Dopamine receptor blockade increases glandular kallikrein-like activity in the neurointermediate lobe of the rat pituitary

The effect of dopamine receptor blockers on glandular kallikrein-like activity in the neurointermediate lobe of the rat pituitary was examined. Male rats were given daily injections of haloperidol (2.5 mg/kg), perphenazine (5 mg/kg) or sulpiride (60 mg/kg) for 7 days. Homogenates of the neurointermediate lobe were prepared. Latent proteases were activated with trypsin and proteolytic activity was measured at 37 degrees C, pH 8.0 using chromogenic peptide substrates. All three dopamine receptor blockers produced about a 100% increase in glandular kallikrein-like activity. The results suggest that glandular kallikrein-like activity in the neurointermediate lobe is under inhibitory control by dopaminergic mechanisms.     

Tissue kallikrein of human seminal plasma is secreted by the prostate gland

Samples of human seminal plasma were subjected to gel filtration, and the eluted fractions were analysed for their contents of tissue kallikrein-like antigen, arginine esterase activity and kininogenase activity. Two peaks of tissue kallikrein-like antigen were detected with apparent molecular masses of about 72 and 48 kDa. As judged by the criteria of molecular mass, immunoreactivity, kininogenase activity, identification of the released kinin as kallidin and inhibition studies, a genuine tissue kallikrein has been identified in the 48-kDa peak. In addition, this peak contains one or more species of immunoreactive tissue kallikrein which differ in molecular mass and enzymatic activities. The 72-kDa peak probably represents the complex of tissue kallikrein with alpha 1-proteinase inhibitor rather than a true high molecular mass tissue kallikrein. The prostate gland was identified as the site of origin of the tissue kallikrein in the seminal fluid by indirect methods and by demonstrating immunoreactive tissue kallikrein in prostatic tissue and secretion.     

Purification of rat urinary kallikrein: comparative studies with rat submandibular gland kallikrein-like serine protease.

A 427-fold purification of rat urinary kallikrein (RUK) was achieved in three steps involving chromatography on columns of DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography on a column of benzamidine-Sepharose. Purified enzyme showed a single band on SDS-PAGE with an estimated molecular weight of 43,000. The amino-terminal sequences of the first 25 residues of RUK resemble the reported sequence for true kallikrein and share 80% identity with rat submandibular gland (RSMG) kallikrein-like serine protease. The RUK is highly reactive towards kallikrein substrates Bz-pro-phe-arg-pNA and DL-val-leu-arg-pNA, and plasmin substrate D-val-leu-lys-pNA. RSMG enzyme is more reactive towards Bz-val-gly-arg-pNA and tosyl-gly-pro-arg-pNA, preferential chromogenic substrates for trypsin-like proteases and thrombin, respectively. Both leupeptin and aprotinin inhibit RUK strongly, but soy bean trypsin inhibitor has no effect on this enzyme. RSMG enzyme is poorly inhibited by any of these inhibitors. The data suggest that although both enzymes are members of tissue kallikrein multigene family, urinary enzyme is a true kallikrein and RSMG enzyme is a kallikrein-like serine protease with different substrate specificity.     

Purification of Rat Urinary Kallikrein: Comparative Studies with Rat Submandibular Gland Kallikrein-Like Serine Protease

Abstract

A 427-fold purification of rat urinary kallikrein (RUK) was achieved in three steps involving chromatography on columns of DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography on a column of benzamidine-Sepharose. Purified enzyme showed a single band on SDS-PAGE with an estimated molecular weight of 43,000. The amino-terminal sequences of the first 25 residues of RUK resemble the reported sequence for true kallikrein and share 80% identity with rat submandibular gland (RSMG) kallikrein-like serine protease. The RUK is highly reactive towards kallikrein substrates Bz-pro-phe-arg-pNA and DL-val-leu-arg-pNA, and plasmin substrate D-val-leu-lys-pNA. RSMG enzyme is more reactive towards Bz-val-gly-arg-pNA and tosyl-gly-pro-arg-pNA, preferential chromogenic substrates for trypsin-like proteases and thrombin, respectively. Both leupeptin and aprotinin inhibit RUK strongly, but soy bean trypsin inhibitor has no effect on this enzyme. RSMG enzyme is poorly inhibited by any of these inhibitors. The data suggest that although both enzymes are members of tissue kallikrein multigene family, urinary enzyme is a true kallikrein and RSMG enzyme is a kallikrein-like serine protease with different substrate specificity.     

