Low Molecular Mass Inhibitors Research Articles

Clinically relevant fusion oncogenes: detection and practical implications.

Mechanistically, chimeric genes result from DNA rearrangements and include parts of preexisting normal genes combined at the genomic junction site. Some rearranged genes encode pathological proteins with altered molecular functions. Those which can aberrantly promote carcinogenesis are called fusion oncogenes. Their formation is not a rare event in human cancers, and many of them were documented in numerous study reports and in specific databases. They may have various molecular peculiarities like increased stability of an oncogenic part, self-activation of tyrosine kinase receptor moiety, and altered transcriptional regulation activities. Currently, tens of low molecular mass inhibitors are approved in cancers as the drugs targeting receptor tyrosine kinase (RTK) oncogenic fusion proteins, that is, including ALK, ABL, EGFR, FGFR1-3, NTRK1-3, MET, RET, ROS1 moieties. Therein, the presence of the respective RTK fusion in the cancer genome is the diagnostic biomarker for drug prescription. However, identification of such fusion oncogenes is challenging as the breakpoint may arise in multiple sites within the gene, and the exact fusion partner is generally unknown. There is no gold standard method for RTK fusion detection, and many alternative experimental techniques are employed nowadays to solve this issue. Among them, RNA-seq-based methods offer an advantage of unbiased high-throughput analysis of only transcribed RTK fusion genes, and of simultaneous finding both fusion partners in a single RNA-seq read. Here we focus on current knowledge of biology and clinical aspects of RTK fusion genes, related databases, and laboratory detection methods.

NMR study of complexes between low molecular mass inhibitors and the West Nile virus NS2B–NS3 protease

The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.

Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey ( Meleagris gallopavo)

The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.

Polymeric and Low Molecular Mass Efflux Pump Inhibitors for Oral Drug Delivery

Transmembrane located transporter proteins can be responsible for the low bioavailability of orally administered drugs. Drug delivery systems which can overcome this barrier caused by efflux pumps are therefore highly on demand. Within the current review, intestinal located efflux transporters, methods to identify efflux pump substrates and inhibitors as well as strategies to minimize efflux pump mediated transport of drugs are discussed. Methods include in silico screening, transport and accumulation studies and monitoring of the ATPase activity. An emphasis has been placed on efflux pump inhibitors including low molecular mass inhibitors such as cyclosporine, PSC833 or KR30031 and polymeric inhibitors such as myrj, thiomers and cremophor EL. Also formulation approaches to circumvent intestinal segments with high efflux pump expression are briefly addressed.

Structural Basis for a High Affinity Inhibitor Bound to Protein Kinase MK2

The Ser/Thr protein kinase MAPKAP kinase 2 (MK2) plays a crucial role in inflammation. We determined the structure of the kinase domain of MK2 in complex with a low molecular mass inhibitor in two different crystal forms, obtained from soaking and co-crystallization. To our knowledge, these are the first structures of MK2 showing the binding mode of an inhibitor with high binding affinity (IC50 8.5 nM). The two crystal forms revealed conformational flexibility in the binding site and extend the experimental basis for rational drug design. Crystal form-1 contained one MK2 molecule per asymmetric unit. Form-2 contained 12 molecules, which arrange into two different types of MK2 trimers. One of them may serve as a model for an intermediate state during substrate phosphorylation, as each MK2 monomer places its activation segment into the substrate peptide binding groove of the trimer neighbor.

Using surface plasmon resonance to directly measure slow binding of low-molecular mass inhibitors to a VanX chip

