Interaction between human pancreatic elastase and plasma protease inhibitors

1 ml of human serum inhibits about 0.9 mg of purified human pancreatic elastase owing to complexation with alpha 1-antitrypsin and alpha 2-macroglobulin. On addition to serum, elastase is preferentially bound by alpha 2-macroglobulin. The complexes between elastase and alpha 1-antitrypsin and alpha 2-macroglobulin, respectively, migrate as alpha 2-globulin on agarose gel electrophoresis. Elastase bound by alpha 1-antitrypsin is precipitated by antibodies against enzyme as well as inhibitor, while the alpha 2-macroglobulin-bound elastase is only precipitated by antibodies against the inhibitor. The molar combining ratio for elastase/alpha 1-antitrypsin is 1:1 and for elastase/alpha 2-macroglobulin 2:1. The elastase bound by alpha 2-macroglobulin retains its activity against low molecular weight substrates, while that bound by alpha 1-antitrypsin is enzymologically inactive.     

Serum elastase 1 and immunoreactive trypsin in chronic pancreatic disease: is there any relationship with trypsin inhibitors?

In order to investigate modifications of serum levels of elastase 1, immunoreactive trypsin, alpha 1-antitrypsin and alpha 2-macroglobulin in chronic pancreatic disease, and to speculate on the possible relationships among these parameters, the enzymes and inhibitors were assayed in the sera of 33 control subjects, 34 pancreatic cancer, 28 chronic pancreatitis and 36 extra-pancreatic diseases. An increase of elastase 1, alpha 1-antitrypsin and alpha 2-macroglobulin was detected in pancreatic cancer, chronic pancreatitis and extra-pancreatic diseases; no changes were found for serum immunoreactive trypsin. Multiple regression analyses showed that only 7% of elastase 1 was explained by inhibitors with alpha 1-antitrypsin playing a major role. Inhibitors did not influence immunoreactive trypsin. Our data indicate that the variations of the serum levels of proteases and antiproteases in chronic pancreatic disease are probably independent of each other.     

The in vitro interactions of rat pancreatic elastase and normal and inflammatory ray serum.

The partition of labelled rat pancreatic elastase (EC 3.4.21.11) between the different protease inhibitors of rat plasma was studied at different levels of saturation of the inhibitors of rat plasma was studied at different levels of saturation of the inhibitor capacity of plasma with the enzyme. The reaction mixtures were analysed by immunoelectrophoretic methods utilizing specific antisera against the different inhibitors and by gel filtration on Sephadex G-200. Rat serum was shown to contain four elastase binding proteins. alpha 1-antitrypsin, alpha 1-macroglobulin and alpha 2-acute phase protein and alpha 1-inhibitor 3 which exhibits immunologic cross-reaction with human inter-alpha-trypsin inhibitor and is of similar molecular weight. With minute amounts of labelled elastase the partition among the binding protein was alpha 1-macroglobulin 60%, alpha 1-antitrypsin 24% and alpha 1-I3 16%. The 60% value of alpha 1-M bound radioactivity in normal serum corresponds to the sum of alpha 1-M and alpha 2-AP labelling in inflammatory serum.     

Retroviral mediated transfer and expression of human alpha 1-antitrypsin in cultured cells

Genetic deficiency of alpha 1-antitrypsin in man is a predisposing factor to emphysema and a disorder potentially correctable by somatic gene therapy. A full-length human alpha 1-antitrypsin cDNA was cloned into a retroviral vector and introduced into cells which package the recombinant gene in a retroviral capsule. Cells infected with the recombinant retrovirus express human alpha 1-antitrypsin mRNA and protein. The recombinant protein is glycosylated, secreted and exhibits anti-protease activity against human neutrophil elastase.     

In vitro interaction of porcine serum and colostrum protease inhibitors with pancreatic trypsin, chymotrypsin and elastase

The partition of 125I-labelled pancreatic trypsin, chymotrypsin and elastase between the inhibitors, alpha 2-macroglobulin f and s, alpha 1-protease inhibitor, alpha 2-antitrypsin, inter-alpha-trypsin inhibitor and the specific sow colostrum protease inhibitor, was studied in vitro by gradually increasing the concentration of these proteases in blood serum from adult and newborn pigs. As revealed by immunoelectrophoresis in combination with autoradiography, differences were noted in the abilities of the various protease inhibitors to interact with and to form complexes with the three proteases, resulting in changes in location, height and numbers of precipitates. Among the serum inhibitors, alpha 2-macroglobulins showed the highest relative affinity to all three proteases, while alpha 1-protease inhibitor showed a high relative affinity only for chymotrypsin. Serum alpha 2-antitrypsin complexed only with trypsin, with a low relative affinity. alpha 2-Antitrypsin also interacted with chymotrypsin and elastase, but without forming complexes. When complexes of sow colostrum protease inhibitor and trypsin were added to the serum from neonatal pigs, these complexes remained stable. The results obtained from these in vitro studies, indicating differences in the relative affinities of the inhibitors to the various proteases, give some information about the role of the inhibitors in vivo, both in adult and in neonatal pigs.     