Characterization of cerastobin, a thrombin-like enzyme from the venom of Cerastes vipera (Sahara sand viper)

Cerastobin, a thrombin-like enzyme, was isolated from the venom of Cerastes vipera (Sahara sand viper) in homogeneous form. Cerastobin had a molecular weight of 38,000 with 348 amino acid residues. It had an isoelectric point of 7.7 (a pH optimum of 7.9 and a temperature optimum of 45 degrees C). Cerastobin hydrolyzed arginine-containing synthetic substrates such as TAME, BAME, and BAEE, but BAPNA was not hydrolyzed. Cerastobin had thrombin-like activity, producing fibrin from fibrinogen and also hydrolyzing chromogenic substrates for thrombin such as 2AcOH.H-D-CHG-But-Arg-pNA (CBS 34.47) and H-D-Phe-Pip-Arg-pNA (S-2238). It showed kallikrein-like activity and hydrolyzed kallikrein substrates 2AcOH.H-D-Phe-Gly-Arg-pNA (CBS 33.27) and H-D-Pro-Phe-Arg-pNA (S-2302). It produced bradykinin from bradykininogen, as uterus contraction was observed. A serine inhibitor, DFP, exerted a pronounced inhibitory effect, suggesting that cerastobin is a serine-type protease. The sequence of 37 residues from the amino-terminal end was investigated. The amino-terminal amino acid was valine as it is in most other thrombin-like enzymes. The amino acid sequence of cerastobin was similar to that of thrombin in some residues and had some homology with that of kallikrein. However, cerastobin showed a high degree of homology to thrombin-like enzymes isolated from various snake venoms. Factor X was partially degraded by cerastobin. It was also found that antithrombin III was degraded by the enzyme. The alpha and beta chains of fibrin monomer were preferentially hydrolyzed by cerastobin, but the gamma chain was quite resistant.     

Purification and characterization of a serine protease (esterase B) from rat submandibular glands

A new protease has been purified to homogeneity from rat submandibular gland homogenate by using DEAE-Sephadex chromatography, chromatofocusing, aprotinin-Sepharose affinity chromatography, and high-performance liquid chromatography. The enzyme has been named esterase B, since it represents the second major esterolytic peak on DEAE-Sephadex chromatography of submandibular gland homogenate. It is an acidic protein (pI = 4.45) with an apparent molecular weight of 27 000. It is heat-stable and has an optimum pH of 9.5. Esterase B hydrolyzed the synthetic substrates tosyl-L-arginine methyl ester and Val-Leu-Arg-p-nitroanilide (S2266). It also cleaved dog plasma kininogen to produce a kinin, identified as bradykinin on reverse-phase high-performance liquid chromatography. Esterase B, however, is only a weak kininogenase, since it had only 5% of the kininogenase activity of equimolar concentrations of glandular kallikrein and had no effect on rat mean blood pressure or on the isolated rat uterus. Esterase B activated plasminogen and had caseinolytic activity. It was inhibited by aprotinin, soybean trypsin inhibitor, lima bean trypsin inhibitor, phenylmethanesulfonyl fluoride, antipain, leupeptin, and p-tosyl-L-lysine chloromethyl ketone. On double immunodiffusion, when reacted with kallikrein and tonin antisera, esterase B showed partial identity with kallikrein but not with tonin. On immunoelectrophoresis against kallikrein antisera, esterase B formed a precipitin arc at a position different from that of kallikrein. Esterase B appears to be a trypsin-like serine protease having some homology with glandular kallikrein.     

