Conformation of the reactive site loop of alpha 1-proteinase inhibitor probed by limited proteolysis.

Elucidation of the reactive site loop (RSL) structure of serpins is essential for understanding their inhibitory mechanism. Maintenance of the RSL structure is likely to depend on its interactions with a dominant unit of secondary structure known as the A-sheet. We investigated these interactions by subjecting alpha 1-proteinase inhibitor to limited proteolysis using several enzymes. The P1-P10 region of the RSL was extremely sensitive to proteolysis, indicating that residues P3'-P13 are exposed in the virgin inhibitor. Following cleavage eight or nine residues upstream from the reactive site, the protein noncovalently polymerized, sometimes forming circles. Polymerization resulted from insertion of the P1-P8 or P1-P9 region of one molecule into the A-sheet of an adjacent proteolytically modified molecule. The site of cleavage within the RSL had a distinct effect on the conformational stability of the protein, such that stability increased as more amino acids insert into the A-sheet. We conclude that the A-sheet of virgin alpha 1-proteinase inhibitor resembles that of ovalbumin, except that it contains a bulge where two or three RSL residues are inserted. Insertion of seven or eight RSL residues, allowed by proteolytic cleavage of the RSL, causes expansion of the sheet. It is likely that the RSL of alpha 1-proteinase inhibitor and several serpins exhibits significantly more mobility than is common among other protein inhibitors of serine proteinases.     

Primary structure of the reactive site of human C1-inhibitor

Human C1-inhibitor (C1-Inh) forms an equimolar complex with complement proteinase C1s that is resistant to dissociation by sodium dodecyl sulfate. The formation of this stable complex results in the cleavage of a peptide bond near the carboxyl terminus of the inhibitor and, whereas the bulk of C1-Inh remains covalently bound to the light chain of C1s, the postcomplex inhibitor peptide can be isolated under denaturing conditions. We have sequenced the amino-terminal region of this peptide and deduced that it represents the carboxyl-terminal side of the reactive site of C1-Inh. Limited proteolysis of C1-Inh by Crotalus atrox protease results in an active derivative lacking an amino-terminal peptide of 36 residues. Further proteolysis of this derivative with Pseudomonas aeruginosa elastase inactivates the inhibitor and a peptide is released. The amino-terminal sequence of this peptide overlaps with that of the postcomplex peptide and indicates that the residue imparting primary specificity to the inhibitor is arginine.     

S-ovalbumin, an ovalbumin conformer with properties analogous to those of loop-inserted serpins.

Most serpins are inhibitors of serine proteinases and are thought to undergo a conformational change upon complex formation with proteinase that involves partial insertion of the reactive center loop into a beta-sheet of the inhibitor. Ovalbumin, although a serpin, is not an inhibitor of serine proteinases. It has been proposed that this deficiency arises from the presence of a charged residue, arginine, at a critical point (P14) in the reactive center region, which prevents loop insertion into the beta-sheet and thereby precludes inhibitory properties. To test whether loop insertion is prevented in ovalbumin we have examined the properties of two forms of ovalbumin: the native protein and S-ovalbumin, a form that forms spontaneously from native ovalbumin and has increased stability. Calorimetric measurements showed that S-ovalbumin was more stable than ovalbumin by about 3 kcal mol-1. CD spectra, which indicated that S-ovalbumin had less alpha-helix than native ovalbumin, and 1H NMR spectra, which indicated very similar overall structures, suggest limited conformational differences between the two forms. From comparison of the susceptibility of the reactive center region of each protein to proteolysis by porcine pancreatic elastase and by subtilisin Carlsberg, we concluded that the limited native-to-S conformational change specifically affected the reactive center region. These data are consistent with a structure for S-ovalbumin in which part of the reactive center loop has inserted into beta-sheet A to give a more stable structure, analogously to other serpins. However, the rate of loop insertion appears to be very much lower than for inhibitory serpins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inactivation of human plasma alpha 1-antichymotrypsin by microbial proteinases

Incubation of human plasma alpha 1-antichymotrypsin with proteinases from various microbial sources resulted in the enzymatic inactivation of the inhibitor as determined by loss of inhibitory activity against alpha-chymotrypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reaction products indicated that intact alpha 1-antichymotrypsin (Mr 67000) had been converted to an inactive form (63000) by limited proteolysis. No stable proteinase/inhibitor complexes were detected, and no random proteolysis of the inactivated inhibitor occurred even after prolonged incubation with the proteinases. Metallo- and serine proteinases from several microbial sources all readily inactivated alpha 1-antichymotrypsin. Since alpha 1-antichymotrypsin is also an early stage acute phase reactant, its inactivation may be important in disrupting bodily defense mechanisms.     

