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.     

Serine proteinase inhibitors from Vipera ammodytes venom. Isolation and kinetic studies

Three protein inhibitors of serine proteinases were isolated from the crude venom of the long-nosed viper Vipera ammodytes ammodytes by ion-exchange and gel chromatography. Two of them strongly inhibit trypsin (Ki = 3.4 X 10(-10) and 5.6 X 10(-10) M), while the third one primarily inhibits chymotrypsin (Ki = 4.3 X 10(-9) M). Their Mr values are close to 7000, and pI is 9.8 in both trypsin inhibitors and 10.0 in the chymotrypsin inhibitor. The N-terminal group in the former inhibitors is blocked; arginine is the N-terminal amino acid in the latter. Besides trypsin and alpha-chymotrypsin, the trypsin inhibitors also inhibit plasmin, human plasma kallikrein and porcine pancreatic kallikrein. The chymotrypsin inhibitor inhibits trypsin and human plasma kallikrein only weakly and does not inhibit plasmin and porcine pancreatic kallikrein. According to their properties, all three inhibitors belong to the Kunitz-pancreatic trypsin inhibitor family of inhibitors.     

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.     

Proteinase inhibitors of horse seminal plasma. A high molecular mass, acid-soluble proteinase inhibitor

Horse seminal plasma does not possess a proteinase inhibitor corresponding to human HUSI-I (human seminal plasma inhibitor). Instead a protein complex of high relative molecular mass (Mr) containing proteinase inhibitory activity was detected, which was called horse seminal plasma protein complex or HSPC. The compound had a broad enzyme-inhibiting spectrum. Its Mr was estimated to be 800 000 and it was composed of 7 different polypeptides with Mr values ranging from 11 000 to 30 000. Its carbohydrate content was between 3.5% and 5%. Despite the high molecular mass, the complex was soluble in diluted perchloric acid and did not lose its biological activity. The high recovery of seminal plasma protein (69%) after perchloric acid treatment, the unaltered immunoelectrophoretic precipitation pattern of the perchloric acid soluble part of seminal plasma, and the similarity of the polypeptide patterns of unfractionated seminal plasma and HSPC suggest that HSPC is one of the major components of horse seminal plasma. In addition to HSPC, horse seminal plasma contained a group of three electrophoretically distinguishable proteinase inhibitors, corresponding roughly to a Mr of 6500. They inhibited only trypsin. The similar Mr values and the identical narrow enzyme specificity suggest that they are isoinhibitors and may be analogues of human HUSI-II (human seminal plasma inhibitor). The lack of a HUSI-I analog in the horse is discussed in relation to a previously made observation that horse tracheobronchial fluid contains no detectable perchloric acid-soluble proteinase inhibitors.     

Trypsin inhibitors of buffalo seminal plasma.

Two trypsin inhibitors from acid-treated buffalo seminal plasma were purified by gel filtration and affinity chromatography. These acid-stable trypsin inhibitors having charge heterogeneity were homogeneous with respect to size as revealed by gel filtration and SDS-PAGE. Gel filtration data suggest molecular weight value of 9,900 Da for inhibitor I and 10,900 Da for inhibitor II. Molecular weight estimated by SDS-PAGE was found to be 10,600 Da and 11,200 Da for inhibitors I and II, respectively. The hydrodynamic properties such as Stokes radii (1.58 nm and 1.62 nm); intrinsic viscosity (2.5725 ml/g and 2.5025 ml/g) and diffusion coefficient (13.499 x 10(-11) m2/sec. and 13.166X10(-11) m2/sec) respectively for inhibitor I and II were determined by analytical gel filtration. These inhibitors were fairly thermostable and could not be stained by PAS reagent. Both the inhibitors were found to inhibit buffalo acrosin but not bovine chymotrypsin.     

Isolation and identification of fetuin-B-like protein from rainbow trout seminal plasma and its localization in the reproductive system

Seminal plasma of rainbow trout (Oncorhynchus mykiss, Salmonidae) contains an inhibitory system consisting of three fractions (I-III) characterized by different electrophoretic migration rates. Using a two-step isolation procedure we purified (20- and 43-fold to homogeneity) and characterized the two subforms of inhibitor I (Ia and Ib). On the basis of the homology alignment of the amino acid sequences, inhibitor I was classified to the family of cysteine proteinase inhibitors - fetuins. The molecular masses were determined to be 61,146.5Da and 63,096.0Da, and the isoelectric points were estimated to be 6.04 and 6.22 for inhibitor Ia and Ib. Both inhibitors were glycoproteins with a carbohydrate content about 13% for inhibitor Ia and 19% for inhibitor Ib. The equilibrium association constant of inhibitor Ib with cod trypsin was determined to be 7.1×10(8)M(-1). Except for the cod trypsin inhibition, the inhibitor Ib effectively inhibited papain belonging to the cysteine proteainases. Comparative studies of the distribution of inhibitor I and the previously described inhibitor II were performed. The presence of inhibitor I in the seminal plasma was a common feature of several Salmoniformes, which was contrary to inhibitor II detected in seminal plasma of other fish families. Inhibitors I and II showed different expression patterns in the testes and spermatic duct of the rainbow trout.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