A Protease Activity Displaying Some Thrombin-like Characteristics in Conditioned Medium of Zinnia Mesophyll Cells Undergoing Tracheary Element Differentiation

The normal development of tracheary elements (TE) requires a selective degradation of the cytoplasm without loss of the extracellular wall that remains behind as the water-conducting units of xylem. Using zinnia-(Zinnia elegans L. cv. Green Envy) cultured mesophyll cells that synchronously transdifferentiate into TEs, extracellular and intracellular proteases, respectively, have been shown to both trigger death and to execute autolysis as the final component of a programmed cell death (PCD). We report here the appearance in the medium of an unusual proteolytic activity correlated with the PCD process just prior to the autolysis. The activity has a pH optimum of 5.5–6.0 and displays some thrombin characteristics. This protease activity has 1) a 10-fold higher affinity towards a thrombin-specific chromogenic substrate than toward a trypsin-specific chromogenic substrate; 2) a 1000-fold lower sensitivity to soybean trypsin inhibitor (STI) compared to trypsin; and 3) limited ability to cleave the protease-activated receptor-1, the native thrombin substrate. However, the addition of partially purified fraction containing the thrombin-like protease activity to the medium of PCD-competent cells does not prematurely trigger PCD, and the thrombin-specific peptide inhibitor phenylalanine-proline-aspartic acid-chloromethylketone fails to inhibit PCD or tracheary element (TE) formation. This suggests that this protease activity may play a role within the cells in execution of the autolysis or in the collapse of the tonoplast rather than as an extracellular proteolytic activity participating in the chain of events leading to cell death. Online publication: 7 April 2005     

Relationship between protein and proteolytic activity in the midgut of mosquitoes.

In female mosquitoes anal injections (enemas) of nutrient solutions were administered in measured amounts to allow direct comparison of protease activities.The amount of protein ingested had a pronounced effect upon the rate of protein digestion, but had little influence upon the rate of protease secretion. Maximal protease activity increased only slightly with increasing meal size and always coincided with the digestion of about 80 per cent of the protein ingested.The use of the enema technique provided an experimental means to reject clearly a neural stimulus for protease secretion. Proof is given for a secretagogue stimulus: the presence of globular proteins with a minimal molecular weight is required for protease secretion.Despite antitryptic factors which are known to occur in different titres in vertebrate bloods, no inhibition was observed in vivo when proteases were recorded after enemas of blood or plasma from several hosts into Aedes aegypti, Anopheles quadrimaculatus, and Culex pipiens quinquefasciatus.Mosquito trypsin was shown to account for about 75 per cent of the proteolytic activity in midgut homogenates. Chymotrypsin was present although with very low activity. Calcium did not stimulate mosquito trypsin as it does mammalian trypsin. Between 22 and 32°C a Q₁₀ of 2·0 was observed for proteases as well as for protein digestion.     

Characterization of the necrotic protein that regulates the Toll-mediated immune response in Drosophila

Necrotic (Nec) is an important component of the proteolytic cascade that activates the Toll-mediated immune response in Drosophila. The Nec protein is a member of the serpin (SERine Protease INhibitor) superfamily and is thought to regulate the cascade by inhibiting the serine protease Persephone. Nec was expressed in Escherichia coli, and the purified protein folded to the active native conformation required for protease inhibitory activity. Biochemical analysis showed that Nec had a broad inhibitory specificity and inhibited elastase, thrombin, and chymotrypsin-like proteases. It did not inhibit trypsin or kallikrein. These data show that Necrotic is likely to inhibit a wide range of proteases in Drosophila and that Nec has the specificity requirements to act as the physiological inhibitor of Persephone in vivo.     

