Fluorescence polarization studies on the interaction of active site modified chymotrypsins with alpha1-protease inhibitor.

Fluorescence polarization has been used to study the interaction of human alpha1-protease inhibitor (alpha1 PI; also called alpha1-antitrypsin) with two active site modified chymotrypsins (CT), dehydroalaninyl-195-alpha-CT (AnhCT) and N-methylhistidinyl-57-alpha-CT (MeCT). For the reaction of the fluorescein-labeled AnhCT (FAnhCT) with alpha 1 PI (Pi type MM, the predominant allelic form), a Kassoc of 1.8 x 10(7) M-1 was obtained by Scatchard analysis, which also indicated 1.3 binding sites. An alternate analysis using a direct dissociation plot, which assumes 1:1 binding, gave a Kassoc of 2.2 x 10(7) M-1. Fluorescein-labeled MeCT (FMeCT) binds somewhat more weakly to alpha PT (Kassoc = 1.2 x 10(6) M-1; 0.87 binding site). Similar results were obtained by using the proflavin displacement method to determine the binding constant for MeCT with alpha 1 PI (Kassoc = 1.0 x 10(6) M-1). With alpha 1 PI (ZZ type) in which the serum level is reduced and there is a strong tendency to develop chronic obstructive pulmonary disease, the Kassoc found by the fluorescence polarization method was similar to that for alpha 1 PI (MM type) for both CT derivatives. Alpha 1 PI (MM type), modified by oxidation with N-chlorosuccinimide, shows a reduced binding affinity for FAnhCT (Kassoc = 6.5 x 10(5) M-1) and no measurable binding with FMeCT (Kassoc less than 1 x 10(4) M-1). Previous studies have demonstrated that bovine CT forms very stable complexes with alpha 1 PI. In contrast, complexes formed with both active site modified CT derivatives undergo rapid dissociation as shown by the drop in the polarization value on dilution or on the addition of excess unlabeled chymotrypsin derivative. This weakened association suggests that, for reaction with alpha 1 PI, the enzyme active site serine is important in stabilizing the enzyme-inhibitor complex.

Plasma immunoreactive pancreatic cationic trypsinogen in cystic fibrosis: a sensitive indicator of exocrine pancreatic dysfunction.

Plasma immunoreactive cationic trypsin(ogen) levels were determined in 32 control subjects and 43 patients with varying degrees of pancreatic insufficiency including 35 with cystic fibrosis (CF) and eight with Shwachman's syndrome. In six CF infants less than 2 years of age, plasma trypsin(ogen) levels were significantly elevated (97.3 +/- 62.2 ng/ml) above the normal range for nine controls (7.0 +/- 5.9 ng/ml; P less than 0.025). Four of these infants had steatorrhea, three of whom had undetectable duodenal trypsin activity after stimulation with secretin-cholecystokinin. In two CF infants, molecular size fractionation by gel filtration of plasma followed by radioimmunoassay of the column fractions demonstrated that trypsinogen was the only immunoreactive species in the circulation. In contrast, in older CF patients with steatorrhea (mean age, 15.3 +/- 4.6 years), plasma cationic trypsin(ogen) levels were undetectable or low (1.1 +/- 1.7 ng/ml). This finding clearly distinguished them from older CF patients without steatorrhea (mean age, 14.3 +/- 3.9 years) in whom cationic trypsin(ogen) levels were significantly higher (23.3 +/- 17.6 ng/ml; P less than 0.01). The mean trypsin(ogen) concentration in the older CF patients without steatorrhea did not differ from the mean value for 23 normal subjects of similar age. Plasma cationic trypsin(ogen) levels in two Schwachman's patients with steatorrhea (0.19 and 0.86 ng/ml) were significantly lower than the values found in six Shwachman's patients without steatorrhea (5.9 +/- 2.3 ng/ml; P less than 0.025). Furthermore, in nine older CF patients and eight Schwachman's patients, circulating trypsin(ogen) levels were highly correlated with duodenal trypsin output after secretin-cholecystokinin stimulation (r = 0.946, P less than 0.01; r = 0.899, P less than 0.01, respectively). These results suggest that in CF infants high levels of circulating trypsin(ogen) persist even in those with Shwachman's syndrome, however, circulating trypsin(ogen) accurately reflects residual pancreatic function.