Secretion and immunochemical properties of the trypsin inhibitor from bovine colostrum.

A trypsin inhibitor was isolated from bovine colostrum by affinity chromatography. Immunoelectrophoresis detected two immunogenic components in the isolated inhibitor, but only one of these was specific for the inhibitor; the other one was identical with an antigen present in liver, kidney, spleen, adrenal, thyroid, thymus, brain, ovarian, testicular and udder tissue and in bull seminal plasma. Using immunoabsorption and immunofluorescence it was shown that the antigens specific for the trypsin inhibitor of colostrum could be demonstrated only in the tissue of an udder that is secreting colostrum. The inhibitor is secreted by the secretory epithelium of the milk alveoli of the udder, during the period when the latter secretes colostrum. This inhibitor was not detected in the milk. Cross-reaction between antisera to colostral inhibitor and basic pancreatic inhibitor or seminal plasma inhibitors yielded negative results. Antiserum to bovine colostral inhibitor showed a positive reaction with inhibitor isolated from porcine colostrum.     

Purification of 5'-nucleotidase from human seminal plasma.

5'-Nucleotidase from human seminal plasma was purified to electrophoretic homogeneity and some of its kinetic and molecular properties compared with those of 5'-nucleotidase from bull seminal plasma. The purification of the enzyme was achieved by using the same affinity chromatography media (Con A-Sepharose and AMP-Agarose or ADP-Agarose) previously used for the purification of bull seminal plasma 5'-nucleotidase (Fini, C., Ipata, P.L., Palmerini, C.A. and Floridi, A. (1983) Biochim. Biophys. Acta 748, 405-412). However, in the present purification procedure no detergent was used as it had been necessary for the purification of the bovine enzyme. The experimental data reveal some main differences between these two enzymes; first, the human enzyme seems to be constituted of a single polypeptide chain of about 71 kDa, while the 5'-nucleotidase of bull seminal plasma, in non denaturing detergent solutions, is a homodimer of about 160 kDa. Another most remarkable difference is that the human enzyme does not seem to contain a phosphatidylinositol anchoring system like the one present in the bovine enzyme and in 5'-nucleotidase of different sources (Low, M.G. (1987) Biochem. J. 244, 1-13). Finally, the AMPase activity of 5'-nucleotidase from human seminal plasma is not affected by dithiothreitol which, on the contrary, is a powerful inhibitor of the bovine enzyme causing the dissociation of its subunits which are held together by disulphide bridges (Fini, C., Minelli, A., Camici, M. and Floridi, A. (1985) Biochem. Biophys. Acta 827, 403-409).     

Human mesotrypsin defies natural trypsin inhibitors: from passive resistance to active destruction

More than twenty years ago Rinderknecht et al. identified a minor trypsin isoform resistant to natural trypsin inhibitors in the human pancreatic juice. At the same time, Estell and Laskowski found that an inhibitor-resistant trypsin from the pyloric caeca of the starfish, Dermasterias imbricata rapidly hydrolyzed the reactive-site peptide bonds of trypsin inhibitors. A connection between these two seminal discoveries was made recently, when human mesotrypsin was shown to cleave the reactive-site peptide bond of the Kunitz-type soybean trypsin inhibitor, and degrade the Kazal-type pancreatic secretory trypsin inhibitor. These observations indicate that proteases specialized for the degradation of protease inhibitors are ubiquitous in metazoa, and prompt new investigations into their biological significance. Here we review the history and properties of human mesotrypsin, and discuss its function in the digestive degradation of dietary trypsin inhibitors and possible pathophysiological role in pancreatitis.     