Photoreduction and incorporation of iron into ferritins.

The characteristics of a new kallikrein-binding protein in human serum and its activities were studied. Both the kallikrein-binding protein and alpha 1-antitrypsin form 92 kDa SDS-stable and heat-stable complexes with human tissue kallikrein. In non-SDS/PAGE, the mobility of these complexes differ. Complex-formation between kallikrein and the binding protein is inhibited by heparin, whereas that between kallikrein and alpha 1-antitrypsin is heparin-resistant. In normal or alpha 1-antitrypsin-deficient-serum, the amount of 92 kDa SDS-stable complex formed upon addition of kallikrein is not related to serum alpha 1-antitrypsin levels. The rate of complex-formation between kallikrein and the binding protein is 12 times higher than that between kallikrein and alpha 1-antitrypsin. Purified alpha 1-antitrypsin, which exhibits normal elastase binding, has a kallikrein-binding activity less than 5% of that of serum. Binding of tissue kallikrein in serum is not inhibited by increasing elastase concentrations, and elastase binding in serum is not inhibited by excess tissue kallikrein. A specific monoclonal antibody to human alpha 1-antitrypsin does not bind to either 92 kDa endogenous or exogenous kallikrein complexes isolated from human serum. The studies demonstrate a new tissue kallikrein-binding protein, distinct from alpha 1-antitrypsin, is present in human serum.     

Kinetics of association of human proteinases with human alpha 2-macroglobulin

Association rates have been determined for the interaction of human alpha 2-macroglobulin with human neutrophil elastase, cathepsin G, and human plasma kallikrein. Both of the neutrophil enzymes are rapidly inactivated by this inhibitor; however, the inactivation of plasma kallikrein is much slower. Comparison of the rates of inactivation with those already established for other inhibitors clearly indicate that alpha 1-proteinase inhibitor is the controlling inhibitor for neutrophil elastase and alpha 1-antichymotrypsin for cathepsin G, alpha 2-macroglobulin acting only as a secondary inhibitor. The control of plasma kallikrein would appear to be rather poor since neither alpha 2-macroglobulin nor C1-inhibitor appears to react very rapidly with this proteinase. Thus, a primary role for alpha 2-macroglobulin in directly inactivating proteinases in blood, under normal physiological conditions, remains to be established.     

Different susceptibility of elastase inhibitors to inactivation by proteinases from Staphylococcus aureus and Pseudomonas aeruginosa.

Neutrophil elastase is thought to contribute to the lung pathology in patients with cystic fibrosis (CF). Therefore, intrapulmonary application of elastase inhibitors might be beneficial for these patients. Inactivation of such inhibitors by bacterial proteinases, however, is an important consideration in this therapy. We studied the effects of Staphylococcus aureus proteinase (STAP) and Pseudomonas aeruginosa elastase (PsE) on native (alpha 1-AT) and recombinant (rAAT) alpha 1-antitrypsin, recombinant secretory leukocyte proteinase inhibitor (rSLPI) and the leech inhibitor eglin C. All inhibitors were inactivated by these bacterial proteinases showing pronounced differences in their susceptibilities to proteolytic cleavage. Comparing the turnover rate (mol of inhibitor inactivated by one mol bacterial proteinase/min), rAAT and alpha 1-AT were approximately 20,000-fold more susceptible to STAP than rSLPI and 50,000-fold more susceptible than eglin C. Pseudomonas aeruginosa elastase inactivated all inhibitors more rapidly than STAP. rAAT and alpha 1-AT were 13-fold and 17,000-fold more susceptible than rSLPI and eglin C, respectively. Incubation of the rAAT-elastase complex with equimolar amounts of STAP did not result in release of elastase activity. Upon simultaneous addition of STAP and leukocyte elastase to rAAT, there was undisturbed elastase inhibition indicating that complex formation with elastase proceeded at a faster rate than inactivation of rAAT by the bacterial proteinase. From these results of inactivation in vitro and considering the immunogenic potential of the inhibitors studied here, we conclude that rSLPI may be the appropriate choice for anti-elastase therapy in CF.     

