enzyme-inhibitor complex can be dissociated in alkaline solutions, with the release of a large fragment of a-1-antitrypsin which has an amino-terminal threonine residue and a molecular weight between 46 000 and 50 000, and a small peptide. During this base-catalyzed hydrolysis, a single new carboxyl-terminal lysine residue is formed. This residue must be the carboxyl-terminal residue of the amino-terminal peptide of a-1-antitrypsin. If the trypsin inhibitor, phenylmethanesulfonyl fluoride, is not added, catalytically active trypsin can be measured after a-l-antitrypsin-trypsin complexes are dissociated at high pH. These findings support the hypothesis that trypsin reacts with a-1-antitrypsin at a Lys-Thr bond and that no peptides of the reactants have been lost from the intact complex. Available evidence suggests that the a-l-antitrypsin-trypsin complex is analogous to the tetrahedral or acyl intermediates which form only transiently between trypsin and its substrates.Attempts to elucidate the amino acids at the inhibitory site in a-1-antitrypsin by modification of specific amino acids have produced ambiguous results. Reagents which are reported to a-1-ANTITRYPSININTERACTION WITH TRYPSIN VOL.1 7, no. 3, 19 7 8 393 supports an alternative hypothesis which explains all of the existing data.

a-l-Antitrypsin is a serum protein that inhibits many proteolytic enzymes. Recently, it was suggested that the a-l-antitrypsin-trypsin complex is an acyl ester analogous to the acyl intermediate that forms between trypsin and its substrates. In previous work we showed that the a-l-antitrypsinsin complex can be split at high pH, releasing a component of a-l-antitrypsin. This component had a new carboxyl-terminal lysine, and it had lost a peptide of about 4000 daltons. In order to determine whether the a-l-antitrypsin is bound to trypsin through the new carboxy-terminal lysine, as would be expected if the above hypothesis is correct, we split the complex in the presence of 180H. When the new carboxy-terminallysine was cleaved with carboxypeptidase B, singly labeled, doubly labeled, and unlabeled lysine were recovered. These data support the hypothesis that the a-l-antitrypsin-trypsin complex is an acyl ester or a tetrahedral precursor that is transformed into the acyl ester form at high pH. If other enzymes are bound by a similar mechanism, the methods used may be useful in determining which amino acids on a-l-antitrypsin bind covalently to each enzyme.Serine proteases cleave proteins by binding to the amino acid for which they are specific and forming a series of intermediates including a tetrahedral adduct, an acyl ester, and, finally, two fragments of the protein and the free enzyme (1-4).On the basis of many structural studies of protein inhibitors of proteolytic enzymes, Bode and his colleagues (5) concluded that the complexes that have been studied by x-ray crystallography are intermediates in peptide hydrolysis (6, 7). Soybean trypsin inhibitor is much larger than pancreatic trypsin inhibitor and folds differently. However, analysis of the complex between porcine trypsin and soybean trypsin inhibitor showed a closely similar conformation of the contact segment. A tetrahedral adduct formed in both cases. Studies of the interaction of trypsin with a-l-antitrypsin, a human serum protein, have suggested that the catalytic site of the enzyme interacts with the active site of this protease inhibitor (8). Moroi and Yamasaki (9) found that the enzyme-inhibitor complex had a lower molecular weight than the constituent reactants, that a-iantitrypsin released from the complex by hydrazine had lost a peptide, and that the amino-terminal residues of the complex were identical to those of trypsin and a-l-antitrypsin, indicating that a-l-antitrypsin had lost a peptide from its carboxyl end. The findings suggest that a-1-antitrypsin is bound to trypsin through an acyl ester bond. Characteristics of this type of bond have been studied with low molecular weight acyl esters.Alkaline hydrolysis of carboxyl esters in aqueous solution is known to follow second-order kinetics and to involve acyloxygen fission (11). Alkaline hydrolysis of n-amyl acetate in solvent water enriched in 180 showed that the oxygen from the medium does not appear in the alcohol produced but resides in the acid (12) and, therefore, that the mec...

A B S T R A C T Neutrophil turnover was studied in the blood and alveoli of normal rabbits. Blood neutrophil turnover was examined by two different methods. In the first method, donor rabbit neutrophils were labeled in vivo by injecting tritium-labeled thymidine intravenously. After 72 h recipient rabbits received blood from the donors. The decline of the specific radioactivity of blood neutrophils was used to determine that their half-life was 4.03 h. In the second method, rabbit peritoneal exudative neutrophils were elicited with oyster glycogen. These cells were labeled with "'Indium oxine and infused into the blood of recipient animals. By their decline in specific radioactivity, the half-life of the blood neutrophil was 4.08 h. These halflives are not significantly different.Lung lavage was performed on the animals that received the "'Indium-labeled neutrophils and the turnover time of the lung neutrophil was found to be 2.63 h. The turnover of the alveolar neutrophil pool accounted for only 0.19% of the total turnover of the blood neutrophils. Therefore, the lung appears to contribute only minimally to the total capacity of the body to dispose of neutrophils.

No abstract