Kallistatin: a novel human tissue kallikrein inhibitor. Purification, characterization, and reactive center sequence.

A novel human tissue kallikrein inhibitor designated as kallistatin has been purified from plasma to apparent homogeneity by polyethylene glycol fractionation and successive chromatography on heparin-Agarose, DEAE-Sepharose, hydroxylapatite, and phenyl-Superose columns. A purification factor of 4350 was achieved with a yield of approximately 1.35 mg per liter of plasma. The purified inhibitor migrates as a single band with an apparent molecular mass of 58 kDa when analyzed on SDS-polyacrylamide gel electrophoresis under reducing conditions. It is an acidic protein with pI values ranging from 4.6 to 5.2. No immunological cross-reactivity was found by Western blot analyses between kallistatin and other serpins. Kallistatin inhibits human tissue kallikrein's activity toward kininogen and tripeptide substrates. The second-order reaction rate constant (ka) was determined to be 2.6 x 10(4) M-1 s-1 using Pro-Phe-Arg-MCA. The inhibition is accompanied by formation of an equimolar, heat- and SDS-stable complex between tissue kallikrein and kallistatin, and by generation of a small carboxyl-terminal fragment from the inhibitor due to cleavage at the reactive site by tissue kallikrein. Heparin blocks kallistatin's complex formation with tissue kallikrein and abolishes its inhibitory effect on tissue kallikrein's activity. The amino-terminal residue of kallistatin is blocked. Sequence analysis of the carboxyl-terminal fragment generated from kallistatin reveals the reactive center sequence from P1' to P15', which shares sequence similarity with, but is different from known serpins including protein C inhibitor, alpha 1-antitrypsin, and alpha 1-antichymotrypsin. The results show that kallistatin is a new member of the serpin superfamily that inhibits human tissue kallikrein.     

Serpins of oat (Avena sativa) grain with distinct reactive centres and inhibitory specificity

Most proteinase inhibitors from plant seeds are assumed to contribute to broad-spectrum protection against pests and pathogens. In oat (Avena sativa L.) grain the main serine proteinase inhibitors were found to be serpins, which utilize a unique mechanism of irreversible inhibition. Four distinct inhibitors of the serpin superfamily were detected by native PAGE as major seed albumins and purified by thiophilic adsorption and anion exchange chromatography. The four serpins OSZa-d are the first proteinase inhibitors characterized from this cereal. An amino acid sequence close to the blocked N-terminus, a reactive centre loop sequence, and the second order association rate constant (ka') for irreversible complex formation with pancreas serine proteinases at 24 degrees C were determined for each inhibitor. OSZa and OSZb, both with the reactive centre scissile bond P1-P1' Thr downward arrow Ser, were efficient inhibitors of pancreas elastase (ka' > 105M-1 s-1). Only OSZb was also an inhibitor of chymotrypsin at the same site (ka' = 0.9 x 105M-1 s-1). OSZc was a fast inhibitor of trypsin at P1-P1' Arg downward arrow Ser (ka' = 4 x 106M-1 s-1); however, the OSZc-trypsin complex was short-lived with a first order dissociation rate constant kd = 1.4 x 10-4 s-1. OSZc was also an inhibitor of chymotrypsin (ka' > 106M-1 s-1), presumably at the overlapping site P2-P1 Ala downward arrow Arg, but > 90% of the serpin was cleaved as substrate. OSZd was cleaved by chymotrypsin at the putative reactive centre bond P1-P1' Tyr downward arrow Ser, and no inhibition was detected. Together the oat grain serpins have a broader inhibitory specificity against digestive serine proteinases than represented by the major serpins of wheat, rye or barley grain. Presumably the serpins compensate for the low content of reversible inhibitors of serine proteinases in oats in protection of the grain against pests or pathogens.     