Immunoreactive forms of cationic trypsin in plasma and ascitic fluid of dogs in experimental pancreatitis.

A canine model of bile-induced pancreatitis has been employed to investigate time-dependent changes in the molecular forms of trypsin in blood and ascitic fluid in this disease. The distribution of immunoreactive trypsin as trypsinogen and trypsin bound to plasma inhibitors in ascitic fluid and plasma during the course of the disease has been investigated by means of a radioimmunoassay for canine pancreatic cationic trypsin. In addition, trypsinlike amidase activity was determined in plasma and ascitic fluid using Z-Gly-Gly-Arg-beta-Nap as substrate. Early plasma and ascitic fluid samples in four dogs that died contained primarily trypsinogen, while extensive activation of trypsinogen to alpha 2-macroglobulin and alpha 1-protease inhibitor-bound trypsin occurred in the course of the disease. A fifth dog survived and showed little activation of trypsinogen. In the four dogs that died, the levels of trypsinlike amidase activity in the ascitic fluid were substantial throughout the course of the disease. The plasma levels of trypsinlike activity in these animals were much lower, but increased during the disease process. The dog that survived had lower concentrations of trypsinlike activity in ascitic fluid and plasma. These results suggest that activation of trypsinogen resulting in inhibitor-bound forms of trypsin in ascitic fluid and plasma is important in the pathogenesis of acute pancreatitis.

Clearance and metabolism of circulating pancreatic proelastase in the rat.

A rat model has been employed to study the mechanism of clearance of pancreatic proelastase from the circulation. The clearance of 125I-labeled proelastase was shown to be biphasic, with a half-life for clearance of free 25,000-dalton proelastase of approximately 7-10 min. A slow component of clearance possibly due to proelastase associated with plasma protease inhibitors was also observed. At 10 min after injection of 125I-labeled proelastase into the circulation, the major fraction of the 125I was found to be localized in the kidney. However, appearance of 125I in urine is slow, with 16 h being required for excretion of 50% of the injected 125I as acid-soluble material. Subcellular localization experiments on homogenates of kidneys removed at 10 min postinjection revealed that the majority of the 125I-labeled material was associated with the mitochondrial and lysosomal fraction. Sucrose-gradient centrifugation studies of this fraction demonstrated that the labeled material passes from the membrane fraction to the lysosomal fraction with time. These data demonstrate that the rat kidney constitutes the major site for both circulatory clearance and catabolism of circulating pancreatic proelastase.

Clearance of circulating anionic and cationic pancreatic trypsinogens in the rat.

The kinetics and mechanism of clearance of pancreatic cationic and anionic trypsinogens from the circulation have been investigated in a rat model. 125I-labeled rat cationic trypsinogen is cleared from the bloodstream with a half-life of approximately 4 min. In contrast, 125I-labeled rat anionic trypsinogen has a half-life in the circulation of approximately 55-60 min. The major site of clearance for both enzymes is the kidney. Neither zymogen binds to plasma proteins to a significant extent over a period of three half-lives. The relative rates of clearance of these zymogens from the circulation appears to correlate with their respective isoelectric points.

Interaction of chymotrypsinogens with alpha 1-protease inhibitor.

In a previous report [Largman, C., Brodrick, J.W., Geokas, M.C., Sischo, W.M., & Johnson, J.H. (1979) J. Biol. Chem. 254, 8516-8523] it was demonstrated that human proelastase 2 and alpha 1-protease inhibitor react slowly to form a complex that is stable to denaturation with sodium dodecyl sulfate and beta-mercaptoethanol and that the zymogen can be recovered from the isolated complex following dissociation by hydroxylamine. The present report demonstrates that bovine chymotrypsinogen A reacts with human alpha 1-protease inhibitor in a very similar manner. The rate of complex formation was measured by two methods. In the first, the reaction was followed by determining the loss of the inhibitory activity of alpha 1-protease inhibitor as a function of time. A second-order rate constant for complex formation formation (pH 7.6, 36 degrees C) of 12.9 +/- 2.4 M-1s-1 was obtained. In the second procedure, the reaction of fluorescein isothiocyanate labeled chymotrypsinogen A with alpha 1-protease inhibitor was measured by fluorescence polarization. A second-order rate constant (pH 7.6, 37 degrees C) of 13.9 +/- 2.1 M-1s-1 was obtained. The rate of complex formation is approximately 10(-5) of that measured for the reaction of bovine chymotrypsin with alpha 1-protease inhibitor. Dissociation of the complex was not observed after dilution or the addition of excess bovine alpha-chymotrypsin. As judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments, human chymotrypsinogens I and II react with alpha 1-protease inhibitor at rates that are approximatley equivalent to that determined for bovine chymotrypsinogen A. In contrast, bovine trypsinogen reacts very slowly with alpha 1-protease inhibitor, at a rate that is at most 10(-2) of that of bovine chymotrypsinogen A. These results suggest that zymogens react with alpha 1-protease inhibitor by virtue of partially formed active sites and that the potential active-site specificity of the zymogen in part determines the rate of complex formation.