VanX, a d,d-dipeptidase, is one of five gene products responsible for vancomycin resistance in pathogenic bacteria and is an attractive drug target in circumventing clinical drug resistance. Our previous combinatorial search of VanX substrates in a dipeptide library of d-X 1- d-X 2 (19 2 = 361) forms has led to the discovery of three new compounds ( d-Ala- d-Phe, d-Ala- d-Tyr, and d-Ala- d-Trp) having higher k cat/ K M values than those of its natural substrate, d-Ala- d-Ala. Based on structures of newly identified substrates, two representative transition state analogs of substrates, d-Ala(P,O) d-Phe ( 6a) and d-Ala(P,O) d-Ala ( 6b) dipeptide phosphonates, used as VanX inhibitor were rationally designed and chemically synthesized. In the synthesis, eight synthetic steps in total were employed for preparing each VanX inhibitor, and their overall isolated yields were 21 and 11% for 6a and 6b, respectively. Binding interactions of d-Ala(P,O) d-Phe ( 6a) and d-Ala(P,O) d-Ala ( 6b) with VanX were confirmed unambiguously and measured quantitatively by surface plasmon resonance. The result reveals that both dipeptide phosphonates are slow-binding inhibitors of VanX (for 6a, k on = 1.18 × 10 3 M −1 s −1, k off = 2.31 × 10 −3 s −1, K D = 1.96 μM, χ 2 = 0.0737; for 6b, k on = 1.09 × 10 3 M −1 s −1, k off = 1.80 × 10 −2 s −1, K D = 16.5 μM, χ 2 = 0.0599). This suggests that only a fraction of the conformers of the inhibitors in solution adopts a conformation best suited for binding interaction with VanX and that the VanX–inhibitor complex may concomitantly undergo a conformational isomerization from an initial but fast weak-binding adduct to slowly convert to a tight-binding complex with a more stable bound geometry. Moreover, in comparison with 6b, an additional aromatic interaction of 6a with the Phe79 residue in the active site of the enzyme, through an energetically favorable face-to-face offset stacked orientation, may account for its higher affinity than 6b to VanX.

In vivo comparison of various polymeric and low molecular mass inhibitors of intestinal P-glycoprotein

Several polymers have been reported to modulate drug absorption by inhibition of intestinal P-glycoprotein (P-gp). The aim of the present study was to provide a direct in vivo comparison of delivery systems based on Pluronic P85, Myrj 52 and chitosan-4-thiobutylamidine (Ch-TBA) in vivo in rats, using rhodamine-123 (Rho-123) as representative P-gp substrate. Furthermore, the postulated low molecular mass P-gp inhibitors 6-mercaptopurine and reduced glutathione (GSH) were evaluated in vitro and in vivo. In vitro, the permeation enhancing effect of 6-mercaptopurine, GSH, Pluronic P85, Myrj 52, and the combination of Ch-TBA with GSH was evaluated by using freshly excised rat intestinal mucosa mounted in Ussing-type diffusion chambers. In comparison to buffer only, Rho-123 transport in presence of 100 microm 6-mercaptopurine, 0.5% (w/v) GSH, 0.5% (w/v) Pluronic P85, 0.5% (w/v) Myrj 52 and the combination of 0.5% (w/v) Ch-TBA/ 0.5% (w/v) GSH, was 2.1, 1.6, 1.9, 1.8, 3.0-fold improved, respectively. In vivo in rat, enteric-coated tablets based on Pluronic P85, Myrj 52 or Ch-TBA/GSH increased the area under the plasma concentration time curve (AUC(0-12)) of Rho-123 1.6-fold, 2.4-fold, 4.3-fold, respectively, in comparison to control only. Contrariwise, the low molecular mass excipients 6-mercaptopurine and GSH showed no significant effect in vivo at all. This in vivo study showed that polymeric P-gp inhibitors and especially the delivery system based on thiolated chitosan significantly increased the oral bioavailability of P-gp substrate Rho-123.

The natural mutation by deletion of Lys9 in the thrombin A‐chain affects the pKa value of catalytic residues, the overall enzyme's stability and conformational transitions linked to Na+ binding