Plasminogen activator inhibitor 2 (PAI-2) is not inactivated by exposure to oxidants which can be released from activated neutrophils

Purified recombinant human monocyte plasminogen activator inhibitor 2 (PAI-2) retained inhibitory activity after exposure to a number of oxidants, including hypochlorite anion (OCl-), chloramine-T (CT) and hydrogen peroxide (H2O2). Analysis of PAI-2 exposed to oxidants by gel filtration chromatography and SDS-PAGE indicated that although the protein could no longer be detected by silver staining, this was not due to fragmentation of the PAI-2 molecule. The sensitivity of a number of serine protease inhibitors (serpins), (eg. alpha 1 proteinase inhibitor (alpha 1PI) and plasminogen activator inhibitor 1 (PAI-1] to oxidative inactivation has been attributed to oxidation of reactive site methionine residues and/or tertiary structural modifications. The relevance of these phenomena and the potential for PAI-2 to be used as a therapeutic inhibitor of urokinase (uPA)-dependent proteolysis during inflammation and tumour metastasis is discussed.     

Characterisation of a highly specific, endogenous inhibitor of cysteine protease from Staphylococcus epidermidis, a new member of the staphostatin family

Staphostatins, a novel family of cysteine protease inhibitors with a unique mechanism of action and distinct protein fold has recently been discovered. In this report we describe the properties of Staphylococcus epidermidis staphostatin A (EcpB), a new member of the family. As for other staphostatins, the recombinant S. epidermidis staphostatin A exerted very narrow inhibitory specificity, limited to cysteine protease from the same species. The closely related proteases from S. aureus cleaved the inhibitor at the reactive site peptide bond and inactivated it. The EcpB homologue, S. aureus staphostatin A (ScpB), was also susceptible to proteolytic cleavage at the same site by non-target cysteine proteases. Conversely, S. aureus staphostatin B (SspC) was resistant to such proteolysis. The difference in the susceptibility of individual inhibitors to proteolytic cleavage at the reactive site suggests subtle variations in the mechanism of interaction with cysteine proteases.     

Kinetics and physiologic relevance of the inactivation of alpha 1-proteinase inhibitor, alpha 1-antichymotrypsin, and antithrombin III by matrix metalloproteinases-1 (tissue collagenase), -2 (72-kDa gelatinase/type IV collagenase), and -3 (stromelysin).

Serpins encompass a superfamily of proteinase inhibitors that regulate many of the serine proteinases involved in inflammation and hemostasis. In vitro, many serpins are catalytically inactivated by proteinases that they do not inhibit, leading to the concept of proteolytic down-regulation of serpin inhibitory capacity. The extent to which down-regulation of serpin activity occurs in vivo is debated, since little is known of the rates at which the process occurs. To address this debate, we have measured the rates of inactivation of three serpins, alpha 1-proteinase inhibitor (alpha 1PI), alpha 1-antichymotrypsin (alpha 1ACT), and antithrombin III (ATIII), by three human matrix metalloproteinases (MMPs-1, -2, and -3) thought to be involved in tissue destruction and repair. Our object was to establish a working kinetic model which can be used to predict whether serpin inactivation by these proteinases is likely to occur in vivo. We determined the rates of inactivation of these three serpins by each of the MMPs and compared these to rates of inhibition of the MMPs by an endogenous inhibitor, alpha 2-macroglobulin. An equation designed to predict the extent of substrate hydrolyzed by an enzyme in the presence of an enzyme inhibitor gave the following predictions of the inactivation in vivo: (i) ATIII is unlikely to be inactivated by the MMPs. (ii) MMP-2 (72-kDa gelatinase/type IV collagenase) is unlikely to inactivate any of the three serpins. (iii) MMP-1 (tissue collagenase) will inactivate alpha 1PI and alpha 1ACT only when its concentration saturates that of its controlling inhibitors. (iv) MMP-3 (stromelysin) may inactivate small amounts of alpha 1PI and more significant amounts of alpha 1ACT, even in the presence of its controlling inhibitors. Any physiologic or pathologic inactivation of these serpins by these MMPs that occurs in vivo will probably be due to MMP-3, and will likely only take place in tissues and inflammatory loci where the concentration of MMP inhibitors is depressed.     

