Capillary Permeability-increasing Activity Research Articles

Properties and cDNA cloning of a hyaluronidase from the stonefish Synanceia verrucosa venom

The venoms of two classes of fish, freshwater stingray (members of the genus Potamotrygon) and stonefish (members of the genus Synanceia), contain not only proteinaceous toxins but also hyaluronidases, which are considered as spreading factors that facilitate the tissue diffusion of toxins by degrading hyaluronan. So far, the freshwater stingray Potamotrygon motoro hyaluronidase and the stonefish Synanceia horrida hyaluronidase (SFHYA1) have been purified and characterized, although their spreading activity is still unclear. In this study, a 59 kDa hyaluronidase was partially purified from the stonefish Synanceia verrucosa and shown to be optimally active at pH 6.6, 37 °C and 0.15 M NaCl. Importantly, the S. verrucosa hyaluronidase enhanced the capillary permeability-increasing activity of the S. verrucosa toxin (neoverrucotoxin), providing evidence for its spreading activity. Furthermore, the primary structure of the S. verrucosa hyaluronidase was elucidated by cDNA cloning. The S. verrucosa hyaluronidase (463 amino acid residues) shares as high as 92% sequence identity with SFHYA1 but less than 50% with other hyaluronidases. Nevertheless, one catalytic residue and four substrate positioning residues, which constitute the active site of human hyaluronidases, are conserved in the S. verrucosa hyaluronidase.

Factors in snake venoms that increase capillary permeability.

Capillary permeability increasing (CPI) activity is a phenomenon of the microvasculature caused by many agents such as snake venoms, histamine, 5-hydroxytryptamine (5-HT), prostaglandins and leukotrienes. Since no systematic study has been done to determine what components of snake venom cause CPI activity, a CPI factor from Naja naja atra (Taiwan cobra) venom was isolated using intravenous injections of Evan's blue dye as the indicator of increased permeability and the factor's properties were extensively studied. Cardiotoxin from Naja naja kaouthia (Thailand cobra) and Mojave toxin from Crotalus scutulatus scutulatus (Mojave rattlesnake) venoms demonstrated CPI activity. Postsynaptic neurotoxins from an elapid and a hydrophid and myotoxin a from Crotalus viridis viridis (prairie rattlesnake) showed no CPI activity at the dose studied. The purified CPI active component from Naja naja atra venom was found to have cardiotoxic activity. Therefore, Elapidae cardiotoxins are CPI active factors. However, CPI activity is not due to cardiotoxins alone as the presynaptic neurotoxin, Mojave toxin, also showed CPI activity. Selective inhibitors were used to indicate possible mechanisms of action on the capillaries by Naja naja atra venom and Crotalus scutulatus scutulatus venom. The histamine H1-receptor blockers diphenhydramine, promethazine, and cyproheptadine were effective against both venoms in preventing increased capillary permeability. These results suggested that histamine release activity is the most likely mechanism resulting in CPI activity from these venoms.

Isolation and Characterization of a Novel Acid Proteinase, Tropiase from Candida tropicalis IFO 0589

A novel acid proteinase (Tropiase) was isolated from Candida tropicalis IFO 0589 by DE52-cellulose, and DEAE-Cosmogel column chromatographies. The purified tropiase gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The enzyme preparation had a molecular weight of 23,900, isoelectric point of pH 5.1, optimum pH range of 7 to 9 and possessed 208 amino acid residues. The enzyme hydrolyzed casein, fibrinogen, keratin and collagen. The purified tropiase demonstrated hemorrhagic and capillary permeability-increasing activities. Inhibition of tropiase occurred with leupeptin and N-bromosuccinimide, however, no inhibition was observed with alpha(2)-macroglobulin, soybean trypsin inhibitor, benzamidine-HCl or diisopropyl fluorophosphate.

Capillary permeability-increasing enzyme-2 from the venom of Agkistrodon caliginosus (kankoku-mamushi): Activity resulting from the release of peptides from fibrinogen

When a mixture of the purified capillary permeability-increasing enzyme-2 from Agkistrodon caliginosus venom and rabbit plasma was injected into the depilated skin of the back of rabbits, the capillary permeability-increasing activity was much greater than that induced by injection of the enzyme alone. To isolate the active substance, the mixture was subjected to reversed-phase high-performance liquid chromatography. Among the active peptides, five peptides were purified and their amino acid sequences were analyzed. These peptides were derived from the amino-terminal portion of the Eβ-chain of fibrinogen. After the enzyme was incubated with rabbit fibrinogen, the three peptides were purified from the incubation mixture by high-performance liquid chromatography, and their amino acid compositions and amino acid sequences were determined. From these results, it was determined the peptides were derived from the amino-terminal portion of the Bβ-chain of fibrinogen. This result indicates that the increase in capillary permeability induced by the enzyme is due to the peptides released from rabbit fibrinogen.