The catalytic competence of the natural thrombin mutant with deletion of the Lys9 residue in the A-chain (deltaK9) was found to be severely impaired, most likely due to modification of the 60-loop conformation and catalytic triad geometry, as supported by long molecular dynamics (MD) simulations in explicit water solvent. In this study, the pH dependence of the catalytic activity and binding of the low-molecular mass inhibitor N-alpha-(2-naphthylsulfonyl-glycyl)-4-amidinophenylalanine-piperidine (alpha-NAPAP) to the wild-type (WT) and deltaK9 thrombin forms were investigated, along with their overall structural stabilities and conformational properties. Two ionizable groups were found to similarly affect the activity of both thrombins. The pKa value of the first ionizable group, assigned to the catalytic His57 residue, was found to be 7.5 and 6.9 in ligand-free deltaK9 and WT thrombin, respectively. Urea-induced denaturation studies showed higher instability of the deltaK9 mutant compared with WT thrombin, and disulfide scrambling experiments proved weakening of the interchain interactions, causing faster release of the reduced A-chain in the mutant enzyme. The sodium ion binding affinity was not significantly perturbed by Lys9 deletion, although the linked increase in intrinsic fluorescence was lower in the mutant. Essential dynamics (ED) analysis highlighted different conformational properties of the two thrombins in agreement with the experimental conformational stability data. Globally, these findings enhanced our understanding of the perturbations triggered by Lys9 deletion, which reduces the overall stability of the molecule, weakens the A-B interchain interactions, and allosterically perturbs the geometry and protonation state of catalytic residues of the enzyme.

Crystal Structures of Calpain–E64 and –Leupeptin Inhibitor Complexes Reveal Mobile Loops Gating the Active Site

The endogenous calpain inhibitor, calpastatin, modulates some patho-physiological aspects of calpain signaling. Excess calpain can escape this inhibition and as well, many calpain isoforms and autolytically generated protease core fragments are not inhibited by calpastatin. There is a need, therefore, to develop specific, cell-permeable calpain inhibitors to block uncontrolled proteolysis and prevent tissue damage during brain and heart ischemia, spinal-cord injury and Alzheimer's diseases. Here, we report the first high-resolution crystal structures of rat mu-calpain protease core complexed with two traditional, low molecular mass inhibitors, leupeptin and E64. These structures show that access to a slightly deeper, but otherwise papain-like active site is gated by two flexible loops. These loops are divergent among the calpain isoforms giving a potential structural basis for substrate/inhibitor selectivity over other papain-like cysteine proteases and between members of the calpain family.

Evidence for cAMP-dependent protein kinase in the dinoflagellate, Amphidinium operculatum

A cAMP dependent protein kinase (PKA) was identified in the dinoflagellate Amphidinium operculum. In vitro kinase activity towards kemptide, a PKA-specific substrate, was not detectable in crude lysates. However, fractionation of dinoflagellate extracts by gel filtration chromatography showed PKA-like activity toward kemptide at approximately 66 kDa. These findings suggest that possible low molecular mass inhibitors in crude lysates were removed by the gel filtration chromatography. Pre-incubation of extracts with cAMP prior to chromatography resulted in an apparent molecular mass shift in the in vitro kinase assay to 40 kDa. An in-gel kinase assay reflected activity of the free catalytic subunit at approximately 40 kDa. Furthermore, western blotting with an antibody to the human PKA catalytic subunit confirmed a catalytic subunit with a mass of approximately 40 kDa. Results from this study indicate that the PKA in A. operculatum has a catalytic subunit of similar size to that in higher eukaryotes, but with a holoenzyme of a size suggesting a dimeric, rather than tetrameric structure.

Cathepsin B and cysteine proteinase inhibitors in human lung cancer cell lines.

