Interactions of the complex between human urinary trypsin inhibitor and human leukocyte elastase with alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The urinary trypsin inhibitor was recently shown to inhibit human leukocyte elastase. Complexes of human urinary trypsin inhibitor with human leukocyte elastase or human trypsin were produced and subjected to gel filtration. The complexes were found to be sufficiently stable up to 24 h incubation (at least 70% recovery). When human serum was added, elastase and trypsin dissociated from the urinary trypsin inhibitor and associated with alpha 1-proteinase inhibitor or alpha 2-macroglobulin. The addition of alpha 1-proteinase inhibitor to a complex of urinary trypsin inhibitor and leukocyte elastase caused a rapid dissociation of the complex (kdiss = 3.2 X 10(-2) s-1).     

Purification and some properties of a trypsin inhibitor from carp (Cyprinus carpio) muscle

A trypsin inhibitor was purified from carp muscle to apparent homogeneity by the successive chromatographies of DEAE-cellulose, DEAE-Sepharose CL-6B, Con A-Sepharose, Ultrogel AcA 44 and hydroxylapatite. The mol. wt of the inhibitor was estimated to be 58,000 by SDS-polyacrylamide gel electrophoresis or 50,000 by gel filtration. The inhibitor seemed to form a 1:1 stoichiometric complex with trypsin, alpha-chymotrypsin and elastase, respectively. Carp muscle trypsin inhibitor was likely to be identical with serum alpha 1-proteinase inhibitor judging from its glycoprotein nature, mol. wt and the inhibition stoichiometry.     

Inactivation of human thrombin in the presence of human alpha1-proteinase inhibitor.

Both the clotting and esterase activities of thrombin are inhibited by alpha1-proteinase inhibitor (alpha1-antitrypsin). The inhibition is a time-and temperature-dependent reaction which is proportional to the molar ratio of thrombin to inhibitor. Both the active-site serine residue of thrombin and the reactive-site lysine residue of alpha1-proteinase inhibitor are involved. alpha1-Proteinase inhibitor forms a 1:1 complex with thrombin that is comparable with the complex formed with trypsin and other proteinases. Incubation of the inhibitor with excess of thrombin, however, results in inactivation of nearly all the enzyme, even though only as much complex is formed as alpha1-proteinase inhibitor present. A portion of the remaining thrombin apparently aggregates. These results suggest that the mechanism for inhibition of thrombin may not be exactly the same as for trypsin, which is inhibited only to the extent to which complex is formed.     

Stoichiometry of the interaction of human plasma alpha 1-proteinase inhibitor with subtilisin BPN'

Interaction of human plasma alpha 1-proteinase inhibitor (alpha 1PI) with subtilisin BPN' was assessed by spectrophotometric determination of the inhibitory capacity and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). During the course of incubation of the enzyme and the inhibitor (E : I = 1 : 7.5) at pH 8.0 about 17% of the enzyme activity which had been inhibited initially was regenerated, indicating a temporary type of inhibition. The results of the titration experiments indicate that 9.8 mol of the inhibitor is required to inhibit 1 mol of the enzyme completely. However, patterns of 5% disc SDS-PAGE under non-reducing conditions revealed only an equimolar complex (Mr80K) of alpha 1PI with the enzyme and no other higher Mr component than the native inhibitor (Mr 56K). On the other hand, complete dissociation of the complex occurred under reducing conditions, producing an enzymatically modified inhibitor. When 5 21% gradient slab SDS-PAGE was employed, no complex formation was observed under either reducing or non-reducing conditions. With the gradient gel system, dissociation of the equimolar complex produced different forms of the inhibitor, that is, regeneration of an intact alpha 1PI under non-reducing conditions and an enzymatically modified form under reducing conditions. All these results indicate that the complex formed between subtilisin BPN' and human alpha 1PI is not so stable as that of the inhibitor with bovine chymotrypsin and that no covalent bond may be involved in the complex formation. The results also indicate that human alpha 1PI is not an effective inhibitor of subtilisin BPN' and behaves like a substrate for the enzyme.     

