Complete amino acid sequence of bovine colostrum low-Mr cysteine proteinase inhibitor.

The complete amino acid sequence of bovine colostrum cysteine proteinase inhibitor was determined by sequencing native inhibitor and peptides obtained by cyanogen bromide degradation, Achromobacter lysylendopeptidase digestion and partial acid hydrolysis of reduced and S-carboxymethylated protein. Achromobacter peptidase digestion was successfully used to isolate two disulfide-containing peptides. The inhibitor consists of 112 amino acids with an Mr of 12787. Two disulfide bonds were established between Cys 66 and Cys 77 and between Cys 90 and Cys 110. A high degree of homology in the sequence was found between the colostrum inhibitor and human gamma-trace, human salivary acidic protein and chicken egg-white cystatin.

Purification and characterization of a bovine colostrum low molecular weight cysteine proteinase inhibitor.

Large amounts of cysteine proteinase inhibitors were found in bovine colostrum. One had a molecular weight of 90,000, and the other a molecular weight of 10,500. The concentrations of both these inhibitors were highest the day after parturition, and were about one-tenth as much on day 7. The lower molecular weight inhibitor was purified by acid treatment, ammonium sulfate fractionation, gel filtration on Sephadex G-50, CM-Sephadex chromatography and rechromatography on Sephadex G-50. The purified preparation gave a single band on SDS-polyacrylamide gel electrophoresis. This inhibitor contained one tryptophanyl residue and one cystinyl residue, and did not contain a free thiol group. Values obtained for its isoelectric point (pI) were 10.0 and 10.3. This material strongly inhibited cathepsin B, cathepsin H, and papain. the higher molecular weight inhibitor was partially purified. It had a pI of 4.2 and inhibited papain, cathepsin H, and cathepsin B.

Isolation and immunological studies of high and low molecular weight cysteine proteinase inhibitors in bovine serum.

Two kinds of cysteine proteinase inhibitor (Mr 145 000 and Mr 15 500) were purified from bovine serum. These purified inhibitors showed a single band on SDS-polyacrylamide gel electrophoresis, respectively. The isoelectric point of the high molecular weight inhibitor was found to be 4.4 and that of the low molecular weight inhibitor was 8.6. The high molecular weight inhibitor inhibited papain and cathepsin H, but had little activity against cathepsin B. While the low molecular weight inhibitor was a strong inhibitor of papain and cathepsin H and showed a weak inhibition of cathepsin B. These two inhibitors showed different immunological reactivities.

Purification of porcine pancreatic kallikrein using various affinity chromatographies and intestinal absorption of the purified kallikrein.

For the simple purification of porcine pancreas kallikrein, various affinity chromatographies using L-prolyl-L-phenylalanyl-L-arginine (Pro-Phe-Arg-OH), tosyl-L-arginine (Tos-Arg-OH) and acetyl-L-phenylalanyl-L-arginine (Ac-Phe-Arg-OH) as ligands were examined. The purification was performed as follows using the extract from acetone powder of porcine pancreas as starting material; ammonium sulfate fractionation (30-80%), affinity chromatography (repeated) and gel filtration on Sephadex G-75, respectively. With these purification techniques, porcine pancreas kallikrein was purified about 2000-fold, respectively. From the results, these affinity chromatographies were considered to be effective for the purification of porcine pancreas kallikrein. Intestinal absorption of the purified kallikrein was also investigated using a rabbit. After the administration of the purified kallikrein into the intestinal tract, peripheral blood was taken from the ear vein and the plasma was assayed for Pro-Phe-Arg-7-amino-4-methylcoumarinamide hydrolytic activity. The concentration of the kallikrein in the blood was maximal at 3 hr after the administration and the absorption at this time amounted to about 0.07%.

Purification and properties of thiol protease inhibitor from rat liver cytosol.

Thiol protease inhibitors were found in the cytosol fractions of various rat tissues. An inhibitor, named cytosol thiol protease inhibitor, was purified from rat liver cytosol by acid treatment and column chromatographies on Sephadex G-50, DEAE-Sephadex and Sephadex G-75. The purified inhibitor gave a single protein band on sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the inhibitor was found to be 12 400 by gel filtration on Sephadex G-75 and SDS-polyacrylamide gel electrophoresis, and its isoelectric point was found to be 5.04. This inhibitor inhibited rat liver lysosomal cathepsin B, B2, C, H and L and papain, but not cathepsin A or D, trypsin or chymotrypsin. The inhibitor caused noncompetitive inhibition of the hydrolytic activity of cathepsin H on alpha-N-benzoyl-DL-arginine 2-naphthylamide and its Ki value was 4.08 . 10(-8) M. Heat treatment at 80 degrees C for 10 min reduced the activity 40%.

Synthetic inhibitors of trypsin, plasmin, kallikrein, thrombin, C1r-, and C1 esterase.

p-Carbethoxyphenyl episol-guanidinocaproate and p-(p'-guanidinobenzoyloxy)-phenyl derivatives were prepared, and their inhibitory effects on trypsin, plasmin, plasma kallikrein, thrombin, C1r- and C1 esterase were examined. Among the various inhibitors tested, p-nitrophenyl p'-guanidinobenzoate, N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzoyl glycolate and N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzilcarbonyloxy glycolate were the most effective inhibitors of trypsin, plasmin, plasma kallikrien and thrombin, and they strongly inhibited the esterolytic activities of C1r- and C1 esterase.