Aprotinin and a seminal plasma factor inhibit the motility of demembranated reactivated rabbit spermatozoa

The demembranation and reactivation of ejaculated rabbit spermatozoa have been studied. ATP, Mg, glutamate, dithiothreitol (DTT), and Tris-HCl were found to be essential for a good reactivation. With this experimental model, we investigated the effects of protease inhibitors on the reinitiation of movement by ATP and on the movement of already motile spermatozoa. Soybean trypsin inhibitor (STI) prolonged the length of reactivation by 7- to 8-fold, whereas pepstatin, antithrombin III, phenylmethylsulfonyl fluoride (PMSF), and alpha-1-antitrypsin had no significant effect. Aprotinin (1.5 micrograms/ml) and leupeptin (50 micrograms/ml) completely prevented the reinitiation of movement by ATP; aprotinin at the same concentration even blocked the movement of motile spermatozoa. A tissue-specific seminal plasma factor could also prevent both the reinitiation of movement and the movement of motile spermatozoa. However, it took 2-3 times the amount of seminal plasma to stop the movement than to prevent the reinitiation of movement. The inhibitor in the seminal plasma is most probably not a protease or an aprotinin-like protease inhibitor since a partially purified preparation of the seminal plasma inhibitor does not hydrolyze a trypsin substrate, is not inhibited by protease inhibitors and has no significant capacity to inhibit trypsin. The data suggest that aprotinin and the seminal plasma inhibitor block movement through different mechanisms. Aprotinin and the seminal plasma inhibitor represent two new tools to study the regulation of sperm movement.     

Kallikrein inhibitors.

So far the Cl inactivator, alpha 2-macroglobulin, antithrombin III (in the presence of heparin), and alpha 1-antitrypsin have been identified as inhibitors of plasma kallikrein; alpha 1-antitrypsin reacts slowly also with tissue kallikreins. Of the various naturally occurring kallikrein inhibitors the basic trypsin-kallikrein inhibitor of bovine organs, aprotinin (the active substance of Trasylol), has attained by far the most interest. This inhibitor, which is produced by mast cells, has unusual properties due to its compact tertiary structure. Additional topics of aprotinin and structurally related inhibitors discussed are the mechanism of enzyme-inhibitor complex formation, the production of chemical mutants of aprotinin, the structural basis of kallikrein inhibition, and selected aspects regarding aprotinin medication.     

Detection of protease inhibitors by a reverse zymography method, performed in a tris(hydroxymethyl)aminomethane-Tricine buffer system

A new detecting method for protease inhibitors, especially for low-molecular-weight inhibitors, is reported. Inhibitor samples were separated on a protein substrate-SDS-polyacrylamide gel in a Tris-Tricine buffer system that improves the separation and identification of peptides and low-molecular-weight proteins. After electrophoresis, the gel was incubated with the target proteases to hydrolyze the background protein substrate. The inhibitor bands, which were protected from proteolysis by the target proteases, were stained. Standard low-molecular-weight inhibitors, such as pepstatin A for pepsin or matrix metalloproteases inhibitor I for collagenase, as well as larger inhibitors, such as soybean trypsin inhibitor or aprotinin for tryspin and cystatin C for papain, were demonstrated by this method and showed clear blue inhibitor bands in the white background when the gels were treated with the target proteases. Some significant applications of this method are introduced. This method is an ideal system for discovering new protease inhibitors in small natural samples.     

Semisynthetic engineering of proteinase inhibitor homologues

A semisynthetic approach to modulate the inhibitory specificity of aprotinin, the Kunitz trypsin inhibitor from bovine mast cells, is described. By the use of peptide-chemical procedures a single amino acid of its reactive site can be replaced by any other coded or non-coded amino acid. Thus, a series of aprotinin homologues have been prepared which demonstrate the individual contribution of a single side chain to the inhibition of a particular target proteinase and enable specific inhibitors to be designed.     