Some properties of a capillary permeability-increasing enzyme-2 from the venom of Agkistrodon caliginosus (Kankoku-Mamushi)

K. Shimokawa and H. Takahashi. Some properties of a capillary permeability-increasing enzyme-2 from the venom of Agkistrodon caliginosus (Kankoku-Mamushi). Toxicon 31, 1221–1227, 1993.—Capillary permeability-increasing enzyme-2 (CPI-enzyme-2) consists of a single polypeptide chain with an isoelectric point of pH 3.5. The enzyme is composed of 369 amino acid residues, based on a mol. wt of 44,000, and contains 20.3% carbohydrate. Both arginine ester hydrolytic and capillary permeability-increasing activities of the enzyme were inhibited by treatment with diisopropylfluorophosphate, indicating that the enzyme is a serine proteinase. The N-terminal amino acid sequence shows homology with that of CPI-enzyme-1 and similarity to those of batroxobin or kallikrein-like enzyme from other snake venoms.

Isolation and characterization of fibrinogenase from candida albicans NH-1

1.1. Fibrinogenase was isolated from Candida albicans NH-1 by DEAE-Cellulose, Sephadex G-75 and Sephadex G-100 column chromatographies.2.2. The purified fibrinogenase gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.3.3. The enzyme preparation had a molecular weight of 13,000, isoelectric point of pH 4.2 and possessed 117 amino acid residues.4.4. The purified fibrinogenase possessed capillary permeability-increasing activity.5.5. The enzyme hydrolyzed fibrinogen, casein, hide powder azure, azocoll hydrolytic activities and also hydrolyzed the oxidized B chain of insulin. The cleavage sites in the oxidized B chain of insulin were identified as Asp(3)-Glu(4), Glu(13)-Ala(14), Ala(14)-Leu(15), Tyr(16)-Leu(17), Arg(22)-Gly(23), Phe(25)-Tyr(26) and Tyr(26)-Thr(27).6.6. Fibrinogenase activity of this preparation was inhibited by α2-macroglobulin antithrombin-III, o-phenanthroline, disodium ethylenediaminetetra acetic acid and dithiothreitol.

Purification of a kininogenase (kininogenase-2) from the venom of Agkistrodon caliginosus (Kankoku-Mamushi)

From the partially purified capillary permeability-increasing enzyme obtained from A. caliginosus venom, another kininogenase (kininogenase-2) was purified by gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50, S-Sepharose Fast Flow and Q-Sepharose Fast Flow. The purified enzyme was homogeneous by polyacrylamide gel electrophoresis at pH 8.3 and SDS-gel electrophoresis. The kininogenase-2 had arginine ester hydrolytic and capillary permeability-increasing activities, and did not show any caseinolytic or clotting activity in a similar manner to a previously purified kininogenase (kininogenase-1). The purified kininogenase-2 liberated bradykinin on incubation with purified bovine high mol. wt kininogen. The rate of bradykinin release from the kininogen by kininogenase-2 was slower than that by the kininogenase-1, although both enzymes rapidly cleaved the peptide bonds in the kininogen molecule.

Comparative study of three phospholipase A 2s from the venom of Vipera aspis

1. 1. Three phospholipase A 2s, PLA 2-I, PLA 2-II and PLA 2-III, were isolated from Vipera aspis venom by gel filtration and ion exchange chromatography. 2. 2. Purified PLA 2-I, -II and -III have mol. wts of 30,200, 16,000 and 13,500, and isoelectric points of 9.45, 7.65 and <4.1, respectively. 3. 3. PLA 2-I consists of an acidic subunit (mol. wt 13,700, pI: <3.5) and a basic subunit (mol. wt 16,500, pI: 10.6), which can be separated under highly acidic conditions. 4. 4. PLA 2-I possessed lethal activity and LD 50 for this preparation was estimated to be 0.288 (0.209–0.397) μg/g, while lethality was not observed when PLA 2-II, -III or each subunit of PLA 2-I were administered. 5. 5. Capillary permeability-increasing activity was found in the samples which possessed basic isoelectric points. Additionally, PLA 2-I and its basic subunit drastically prolonged activated partial thromboplastin time of platelet rich plasma. 6. 6. Intramuscular injections of PLA 2-I, -II and -III increased serum creatine phosphokinase activity in mice, indicating that damage in muscle was caused by these enzymes. 7. 7. NH 2-terminal sequences of the three PLA 2-s were compared with other phospholipase A 2s from snake venoms. Furthermore, antigenicities were tested using antiserum prepared against each sample.