Isolation of an enzymatically active tissue kallikrein from human seminal plasma by immunoaffinity chromatography

A tissue kallikrein from human seminal plasma was isolated by immunoaffinity chromatography and characterized. Its molecular mass was determined by gel filtration to be approximately 40000 Da. The enzyme preparation liberates kinin from human HMW kininogen (specific activity: 0.594 HMW kininogen-U/mg), lowers the blood pressure of dogs after intravenous injection (specific activity: 1740 biol. kallikrein unit/mg) and is strongly inhibited by aprotinin but not by soybean trypsin inhibitor. N alpha-Acetyl-L-phenylalanyl-L-arginine ethyl ester, D-valyl-L-leucyl-L-agrine ethyl ester and N-benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester are cleaved with identical rates by the enzyme from human seminal plasma and human urinary kallikrein.     

New human colorectal carcinoma cell lines that secrete proteinase inhibitors in vitro

Two new human cell lines, RCM-1 and CoCM-1, have been established from primary colorectal adenocarcinomas. Both cell lines were unique in that the cultures secreted trypsin inhibitors in vitro. The activities of these inhibitors were accumulated in serum-free media of both cell lines over a period of several days. Two inhibitors (PI-1 and PI-2) were isolated from serum-free conditioned medium in which RCM-1 was grown by anion-exchange and gel filtration high-performance liquid chromatography. PI-1 inhibited trypsin and chymotrypsin strongly, and pancreatic elastase weakly. Its molecular weight was about 57 kilodaltons (Kd) as determined by gel filtration chromatography. It cross-reacted with the antiserum elicited against human alpha 1-antitrypsin in double immunodiffusion. PI-1 corresponding to alpha 1-antitrypsin was also demonstrated immunohistochemically in both cell lines. PI-2 inhibited trypsin strongly, and chymotrypsin, kallikrein and plasmin weakly. It had higher molecular weight (200-300 Kd) than that of PI-1, and did not cross-react with antisera against human alpha 1-antitrypsin, alpha 2-macroglobulin, alpha 1-antichymotrypsin, alpha 2-plasmin inhibitor, inter-alpha-trypsin inhibitor and urinary trypsin inhibitor. RCM-1 and CoCM-1 are the first colorectal adenocarcinoma cell lines that secrete functionally active trypsin inhibitors, including alpha 1-antitrypsin in vitro, and are useful for the study of tumor-cell derived proteinase inhibitors.     

Isolation and characterization of two proteinase inhibitors from the male reproductive tract of mice

Low molecular weight, acid-stable proteinase inhibitors from epididymal and seminal vesicle homogenates were isolated and characterized. The isolation procedure consisted of gel filtration, trypsin affinity, and ion exchange chromatography. The inhibitor from seminal vesicle homogenates has a molecular weight of approximately 6,200, and that of the epididymal inhibitor was estimated at 4,000. Antiserum directed against the seminal vesicle inhibitor did not react with epididymal components. The epididymal inhibitor shows competitive, whereas the seminal vesicle inhibitor shows noncompetitive inhibition against trypsin on double reciprocal plots. Both inhibitors are effective against trypsin and acrosin but not against chymotrypsin, kallikrein, thrombin, or plasmin. To verify site of origin and to investigate androgen dependency of the epididymal inhibitor, mice were efferentiectomized, orchiectomized, or orchiectomized with androgen supplementation. Gel filtration profiles of acid-treated epididymal homogenates from normal and efferentiectomized animals show inhibitor peaks in the same regions. The concentration of acid-stable inhibitor from epididymal homogenates decreased with orchiectomy but returned to normal values when exogenous androgen was supplied. These observations suggest that the low molecular weight inhibitor in the epididymal homogenates is distinct from that in the seminal vesicles. Furthermore, the inhibitor associated with epididymal homogenates is androgen-dependent, and the epididymis is the site of origin of this inhibitor.     

Human ovarian tumor-associated trypsin. Its purification and characterization from mucinous cyst fluid and identification as an activator of pro-urokinase

In search of the target protease for the tumor-associated trypsin inhibitor TATI we recently identified a trypsin-like protease in cyst fluid of mucinous ovarian tumors (Stenman, U.-H., Koivunen, E., and Vuento, M. (1988) Biol. Chem. Hoppe-Seyler 369, 9-14). We have now purified this protease and demonstrate that it represents isoenzyme forms of trypsinogen, here called tumor-associated trypsin(ogen)s (TAT). The purification procedure comprised batchwise anion exchange chromatography, immunoaffinity chromatography with antibodies to trypsin, and separation of the two isoenzymes by reverse phase chromatography. In sodium dodecyl sulfate (SDS)-gel electrophoresis, the TAT-1 and TAT-2 isoenzymes have relative molecular weights (Mr) of 25,000 and 28,000, respectively, TAT-2 being the major component. The amino-terminal amino acid sequences correspond to those of pancreatic trypsinogen-1 and -2, respectively, and activation of the zymogens results in cleavage of a NH2-terminal activation peptide of 8 residues characteristic of trypsinogen. Isoelectric focusing in the presence of urea gives pI values of about 5 and 4 for TAT-1 and -2, respectively. The substrate specificities of the two TAT isoenzymes are very similar to, but not identical with, those of trypsin-1 and trypsin-2, respectively, suggesting slight differences in substrate binding site. TAT was found to be an efficient activator of pro-urokinase. Hence, TAT could take part in the protease cascade associated with tumor invasion.     