Enhancement of transformed cell growth in agar by serine protease inhibitors

We investigated the effects of three serine protease inhibitors (leupeptin, soybean trypsin inhibitor, and aprotinin) on the serum-free growth of two transformed cell lines in soft agar. Aprotinin markedly enhanced the growth of rat embryo fibroblasts that had been transformed by polyoma middle T antigen (PyMLV-REF52), while having only a slight effect on the colonial growth of SV40 transformed Balb/c 3T3 cells (SV3T3-Aga). Leupeptin and soybean trypsin inhibitor, on the other hand, significantly enhanced the growth of SV3T3-Aga cells while having little effect on PyMLV-REF52 growth. We observed no stimulatory effect of any of the protease inhibitors on serum-free monolayer growth. Under conditions of excess aprotinin, PyMLV-REF52 cells were found to be unresponsive to epidermal growth factor (EGF) at a concentration that would normally stimulate agar colony growth. However, aprotinin was not capable of supporting colony formation with transforming growth factor-beta. These results indicate that aprotinin acts primarily as a protease inhibitor in spite of its structural homology to EGF and that EGF may promote the soft agar growth of these cell lines either by inhibiting proteolysis directly or by enhancing the synthesis of a serine protease inhibitor.     

Synthesis, cloning and expression of recombinant aprotinin

Synthetic DNA fragments containing the coding sequence for the serine proteinase inhibitor aprotinin, also known as bovine pancreatic trypsin inhibitor (BPTI) a Kunitz type inhibitor were fused to form a synthetic aprotinin gene by the method of Khorana and cloned into E. coli. The synthetic gene is characterized by the presence of certain restriction sites. These restriction sites are unique within the used cloning system. Therefore, a great number of modifications can be achieved easily by exchange of appropriate restriction fragments. Using this method the variant [Glu52]aprotinin was obtained starting from the aprotinin gene. Both genes were successfully expressed in E. coli as fusion proteins with beta-galactosidase using vector pUR 278. No translation products could be detected in four other expression system (pUR 108, pDR 540, pKK 223-3 and pUC 8). [Glu52]aprotinin was purified and renatured after cyanogen bromide cleavage of the fusion protein. This recombinant [Glu52]aprotinin shows exactly the same trypsin-inhibitory profile as natural aprotinin.     

Semiquantitative determination of low molecular weight proteinase inhibitors in the presence of high molecular weight inhibitors by a modified radial diffusion assay

The radial diffusion assay is very suitable for the determination of proteinase inhibitors in biological fluids. By combining radial diffusion and ultrafiltration, it has become possible to directly determine low molecular weight proteinase inhibitors in mixtures with inhibitors of higher molecular weight. By this modification the inhibitor solutions to be investigated are not pipetted into wells as usually, but are applied on small pieces of dialysis membranes lying on the gel. The exclusion limit of the membrane must be of a magnitude that the inhibitors of higher molecular weight are retained, whereas the inhibitors of lower molecular weight can diffuse into the gel. The modified method can be used for the direct determination of e.g. aprotinin (Mr 6500) in the presence of alpha 1-proteinase inhibitor (Mr 54,000), ovoinhibitor (Mr 50,000) and ovomucoid (Mr 27,000), respectively. The modified method is suitable for the direct determination of low molecular weight inhibitors of trypsin and papain in serum, synovial fluid and saliva. Tissue extracts containing 4 M guanidine hydrochloride or 6 M urea can be investigated directly, too.     

Characterization and sequence determination of six aprotinin homologues from bovine lungs

Six Kunitz inhibitors, which are dissimilar to aprotinin, can be isolated from bovine lungs. These homologues cannot be distinguished from aprotinin, in respect to their inhibitory specificity. They have, however, different amino-acid compositions and a different degree of basicity. The entire primary structures of these inhibitors were elucidated by automated Edman sequencing. Besides the known Glp-1-aprotinin another aprotinin homologue (des-Ala58-aprotinin) was isolated, which could result from a different proteolytic processing of the bovine aprotinin precursor. The other homologues can be denoted as aprotinin isoinhibitors, showing several amino-acid replacements compared to aprotinin and which also appear in the area of the contact region.     