Differential inhibition of serine proteinases by rabbit alpha 1-proteinase inhibitors F and S

Inhibition of six serine proteinases (bovine trypsin and chymotrypsin, equine leucocyte proteinases type 1 and 2A, porcine pancreatic elastase type III and rabbit plasmin) by rabbit alpha 1-proteinase inhibitors F and S was studied. In each case examined, the F form reacted more rapidly. The number of moles of an enzyme inhibited by one mole of alpha 1-proteinase inhibitor in a complete reaction (molar inhibitory capacity) ranged from 0.26 (leucocyte proteinase type 1) to 1.01 (trypsin). More significantly, however, the molar inhibitory capacities of both alpha 1-proteinase inhibitors differed for the same enzymes. The highest F/S inhibitory ratio was recorded with chymotrypsin (1.88), and the lowest with elastase (0.69). These differences in molar inhibitory capacities are likely to reflect the dual nature of the reaction between the inhibitor and a proteinase, that is, either complex formation or inactivation of alpha 1-proteinase inhibitor without enzyme inhibition. No evidence was obtained to suggest that differential reactivity and differential inhibitory capacity are interdependent. The observations are consistent with the view that rabbit alpha 1-proteinase inhibitors F and S are closely related yet functionally distinct proteins.     

Isolation and characterization of alpha2-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis.

A procedure is presented for purifying a novel proteinase inhibitor in human plasma whose apparent unique biological property is to inhibit efficiently the lysis of fibrin clots induced by plasminogen activator. The final product is homogeneous as judged by disc gel electrophoresis, and immunoelectrophoresis. Its molecular weight estimated by sodium dodecyl sulfate gel electrophoresis or sedimentation equilibrium is 67,000 and 63,000, respectively. The inhibitor is a glycoprotein consisting polypeptide chain containing 11.7% carbohyrate. It migrates in the alpha2-globulin region in immunoelectrophoresis. The inhibitor is chemically and immunologically different from all the other known inhibitors in plasma. Inhibition of plasmin by the inhibitor is almost instantaneous even at 0 degrees, in contrast to the slow inhibition of urokinase (plasminogen activator in urine). Plasminogen activation by urokinase-induced clot lysis is inhibited by the inhibitor mainly through a mechanism of instantaneous inhibition of plasmin formed and not through the inhibition of urokinase. The inhibitor also inhibits trypsin. Consequently, it is suggested that this newly identified inhibitor is named alpha2-plasmin inhibitor or alpha2-proteinase inhibitor. A specific antibody directed against the inhibitor neutralizes virtually all inhibitory activity of plasma to activator-induced clot lysis. Immunochemical quantitation of the inhibitor was specific antiserum to the inhibitor and the purified inhibitor as a standard indicates that the concentration of the inhibitory in the serum of a healthy man is in or near the range of 5 to 7 mg/100 ml, which is the lowest concentration among the concentration of the proteinase inhibitors in plasma. The inhibitor and plasmin, trypsin, or urokinase form a complex which cannot be dissociated with denaturing and reducing agents. The formation of the enzyme-inhibitor complex occurs on a 1:1 molar basis and is associated with the cleavage of a unique peptide bone, which is most clearly demonstrated in the interaction of the inhibitor and beta-trypsin. In the complex formation between the inhibitor and plasmin, the inhibitor is cross-linked with the light chain which contains the active site of plasmin. It is suggested that, in a fashion analogous to complex formation between alpha1-antitrypsin and trypsin, the cross-links are formed between the active site serine of the enzyme and the newly formed COOH-terminal residue of the inhibitor, with cleavage of a peptide bond.     

Hepatocyte uptake of alpha 1-proteinase inhibitor-trypsin complexes in vitro: evidence for a shared uptake mechanism for proteinase complexes of alpha 1-proteinase inhibitor and antithrombin III

In vivo clearance studies have indicated that the clearance of proteinase complexes of the homologous serine proteinase inhibitors alpha 1-proteinase inhibitor and antithrombin III occurs via a specific and saturable pathway located on hepatocytes. In vitro hepatocyte-uptake studies with antithrombin III-proteinase complexes confirmed the hepatocyte uptake and degradation of these complexes, and demonstrated the formation of a disulfide interchange product between the ligand and a cellular protein. We now report the results of in vitro hepatocyte uptake studies with alpha 1-proteinase inhibitor-trypsin complexes. Trypsin complexes of alpha 1-proteinase inhibitor were prepared and purified to homogeneity. Uptake of these complexes by hepatocytes was time and concentration-dependent. Competition experiments with alpha 1-proteinase inhibitor, alpha 1-proteinase inhibitor-trypsin, and antithrombin III-thrombin indicated that the proteinase complexes of these two inhibitors are recognized by the same uptake mechanism, whereas the native inhibitor is not. Uptake studies were performed at 37 degrees C with 125I-alpha 1-proteinase inhibitor-trypsin and analyzed by sodium dodecyl sulfate-gel electrophoresis in conjunction with autoradiography. These studies demonstrated time-dependent uptake and degradation of the ligand to low molecular weight peptides. In addition, there was a time-dependent accumulation of a high molecular weight complex of ligand and a cellular protein. This complex disappeared when gels were performed under reducing conditions. The sole cysteine residue in alpha 1-proteinase inhibitor was reduced and alkylated with iodoacetamide. Trypsin complexes of the modified inhibitor were prepared and purified to homogeneity. Uptake and degradation studies demonstrated no differences in the results obtained with this modified complex as compared to unmodified alpha 1-proteinase inhibitor-trypsin complex. In addition, the high molecular weight disulfide interchange product was still present on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized cells. Clearance and clearance competition studies with alpha 1-proteinase inhibitor-trypsin, alkylated alpha 1-proteinase inhibitor-trypsin, antithrombin III-thrombin, and anti-thrombin III-factor IXa further demonstrated the shared hepatocyte uptake mechanism for all these complexes.     

