Peculiarities of substrate hydrolysis by endopeptidases from hepatopancreas of king crab

Kinetic parameters of hydrolysis of peptide and protein substrates by psychrophilic endopeptidases from hepatopancreas of the king crab Paralithodes camtschaticus (PC), in particular, by trypsin, collagenolytic protease, and metalloprotease, were measured at different temperatures. The PC trypsin was shown to hydrolyze Bz-Arg-pNA in the temperature range studied (4–37°C) 19 times more effectively than bovine trypsin. The rate constants of hydrolysis of Glp-Ala-Ala-Leu-pNA by the PC collagenolytic protease increased approximately by one order of magnitude along with temperature decrease, while K _m decreased by 3.5 times. The effective values of K _m for the hydrolysis of azocasein by the metalloprotease insignificantly depend on temperature. We proposed that electrostatic interactions of negative charges around the cavity of active site are critical for the effective hydrolysis of substrates by endopeptidases of the PC hepatopancreas.     

Isolation and primary structure of trypsin from the red king crab Paralithodes camtschaticus

Trypsin from hepatopancreas of the Paralithodes camtschaticus crab was isolated in homogeneous state by successive ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Agarose modified with peptide ligands from trypsin hydrolysate of salmin, and ion-exchange chromatography on a Mono Q column. The total yield of the protein was 64%. Its N-terminal amino acid sequence was determined (IVGGTEVTPG-). A sample of amplified total cDNA of hepatopancreas of king crab was obtained. The cDNA fragment containing the complete coding part of the gene was isolated on the basis of the known N-terminal amino acid sequence of the mature form of the trypsin. The polypeptide chain of the proenzyme consists of 266 aa. The mature trypsin involves 237 aa, which corresponds to its molecular mass of 24.8 kDa. A comparison of the amino acid sequence of the king crab trypsin with those of trypsins from other species of crustaceans demonstrated their high structural homology. The trypsin from the Penaeus vannamei shrimp appeared to be the most close in its primary structure to that of the king crab (70% identity).     

Determination with a microelectrode of membrane potential of giant human erythrocytes obtained by La(3+)-induced fusion

The effect of La3+ on the fusion of erythrocytes of blood stored for a week at +4 degrees C was studied. It was shown that the fusion of erythrocytes begins after one day of storage of blood. The most intensive fusion of erythrocytes was observed on day 4 of blood storage. As a result, giant cells with a size of 100 microns and more arise. The electrical potential of giant cells was measured using a microelectrode and was -6.6 +/- 1.5 mV.     

Isolation and Primary Structure of Trypsin from the King Crab Paralithodes camtschaticus

Trypsin from hepatopancreas of the crab Paralithodes camtschaticuswas isolated in homogeneous state by successive ion-exchange chromatography on DEAE-Sephadex, affinity chromatography on Agarose modified with peptide ligands from trypsin hydrolysate of salmin, and ion-exchange chromatography on a Mono Q column. The total yield of the protein was 64%. Its N-terminal amino acid sequence was determined (IVGGTEVTPG-). A sample of amplified total cDNA of hepatopancreas of king crab was obtained. A cDNA fragment containing the complete coding part of the gene was isolated on the basis of the known N-terminal amino acid sequence of the mature form of the trypsin. The polypeptide chain of the proenzyme consists of 266 aa. The mature trypsin involves 237 aa, which corresponds to its molecular mass of 24.8 kDa. A comparison of the amino acid sequence of the king crab trypsin with those of trypsins from other species of crustaceans demonstrated their high structural homology. The trypsin from the shrimp Penaeus vannamei appeared to be closest in primary structure to that of the king crab (65% identity).     

Characteristics of substrate hydrolysis by endopeptidases from the hepatopancreas of the king crab

Kinetic parameters of hydrolysis of peptide and protein substrates by psychrophilic endopeptidases from hepatopancreas of the king crab Paralithodes camtschaticus (PC), in particular, by trypsin, collagenolytic protease, and metalloprotease, were measured at different temperatures. The PC trypsin was shown to hydrolyze Bz-Arg-pNA in the temperature range studied (4-37 degrees C) 19 times more effectively than bovine trypsin. The rate constants of hydrolysis of Glp-Ala-Ala-Leu-pNA by the PC collagenolytic protease increased approximately by one order of magnitude along with temperature decrease, while Km decreased by 3.5 times. The effective values of Km for the hydrolysis of azocasein by the metalloprotease insignificantly depend on temperature. We proposed that electrostatic interactions of negative charges around the cavity of active site are critical for the effective hydrolysis of substrates by endopeptidases of the PC hepatopancreas.