitors; D005340:Fibrinogen; D003094:Collagen; D014357:Trypsin