Kallikrein (kininogenase) in the mouse nephron: effect of dietary potassium

Kininogenase activity of kallikrein was measured in microdissected mouse nephron segments using kininogen from dog plasma and a radioimmunoassay for bradykinin. When single nephron segments were examined, results showed a large scatter. This was found to be due to heterogeneity of distal convoluted tubules (DCT) from different nephrons, since replicate measurements in pools of DCT structures did not show this degree of variation. Nearly 20% of activity was accessible to extracellular substrate when freshly dissected segments were incubated in isoosmotic media. Freezing and thawing which markedly releases activity of intracellular enzymes, did not significantly elevate kininogenase activity. On the other hand deoxycholate and trypsin treatment increased tubular kininogenase activity in an additive fashion. A detailed analysis of microdissected tubule fragments revealed that kallikrein is concentrated in late distal convoluted tubule before entering a branching point (connecting tubule). In contrast initial portions of distal convoluted tubules and cortical collecting tubules contained only little kallikrein activity. Potassium rich diet increased basal and total activity 5-fold, when compared to a potassium poor diet.     

Isolation of a crotalase-like protease with alpha-fibrinogenase activity from the western diamondback rattlesnake, Crotalus atrox.

Venom toxins were isolated from rattlesnake (Crotalus atrox) venom by cation-exchange chromatography. Seven major fractions could be obtained by single-step ion-exchange chromatography with two fractions showing essentially apparent homogeneity by SDS-gel electrophoresis. All fractions showed various extents of specific proteolytic activity against alpha- or beta-chains of fibrinogen molecules. Further characterization of one of the purified fractions with alpha-fribrinogenase activity indicated that it is a single-chain thrombin-like protease with a molecular mass of about 30 kDa. It is relatively heat stable, inhibited by phenylmethanesulfonyl fluoride, N alpha-p-tosyl-L-phenylalanine chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone but not by soybean trypsin inhibitor and beta-mercaptoethanol. Amino acid analysis showed that the enzyme possesses an amino acid composition very similar to thrombin and crotalase characterized before from the closely related snake venoms. N-Terminal sequence analysis of the enzyme corroborated the close similarity between this enzyme and those sequences of crotalase and kallikrein-like enzymes characterized from the same Crotalidae snake family. This study is in contrast to the previous reports which indicated a lack of thrombin- and crotalase-like enzyme in the venom of Western diamondback rattlesnake.     

Human plasma kallikrein. Purification and preliminary characterization

A method is described for the convenient purification of the protease plasma kallikrein from human Cohn fraction IV-1. The enzyme was produced by endogenous activation after acid treatment to remove an inhibitor and was concentrated by the successive use of affinity adsorbents prepared by the immobilization of soybean trypsin inhibitor and aminobenzamidine. The esterase- and kinin-producing activities were enriched about 1100-fold from fraction IV-1.Several properties of plasma kallikrein strengthen the impression that it is related to trypsin, namely, competitive inhibition by benzamidine and the formation of a stable p-guanidinobenzoyl acyl enzyme intermediate. Inactivation by affinity labeling with Z-LysCH₂Cl was successful in contrast to the inertness of Tos-LysCH₂Cl.     

Activation of phospholipase A2 of human spermatozoa by proteases

The effect of various proteases (kallikrein, plasmin, and trypsin) on sperm phospholipase A₂ activity (PA₂: EC 3.1.1.4) has been studied. The addition of trypsin to spermatozoa, isolated and washed in the presence of the protease inhibitor benzamidine, increased PA₂ activity optimally with trypsin concentrations of 1.0–1.5 units/assay. In kinetic studies, all of the above proteases stimulated the deacylation of phosphatidylcholine (PC); in fresh spermatozoa, trypsin showed a higher activation potential than kallikrein or plasmin. In the presence of benzamidine, the activity remained at basal levels. Endogenous protease activity due to acrosin (control) resulted in an increase in PC deacylation compared to the basal level. The maximum activation time of PA₂ activity by proteases was 30 min. Natural protease inhibitors (soybean trypsin inhibitor and aprotinin) kept the PA₂ activity at basal levels and a by-product of kallikrein, bradykinin, did not significantly affect the control level. Protein extracts of fresh spermatozoa exhibited the same pattern of PA₂ activation upon the addition of proteases, thus indicating that the increase in PA₂ activity was not merely due to the release of the enzyme from the acrosome. All of these findings suggest the presence of a precursor form of phospholipase A₂ that can be activated by endogenous proteases (acrosin) as well by exogenous proteases present in seminal plasma and in follicular fluid (plasmin, kallikrein). Thus, this interrelationship of proteases and prophospholipase A₂ could activate a dormant fusogenic system: the resulting effect would lead to membrane fusion by lysolipids, key components in the acrosome reaction.     