Effect of protease inhibitors on androgen receptor analysis in rat prostate cytosol

Prostate androgen receptors are liable to proteolytic digestion during in vitro analysis; thus, various proteolytic enzyme inhibitors were tested for their ability to improve the androgen receptor assay. The serine (phenylmethylsulfonylflouride, aprotinin, p-aminobenzamidine) and thiol-senine (leupeptin, bacitracin) protease inhibitors individually present in the homogenization buffer significantly increased the measurable androgen binding sites by 30-35% in rat prostate cytosol as determined by saturation analysis with [3H]-17 beta-hydroxy-17-methyl- 4,9 11-estratrien-3-one (R-1881) for 20 hr at 4 degrees C. The apparent binding affinity was also increased by these compounds. Various combinations were tried and aprotinin/bacitracin was found to be additive in effect. This combination was also shown to prevent receptor degradation as determined by sucrose density gradient centrifugation. The carboxyl protease inhibitor, pepstatin A, was ineffective in improving the receptor assay. Rabbit bile, an inhibitor of seminin, interfered with receptor binding thus rendering it ineffective for use in saturation analysis. The results show that the use of serine-thiol protease inhibitors significantly improves the cytosol androgen receptor yield and assay sensitivity; therefore, we recommend routine inclusion of these compounds(s) in the homogenization buffer for androgen receptor assays.     

Entrapment of protein protease inhibitors in red blood cells.

Aprotinin and alpha 1-proteinase inhibitor have been encapsulated in human red blood cells (RBC) by a dialysis technique that involves transient hypotonic haemolysis followed by isotonic resealing. Both protease inhibitors can be encapsulated to a considerable extent. These molecules are released only by haemolysis of the cells and that excludes the possibility of using loaded erythrocytes for a slow release of the inhibitor(s) in the blood stream. However, the stability of the two inhibitors, the evidence for the binding of aprotinin to RBC components, and the results showing inhibition of endogenous proteolytic activity indicate that the inhibitors may be valuable in blocking, at least partially, undesired intraerythrocytic proteolytic reactions.     

Stage-specific inhibition of Xenopus embryogenesis by aprotinin, a serine protease inhibitor.

We examined the effects of various protease inhibitors on Xenopus laevis embryogenesis. Aprotinin, a serine protease inhibitor, was found to inhibit embryogenesis markedly, but other protease inhibitors had virtually no effect. The inhibitory effect of aprotinin was specific for embryos at the blastula or gastrula stage. These results suggest that an aprotinin-sensitive protease involved in embryonic development is secreted from the embryos or appears on the surface of embryonic cells at these stages. We found that various serine proteases are in fact secreted from the embryos with their development and that some of them are sensitive to aprotinin.     

Expression, purification and characterization of aprotinin and a human analogue of aprotinin

Aprotinin is a Kunitz-type inhibitor with a relatively broad specificity. It has been shown to be clinically useful for the management of hemorrhagic complications. In this report, small ubiquitin-related modifier (SUMO) linked with a hexa-histidine tag was used as a fusion partner for the production of recombinant aprotinin and a human aprotinin analogue (cloned form human cDNA library). Both fusion proteins were overexpressed mainly as inclusion bodies in Escherichia coli and accounted for approximately 28% of the total cell proteins. After purification by Ni-Sepharose affinity chromatography and renaturation, the fusion proteins were cleaved with SUMO protease 1. Aprotinin and its analogue were separated from the fusion partner by the subtractive chromatography using Ni-Sepharose and then further purified with CM-cellulose. Kinetic studies demonstrated that the amidolytic activity of plasmin was competitively inhibited by recombinant aprotinin with a K_i of 8.6 ± 2.4 nM, which was similar to the K_i (7.5 ± 2.7 nM) of natural aprotinin. The K_i of human aprotinin analogue was 22.7 ± 6.5 nM. The expression strategy described in this study allows convenient high yield and easy purification of small recombinant protease inhibitors with complete native sequences.     

Pyroglutamyl-aprotinin, a new aprotinin homologue from bovine lungs--isolation, properties, sequence analysis and characterization using 1H nuclear magnetic resonance in solution

A new Kunitz-inhibitor, which is different from aprotinin was extracted from bovine lungs with methanol, further purified by affinity chromatography on trypsin-Sepharose CL-6B and by repeated cation exchange chromatography on CM-Sephadex C-25. The inhibitor, which is less basic than aprotinin was characterized by polyacrylamide gel electrophoresis and ion-exchange HPLC. The N-terminus is blocked by pyroglutamic acid (Glu-1). After enzymatic removal of this residue with pyroglutamate aminopeptidase, complete identity with the primary structure of aprotinin was established by sequencing the inhibitor, which had been oxidized with performic acid, and by sequencing a tryptic fragment. The occurrence of the inhibitor, which can be denoted as pyroglutamyl-aprotinin or Glu-1-aprotinin, but which cannot be distinguished from aprotinin regarding its inhibitory specificity, is obviously the result of a different proteolytic processing of the bovine aprotinin precursor. By using CD and NMR-techniques it was shown that the N-terminus of the inhibitor is blocked, and that the conformation and the internal mobility correspond with those of aprotinin.     