The molecular stoichiometry of trypsin inhibition by human alpha-1-proteinase inhibitor

The stoichiometry of interaction of human alpha-1-proteinase inhibitor with porcine trypsin has been determined using a highly purified preparation of inhibitor. In contrast to the reports of others, one mole of alpha-1-proteinase inhibitor was found to inhibit two moles of trypsin. Disc gel electrophoresis indicates that the 2:1 complex is preferentially formed even when free alpha-1-proteinase inhibitor is still present.     

The major plasma kallikrein inhibitor of guinea pig plasma.

A plasma kallikrein inhibitor in guinea pig plasma (KIP) was purified to homogeneity. KIP is a single chain protein and the apparent molecular weight is estimated to be 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In amino acid composition, KIP is similar to human and mouse alpha 1-proteinase inhibitors and mouse contrapsin. KIP forms an equimolar complex with plasma kallikrein in a dose- and time-dependent fashion. The association rate constants for the inhibition of guinea pig plasma kallikrein by KIP, alpha 2-macroglobulin, C1-inactivator and antithrombin III were 2.5 +/- 0.3.10(4), 2.4 +/- 0.4.10(4), 6.6 +/- 0.5.10(4) and 9.1 +/- 0.6.10(2), respectively. Comparison of the association rate constants and the normal plasma concentrations of the four inhibitors demonstrates that KIP is ten-times as effective as alpha 2-MG and other two inhibitors are marginally effective in the inhibition of kallikrein. KIP inhibits trypsin and elastase rapidly, and thrombin and plasmin slowly, but is inactive for chymotrypsin and gland kallikrein. These results suggest that KIP is the major kallikrein inhibitor in guinea pig plasma and the proteinase inhibitory spectrum is unique to KIP in spite of the molecular similarity to alpha 1-proteinase inhibitor.     

Behaviour of serum immunoreactive trypsin after serum activation by enterokinase in normal and cystic fibrosis patients. Evidence of a trypsin-alpha 1-proteinase inhibitor complex in some cystic fibrosis patients

Serum immunoreactive trypsin (IRT) concentrations are elevated in newborn children with cystic fibrosis (CF) and subsequently fall, in most cases, to values below normal. To evaluate the molecular form(s) of IRT present in serum, we have performed serum activation by enterokinase and have measured serum IRT before and after activation. This approach is based on the postulate that enterokinase converts trypsinogen into trypsin, and this trypsin would then be mainly trapped by alpha 2-macroglobulin, thus escaping the assay. This assumption was confirmed in the 28 controls studied, where the mean percentage (+/- S.D.) of IRT recovery after serum activation was 13.7 +/- 2.9. Previous inhibition of alpha 2-macroglobulin by methylamine raised the recovery over 85%, confirming that most of the serum IRT present in controls was in the form of trypsinogen. Identical results were obtained in the serum of 10 obligate heterozygotes and in 57 out of 80 CF patients. In 23 CF patients the mean percentage of IRT recovery after serum activation was 41.6 +/- 17.6. Gel-filtration studies were performed on the sera of the CF patients showing an abnormal increase in the IRT recovery after serum activation. We could demonstrate that IRT was distributed in two fractions: one eluted with the Mr 25,000 protein as usually found in controls and other CF sera, and the other eluted with the Mr 75,000 protein corresponding to a complex of trypsin with alpha 1-proteinase inhibitor. These results show that, in these sera, active trypsin has been directly released in blood. These findings suggest that in some patients with CF, subclinical attacks of acute pancreatitis may occur.     