Trypsin reactivity site of the Vicia angustifolia proteinase inhibitor

Trypsin [EC 3.4.21.4] modified (reactive site cleaved) Vicia angustifolia proteinase inhibitor was prepared at pH 3 with a catalytic amount of trypsin and purified using columns of Sephadex G-50 and DEAE-Sephadex A-25. The modified inhibitor, which still retained antitryptic activity, lost its activity upon treatment with carboxypeptidase B or citraconic anhydride. End-group analyses revealed that the carboxyl-terminal Arg and the amino-terminal Ser residues were newly exposed end-groups in the modified inhibitor. It takes a much longer incubation time (about 1 h) to exhibit the maximal inhibitory activity against trypsin. Reduction and carboxymethylation of the modified inhibitor produced two fragments on Sephadex G-50 chromatography. The smaller fragment consisted of about 32 amino acid residues and possessed a new carboxyl-terminal Arg residue. The larger fragment consisted of about 80 residues and possessed a Ser residue at its amino-terminus. These results indicate that the small fragment was derived from the amino-terminal portion of the modified inhibitor and the large fragment from the carboxyl-terminal. It is also concluded that an Arg-Ser bond is the reactive site as well as the inhibitory site of the V. angustifolia inhibitor against trypsin. The sequence around the antitryptic site exhibits high degrees of homology with other double-headed inhibitors of legume origin, such as the Bowman-Birk inhibitor, lima beam inhibitor, and the major inhibitor in chick-peas.     

Conversion of antithrombin from an inhibitor of thrombin to a substrate with reduced heparin affinity and enhanced conformational stability by binding of a tetradecapeptide corresponding to the P1 to P14 region of the putative reactive bond loop of the inhibitor.

A synthetic tetradecapeptide having the sequence of the region of the antithrombin chain amino-terminal to the reactive bond, i.e. comprising residues P1 to P14, was shown to form a tight equimolar complex with antithrombin. A similar complex has previously been demonstrated between alpha 1-proteinase inhibitor and the analogous peptide of this inhibitor (Schulze, A. J., Baumann, U., Knof, S., Jaeger, E., Huber, R. and Laurell, C.-B. (1990) Eur. J. Biochem. 194, 51-56). The antithrombin-peptide complex had a conformation similar to that of reactive bond-cleaved antithrombin and, like the cleaved inhibitor, also had a higher conformational stability and lower heparin affinity than intact antithrombin. These properties suggest that the peptide bound to intact antithrombin at the same site that the P1 to P14 segment of the inhibitor occupies in reactive-bond-cleaved antithrombin, i.e. was incorporated as a sixth strand in the middle of the major beta-sheet, the A sheet. The extent of complex formation was reduced in the presence of heparin with high affinity for antithrombin, which is consistent with heparin binding and peptide incorporation being linked. Antithrombin in the complex with the tetradecapeptide had lost its ability to inactivate thrombin, but the reactive bond of the inhibitor was cleaved as in a normal substrate. These observations suggest a model, analogous to that proposed for alpha 1-proteinase inhibitor (Engh, R.A., Wright, H.T., and Huber, R. (1990) Protein Eng. 3, 469-477) for the structure of intact antithrombin, in which the A sheet contains only five strands and the P1 to P14 segment of the chain forms part of an exposed loop of the protein. The results further support a reaction model for serpins in which partial insertion of this loop into the A sheet is required for trapping of a proteinase in a stable complex, and complete insertion is responsible for the conformational change accompanying cleavage of the reactive bond of the inhibitor.     