Interactions in vitro and in vivo between human and porcine cationic pancreatic elastase and plasma protease inhibitors

Trace amounts of porcine pancreatic elastase mixed with porcine serum, or injected intravenously into the pig, were found to be bound mainly to alpha 1- and alpha 2-macroglobulin (90%). Alpha 1-macroglobulin approached saturation with elastase before significant binding to alpha 2-macroglobulin was demonstrable. Human pancreatic cationic elastase showed in human serum preferential binding to alpha 2-macroglobulin, but the elastase was also bound by alpha 1-protease inhibitor and by alpha 1-antichymotrypsin. The porcine elastase-alpha 1 alpha 2-macroglobulin complexes injected intravenously or formed in vivo in the pig were rapidly eliminated from the blood stream following a first order reaction with t 1/2 = 8 min. Porcine alpha 1-protease-inhibitor-bound elastase disappeared considerably more slowly.     

Antiproteinase activity of alpha 2-macroglobulin

Blood serum separation by the method of gel filtration on Sephadex G-200 with the subsequent immunochemical determination of the quantitative content of basic proteolysis inhibitors permitted isolating the alpha 2-macroglobulin fraction while alpha 1-antitrypsin and alpha 1-antichymotrypsin separation was a failure. The immunochemical analysis of the antienzymic activity of the isolated inhibitors showed that 32.3 +/- 3.5% of the introduced kallikrein, 18.7 +/- 0.6% of trypsin and 14.4 +/- 4.1% of chymotrypsin were bound in the zone of alpha 2-macroglobulin. The rest of antienzymic activity was localized in the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin. After a preliminary saturation of blood serum with trypsin in the amount equivalent to its antitryptic capacity (200 micrograms/ml) the ability of alpha 2-macroglobulin to bind kallikrein and chymotrypsin lowers considerably (by 69 and 72%, respectively). In the zone of alpha 1-antitrypsin and alpha 1-antichymotrypsin a decrease in the ability to bind kallikrein and chymotrypsin amounted to 44 and 12% respectively. Thus, alpha 2-macroglobulin being bound with trypsin looses considerably its ability to bind other enzymes.     

Levels of alpha 1-antitrypsin and alpha 2-macroglobulin in blood serum and its antitryptic capacity

Immunochemical analysis in combination with gel filtration and isoelectric focusing made it possible to state that in blood serum of healthy people 81.3 +/- 0.5% of administered trypsin is bound with alpha 1-antitrypsin and 18.7 +/- 0.6%--with alpha 2-macroglobulin. The latter is functionally heterogeneous, only 40% of it is bound with trypsin and in the formed complex the antigenic properties of trypsin and alpha 2-macroglobulin are lost. A great number of blood serum alpha 1-antitrypsin cannot fix trypsin. The content of such alpha 1-antitrypsin rises sharply with pathology available. In the immunochemical estimation of the organism inhibitory potential relative to proteolytic enzymes not only the amount of the inhibitor but also its functional activity should be taken into account. The data of immunochemical research of the blood serum isoelectrophoregrams show that the most considerable changes under conditions of pathology occur in alpha 2-macroglobulin.     

Identification and characterization of trypsin, chymotrypsin and elastase inhibitors in porcine serum.

Characterization of the trypsin-, chymotrypsin- and elastase-inhibiting properties of porcine serum was carried out by gel filtration on Ultrogel, AcA 44, and agarose gel electrophoresis with subsequent processing for protease-inhibiting activity. Moreover, by allowing the fractions obtained from gel filtration to react with antibodies to porcine serum protease inhibitors, the specific inhibiting properties of these inhibitor molecules were identified. At least six protease inhibitors were identified and partially characterized in porcine serum. Two alpha 2 -macroglobulins (alpha 2 Mf and alpha 2 Ms), homologues to human alpha 2 -macroglobulin, with slightly different electrophoretic mobilities, were both found to exhibit trypsin, chymotrypsin and elastase inhibiting activity. Alpha 1 -Protease inhibitor (Mr 51 000), a homologue to human alpha 1 -protease inhibitor (alpha 1 -antitrypsin), also showed trypsin-, chymotrypsin- and elastase-inhibiting properties. Inter-alpha-trypsin inhibitor (Mr 162 000 and 129000), a porcine serum counterpart to human inter-alpha-trypsin inhibitor, showed trypsin- and chymo-trypsin-inhibiting properties. In addition, a specific trypsin inhibitor, alpha 2 -antigrypsin (Mr 58 000), and a specific elastase inhibitor, beta-elastase inhibitor, were characterized in porcine serum, and these seem to have no counterparts in human serum.     