In vivo metabolism of inter-alpha-trypsin inhibitor and its proteinase complexes: evidence for proteinase transfer to alpha 2-macroglobulin and alpha 1-proteinase inhibitor

Inter-alpha-trypsin inhibitor was purified by a modification of published procedures which involved fewer steps and resulted in higher yields. The preparation was used to study the clearance of the inhibitor and its complex with trypsin from the plasma of mice and to examine degradation of the inhibitor in vivo. Unlike other plasma proteinase inhibitor-proteinase complexes, inter-alpha-trypsin inhibitor reacted with trypsin did not clear faster than the unreacted inhibitor. Studies using 125I-trypsin provided evidence for the dissociation of complexes of proteinase and inter-alpha-trypsin inhibitor in vivo, followed by rapid removal of proteinase by other plasma proteinase inhibitors, particularly alpha 2-macroglobulin and alpha 1-proteinase inhibitor. Studies in vitro also demonstrated the transfer of trypsin from inter-alpha-trypsin inhibitor to alpha 2-macroglobulin and alpha 1-proteinase inhibitor but at a much slower rate. The clearance of unreacted 125I-inter-alpha-trypsin inhibitor was characterized by a half-life ranging from 30 min to more than 1 h. Murine and human inhibitors exhibited identical behavior. Multiphasic clearance of the inhibitor was not due to degradation, aggregation, or carbohydrate heterogeneity, as shown by competition studies with asialoorosomucoid and macroalbumin, but was probably a result of extravascular distribution or endothelial binding. 125I-inter-alpha-trypsin inhibitor cleared primarily in the liver. Analysis of liver and kidney tissue by gel filtration chromatography and sodium dodecyl sulfate gel electrophoresis showed internalization and limited degradation of 125I-inter-alpha-trypsin inhibitor in these tissues. No evidence for the production of smaller proteinase inhibitors from 125I-inter-alpha-trypsin inhibitor injected intravenously or intraperitoneally was detected, even in casein-induced peritoneal inflammation. No species of molecular weight similar to that of urinary proteinase inhibitors, 19,000-70,000, appeared in plasma, liver, kidney, or urine following injection of inter-alpha-trypsin inhibitor.     

Heparin strongly decreases the rate of inhibition of neutrophil elastase by alpha 1-proteinase inhibitor

Heparin depresses the second-order rate constant ka for the inhibition of neutrophil elastase by alpha 1-proteinase inhibitor. High molecular mass heparin decreases ka from 1.3 x 10(7) M-1 s-1 to a limit of 4.6 x 10(4) M-1 s-1. Low molecular mass heparin is about 7-fold less effective. Dermatan sulfate and chondroitin sulfate are less efficient. Heparin preparations used in clinical care also strongly depress ka when tested at concentrations corresponding to their clinical efficacy. Heparin also decreases the ka for the elastase/eglin c and the cathepsin G/alpha 1-proteinase inhibitor systems but not that for the alpha 1-proteinase inhibitor/pancreatic elastase or trypsin pairs. These results, together with Sepharose-heparin binding studies, indicate that the ka-depressing effect of the polymer is related to its ability to form a tight complex with elastase but not with alpha 1-proteinase inhibitor. One mol of high molecular mass heparin binds 3 mol of neutrophil elastase with a Kd of 3.3 nM. Low molecular mass heparin binds elastase with a 1:1 stoichiometry and a Kd of 89 nM. For both heparins ka is lowest when elastase is fully saturated with heparin. From this we conclude that heparin decreases ka, because the heparin-elastase complex is able to slowly react with alpha 1-proteinase inhibitor and not because the inhibitor slowly dissociates the heparin-elastase complex. These findings may have important pathophysiological bearing.     

Studies on the interaction between leukocyte elastase, antileukoproteinase and the plasma proteinase inhibitors alpha 1-proteinase inhibitor and alpha 2-macroglobulin

The dominating inhibitor of leukocyte elastase in human respiratory tract secretions is a low molecular mass inhibitor, designated antileukoproteinase. An equimolar antileukoproteinase-elastase complex was produced and subjected to gel filtration after differing time intervals and was found to be stable. On addition to human serum, however, elastase dissociated from antileukoproteinase and formed a complex with alpha 1-proteinase inhibitor. A small amount of elastase was also found bound to alpha 2-macroglobulin. Antileukoproteinase was capable of inhibiting elastase bound to alpha 2-macroglobulin. This inhibition was more complete and more rapid when the alpha 2-macroglobulin-elastase complex was in a molar ratio of 1:1 than in a ratio of 1:2.     