Inhibition of human pancreatic elastase 2 by peptide chloromethyl ketones.

The inactivation of human pancreatic elastase 2 (EC 3.4.21.11) by a series of peptide chloromethyl ketones has been investigated. Among a series of compounds with the structure X-Ala-Ala-Pro-Y-CH2Cl (where X=acetyl-, succinyl-, methylsuccinyl-, or H-), the kinetic parametrs for inhibition of elastas 2 depend markedly on the amino acid (Y) in the P1 position. Succinyl-Ala-Ala-Pro-Leu-CH2Cl was found to be an extremely effective inhibitor of human elastase 2, qith a first-order rate constant for covalent bond formation (k3) of 0.033s-1 and a dissociation constant, Ki, for the enzyme inhibitor complex of 7.4 . 10(-7) M. The second-order rate constant k3/Ki for inhibition of elastase 2 by the analogous compound containing a free amino group in place of the succinyl moiety is 150 times lower than that found for the succinyl or acetyl derivative, suggesting that the presence of a positive charge at this position reduces the proper binding of the inhibitor to the enzyme.

Human pancreatic proelastase 2. Sequence of the activation peptide.

The N-terminal sixteen residues of the amino acid sequence of reduced and alkylated human pancreatic proelastase 2 have been established, and N-terminal amino acid residue has been shown to be carboxymethylcysteine. A peptide containing an amino acid sequence corresponding to the first twelve residues of proelastase 2 was isolated following activation of proelastase 2 with trypsin, performic acid oxidation, and gel filtration. This peptide was not released prior to performic acid oxidation, suggesting that it remains attached to the major peptide chain via a disulfide bond containing the N-terminal half-cysteine in a manner similar to that found for chymotrypsinogens. However, the amino acid sequence of the activation peptide is not strongly homologous to either porcine chymotrypsinogen A or porcine proelastase 1.

Demonstration of a pancreatic proelastase 2-alpha 1-protease inhibitor complex in normal human plasma.

A peak of immunoreactive pancreatic elastase 2 with a molecular weight consistent with that of a complex of elastase 2 and alpha 1-protease inhibitor (also referred to as alpha 1-antitrypsin) can be detected by radioimmunoassay in normal human serum or plasma (Geokas et al., J. Biol. Chem. 252:61-67, 1977). This material has been purified by gel filtration on Sephadex G-200 and by ion-exchange chromatography on DEAE-cellulose. The alpha 1-protease inhibitor-bound immunoreactive elastase 2 has been dissociated by incubation with hydroxylamine, and the resulting immunoreactive product isolated by gel filtration on Sephadex G-100. The dissociated immunoreactive elastase 2 was shown by affinity chromatography on turkey egg white inhibitor-bound agarose, before and after activation by bovine trypsin, to consist only of proelastase 2. A second peak of immunoreactive material associated with the high molecular weight fraction of plasma has been shown to result from a specific interaction of the 125I-labeled phenylmethanesulfonyl-elastase 2 employed as tracer in the radioimmunoassay with alpha 2-macroglobulin, resulting in apparent immunoreactivity. These results demonstrate that all of the detectable immunoreactive pancreatic elastase 2 in normal human plasma is proelastase 2 bound to alpha 1-protease inhibitor.

Substrate specificity of human pancreatic elastase 2.