Cell lines derived from human squamous cell (EPCL), large cell (LCLC), and small cell lung cancer (SCLC) lines were investigated for the expression of cathepsin B (Cat B) and cysteine proteinase inhibitors (CPIs). The EPLC and LCLC lines expressed 5- to 50-fold more Cat B activity and contained more mature Cat B of M(r) 27-29 kDa (> 2.5 microg/mg total protein) than the SCLC lines (< 1.0 microg/mg total protein). The LPLC lines also secreted the highest amounts of Cat B precursor of M(r) about 46 kDa. Inhibitory activities against Cat B and papain were associated with high molecular mass (HMM) and low molecular mass (LMM) inhibitory proteins, both in cell extracts and in media. About 75% of the inhibitory activity was associated with HMM inhibitors, the majority of which were kininogens (M(r) > or = 67 kDa). The LMM inhibitors of M(r) 10-15 kDa were cystatin C and stefins A and B, which were quantitated by ELISA: stefins A and B were present in cell extracts and medium in similar concentrations (5-200 ng/10(6) cells), while 80-99% of the cystatin C was released in the medium (10-195 ng/10(6) cells). Phorbol ester (PMA), which induces protein-kinase C mediated signal transduction and enhances cellular differentiation in many non-small cell lung cancer (NSCLC) cell lines, increased intracellular Cat B activity and Cat B protein as well as its secretion in some cell lines but not in others, regardless of their histological type. PMA significantly (P < 0.049) decreased intracellular stefin A concentrations in two EPLC lines and non-significantly in two LCLC lines. PMA decreased secretion of stefin A in all EPLC lines, but not in LCLC lines, while IGF-I significantly increased stefin B secretion in both SCLC lines. These data showed that lung tumor cells produce both cysteine proteinases and cystatins. As the antagonistic molecules are regulated differently in histologically different types of lung tumor cells, it is possible that an imbalance between the proteinases and their specific inhibitors plays a role in progression of certain types of lung tumors in vivo.

Facile synthesis of aryl(difluoromethyl)phosphonates through CuBr-mediated cross coupling reactions of [(diethoxyphosphinyl)difluoromethyl]zinc bromide with aryl iodides

The CuBr-promoted coupling reaction of [(diethoxyphosphinyl)difluoromethyl]zinc bromide 6 with aryl iodides in either DMF or DMA was examined to give a series of aryl(difluoromethyl)phosphonates. The coupling reaction was applicable to the selective synthesis of α,α-difluoro-4- iodobenzylphosphonale 11e, a useful intermediate for the preparation of F 2Pmp. 1-[(Diethylphosphono)difluoromethyl] naphthalene 15 and the regioisomer 16 were obtained in high yields from 1- and 2-iodonaphthalene, respectively. The phosphonate 16 was transformed to the free acid 4a, a low molecular-mass inhibitor of protein phosphatases.

Calcium requirement and inhibitor spectrum for intracellular HIV type 1 gp160 processing in cultured HeLa cells and CD4+ lymphocytes: similarity to those of viral envelope glycoprotein maturase.

We recently purified the calcium-independent processing protease named viral envelope glycoprotein maturase (VEM), that converts human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein precursor gp160 to gp120 and gp41, from the human CD4+ T cell line, Molt-4 clone 8 [Kido, H., Kamoshita, K., Fukutomi, A., and Katunuma, N. (1993) J. Biol. Chem. 268, 13406-13413]. In this report, we deal with the inhibitor specificity and calcium requirement for intracellular gp160 processing in cultured HeLa cells and human CD4+ lymphocytes. Processing of gp160 in these cells infected with recombinant vaccinia virus encoding the gp160 gene was not affected by intracellular calcium depletion induced by the calcium ionophore A23187 and EGTA or by intracellular calcium administration. Processing of gp160 by the purified VEM in vitro was not inhibited by EDTA, EGTA, or the metallo-protease inhibitor phosphoramidon, but was specifically inhibited by a substrate analog, decanoyl-RVKR-chloromethylketone, and the trypsin-type protease inhibitors aprotinin, HI-30, and diisopropyl fluorophosphate (DFP). It was also inhibited by E-64 and thiol reagents. But intracellular gp160 processing was inhibited only by permeable, low molecular mass inhibitors of VEM, such as DFP, E-64, and thiol reagents. Syncytium formation induced by cell surface gp120 was also inhibited by permeable inhibitors of VEM. Taken together, our results indicate that calcium ions may not be essential for intracellular gp160 processing and so HIV-1 gp160 induced by recombinant vaccinia virus may be processed mainly by a protease(s) that does not require calcium ions, such as VEM in these cells.

Relationship between two types of mouse sperm surface sites that mediate binding of sperm to the zona pellucida.