Isolation of basic acrosin inhibitor from bull seminal plasma (BUSI II).

An acrosin inhibitor was isolated from bull seminal plasma by gel filtration on Sephadex G-50 fine and ion-exchange chromatography on CM-Sephadex. The inhibitor is a basic polypeptide (pl greater than or equal to 10.5) of molecular weight 6 200 (calculated from amino acid composition). Its N-terminal amino group is blocked. The inhibitor is not strictly specific in its effect since it also inhibits trypsin and to a lesser degree chymotrypsin, in addition to bull and boar acrosin.     

The amino acid sequence of the double-headed protein proteinase inhibitor from dog submandibular glands, I. Structural homology to the pancreatic secretory trypsin inhibitors (author's transl)]

The primary structure of the broad specificity proteinase inhibitor from dog submandibular glands was elucidated. The inhibitor consists of a single polypeptide chain of 117 amino acids which is folded into two domains (heads) connected by a peptide of three amino acid residues. Both domains I and II show a clear structural homology to each other as well as to the single-headed pancreatic secretory trypsin inhibitors (Kazal type). The trypsin reactive site (-Cys-Pro-Arg-Leu-His-Glx-Pro-Ile-Cys-) is located in domain I and the chymotrypsin reactive center (-Cys-Thr-Met-Asp-Tyr-Asx-Arg-Pro-Leu-Tyr-Cys-) in domain II, cf. the Figure. The inhibitor is thus double-headed with two independent reactive sites. Whereas head I is responsible for the inhibition of trypsin and plasmin, head II is responsible for the inhibition of chymotrypsin, subtilisin, elastase and probably also Aspergillus oryzae protease and pronase. Remarkably, the structural homology exists also to the single-headed acrosin-trypsin inhibitors from seminal plasma[12] and the Japanese quail inhibitor composed of three domains[13].     

The interaction in human plasma of antiplasmin, the fast-reacting plasmin inhibitor, with plasmin, thrombin, trypsin and chymotrypsin.

The inhibition of plasmin, (EC 3.4.21.7), thrombin (EC 3.4.21.5), trypsin (EC 3.4.21.4) and chymotrypsin (EC 3.4.21.1) by antiplasmin, the recently described fast-reacting plasmin inhibitor of human plasma, was studied. To determine the quantitative importance of antiplasmin relative to the other plasma protease inhibitors, enzyme inhibition assays were performed on whole plasma and on plasma specifically depleted in antiplasmin, after addition of excess enzyme. Plasmin was the only enzyme for which the inhibitory capacity of antiplasmin-depleted plasma was lower than that of normal plasma. To determine the affinity of the enzymes for antiplasmin, as compared to the other inhibitors, various amounts of enzymes were added to normal plasma and the formation of enzyme-antiplasmin complexes studied by crossed immunoelectrophoresis using specific antisera against antiplasmin. Plasmin and trypsin, but not thrombin or chymotrypsin formed complexes with antiplasmin. It is concluded that antiplasmin is the only fast-reacting plasmin inhibitor of human plasma. It is also a fast-reacting inhibitor of trypsin but only accounts for a very small part of the fast-reacting trypsin-inhibitory activity of plasma. This can be explained by the low concentration of antiplasmin (1 muM) in normal plasma, compared to the other inhibitors (e.g. alpha1-antitrypsin: 40-80 muM).     

Isolation and characterization of a new trypsin inhibitor from Crotalaria paulina seeds

A new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Purification of the inhibitor was carried out by gel filtration, ion-exchange chromatography, and subsequent reversed-phase HPLC. The presence of a single polypeptide chain, with a molecular mass of 20 kDa and isoelectric point 4.0, was detected. The trypsin inhibitor had a Ki value of 4.5 x 10(-8) M and was capable of acting on human, bovine, and porcine trypsin and weakly on bovine chymotrypsin. Amino acid analysis showed that CPTI has a high content of aspartate, glutamate, leucine, serine, and glycine, having 177 amino acid residues in its composition. These data suggest that the protein belongs to the Kunitz-type trypsin inhibitors.