Biochemical and physiological studies on a kallikrein-like enzyme from the venom of Crotalus viridis viridis (Prairie rattlesnake)

A kallikrein-like enzyme was isolated from Crotalus viridis viridis (Prairie rattlesnake) venom by Sephadex G-50, DEAE-Sephacel and heparin-Sepharose CL-6B column chromatography. The purified enzyme has a molecular mass of 32 kDa and an isoelectric point of 5.4. The enzyme catalyzed the hydrolysis of arginine esters, kallikrein substrates ProPheArgMCA and ZPheArgMCA. The specificity of the enzyme's substrate requirement is demonstrated by the fact that no proteolytic activity was detected against either dimethyl casein or fibrinogen. The enzyme also cleaves kininogen analogs to release bradykinin. Although the enzyme induced contraction of the isolated rat uterus directly at high concentrations, more forceful contractions resulted when the reaction mixture of the enzyme and bovine plasma was applied to the uterus. The reaction mixture of 5 · 10 −11 M of the enzyme and plasma caused contractions equal to that of 10 −9 M of bradykinin. Additionally the enzyme demonstrated capillary permeability-increasing activity and hypotensive activity on the anaesthetized rat, suggesting that the enzyme releases the dilator of the wall of capillaries from plasma. Uterine contraction, capillary permeability-increasing activity and arginine esterolytic activity were inhibited by diisopropyl fluorophosphate, indicating that the serine hydroxyl group is essential for enzymatic and biological activities. It was demonstrated that the NH 2-terminal region of the enzyme has significant similarities in sequence with kallikrein-like enzymes from other snake venoms and porcine pancreatic kallikrein.

Purification and characterization of a lethal factor in venom from the crown-of-thorns starfish ( Acanthaster planci)

K. Shiomi, S. Yamamoto, H. Yamanaka and T. Kikuchi. Purification and characterization of a lethal factor in venom from the crown-of-thorns starfish ( Acanthaster planci). Toxicon 26, 1077–1083, 1988. — A lethal factor in venom of the crown-of-thorns starfish ( Acanthaster planci) was obtained in an electrophoretically pure state by chromatography on CM-cellulose and Sephadex G-100. The purified lethal factor is a basic (pI 10.6) glycoprotein (carbohydrate content 3.5%). The mol. wt was estimated to be 20,000 by gel filtration or 25,000 by SDS-disc electrophoresis, suggesting that the lethal factor has no subunit structure. Despite its basicity, the lethal factor was richer in acidic amino acids than in basic amino acids. The lethal factor had an ld 50 of 0.43 mg/kg (i.p. injection into mice). Hemolytic, edema-forming and capillary permeability-increasing activities, though very weak, were also exhibited by the lethal factor, while hemorrhagic and phospholipase A activities were not present.

Physiological and biochemical properties of A kallikrein-like enzyme from the venom of Vipera aspis aspis (aspic viper)

Y. Komori and H. Sugihara. Physiological and biochemical properties of a kallikrein-like enzyme from the venom of Vipera aspis aspis (aspic viper), Toxicon 26, 1193–1204, 1988.—A kallikrein-like enzyme was isolated from the venom of Vipera aspis aspis by Sephadex G-75, Q-Sepharose and Heparin-Sepharose CL-6B column chromatography. The purified enzyme is a glycoprotein with a mol.wt of 43,000 and an isoelectric point of 4.1. The enzyme possesses arginine ester hydrolase activity, but no proteolytic activity against either dimethylcasein or fibrinogen. The reaction mixture of the enzyme and bovine plasma induced contraction of the isolated rat uterus, suggesting that the enzyme releases kinin from the plasma constituent. The amount of enzyme, which releases an equal amount of kinin corresponding to 1 nmole of bradykinin per min, is 2.36 mg. Additionally, the kallikrein-like enzyme demonstrated capillary permeability-increasing activity and hypotensive activity. A synthetic kininogen analog, Ser-Leu-Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ser-Val-Gln-Val-Ser, was cleaved by the enzyme to release bradykinin and kallidin, also indicating that the enzyme has a kallikrein-like activity. Uterine contraction, capillary permeability-increasing activity and arginine ester hydrolase activity were inhibited by diisopropyl fluorophosphate, suggesting that the serine hydroxyl group is essential for enzymatic and biological activities. Antithrombin III and heparin, serine-protease inhibitors found in plasma had no inhibitory effect on these activities of the purified enzyme. The amino acid sequence of the NH 2 terminal region of the enzyme has similarities with kallikrein-like enzynes from other snake venoms and with porcine pancreatic kallikrein.