Fibrinogenolytic proteases isolated from the snake venom of Taiwan habu: serine proteases with kallikrein-like and angiotensin-degrading activities

Two venom proteases with fibrinogenolytic activity were isolated from the venom of Taiwan habu (Trimeresurus mucrosquamatus), one major crotalid snake species in Taiwan. The purified enzymes showed a strong beta-fibrinogenolytic activity, cleaving the beta-chain of fibrinogen molecules specifically. They also showed strong kallikrein-like activity in vitro, releasing bradykinin from kininogen. The purified enzymes did not coagulate human plasma, yet decreasing fibrinogen levels in plasma and prolonging bleeding without formation of fibrin clots, indicating that both proteases have specificities different from thrombin and the thrombin-like proteases of snake venom reported previously. They also exhibit amidase activity against N-benzoyl-Pro-Phe-Arg-p-nitroanilide, which is a specific synthetic substrate for kallikrein-like proteases. Their stability at high temperatures was examined and found to be more stable when compared with ancrod and thrombin. Intravenous injection of either protease was shown to lower blood pressure in experimental rats. Most noteworthy is the observation that the proteases can cleave angiotensin I and release bradykinin from plasma kininogen in vitro, which is a strong vasodilator and probably responsible for the in vivo hypotensive effect of these venom proteases.     

Snake venom proteases affecting hemostasis and thrombosis

The structure and function of snake venom proteases are briefly reviewed by putting the focus on their effects on hemostasis and thrombosis and comparing with their mammalian counterparts. Up to date, more than 150 different proteases have been isolated and about one third of them structurally characterized. Those proteases are classified into serine proteases and metalloproteinases. A number of the serine proteases show fibrin(ogen)olytic (thrombin-like) activities, which are not susceptible to hirudin or heparin and perhaps to most endogenous serine protease inhibitors, and form abnormal fibrin clots. Some of them have kininogenase (kallikrein-like) activity releasing hypotensive bradykinin. A few venom serine proteases specifically activate coagulation factor V, protein C, plasminogen or platelets. The venom metalloproteinases, belonging to the metzincin family, generally show fibrin(ogen)olytic and extracellular matrix-degrading (hemorrhagic) activities. A few venom metalloproteinases show a unique substrate specificity toward coagulation factor X, platelet membrane receptors or von Willebrand factor. A number of the metalloproteinases have chimeric structures composed of several domains such as proteinase, disintegrin-like, Cys-rich and lectin-like domains. The disintegrin-like domain seems to facilitate the action of those metalloproteinases by interacting with platelet receptors. A more detailed analysis of snake venom proteases should find their usefulness for the medical and pharmacological applications in the field of thrombosis and hemostasis.     

Expression, purification and characterization of the second Kunitz-type protease inhibitor domain of the human WFIKKN protein.

Recently we have described a novel secreted protein (the WFIKKN protein) that consists of multiple types of protease inhibitory modules, including two tandem Kunitz-type protease inhibitor-domains. On the basis of its homologies we have suggested that the WFIKKN protein is a multivalent protease inhibitor that may control the action of different proteases. In the present work we have expressed the second Kunitz-type protease inhibitor domain of the human protein WFIKKN in Escherichia coli, purified it by affinity chromatography on trypsin-Sepharose and its structure was characterized by CD spectroscopy. The recombinant protein was found to inhibit trypsin (Ki = 9.6 nm), but chymotrypsin, elastase, plasmin, pancreatic kallikrein, lung tryptase, plasma kallikrein, thrombin, urokinase or tissue plasminogen activator were not inhibited by the recombinant protein even at 1 microm concentration. In view of the marked trypsin-specificity of the inhibitor it is suggested that its physiological target may be trypsin.     

Inhibition of proteases in coagulation, kinin-forming and complement systems by alpha2-plasmin inhibitor.