Vascular localization of bovine pancreatic trypsin-inhibitor-related molecular forms in bovine spleen

Bovine spleen proteic inhibitors of serine proteases, belonging to the bovine pancreatic trypsin inhibitor (BPTI or aprotinin) family, have been localized, using immunocytochemical techniques, in the smooth muscle cells of some bovine spleen blood vessels. This vascular localization also occurs in a variety of bovine organs and differs from that of BPTI itself which is found exclusively in bovine mast cells, in agreement with previous reports. These data would be in favour of a possible involvement of one or more BPTI-type inhibitors in vascular processes by acting at the level of the smooth muscle cells, the tissue responsible for vasodilation/vasoconstriction events.     

Aprotinin inhibits the hormone binding of the estrogen receptor from calf uterus

Micromolar concentrations of the proteinase inhibitor, aprotinin, produced a dose-dependent inhibition in the binding capacity of the estrogen receptor from calf uterus. Aprotinin inhibition was greater at 28 degrees C than at 4 degrees C and only occurred when conditions allowed the receptor transformation. When aprotinin was tested in the presence of transformation inhibitors, its effect was no longer seen. The binding capacity of the highly purified estrogen-binding subunit was similarly inhibited.     

Characteristics of binding of zwitterionic liposomes to water-soluble proteins

The interactions of zwitterionic phospholipids phosphatidylcholine and phosphatidylethanolamine with protein proteinase inhibitors aprotinin and Bowman-Birk soybean proteinase inhibitor have been investigated. An increase in the hydrophobicity of the liposome surface was shown to be an important factor for the formation of proteoliposomes. According to ³¹P-NMR spectra, incorporation of the proteins into the liposomes does not influence the structural organization of the surface of the liposomes. Increasing the ionic strength does not inhibit the process of proteoliposome formation. Fluorescence assay of the complexes of anthracene-labeled phospholipids with the rhodamine B-labeled protein showed that after the encapsulation into the liposomes, the protein is located inside the particles and between the bilayers. Also, the effect of phospholipids with saturated fatty acid residues on the protein-lipid interaction was studied by differential scanning calorimetry. The results indicate that water-soluble proteins efficiently interact with zwitterionic phospholipids, and the encapsulation of the proteins into the liposomes is provided by electrostatic and hydrophobic forces (in the case of aprotinin) or predominantly by hydrophobic forces (Bowman-Birk soybean proteinase inhibitor).     

Immunolocalisation of BPTI-like serine proteinase inhibitory proteins in mast cells, chondrocytes and intervertebral disc fibrochondrocytes of ovine and bovine connective tissues. An immunohistochemical and biochemical study

A polyclonal anti-bovine pancreatic trypsin inhibitor (BPTI) IgY was raised in chickens immunised with aprotinin. The anti-BPTI IgY was subsequently isolated from egg yolks and purified to homogeneity by affinity chromatography on immobilised aprotinin and by Superose 6 size exclusion fast protein liquid chromatography (FPLC). Immunoblotting with the chicken IgY demonstrated its specificity for BPTI; 3.9 ng BPTI could be detected by this technique. There was no crossreactivity against alpha1-proteinase inhibitor (human and sheep), inter-alpha-trypsin inhibitor (human and sheep), secretory leucocyte proteinase inhibitor or a range of serine proteinase inhibitory proteins (SPIs) isolated from plant sources (soybean and lima bean trypsin inhibitor, potato trypsin and chymotrypsin inhibitors) or serum SPIs (antithrombin-III, alpha2-macroglobulin). Immunoblotting using the anti-BPTI IgY identified the 6- to 12- and 58-kDa forms of endogenous ovine cartilage SPIs in cartilage extracts, confirming the interrelationship of the ovine cartilage SPIs with BPTI. BPTI-domain SPIs were immunolocalised within mast cells of ovine and bovine duodenum, lung and pancreas, and in ovine and bovine bronchial cartilage chondrocytes, chondrocytes of the superficial and intermediate zones of articular cartilage and in the fibrochondrocytes/chondrocytes of the nucleus     

Aprotinin binding to amyloid fibrils.

