Inter-alpha-trypsin inhibitor is a complex of three different protein species.

We found that, in contrast to earlier reports, inter-alpha-trypsin inhibitor of human serum is not a single-chain glycoprotein, but a protein complex. Three different protein components which are encoded on three independent mRNAs could be demonstrated by sequencing of cloned cDNA as well as of peptides obtained from purified inter-alpha-trypsin inhibitor. Accordingly, after fractionation of plasma by ammonium sulfate precipitation, ion-exchange chromatography, zinc-chelate chromatography, and SDS-polyacrylamide gel electrophoresis three proteins can be detected in Western blots using antibodies directed against inter-alpha-trypsin inhibitor.

Isolation and characterization of porcine alpha 1-proteinase inhibitor. Leukocyte elastase-inhibitor complexes in porcine blood, I.

alpha 1-Proteinase inhibitor was purified from porcine blood by ammonium sulphate and Cibachron Blue-Sepharose fractionation, ion exchange chromatography on DEAE-Cellulose, gel filtration on Sephadex G-25, and zinc chelating chromatography. Thus, an inhibitor preparation with a specific activity of 1.62 IU/mg protein (enzyme: trypsin; substrate: BzArgNan) was obtained. In sodium dodecyl sulphate gel electrophoresis one protein band corresponding to a molecular mass of 67.6 kDa was found. On isoelectric focusing 6 protein bands with isoelectric points of 3.80, 3.90, 4.05, 4.20, 4.25 and 4.45 were separated. The amino acid composition was determined. The association rate constants for the inhibition of various serine proteinases were measured.

Purification and properties of guinea-pig submandibular-gland kallikrein.

Guinea-pig submandibular kallikrein has been purified from the glands to electrophoretic homogeneity by conventional procedures. The enzyme is active as a kininogenase, releasing kallidin at a rate of 462 micrograms/min per mg of protein from bovine kininogen, and proved potently hypotensive in the guinea pig and in the dog, properties which indicate its tissue kallikrein nature. The specific activity determined on the substrate N-alpha-benzoyl-L-arginine ethyl ester (11.1 mumol/min per mg of protein) is much lower than that measured with N-acetyl-L-phenylalanyl-L-arginine ethyl ester (483 mumol/min per mg of protein). The latter value is of an order of magnitude comparable with the specific activities of other tissue kallikreins determined with this sensitive kallikrein substrate. The enzyme is a glycoprotein consisting of 237 amino acid residues and containing three to four glucosamine molecules. Its amino acid composition is not identical with that reported for guinea-pig coagulating-gland kallikrein, but is remarkably similar to that of the porcine tissue kallikreins. Apparent Mr values are 29000 (sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) or 34000 (gel filtration). The amino acid sequence of the first 31 N-terminal residues was determined and was found to be closely homologous with that of other tissue kallikreins.

Substrate specificity of porcine pancreatic kallikrein.

The primary specificity of porcine pancreatic kallikrein is directed predominantly against arginyl and much less so against lysyl bonds. In addition, the enzyme exhibits pronounced secondary specificity for a bulky residue, preferentially phenylalanine, in position P2 of substrates. This feature is found also in porcine submandibular and urinary and in human urinary kallikrein, but not in bovine trypsin. Residues in P3 and P1' and P1' to P3' also affect hydrolysis by pancreatic kallikrein distinctly more than tryptic hydrolysis. The hexapeptide Pro-Phe-Arg-Ser-Val-Gln with the sequence of bovine kininogen around the C-terminus of kinin contains all the structural elements essential for the interaction with kallikrein, and even glutamine appears dispensable. In contrast to ester models for this site, peptidyl methionine esters with the structure of kininogen towards the N-terminus of kinin, notably bulky leucine in P2, are very poor kallikrein substrates, and appear to be of no value as models for the cleavage of kininogen under formation of kallidin.

The isolation and properties of pig submandibular kallikrein.

The kallikrein from pig submandibular glands was highly purified, with an overall yield of 31%. Affinity chromatography on bovine basic pancreatic trypsin inhibitor linked to Sepharose 4B was an especially effective step in the purification procedure, giving a purification factor of 80. The enzyme is a single-chain molecule, occurring, as does pig urinary kallikrein, as a major B-form of apparent mol.wt. 39600 and minor amounts of an A-form of apparent mol.wt. 35900; the two forms can be separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The amino acid composition of pig submandibular kallikrein is very similar to, but not quite identical with, that of the two-chain beta-kallikrein isolated from pig pancreatic autolysates. Submandibular kallikrein contains notably more glucosamine and hexoses than does pancreatic beta-kallikrein. Submandibular kallikrein, and also urinary kallikrein, exhibit an unusual biphasic hydrolysis of substrate esters that is not shared by pancreatic beta-kallikrein. For the submandibular enzyme, the K(m) for the initial reaction phase of the hydrolysis of alpha-N-benzoyl-l-arginine ethyl ester is 0.15+/-0.01mm (mean+/-s.e.m.), but rises to 0.69+/-0.04mm (mean+/-s.e.m.) in the stationary reaction phase; the V(max.) does not differ significantly between the two phases. The esterolytic activities of submandibular and urinary kallikreins on a number of esters of different amino acids resemble each other much more closely than those of pancreatic beta-kallikrein.

Peptide esters and nitroanilides as substrates for the assay of human urinary kallikrein.

Ac-Phe-ArgOMe is hydrolyzed much faster than are Bz-ArgOEt, Z-ArgOMe, or Ac-Gly-ArgOMe by the kallikrein from human urine. The synthesis of Ac-Phe-ArgOEt is described. Hydrolysis of this substrate can be conveniently monitored by a coupled spectrophotometric procedure. Increase in absorbance is linear with time and proportional to the amount of kallikrein up to a deltaA366 of at least 0.22/10 min. This assay for human urinary kallikrein is 46-fold more sensitive than that based on Bz-ArgOEt and 38-fold more sensitive than that with D-Val-Leu-Arg-p-nitroanilide. A number of other arginine p-nitroanilides are hydrolyzed by this enzyme at still lower rates. The assay of human urinary kallikrein with D-Val-Leu-ArgOEt is about a factor of two less sensitive than the assay with Ac-Phe-ArgOEt. This also holds for Z-TyrONp, which displays a rapid spontaneous hydrolysis. Furthermore, the rate of the enzymic reaction with Z-TyrONp drops off rapidly.