The substrate specificity of human pancreatic elastase 2 was investigated by using a series of peptide p-nitroanilides. The kinetic constants, kcat and Km, for the hydrolysis of these peptides revealed that this serine protease preferentially hydrolyzes peptides containing P1 amino acids which have medium to large hydrophobic side chains, except for those which are disubstituted on the first carbon of the side chain. Thus, human pancreatic elastase 2 appears to be similar in peptide bond specificity to the recently described porcine pancreatic elastase 2 [Gertler, A., Weiss, Y., & Burstein, Y. (1977) Biochemistry 16, 2709] but differs significantly in specificity from porcine elastase 1. The best substrates for human pancreatic elastase 2 were glutaryl-Ala-Ala-Pro-Leu-p nitroanilide and succinyl-Ala-Ala-Pro-Met-p-nitroanilide. However, there was little difference among substrates with leucine, methionine, phenylalanine, tyrosine, norvaline, or norleucine in the P1 position. Changes in the hydrolysis rate of peptides with differing P5 residues indicate that this enzyme has an extended binding site which interacts with at least five residues of peptide substrates. The overall catalytic efficiency of human pancreatic elastase 2 is significantly lower than that of porcine elastase 1 or bovine chymotrypsin with the compounds studied.

Molecular forms of immunoreactive pancreatic cationic trypsin in pancreatitis patient sera.

The molecular forms of immunoreactive pancreatic cationic trypsin in sera of patients with acute pancreatic inflammation have been characterized using a radioimmunoassay technique that is capable of detecting trypsinogen as well as trypsin bound to alpha 1-antitrypsin. Trypsin bound to alpha 2-macroglobulin is not immunoreactive under normal assay conditions. However, alpha 2-macroglobulin-bound trypsin can be detected after gel filtration of serum on Bio-Gel A-0.5 m and acid treatment of column fractions. The average serum level of immunoreactive cationic trypsin from 20 patients with acute pancreatic inflammation was 1,590 ng/ml. An average normal value of 26 ng/ml has been obtained previously. Serum samples from 14 patients with pancreatic inflammation were chromatographed under conditions that resolve trypsinogen, alpha 1-antitrypsin-bound trypsin, and alpha 2-macroglobulin-bound trypsin. In each case, the major portion of the immunoreactive material eluted at a position corresponding to free trypsinogen, while a minor fraction of the immunoreactive material appeared to be trypsin bound to alpha 1-antitrypsin. The zymogen nature of the major peak was confirmed in one case by activation with human enteropeptidase. In 11 of 14 patients, acid treatment of the alpha 2-macroglobulin peak yielded immunoreactive trypsin.

Determination of human pancreatic cationic trypsinogen in serum by radioimmunoassay.

A specific radioimmunoassay has been developed for human pancreatic cationic trypsin. The assay has been employed for the determination of immunoreactive forms of pancreatic cationic trypsin in blood. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone (TLCK) to prevent binding of the tracer to the serum inhibitors while maintaining its immunoreactivity. The average normal serum level determined was 26 ng/ml, with a range of 12--41 ng/ml. Eight of nine patients with acute pancreatic inflammation had at least a 15-fold elevation of total serum immunoreactive cationic trypsin. Cationic trypsinogen and cationic trypsin bound to alpha1-antitrypsin cross-react strongly in the radioimmunoassay. Thus it is possible to measure these potential molecular forms of cationic trypsin in serum. When normal human serum was fractionated on Sephadex G-200, all of the immunoreactive material eluted as a single peak of approximately 23,000 mol wt. No cationic trypsin could be detected in association with alpha1-antitrypsin or alpha2-macroglobulin. The 23,000-mol-wt peak was definitively shown to contain trypsinogen by affinity chromatography and by activation with human enteropeptidase. The identification of cationic trypsinogen in blood implies that the zymogen is secreted into the circulation by the pancreas rather than entering the bloodstream via absorption from the intestine.

Demonstration of human pancreatic anionic trypsinogen in normal serum by radioimmunoassay.

A specific radioimmunoassay for human pancreatic anionic trypsin has been developed. The trypsin employed as radioiodinated tracer in the assay was inactivated with tosyl-L-lysine chloromethyl ketone in order to prevent binding of the tracer to the serum inhibitors alpha1-antitrypsin and alpha2-macroglobulin. A normal serum level of immunoreactive anionic trypsin of 5.45 ng/ml was determined. The results of experiments in which serum was fractionated by Sephadex G-200 gel filtration suggest that essentially all of the immunoreactive material in normal human serum is trypsinogen. This finding implies that a small fraction of the zymogens synthesized in the pancreas are released directly into the circulation.