Interactions in vitro and in vivo between rat serum protease inhibitors and anodal and cathodal rat trypsin and chymotrypsin.

Reaction mixtures of increasing amounts of the pancreatic homologous proteases, anodal and cathodal chymotrypsin and trypsin, respectively, and normal rat serum were analyzed by immunoelectrophoretic methods in order to determine their distribution on serum protease inhibitors. This paper concerns three proteins occurring in normal serum and capable of binding protease viz. alpha1-macroglobulin, alpha1-antitrypsin and alpha1-inhibitor 3. The distribution of the enzymes among these protease inhibitors differed significantly from one protease to another. The distribution of the proteases among the serum protease inhibitors following intravenous injection of 125I-labelled proteases corresponded to that in vitro. Complexes formed with alpha1-macroglobulin and alpha1-inhibitor 3 were quickly eliminated irrespective of the enzyme species used, whereas those formed with alpha1-antitrypsin persisted much longer in the circulation.     

Inhibition of human factor Xa by various plasma protease inhibitors

The inhibitory effects of the plasma protease inhibitors antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin on the activity of human factor Xa have been studied using purified proteins. The rate of inhibition was determined by measuring the residual factor Xa activity at timed intervals utilizing the synthetic peptide susbtrate Bz-Ile-Glu(piperidyl)-Gly-Arg-pNA. Kinetic analysis with varying molar concentrations of inhibitors demonstrated that the inhibition of factor Xa by antithromin III, alpha 2-macroglobulin and alpha 1-antitrypsin followed second-order kinetics. Calculated values of the rate constants for the inhibition of factor Xa by antithrombin III, alpha 2-macroglobulin and alpha 1-antitrypsin were 5.8 . 10(4), 4.00 . 10(4) and 1.36 . 10(4) M -1 . min -1, respectively. The plasma concentrations of the inhibitors can be used to assess their potential relative effectiveness against factor Xa. In plasma this was found as alpha 1-antitrypsin greater than antithrombin III greater than alpha 2-macroglobulin in the ratio 4.64: 2.08: 1.0. Cephalin was shown to inhibit the rate of reaction between factor Xa and antithrombin III.     

Differential effects of flavonols on inactivation of alpha1-antitrypsin induced by hypohalous acids and the myeloperoxidase-hydrogen peroxide-halide system

Alpha1-antitrypsin is well known for its ability to inhibit human neutrophil elastase. Pretreatment of alpha1-antitrypsin with hypohalous acids HOCl and HOBr as well as with the myeloperoxidase-hydrogen peroxide-chloride (or bromide) system inactivated this proteinase. The flavonols rutin, quercetin, myricetin, and kaempferol inhibited the inactivation of alpha1-antitrypsin by HOCl and HOBr with rutin having the most pronounced effect. In contrast, these flavonols did not remove the proteinase inactivation by the myeloperoxidase-hydrogen peroxide-halide system. Taurine did not protect against the inactivation of alpha1-antitrypsin by HOCl, HOBr, or the myeloperoxidase-hydrogen peroxide-halide system, while methionine was efficient in all systems. A close association between myeloperoxidase and alpha1-antitrypsin was revealed by native gel electrophoresis and in-gel peroxidase staining. In addition, alpha1-antitrypsin binds to the myeloperoxidase components transferred after SDS-PAGE on a blotting membrane. With this complex formation, myeloperoxidase overcomes the natural antioxidative protective system of plasma and prevents the inactivation of alpha1-antitrypsin.     

Protein abnormalities in adult respiratory distress syndrome, tuberculosis, and cystic fibrosis sera