Inhibitory activities of alpha2-plasmin inhibitor against various proteases were investigated. The inhibitor promptly inhibited the esterolytic activity of alpha-chymotrypsin and progressively inhibited the esterolytic or amidolytic activities of bovine plasma kallikrein, bovine thrombin and bovine activated factor X. Heparin had no effect on the reaction of the inhibitor with thrombin or activated factor X. However, the inhibitor had no effect on the activities of human C-1-esterase, papain and snake venom kininogenase. On the basis of its rapid inhibition of kallikrein, alpha2-plasmin inhibitor is considered to exert some regulating effect on kallikrein activity in plasma.     

Molecular characterization of BjussuSP-I, a new thrombin-like enzyme with procoagulant and kallikrein-like activity isolated from Bothrops jararacussu snake venom

A thrombin-like enzyme, named BjussuSP-I, isolated from Bothrops jararacussu snake venom, is an acidic single-chain glycoprotein with M(r)=61,000, pI approximately 3.8 and 6% sugar. BjussuSP-I shows high proteolytic activity upon synthetic substrates, such as S-2238 and S-2288. It also shows procoagulant and kallikrein-like activity, but is unable to act on platelets and plasmin. These activities are inhibited by specific inhibitors of this class of enzymes. The complete cDNA sequence of BjussuSP-I with 696bp encodes open reading frames of 232 amino acid residues, which conserve the common domains of thrombin-like serine proteases. BjussuSP-I shows a high structural homology with other thrombin-like enzymes from snake venoms where common amino acid residues are identified as those corresponding to the catalytic site and subsites S1, S2 and S3 already reported. In this study, we also demonstrated the importance of N-linked glycans to improve thrombin-like activity of BjussuSP-I toxin.     

Method for determining kallikrein of tissue origin in blood plasma and its clinical significance

Based on a study of the kininogenase activity of the total plasma kallikrein in the presence of 3 concentrations of the soybean inhibitor trypsin (0.5, 1.0, 10.0 micrograms/ml) one can measure at a time the activity of tissue kallikrein (without specifying the source) and the activity of 3 forms of plasma kallikrein, including its adsorption on kaolin that characterizes the conformational structure of the enzyme. Examination of 10 healthy subjects and 136 patients revealed a 10 to 20-fold increase in the content of tissue kallikrein in plasma of 70% of diabetes mellitus patients and a 2.5 to 3-fold elevation in 50% of patients with chronic occupational bronchitis, and in 30% of patients suffering from chronic hepatitis. The method suggested makes it possible to have a better insight into the physiological and pathogenetic role of the kinin system and may be used for laboratory control over the treatment efficacy.     

Detection of serine proteases in extracts of the domestic mite Blomia tropicalis

Previous studies have shown that the domestic mites Dermatophagoides pteronyssinus and D. farinae contain allergens with serine protease activity. These proteolytic allergens include trypsin, chymotrypsin, elastase, kallikrein, and C3/C5 convertase. However, it is not known whether the domestic mite Blomia tropicalis shares with other mite species the serine protease activities. The enzymatic activity present in extracts obtained from food-free B. tropicalis was investigated using specific substrates and inhibitors. Based upon the concentration response and inhibition profiles, and the digestion of specific substrates our data demonstrate that extracts from B. tropicalis exhibit several serine-protease-like activities. The enzyme activities detected in the B. tropicalis extracts are trypsin, elastase, chymotrypsin, kallikrein, C3/C5 convertase, and mast cell protease. Our results also demonstrate that kallikrein and C3/C5 convertase-like activities were not significantly affected by the α₁-antiprotease, a naturally occurring serine protease inhibitor which protects lung mucosa from the enzymatic action. These data strongly suggest that the Echymyopodidae mite B. tropicalis shares at least five serine proteases with members of other mite families, the Glycyphagidae and Pyroglyphidae. In addition, our data demonstrate the potential use of biochemical methods to detect serine proteases for evaluation of mite growth in vitro, or to detect environmental exposures to these enzymes. This revised version was published online in July 2006 with corrections to the Cover Date.