There are at least two binding sites for the mouse egg zona pellucida on the surface of mouse sperm: a site with galactosyltransferase (GT) activity inhibitable by uridine-5'-diphosphate-dialdehyde (UDPd) and alpha-lactalbumin, and a trypsin inhibitor-sensitive (TI) site that hydrolyzes guanidinobenzoate (GB) esters. Characterization of GT activity gave the Km for UDP galactose as 37 microM with N-acetylglucosamine as galactose acceptor, and Vmax as 0.37 pmol/min/10(6) sperm. UDP galactose from 12.5-100 microM inhibited sperm binding to zona-intact eggs in a concentration-dependent manner with close correlation to GT activity (r = 0.95). To assess the independence and spatial relationship of the two types of site, cross-perturbation studies were performed. p-Nitrophenyl-GB, a low molecular mass inhibitor specific for the TI site, had no effect on the enzyme activity of the GT site. Conversely, UDPd, a specific inhibitor of GT, had no effect on GB hydrolysis. Weak inhibitions were found when soybean trypsin inhibitor (SBTI) was included with the GT assay and when GB hydrolysis was assayed in the presence of alpha-lactalbumin or asialo-agalacto-(alpha 1-acid glycoprotein). Acid-solubilized zona protein (ASZP) weakly inhibited the GT reaction, while stronger inhibition was seen with chymotrypsin-solubilized zona protein (CSZP). ASZP inhibited sperm binding to zonae with the same concentration dependence associated with inhibition of GB hydrolysis, but the inhibition of GT enzyme activity was on the same order as that found with SBTI, indicating that ASZP was only binding to the TI site under enzyme assay conditions. The results support the hypothesis that the two types of site are independent in binding their specific zona ligands, but are close enough for steric perturbation of the enzyme activity of one site by macromolecules bound to the other. The different interactions of solubilized zona preparations with the GT site under enzyme assay conditions are an indication that conditions which favor the enzyme activity of the site may interfere with the physiological binding functions of the site.

Low Molecular Mass Inhibitors from Calf Thymus Selective for T-Lymphocyte Proliferation

Endogenous factors preferentially inhibiting T-lymphocyte proliferation were prepared from the acetone precipitate of a 60% ethanol extract from calf thymus and their biochemical properties examined. By ultrafiltration the strongest lymphocyte-selective inhibition was found in the molecular mass range between 1 and 5 kDa. Fast protein liquid chromatography (FPLC) of this fraction on an anion exchange column eluted the lymphocyte inhibitors at 205-265 mM ammonium acetate. Staining procedures following IEF and TLC suggested, that the inhibitors may not be glycoconjugates or amines but small weakly acidic peptides (less than 3 kDa).

Cephalosporin antibiotics can be modified to inhibit human leukocyte elastase.

Several laboratories, including our own have reported the synthesis and activity of certain low relative molecular mass inhibitors of mammalian serine proteases, especially human leukocyte elastase (HLE, EC 3.4.21.37), an enzyme whose degradative activity on lung elastin has been implicated as a major causative factor in the induction of pulmonary emphysema, and which is present in the azurophil granules of human polymorphonuclear leukocytes (PMN). Normally, these granules fuse with phagosomes containing engulfed foreign material (such as bacteria), and HLE, in combination with other lysosomal enzymes, catabolizes the particles. Under certain pathological conditions, however, PMN become attached to host protein (elastin fibres, basement membrane, connective tissue, immune complexes), and in response to this adherence, the granules may fuse with the PMN outer membrane and release their contents, including HLE, directly onto the tissue. Besides emphysema, HLE may also contribute to the pathogenesis of disease states such as adult respiratory distress syndrome, and its potential involvement in rheumatoid arthritis makes HLE inhibitors of considerable interest. It is known that cephalosporin antibiotics (for example, cephalothin (compound I, Table 2)) are acylating inhibitors of bacterial serine proteases which help synthesize the cell wall by performing a transpeptidation reaction on a peptidyl substrate bearing a D-Ala-D-Ala terminus. We now report that neutral cephalosporins (that is, compounds not bearing a free carboxyl at position C-4) can be modified to become potent time-dependent inhibitors of HLE.

Acid-stable low molecular mass proteinase inhibitors in human lung lavage.