Crossed immunoelectrophoresis (X-IEP) revealed several abnormalities in serum proteins from patients with adult respiratory distress syndrome (ARDS), tuberculosis (TB), and cystic fibrosis (CF). The two quite different kinds of pulmonary disease, one acute (ARDS) and the other chronic (TB and CF) exhibited serum changes specific for each disease and abnormalities associated with inflammation and pathogenesis, in general. In ARDS sera, most proteins were extremely low, presumably due to leakage into the lungs through damaged tissue, while the acute-phase proteins, orosomucoid, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, were markedly high when compared to the overall protein pattern. The extremely high alpha 1-antichymotrypsin values were not seen in corresponding TB and CF sera. Numerous TB patients had elevated alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, but only the alpha 1-antitrypsin population mean was significantly different from normal. Gc-globulin, ceruloplasmin, and beta-lipoprotein were higher and alpha 1-lipoprotein and inter-alpha-trypsin inhibitor lower than normal. All other quantitative serum changes were not statistically significant. Surprisingly, all TB patients belonged to the Gc-1-1 genotype in contrast to the Gc-1-1, Gc-1-2, Gc-2-2 polymorphisms of the other populations. CF homozygote sera revealed statistically significant increases in the acute-phase proteins, alpha 1-antitrypsin, alpha 1-antichymotrypsin, and haptoglobin, while orosomucoid, transferrin, IgA, and IgG tended to be higher than normal. The tendency for higher levels of transferrin indicated possible iron deficiency in some patients. In contrast, prealbumin, alpha 1-lipoprotein, and inter-alpha-trypsin inhibitor were significantly depressed in CF patients. CF heterozygotes shared the decrease of alpha 1-lipoprotein with the patients while exhibiting small but significant depressions of alpha 2-macroglobulin and IgG. Though not statistically significant, lowered concentrations of alpha 1-antitrypsin were evident for the heterozygotes.     

The alpha 1-antitrypsin gene and its deficiency states

alpha 1-antitrypsin, a 52 kDa antiprotease, provides the major defense to the lower respiratory tract against the ravages of neutrophil elastase, a powerful serine protease. A variety of mutations in the coding exons of the alpha 1-antitrypsin gene result in 'alpha 1-antitrypsin deficiency', leading to emphysema at an early age. A subset of mutations cause liver disease and a rare mutation is associated with a bleeding diathesis. Preventive treatment for the emphysema associated with alpha 1-antitrypsin deficiency is available in the form of intermittent infusions with alpha 1-antitrypsin, and strategies have been developed to reverse the deficiency state with gene therapy.     

Hormonal regulation of serum alpha 1-antitrypsin and hepatic alpha 1-antitrypsin mRNA in rats

We evaluated the effects of pituitary dependent hormones on alpha 1-antitrypsin in male rats. Hepatic alpha 1-antitrypsin mRNA was measured by in vitro translation and by specific hybridization with a mouse cDNA alpha 1-antitrypsin probe. Hypophysectomy caused a 50-75% decrease in serum elastase inhibitory capacity (measuring functional alpha 1-antitrypsin) and hepatic alpha 1-antitrypsin mRNA content. In hypophysectomized animals, no increase in elastase inhibitory capacity or alpha 1-antitrypsin mRNA levels by translation was found when met-human growth hormone alone or corticosterone, dihydrotestosterone and thyroxine were given together. Growth hormone increased alpha 1-antitrypsin mRNA by hybridization to a small extent. Addition of growth hormone to the combination of corticosterone, dihydrotestosterone, and thyroxine increased serum elastase inhibitory capacity and alpha 1-antitrypsin mRNA. We conclude that growth hormone acts synergistically with the other pituitary dependent hormones to regulate serum and hepatic mRNA levels of alpha 1-antitrypsin.     

Comparison of the inhibition of thrombin by three plasma protease inhibitors.

Human alpha-thrombin is inhibited by the circulating protease inhibitors alpha1-antitrypsin, antithrombin III, and alpha2-macroglobulin. Kinetic analyses of the inhibitor thrombin interactions were carried out utilizing either fibrinogen or the synthetic substrate Bz-Phe-Val-Arg-p-nitroanilide as substrates to determine residual thrombin activity. These studies demonstrated that the inhibition of thrombin by alpha1-antitrypsin, antithrombin III, and alpha2-macroglobulin followed second-order kinetics. The rate constants for the inhibition of thrombin by alpha1-antitrypsin, antithrombin III, and alpha2-macroglobulin are 6.51 +/- 0.38 x 10(3), 3.36 +/- 0.34 x 10(5), and 2.93 +/- 0.02 x 10(4) M-1 min-1, respectively. Comparison of the second-order rate constants and the normal plasma levels of the three inhibitors demonstrates that, under the in vitro conditions utilized, antithrombin III is five times and alpha2-macroglobulin is one-third as effective as alpha1-antitrypsin in the inhibition of thrombin.     

Detection of inhibitory inactive fraction of alpha-1-antitrypsin in human serum

Pseudomonas aeruginosa elastase is not inhibited by human serum alpha-1-antitrypsin, it damages inhibitory activity of alpha-1-antitrypsin activity instead. A simple method, based on electrophoresis in urea containing polyacrylamide gel, is described for the separation of active part of the inhibitor from that inactivated by the bacterial enzyme.