Different low molecular mass ligands have been used to identify amyloid deposits. Among these markers, the dyes Thioflavin T and Congo Red interact specifically with the beta-sheet structure arranged in a cross-beta conformation, which is characteristic of amyloid. However, the molecular details of this interaction remain unknown. When labelled with technetium-99m, the proteinase inhibitor aprotinin has been shown to represent a very important radiopharmaceutical agent for in vivo imaging of extra-abdominal deposition of amyloid in amyloidosis of the immunoglobulin type. However, no information is available as to whether aprotinin binds other types of amyloid fibrils and on the nature and characteristics of the interaction. The present work shows aprotinin binding to insulin, transthyretin, beta-amyloid peptide and immunoglobulin synthetic amyloid fibrils by a specific dot-blot ligand-binding assay. Aprotinin did not bind amorphous precipitates and/or the soluble fibril precursors. A Ka of 2.9 microM-1 for the binding of aprotinin to insulin amyloid fibrils was determined by Scatchard analysis. In competition experiments, analogues such as an aprotinin variant, a spermadhesin and the soybean trypsin inhibitor were tested and results suggest that both aprotinin and the spermadhesin interact with amyloid fibrils through pairing of beta-sheets of the ligands with exposed structures of the same type at the surface of amyloid deposits. An electrostatic component may also be involved in the binding of aprotinin to amyloid fibrils because important differences in binding constants are observed when substitutions V15L17E52 are introduced in aprotinin; on the other hand beta-sheet containing acidic proteins, such as the soybean trypsin inhibitor, are unable to bind amyloid fibrils.     

Effects of Fibrinolytic Inhibitors on Chondrogenesis of Bone-marrow Derived Mesenchymal Stem Cells in Fibrin Gels

The objective of this study was to examine the effect of two fibrinolytic inhibitors, aprotinin and aminohexanoic acid, on chondrogenesis of rabbit bone marrow mesenchymal stem cells (BM-MSCs). Rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand White rabbits. Cell–fibrin constructs were made by mixing a cell–fibrinogen (10⁷ cells/ml; 40 mg/ml fibrinogen) solution with a thrombin (5 IU/ml) solution and then divided into four groups: aprotinin control, aprotinin + transforming growth factor β (TGF-β), aminohexanoic acid control, and aminohexanoic acid + TGF-β. Each of these groups was further treated with three different concentrations of inhibitors and the TGF-β groups were treated with 10 ng/ml of TGF-β1. The chondrogenic gene expressions, DNA content, and glycosaminoglycan content of samples were analyzed after 14 days of culture. The aprotinin groups exhibited significantly higher levels of aggrecan gene expression and glycosaminoglycan content than the aminohexanoic acid groups. However, inhibitor neither influenced gene expression of type II collagen nor proliferation (i.e., DNA content) of BM-MSCs. These findings suggest that fibrinolytic inhibitors used to control degradation of fibrin clot may influence TGF-β-induced chondrogenesis of BM-MSCs.     

Recombinant aprotinin produced in transgenic corn seed: extraction and purification studies

Expression in transgenic plants is potentially one of the most economical systems for large-scale production of valuable peptide and protein products. However, the downstream processing of recombinant proteins produced in plants has not been extensively studied. In this work, we studied the extraction and purification of recombinant aprotinin, a protease inhibitor used as a therapeutic compound, produced in transgenic corn seed. Conditions for extraction from transgenic corn meal that maximize aprotinin concentration and its fraction of the total soluble protein in the extract were found: pH 3.0 and 200 mM NaCl. Aprotinin, together with a native corn trypsin inhibitor (CTI), was captured using a tryspin-agarose column. These two inhibitors were separated using an agarose-IDA-Cu2+ column that proved to efficiently absorb the CTI while the recombinant aprotinin was collected in the flowthrough with purity of at least 79%. The high purity of the recombinant aprotinin was verified by SDS-PAGE and N-terminal sequencing. The overall recombinant aprotinin recovery yield and purification factor were 49% and 280, respectively. Because CTI was also purified, the recovery and purification process studied has the advantage of possible CTI co-production. Finally, the work presented here introduces additional information on the recovery and purification of recombinant proteins produced in plants and corroborates with past research on the potential use of plants as biorreactors.