Resistance of horse alpha 1-proteinase inhibitor to perchloric acid denaturation and a simplified purification procedure resulting therefrom

Addition of perchloric acid (6.4% w/v final concentration) to horse alpha 1-proteinase inhibitor or to horse plasma neither precipitated nor inactivated alpha 1-proteinase inhibitor. None of the isoinhibitors of alpha 1-proteinase inhibitor was altered by dilute perchloric acid. This unexpected behavior led to a simplified procedure for the purification of horse alpha 1-proteinase inhibitor, consisting of removal of the bulk of plasma proteins, by perchloric acid precipitation and by gel filtration on Sephadex G-75 and G-200. The resulting preparations of alpha 1-proteinase inhibitor were immunogenically pure. The simplified purification procedure permitted the immunochemical comparison of the isoinhibitors of alpha 1-proteinase inhibitor, which proved to be immunologically identical.     

Modification of the isoinhibitors of human serum alpha 1-proteinase inhibitor (alpha 1-antitrypsin) by pancreatic proteases

Due to the action of a serum protease, the two most cathodal isoinhibitors of the alpha 1-proteinase inhibitor (alpha 1-PI) are cleaved at the Gly5-Asp6 bond and lack two negative charges. In spite of this, these can bind trypsin and chymotrypsin, showing that the N-terminal pentapeptide is not indispensable for inhibition function. Pancreatic proteases also cleave a bond near the N-terminus in alpha 1-PI, resulting in a loss of two negative charges and a corresponding cathodal shift in the electrofocusing behavior of the isoinhibitors. Trypsin cleaves isoinhibitors near the N-terminus at a large inhibitor excess and unless an additional cleavage takes place, at least two of the new isoinhibitors remain active. An additional cleavage(s), most likely at a distance of 30-40 residues from the C-terminus results in a corresponding decrease of the molecular mass and a loss of inhibition function. Although the C-terminal cleavage peptide does separate from the protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it remains associated with it under conditions of polyacrylamide gel isoelectric focusing. Chymotrypsin also cleaved alpha 1-PI near the N-terminus but this could be observed only at protease excess and the modified isoinhibitors did not form complexes with chymotrypsin. The molecular polymorphism of alpha 1-PI is partly explained by the absence of the N-terminal pentapeptide from some of the isoinhibitors.     

Interactions in vitro and in vivo between anionic and cationic porcine trypsin and porcine plasma proteinase inhibitors

A method for purifying porcine anionic and cationic trypsin is presented. Reaction mixtures with increasing amounts of the two porcine trypsins and porcine serum were studied in vitro to evaluate the relative importance of alpha 1-macroglobulin and alpha 2-macroglobulin as well as alpha 1-proteinase inhibitor in the rapid binding of porcine anionic and cationic trypsin. Porcine cationic trypsin was preferentially bound to alpha 1-macroglobulin, while anionic trypsin exhibited equal binding to both alpha-macroglobulins. Both trypsins were also bound by the alpha 1-proteinase inhibitor but not until alpha 1-macroglobulin approached saturation. Trypsin-alpha-macroglobulin complexes were cleared from plasma with a half-life of 6 min. For trypsin-alpha 1-proteinase inhibitor-complexes the half-life was 120 min. These findings are in accordance with results for other mammalian species, including man.     

Active site distortion is sufficient for proteinase inhibition by serpins: structure of the covalent complex of alpha1-proteinase inhibitor with porcine pancreatic elastase

We report here the x-ray structure of a covalent serpin-proteinase complex, alpha1-proteinase inhibitor (alpha1PI) with porcine pancreatic elastase (PPE), which differs from the only other x-ray structure of such a complex, that of alpha1PI with trypsin, in showing nearly complete definition of the proteinase. alpha1PI complexes with trypsin, PPE, and human neutrophil elastase (HNE) showed similar rates of deacylation and enhanced susceptibility to proteolysis by exogenous proteinases in solution. The differences between the two x-ray structures therefore cannot arise from intrinsic differences in the inhibition mechanism. However, self-proteolysis of purified complex resulted in rapid cleavage of the trypsin complex, slower cleavage of the PPE complex, and only minimal cleavage of the HNE complex. This suggests that the earlier alpha1 PI-trypsin complex may have been proteolyzed and that the present structure is more likely to be representative of serpin-proteinase complexes. The present structure shows that active site distortion alone is sufficient for inhibition and suggests that enhanced proteolysis is not necessarily exploited in vivo.     