Plasma serine proteinase inhibitors (serpins) exhibit major conformational changes and a large increase in conformational stability upon cleavage at their reactive sites

Intact and proteolytically modified human serpins, alpha 1-proteinase inhibitor, antithrombin III, alpha 1-antichymotrypsin, and C1 inhibitor, were compared by circular dichroism, fluorescence spectroscopy, and resistance against unfolding by guanidine HCl. The modified proteins were prepared from the intact and active inhibitors by selective proteolytic cleavage in their reactive site loops and tested for complete loss of activity. Significant differences in the spectral properties between intact and modified inhibitors indicate that a major conformational rearrangement is triggered by the cleavage. This leads to a large increase in conformational stability as demonstrated by large shifts of the transition profiles recorded as a function of guanidine HCl concentration at 20 degrees C by circular dichroism at 220 nm. Intact inhibitors were unfolded in two steps of about equal size centered at 0.8-1.7 and 2.5-3.5 M concentrations of the denaturant, respectively. Under identical conditions modified inhibitors are completely stable, and their denaturation occurs only well above 4 M guanidine HCl in one or two steep transition steps. From the similarity of the spectral changes and shifts in transition profiles for all four serpins studied it is concluded that the conformational changes and stabilization triggered by the modification hit is an important common mechanistic feature of this class of inhibitors. This is supported by the observation that ovalbumin, which is homologous with the serpins but apparently lacks inhibitory activity, exhibits neither spectral changes nor a significant change in stability upon proteolytic modification.     

Characterization of the inactive fragment resulting from limited proteolysis of human alpha1-proteinase inhibitor by Crotalus adamanteus proteinase II.

The inactive 50,000-dalton fragment of human plasma alpha1-proteinase inhibitor resulting from limited proteolysis of the inhibitor by Crotalus adamanteus proteinase II has been isolated and partially characterized. The amino acid composition of the inactivated inhibitor indicates the loss of a peptide fragment from the intact inhibitor. Both intact and inactivated inhibitor contain COOH-terminal lysine. However, the NH2 terminus of the intact inhibitor is Glx, whereas that of inactivated inhibitor is methionine. NH2-terminal analysis of the inactive inhibitor fragment revealed the following sequence: -Met-Phe-Leu-Glu-Ala-Ile-Pro-Met-Ser-Ile-Pro-Pro-Gln-Val-Lys-Phe-Asn. The data show that the venom proteinase has inactivated alpha1- proteinase inhibitor by cleavage of a single bond which differs from that reported for trypsin or papain.     

Analysis of the effects of snake venom proteinases on the activity of human plasma C1 esterase inhibitor, alpha 1-antichymotrypsin and alpha 2-antiplasmin

Incubation of C1 esterase inhibitor with Crotalid, Viperid and Colubrid snake venoms resulted in enzymatic inactivation of the inhibitor. Intact inhibitor (104 kDa) was converted into an active intermediate species of 89 kDa and then a further cleavage resulted in formation of an 86-kDa inactive inhibitor. In contrast, C1 esterase inhibitor did not lose activity during incubation with Elapid venoms; however, the intact inhibitor was gradually converted to an active species of 89 kDa during the incubation. Human alpha 1-antichymotrypsin was inactivated by all venoms tested, including those from the Elapid family. The 67-kDa intact inhibitor was converted by the venom proteinases to an inactive 63-kDa form. The results suggest that this acute-phase plasma protein is readily susceptible to inactivation by venom proteinases. Human alpha 2-antiplasmin (68 kDa) was cleaved to form a 61-kDa active intermediate, which then underwent a second cleavage to produce an inactive 53-kDa product. Elapid venoms had no effect on alpha 2-antiplasmin activity and did not cleave this inhibitor. All inhibitors were inactivated with catalytic amounts of venom proteinases. No stable proteinase-proteinase inhibitor complexes were detected, and no random proteolysis of the inhibitors occurred.     

Alpha 1-proteinase inhibitor is a neutrophil chemoattractant after proteolytic inactivation by macrophage elastase

Mouse macrophage elastase, a metalloproteinase, catalytically inactivates human alpha 1-proteinase inhibitor (alpha 1-PI) by attacking a single peptide bond between Pro357 and Met358, resulting in Mr = 4,200 and 47,800 fragments. We show here that this proteolytically inactivated alpha 1-PI is a potent chemotactic factor for human neutrophils at a concentration of 1 nM. The chemotactic response is equivalent to that elicited by formyl-methionyl-leucyl-phenylalanine. Native alpha 1-PI does not stimulate chemotaxis. Purification of the two fragments of alpha 1-PI that result from proteolysis by macrophage elastase indicated that the Mr = 4,200 fragment is responsible for the chemotactic activity. However, the two proteolysis fragments do not dissociate from each other under physiologic conditions. Therefore, the ability of proteolytically inactivated alpha 1-PI to act as a mediator of inflammation is due to rearrangement of the alpha 1-PI molecule rather than to release of a cleavage fragment.     