Some properties of a proteolytic enzyme from the venom of Agkistrodon caliginosus (kankoku-mamushi) which increases capillary permeability

Y. Ohtani and H. Takahashi. Some properties of a proteolytic enzyme from the venom of Agkistrodon caliginosus (kankoku-mamushi) which increases capillary permeability. Toxicon 26, 181 – 190, 1998. — The molecular weight of the capillary permeability-increasing enzyme from the venom of A. caliginosus was estimated to be about 32,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This enzyme consists of a single polypeptide chain with an isoelectric point of pH 4.80. The enzyme is composed of 266 amino acid residues, based on a molecular weight of 32,000, and contained 11.4% carbohydrate. The enzyme more readily hydrolyzed arginine esters than lysine esters and did not hydrolyze tyrosine ester. Both the arginine ester hydrolytic and capillary permeability-increasing activities were inhibited by treatment with diisopropylfluorophosphate, indicating that the enzyme is an arginine esterase of the serine proteinase type. The substrate specificity of the enzyme toward synthetic chromogenic or fluorogenic substrates for mammalian serine proteinases and the pH and heat stabilities of the enzyme were also investigated.

Purification of a kininogenase from the venom of Agkistrodon caliginosus (Kankoku-Mamushi)

—During the isolation of a capillary permeability-increasing enzyme from the venom of A. caliginosus, a kininogenase was also purified from the venom by gel filtration on Sephadex G-100, ion-exchange chromatographies on CM-Sephadex C-50 and DEAE-Sephadex A-50, and gel filtration on Sephadex G-75. By this procedure, 11 mg of the purified enzyme were obtained from 4 g of the venom. The purified enzyme was homogeneous by polyacrylamide disc gel electrophoresis at pH 8.3 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and did not show any caseinolytic or clotting activity. The purified enzyme released bradykinin from purified bovine high mol.wt kininogen. The capillary permeability was increased by injection of the purified enzyme into the depilated skin of the back of a rabbit. It is supposed that the capillary permeability-increasing activity exerted by the enzyme is due to the release of bradykinin.

Kallikrein-like enzyme from Crotalus ruber ruber (red rattlesnake) venom

1. 1. A kallikrein-like enzyme was isolated and characterized from the venom of Crotalus ruber ruber (red rattlesnake). 2. 2. The kallikrein-like enzyme was shown to be homogeneous as demonstrated by a single band on acrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunodiffusion and reverse-phase (RP) HPLC. 3. 3. The enzyme has a molecular weight of 31,000 and isoelectric point of 4.6. It consists of 271 total amino acid residues, 24% of which are acidic amino acids. 4. 4. Specific esterolytic activities of the kallikrein-like enzyme on N- tosyl- l- arginine methylester (TAME) and N- benzoyl- l- arginine ethylester (BAEE) are 109.5 and 23.6 μmol/min/mg, respectively. 5. 5. The enzyme differs from trypsin as the soybean trypsin inhibitor does not inhibit the enzyme's action. Diisopropylfluorophosphate (DFP) inhibits the enzyme, suggesting that the serine hydroxyl group is important for enzyme activity. 6. 6. The enzyme is not lethal at 15 μg/g in mice and has no hemorrhagic activity, yet the injection of the purified enzyme intradermally, produced capillary permeability-increasing activity as shown by the use of Evans blue dye, and immediate drop in blood pressure. It also contracted the rat uterus.

Capillary permeability-increasing enzyme from the venom of Agkistrodon caliginosus (kankoku-mamushi): activity due to the release of peptide material from a protein in bovine plasma

When a mixture of the purified capillary permeability-increasing enzyme from A. caliginosus venom and bovine plasma or heated bovine plasma was injected into the depilated skin of the back of a rabbit, the capillary permeability-increasing activity was much greater than that induced by injection of the enzyme alone. The substance which increases capillary permeability was extracted from the incubated mixture of bovine plasma and enzyme with 50 – 70% ethanol. Its activity was lost when treated with carboxypeptidase A. Thus, it is supposed that the increase in capillary permeability induced by the enzyme is due to a low molecular weight peptide released from a protein in bovine plasma by the action of the enzyme. No liberation by the enzyme of histamine or anaphylatoxins of the complement system was found.