Human cationic and anionic trypsins: differences of interaction with alpha 1-proteinase inhibitor

Human cationic (trypsin 1) and anionic (trypsin 2) trypsins were obtained by controlled activation of purified trypsinogens 1 and 2, respectively. The interactions of trypsin 1 and trypsin 2 with human alpha 1-proteinase inhibitor (alpha 1PI) were analysed and compared by studies in vitro. The enzymatic activity and inhibitory capacity measurements were assessed using Glp-Gly-Arg-Nan as substrate. The association rate constants showed that the inhibition of trypsin 2 occurred more than 10 times faster than that of trypsin 1. The equimolar complexes obtained between either trypsin and alpha 1PI were visualized by electrophoresis followed by immunoblotting. The inhibition of the two trypsins was temporary i.e. the complexes trypsin 1-alpha 1PI and trypsin 2-alpha 1PI broke down with time yielding inactive alpha 1PI (Mr 50,000) and active enzyme. But the stability time for trypsin 1-alpha 1PI was much larger than that of trypsin 2-alpha 1PI. In vivo, alpha 1PI is not able to control the activity of trypsin 1 except when alpha 2-macroglobulin (alpha 2M) is already saturated. According to the delay times of inhibition calculated from normal concentrations in serum, alpha 1PI inhibits trypsin 2 as fast as alpha 2M inhibits trypsin 1. These results suggest that a significant role can be assigned to alpha 1PI in the inhibition of trypsin 2 in physiological conditions and of trypsin 1 in pathological ones.     

1-deoxynojirimycin impairs oligosaccharide processing of alpha 1-proteinase inhibitor and inhibits its secretion in primary cultures of rat hepatocytes

1-Deoxynojirimycin was found to inhibit oligosaccharide processing of rat alpha 1-proteinase inhibitor. In normal hepatocytes alpha 1-proteinase inhibitor was present in the cells as a 49,000 Mr high mannose type glycoprotein with oligosaccharide side chains having the composition Man9GlcNAc and Man8GlcNAc with the former in a higher proportion. Hepatocytes treated with 5 mM 1-deoxynojirimycin accumulated alpha 1-proteinase inhibitor as a 51,000 Mr glycoprotein with carbohydrate side chains of the high mannose type, containing glucose as measured by their sensitivity against alpha-glucosidase, the largest species being Glc3Man9GlcNAc. Conversion to complex oligosaccharides was inhibited by the drug. In addition, increasing concentrations of 1-deoxynojirimycin inhibited glycosylation resulting in the formation of some alpha 1-proteinase inhibitor with two instead of three oligosaccharide side chains. 5 mM 1-deoxynojirimycin inhibited the secretion of alpha 1-proteinase inhibitor by about 50%, whereas secretion of albumin was unaffected. The oligosaccharides of alpha 1-proteinase inhibitor secreted from 1-deoxynojirimycin-treated cells were characterized by their susceptibility to endoglucosaminidase H, incorporation of [3H]galactose, and [3H]fucose and concanavalin A-Sepharose chromatography. It was found that 1-deoxynojirimycin did not completely block oligosaccharide processing, resulting in the formation of alpha 1-proteinase inhibitor molecules carrying one or two complex type oligosaccharides. Only these alpha 1-proteinase inhibitor molecules processed to the complex type in one or two of their oligosaccharide chains were nearly exclusively secreted. This finding demonstrates the importance of oligosaccharide processing for the secretion of alpha 1-proteinase inhibitor.     

Proteinase inhibitors in rat serum. Purification and partial characterization of three functionally distinct trypsin inhibitors.

Three different serine proteinase inhibitors were isolated from rat serum and purified to apparent homogeneity. One of the inhibitors appears to be homologous to alpha 1-proteinase inhibitor isolated from man and other species, but the other two, designated rat proteinase inhibitor I and rat proteinase inhibitor II, seem to have no human counterpart. alpha 1-Proteinase inhibitor (Mr 55000) inhibits trypsin, chymotrypsin and elastase, the three serine proteinases tested. Rat proteinase inhibitor I (Mr 66000) is active towards trypsin and chymotrypsin, but is inactive towards elastase. Rat proteinase inhibitor II (Mr 65000) is an effective inhibitor of trypsin only. Their contributions to the trypsin-inhibitory capacity of rat serum are about 68, 14 and 18% for alpha 1-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor II respectively.