Isolation carboxypeptidase inhibitor

Several plant proteins have been isolated that inhibit the metalloprotease carboxypeptidase A [205-217] (Table 7), notably potato carboxypeptidase inhibitor PCI [207-217] (Table 7). PCI is a small, cysteine-rich protein with a compact knotted structure determined by 3 disulphide links. The C-terminal region inserts into the active site of the carboxypeptidase. The C-terminal glycine is cleaved and remains trapped in the active site, this representing an example of suicide inactivation [207-216]. 

The crystal structure of the HNL isolated from S. bicolor (SbHNL) was determined in a complex with the inhibitor benzoic acid." The folding pattern of SbHNL is similar to that of wheat serine carboxypeptidase (CP-WII)" and alcohol dehydrogenase." A unique two-amino acid deletion in SbHNL, however, is forcing the putative active site residues away from the hydrolase binding site toward a small hydrophobic cleft, thereby defining a completely different active site architecture where the triad of a carboxypeptidase is missing. 

The enzyme carboxypeptidase A is particularly amenable to structural investigation crystal structures of the enzyme, of complexes of the enzyme with substrates, substrate analogues and inhibitors, and of transition-state analogues are available. To isolate an enzyme-substrate complex for a one-substrate enzyme reaction, or for an enzyme reaction where water is a... 

Actinophyllic acid (3), an indole alkaloid with novel l-azabicyclo-[4.4.2]dodecane and l-azabicyclo[4.2.1]nonane fragments, was isolated from the tree Alstonia actinophylla, collected on the Cape York Peninsula, Queensland, Australia, in 2005 by Quinn, Carroll and coworkers [23]. As a carboxypeptidase U inhibitor (IC50 = 0.84 pM), actinophyllic acid shows potential application for the treatment of cardiovascular disorders [23]. Much effort has been devoted to this unique molecule, including recent synthetic studies by Wood and coworkers [24], but only Overman et al. has accomplished its synthesis [25],... 

Several peptides isolated from the venom of the South American snake Bothrops jararaca are potent ACE inhibitors and were briefly used for the treatment of hypertension, but were soon superseded by surprisingly simple molecules with high inhibitory effect. At the Squibb Institute for Medical Research in Princeton, New Jersey, Miguel A. Ondetti (Plate 32) recognized that ACE is similar in its substrate specificity to the well studied protease car-boxypeptidase A. He designed, therefore, molecules that should fit into the active site of ACE (presumably similar to the active site of carboxypeptidase A) and form complexes with the enzyme. The dipeptide , 2-D-methyl-3-mercapto-propionyl-L-proline, captopril [7] strongly associated with the enzyme and... 

It is sometimes extremely difficult to repeat a biochemical experiment, and the preparation of the crystalline form which we studied ( a= 51.41 A, c = 47.19 A and p = 97°35 ) was just such a problem. Only about two of some thirty preparations crystallized with these cell dimensions. On the other hand most preparations, including the commercial preparation, give cell dimensions of a= 50.9 A, b = 57.9 A, c = 45.0 A and p = 94°40. The activities of these two forms are very different in the solid state, 1/3 and 1/300 of that of the material in solution Proc. Nat. Acad. Sci. 70, 3797 (1973)). The arsanilzao Tyr 248 derivative of the former crystals behaves like the material in solution, but this derivative of the latter crystals is quite different from the material in solution. Vallee continues to discoimt the results of our crystallography, done on the former unit cell, on the basis of his results on the commercial form, which most likely has the latter unit cell. Probably the main difference is in the intermolecular contacts in the two crystalline forms. Since the crystals of the X-ray study are active, the conclusion of this work should be tested on that basis. Our present plan (1977) is to elucidate the structures of the protein inhibitor of carboxypeptidase A isolated from potatoes, and of its complex with the enzyme in the hope that atomic displacements toward the transition state can be recognized. 

Inhibitors of the metallo-carboxypeptidases A and B are rare. The first such inhibitor was isolated from potatoes, and later an inhibitor with similar specificity was isolated from the intestinal worm, Ascaris lumbricoides. The latter inhibitor is difficult to purify, which precludes any nutritional studies in animals. Potato CPI, on the other hand, can represent a significant contribution to the potato proteins, depending upon variety (7). In a survey of 106 commercial and experimental varieties, CPI varied from zero to 846 yg/ml tuber juice (over 4% of the soluble proteins). In Russet Burbank potatoes the levels are about 30% of this value. The isolation of enough CPI from Russet Burbank potatoes for a nutritional study was therefore possible, if a simple efficient method could be developed to isolate it. 

Supplementing chick diets with a protein fraction, isolated from raw Russet Burbank potato tubers, enriched in proteinase inhibitors, had previously been shown to severely depress their growth (5). The inhibitor-rich fraction contained at least six well characterized inhibitors of mammalian pancreatic digestive proteinases trypsin, chymotrypsin, elastase and carboxypeptidases A and B (5). Since the fraction contained an array of proteinase inhibitors it was not known if CPI contributed to the growth depressing activities. 

In the last two years inhibitors for the proteinases A and B, and carboxypeptidase Y have been isolated from yeast (6,9-14) some of their properties are summarized in Table III. Two subgroups of inhibitors for the proteinases A and B, respectively, have been separated according to differences in electrophoretic mobility, i.e. in isoelectric point (see Table III). In early experiments (6), evidence for four different species of proteinase A-inhibitors had been obtained, but only the species 1 2 and 1 3 could be further purified and characterized (6,11). It has been shown recently (Biinning and Holzer in press) that in contrast to Baker s yeast, which contains the two inhibitors for proteinase B (10), in several strains of Saccharomyces cerevisiae only I 2 could be demonstrated and in Saccharomyces carlsbergensis, only 1 1. Single cell cultures from Baker s yeast contain 1 1 1 2 in the same concentration ratio of 1 3 as observed in preparations from commercial Baker s yeast. 

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