Purification and characterization of arginine ester hydrolases from the venom of the Chinese habu snake ( Trimeresurus mucrosquamatus)

An arginine ester hydrolase (ME-5) was isolated from the venom of Trimeresurus mucrosquamatus by column chromatography on Sephadex G-100, CM-Sephadex C-50, DEAE-Sephacel and by isoelectric focusing, obtaining 1.4 mg of purified enzyme from 1 g of crude venom. The enzyme was homogeneous by SDS and non SDS disc electrophoresis on polyacrylamide gel at pH 8.3. ME-5 is a glycoprotein which possesses both TAME hydrolase and capillary permeability-increasing activity, but it did not show clotting or bradykinin-releasing activities. Its molecular weight is approximately 33,000 and its isoelectric point is 6.48. The enzyme is stable to heat treatment and to pH changes between 5 and 9. Trimeresurus mucrosquamatus venom contains five arginine ester hydrolases, designated as ME-1, 2, 3, 4 and 5. Three of the five (ME-3, 4 and 5) are inactivated by DFP, suggesting that the serine hydroxyl group is involved in enzymatic activity. All five arginine ester hydrolases showed capillary permeability-increasing activity, but none of the enzymes showed clotting activity. Their amino acid compositions were determined and all appear to be unique and distinct from those of other snake venoms.

Enzymochemical studies on snake venoms. X. Purification and properties of arginine ester hydrolase (ME-3) in the venom of Trimeresurus mucrosquamatus

One of the TAME hydrolases of Trimeresurus mucrosquamatus venom was isolated by a combination of gel filtration on Sephadex G-100 and ion exchange chromatographies on CM-Sephadex C-50, Aminohexyl-Sepharose 4B and DEAE-Sephacel. From 1 g of the lyophilized venom 1.5 mg of purified arginine ester hydrolase was obtained. The substrate specificity of this enzyme was strictly directed to the hydrolysis of ester linkages to which an arginine residue contributed the carbonyl group. The esterolytic activity was inhibited by benzamidine, N-(p-tosyl)-L-phenylalanine chloromethyl ketone (TPCK) or diisopropyl fluorophosphate (DFP). The purified enzyme was proved to be homogeneous by electrophoresis on polyacrylamide gel and isoelectric focusing. The molecular weight was found to be about 33000. Its isoelectric point was 6.93. The esterolytic activity of the final preparation was 125.7 unit/mg. This enzyme had capillary permeability-increasing activity, but not kininreleasing and clotting activities. This protein was stable to heat treatment, and between pH 4-9. Its Michaelis constant (Km) for TAME and inhibition constants (K1) for benzamidine, TPCK, and DFP were found to be 7.81×10-3 M, 0.67×10-3 M, 0.43×10-3 M, and 3.66×10-3 M, respectively. This enzyme was a glycoprotein.

Relationship between pharmacological actions and enzymatic activities of the venom of Trimeresurus gramineus.

The venom of Trimeresurus gramineus was separated into five fractions, namely α, β, γ, δ and ϵ, by means of starch zone electrophoresis. The most toxic, γ-fraction, which possesses the highest caseinolytic activity, exerts the highest hemorrhagic as well as anticoagulant activities. β-fraction also has a high hemorrhagic activity, but is almost devoid of caseinolytic and anticoagulant activities. EDTA greatly decreases lethality as well as hemorrhagic and caseinolytic activities, thus hemorrhage combined with anticoagulant activity seems to play a leading role in lethality of the venom. Both hemorrhagic and caseinolytic activities are also inhibited by either cysteine or heat treatment. β-, γ- and δ-fractions possess high phospholipase A activity and strongly release histamine from the rat diaphragm preparation. Since the capillary permeability increasing activity (CPIA) of these fractions is inhibited markedly by diphenhydramine and less markedly by methysergide, it appears that phospholipase A plays an important role in CPIA by releasing histamine. However, since DFP also partially inhibits CPIA and the combined use of diphenhydramine, methysergide and DFP completely inhibits CPIA of the venom, the bradykinin-releasing activity of the venom may be also involved. The least toxic, α-fraction, which possesses the highest thrombin-like as well as arginine ester hydrolytic activities, is much weaker in the above-mentioned activities. ϵ-fraction exerts strong local irritation on the rabbit eye, but is almost devoid of other activities.

PRESENCE OF KALLIKREIN IN THE GAMMA-GLOBULIN PERMEABILITY FACTOR OF GUINEA-PIG SERUM.

gamma-Globulin fractions isolated from guinea-pig serum by chromatography on diethylaminoethyl cellulose exhibited all the properties of kallikrein. From a range of amino acid esters tested, only esters of arginine and lysine were hydrolysed; the esterase and capillary permeability-increasing activities of different samples were correlated. Both esterase and permeability-increasing activities could be increased by the addition of certain albumin-rich fractions or of glass beads previously treated with guinea-pig serum.