Full or partial substitution of the reactive center loop of alpha-1-proteinase inhibitor by that of heparin cofactor II: P1 Arg is required for maximal thrombin inhibition

The abundant plasma protein alpha(1)-proteinase inhibitor (alpha(1)-PI) physiologically inhibits neutrophil elastase (NE) and factor XIa and belongs to the serine protease inhibitor (serpin) protein superfamily. Inhibitory serpins possess a surface peptide domain called the reactive center loop (RCL), which contains the P1-P1' scissile peptide bond. Conversion of this bond in alpha(1)-PI from Met-Ser to Arg-Ser in alpha(1)-PI Pittsburgh (M358R) redirects alpha(1)-PI from inhibiting NE to inhibiting thrombin (IIa), activated protein C (APC), and other proteases. In contrast to either the wild-type or M358R alpha(1)-PI, heparin cofactor II (HCII) is a IIa-specific inhibitor with an atypical Leu-Ser reactive center. We examined the effects of replacement of all or part of the RCL of alpha(1)-PI with the corresponding parts of the HCII RCL on the activity and specificity of the resulting chimeric inhibitors. A series of 12 N-terminally His-tagged alpha(1)-PI proteins differing only in their RCL residues were expressed as soluble proteins in Escherichia coli. Substitution of the P16-P3' loop of alpha(1)-PI with that of HCII increased the low intrinsic antithrombin activity of alpha(1)-PI to near that of heparin-free HCII, while analogous substitution of the P2'-P3' dipeptide surpassed this level. However, gel-based complexing and quantitative kinetic assays showed that all mutant proteins inhibited thrombin at less than 2% of the rate of alpha(1)-PI (M358R) unless the P1 residue was also mutated to Arg. An alpha(1)-PI (P16-P3' HCII/M358R) variant was only 3-fold less active than M358R against IIa but 70-fold less active against APC. The reduction in anti-APC activity is desired in an antithrombotic agent, but the improvement in inhibitory profile came at the cost of a 3.5-fold increase in the stoichiometry of inhibition. Our results suggest that, while P1 Arg is essential for maximal antithrombin activity in engineered alpha(1)-PI proteins, substitution of the corresponding HCII residues can enhance thrombin specificity.     

Absence of large-scale conformational change upon limited proteolysis of ovalbumin, the prototypic serpin

1H and 31P NMR spectroscopies have been used to examine the effects of limited proteolysis with subtilisin Carlsberg on the global conformation of ovalbumin and on the local environment of phosphoserine 344, a residue two positions removed from the site of proteolysis. Such limited proteolysis has been shown to result in excision of a hexapeptide from the region of the protein that, in other serine protease inhibitors (serpins), contains the reactive center. Based on the structure of the related serpin alpha 1-antitrypsin, it has been predicted that phosphoserine 344 should undergo a large change in environment upon proteolysis of ovalbumin (L?bermann, H., Tokuoka, R., Deisenhofer, J., and Huber, R. (1984) J. Mol. Biol. 177, 531-550). Proteolysis of ovalbumin produces a small upfield shift (0.15 ppm) of the 31P resonance of phosphoserine 344. In addition, the pKa of phosphoserine 344 is raised by 0.1 pH unit. At pH 8.5, phosphoserine 344 in cleaved ovalbumin (plakalbumin) is as accessible to hydrolysis by Escherichia coli alkaline phosphatase as it is in native ovalbumin. 1H NMR shows that dephosphorylation of serine 344 has an imperceptible effect on the protein's conformation. Similarly, little effect on conformation is seen by 1H NMR upon proteolysis of ovalbumin. These findings suggest that ovalbumin does not undergo a marked conformational change analogous to that inferred for the related members of the serpin superfamily, alpha 1-antitrypsin and antithrombin III, nor do the residues close to the site of proteolysis appear to change environment from that of an exposed loop to a buried strand of beta-sheet. These findings are not consistent with the hypothesis of Carrell and Owen ((1985) Nature 317, 730-732) for the role of the exposed loop in serpins of directly facilitating conformational change upon cleavage of the loop. Instead, it is proposed that cleavage of the exposed loop alters the solvent accessibility of residues formerly covered by the loop and that this provides the thermodynamic impetus for conformational change, perhaps by disruption of a salt bridge crucial to the integrity of the native structure.     