An acid-stable, low molecular mass proteinase inhibitor, bronchial mucus proteinase inhibitor (BMPI), has been isolated from sputum and partially characterised. A single band with a modal molecular mass of 18 700 was observed following SDS-polyacrylamide gel electrophoresis. BMPI inhibited human leukocyte elastase, cathepsin G, trypsin and chymotrypsin, but not porcine pancreatic elastase. Although BMPI had a molecular mass close to the similarly isolated inhibitor of Girard et al. (Girard, F., Tournier, J.M., Polu, J.M. & Sadoul, P. (1980), Bull. Eur. Physiopathol. Respir. 16 (Suppl.) 237-245), and although it showed immunological cross reactivity to the low molecular mass inhibitor of Kramps et al. (Kramps, J.A., Franken, C., Meyer, C.J.L.M. & Dijkman, J.H. (1981) J. Histochem. Cytochem. 29, 712-719), it was found to have an amino-acid profile different to any previously described inhibitor. BMPI was detectable in bronchoalveolar lavage fluid collected from healthy and diseased human lungs. The median molar ratio of BMPI/alpha 1-proteinase inhibitor (alpha 1 PI) observed in these lavage samples was 0.7, which is generally higher than those derived from the data of other authors. This suggests that BMPI is a different protein to those previously described, although its exact relationship to other low molecular mass proteinase inhibitors remains to be determined.

Purification, characterization and sequence determination of a double-headed trypsin inhibitor peptide from Trichosanthes kirilowii (a Chinese medical herb).

A double-headed trypsin inhibitor peptide was isolated and purified from the root of Trichosanthes kirilowii Maxim (Cucurbitaceae), a Chinese medical herb, by 2.5% trichloroacetic acid and heat treatment followed by affinity chromatography with immobilized trypsin and ion-exchange chromatography. This inhibitor, consisting of 41 amino-acid residues with three pairs of disulfide bonds was sequenced. Two active domains were found to be located at two disulfide loops composed of eight (Pos. 17-24) and nine (Pos. 29-37) amino-acid residues, respectively. It inhibits two molecules of trypsin simultaneously and might be regarded as the smallest double-headed trypsin inhibitor (Mr = 4575) so far known. The chemical modification of the inhibitor with cyclohexandione and citraconic anhydride showed that Arg20-Gly21 and Lys30-Leu31 corresponded to the two reactive sites, respectively. The discovery of the Trichosanthes inhibitor is of importance not only for the study on the structure-function relationship of proteinase inhibitor peptides but also for the search for low molecular mass inhibitors of clinical value among Chinese medical herbs.

Isolation of acid-resistant urinary trypsin inhibitors by high performance liquid chromatography and their characterization by N-terminal amino-acid sequence determination.

Two crude fractions of acid-resistant trypsin inhibitors (apparent molecular masses 44 and 20 kDa, respectively) were prepared from human urine by gel permeation chromatography. From both preparations the pure inhibitors were isolated by high performance liquid chromatography (HPLC). Their N-terminal amino-acid sequences were determined and compared with those of HI-30 and HI-14 as isolated by reversible binding to either immobilized trypsin or immobilized chymotrypsin. The N-terminal amino-acid sequence of the high-molecular mass inhibitor UI-I isolated by HPLC was identical with those of HI-30 and UI-C-I isolated via immobilized trypsin or chymotrypsin, respectively. The low-molecular mass inhibitors UI-II and UI-C-II differ from HI-14 by the N-terminal extension Glu-Val-Thr-Lys-when obtained by HPLC or by the extension Thr-Lys-when obtained via immobilized chymotrypsin, respectively. The comparison of these N-termini with the amino-acid sequence of HI-30 (Ala1-...-Val16-Thr-Glu-Val-Thr-Lys-HI-14) defines the low molecular urinary trypsin inhibitors as proteolytic degradation products of the high-molecular urinary inhibitor. Proteolysis may occur at different bonds. The existing discrepancies in molecular architecture and in molecular masses of the urinary trypsin inhibitors are discussed.

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.