Pancreas, bovine

ENZYMATIC ANALYSIS WITH CARBOXYPEPTIDASES. Carboxypeptidases are enzymes that cleave amino acid residues from the C-termini of polypeptides in a successive fashion. Four carboxypeptidases are in general use A, B, C, and Y. Carboxypeptidase A (from bovine pancreas) works well in hydrolyzing the C-terminal peptide bond of all residues except proline, arginine, and lysine. The analogous enzyme from hog pancreas, carboxypeptidase B, is effective only when Arg or Lys are the C-terminal residues. Thus, a mixture of carboxypeptidases A and B liberates any C-terminal amino acid except proline. Carboxypeptidase C from citrus leaves and carboxypeptidase Y from yeast act on any C-terminal residue. Because the nature of the amino acid residue at the end often determines the rate at which it is cleaved and because these enzymes remove residues successively, care must be taken in interpreting results. Carboxypeptidase Y cleavage has been adapted to an automated protocol analogous to that used in Edman sequenators. 

Figure 4.8 Cation-exchange liquid chromatography of basic proteins. Column, Asahipak ES502C eluent, 20 min linear gradient of sodium chloride from 0 to 500 mM in 50 mM sodium phosphate buffer pH 7.0 flow rate, 1 ml min-1 temperature, 30 °C detection, UV 280 nm. Peaks 1, myoglobin from horse skeletal muscle (Mr 17 500, pi 6.8-7.3) 2, ribonuclease from bovine pancreas (Mr 13 700, pi 9.5-9.6) 3, a-chymotrypsinogen A from bovine pancreas (Mr 257 000, pi 9.5) and 4, lysozyme from egg white (Mr 14 300, pi 11.0-11.4). (Reproduced by permission from Asahikasei data)...

Ribonuclease A, Bovine pancreas. Horse heart. AOT/isooctane Solubilization [72]... 

RNase A Bovine pancreas DAB/cyclohexane Activity studies [98]... 

Carbonic anhydrase Flavodoxin Hemoglobin Hexokinase Insulin Rubredoxin Superoxide dismutase Bovine erythrocytes Megasphaera elsdenii Bovine blood Yeast Bovine pancreas M. elsdenii Bovine erythrocytes TOMAC/Rewopal HVS/octanol/ isooctane Solubilization [105]... 

The structural analysis of the trypsin inhibitor from bovine pancreas (BPTI) in complex with trypsin shows that the inhibitor occupies and blocks the substrate binding pocket in a highly complementary maimer (fig. 2.9). In the trypsin-BPTI complex, the catalytically essential Ser-OH of trypsin contacts a CO group of the inhibitor in a manner very similar to the tetrahedral transition state of amide or ester bond hydrolysis (see fig. 2.9b). The inhibitor can be likened to a pseudo-substrate and, as such, is bound with high affinity. The cleavage of the peptide bond is, however, not possible due to other circumstances, such as the fact that water is prevented from reaching the active site with the inhibitor boimd. 

In view of the high stability of the enzyme most samples have been prepared by the procedure described by Kunitz (16) and modified by McDonald (17) to remove all traces of proteolytic activity. During this procedure the minced bovine pancreas is exposed to 0.25 N sulfuric acid, ammonium sulfate precipitation, 10 min at 95°-100° and pH 3, and, finally, reprecipitation. The product can be crystallized it was also shown later to contain a number of components all with ribonuclease activity. A practical summary of all details is given by Kunitz and McDonald (18). 

Cytochrome c (bovine heart) Myoglobin (horse heart) Chymotrypsinogen (bovine pancreas) /3-Lactoglobulin (goat milk)... 

Chymotrypsin Hydrolysis of proteins Bovine pancreas Zonal lysis in cataract removal... 

Trypsin Hydrolysis of proteins Bovine pancreas Wound and ulcer cleansing... 

Figure 10-5. The environment of the metal in a series of zinc metalloproteins. The proteins are (a) human carbonic anhydrase II, (b) thermolysin from Bacillus thermoproteolyticus, and (c) bovine pancreas carboxypeptidase. Each of these enzymes is, essentially, hydrolytic.

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