Selectivity and stereospecificity of the reactions of dichlorodiammineplatinum(II) with three purified plasma proteins

The reactions of cis- and trans-dichlorodiammineplatinum(II) (cis- and trans-DDP) with albumin and two plasma proteinase inhibitors were compared. Reaction with alpha 2-macroglobulin (alpha 2M) resulted in subunit crosslinking and loss of proteinase binding activity. The reaction also modified a receptor recognition site present on each alpha 2M subunit. While more trans-DDP was incorporated into alpha 2M than cis-DDP, cis-DDP was more effective at blocking receptor recognition, alpha 1-proteinase inhibitor was also inactivated by reaction with either cis- or trans-DDP. These reactions resulted in binding of platinum to methionine-358 at the reactive center of this inhibitor. Trans-DDP, however, was less selective and also bound to the single cysteine residue (Cys-232) of alpha 1PI. Reaction of albumin with cis-DDP resulted in incorporation of about 1 mol platinum per mol protein, and this platinum modified the single cysteine (Cys-34) in the molecule. Albumin incorporated twice as much trans-DDP, but the binding did not involve cysteine-34. In general, reactions of cis-DDP with proteins appear to be more selective than those observed for modification with the trans isomer.     

Characterization of a cDNA for human protein C inhibitor. A new member of the plasma serine protease inhibitor superfamily

A cDNA library in lambda-phage lambda gt11 containing DNA inserts prepared from human liver mRNA was screened with monoclonal antibodies to human protein C inhibitor. Six positive clones were isolated from 6 X 10(6) phages and plaque purified. The cDNA in the phage containing the largest insert, which hybridized to a DNA probe prepared on the basis of the amino-terminal amino acid sequence of the mature inhibitor, was sequenced. This cDNA insert contained 2106 base pairs coding for a 5'-noncoding region, a 19-amino acid signal peptide, a 387-amino acid mature protein, a stop codon, and a long 3'-noncoding region of 839 base pairs. Based on the amino acid sequence of the carboxyl-terminal peptide released by cleavage of protein C inhibitor by activated protein C as well as by thrombin, the reactive site peptide bond of protein C inhibitor is Arg354-Ser355. Five potential carbohydrate-binding sites were found in the mature protein. The high homology of the amino acid sequence of protein C inhibitor to the other known inhibitors clearly demonstrates that protein C inhibitor is a member of the superfamily of serine protease inhibitors including alpha 1-antichymotrypsin, alpha 1-antitrypsin, antithrombin III, ovalbumin, and angiotensinogen. Based on the difference matrices for these proteins, we present possible phylogenetic trees for these proteins.     

Reactive site mutants of recombinant protein C inhibitor

Protein C inhibitor (PCI) is a heparin-binding serine proteinase inhibitor (serpin) which is thought to be a physiological regulator of activated protein C (APC). The residues F353-R354-S355 (P2-P1-P1′) constitute part of the reactive site loop of PCI with the R-S peptide bond being cleaved by the proteinase. Changing the reactive site P1 and P2 residues to those of either proteinase nexin-1, α₁-proteinase inhibitor or heparin cofactor II resulted in a decrease in inhibitory activity towards thrombin and APC. Changing the P2 residue F353 → P generated a rPCI which was a better thrombin inhibitor, but was 10-fold less active with APC. While these results support the concept that the P1 and P2 residues are important in the specificity of PCI, they suggest that the reactive site residues are not the only determinant of serpin specificity. Kinetic analysis of the rPCI variants was consistent with PCI operating by a mechanism similar to that proposed for other serpins. In this model an intermediary complex forms between inhibitor and proteinase that can proceed to either cleavage of the inhibitor as substrate or formation of an inactive complex.