Kallikrein inhibitors in rat plasma.

The kallikrein inhibitor contents of human and animal plasma were determined with glandular kallikreins [EC 3.4.21.8]. One ml of plasma could inactivate 20-700 kallikrein units (KU). Rat plasma was the most potent and inactivated 230-700 KU. However, no enzyme capable of inactivating kallikrein could be found in this plasma. Two fractions which inhibited hog pancreatic kallikrein, a fraction corresponding to alpha2-macroglobulin and a fraction which was eluted prior to albumin, were separated from rat plasma by Sephadex G-200 gel filtration. The former inhibitor could inhibit hog pancreatic kallikrein action on Nalpha-benzoyl-L-arginine ethyl ester (BAEE) as well as in the dog vasodilator assay. The other inhibitor was partially purified from rat plasma. One mg of the preparation inhibited 67 KU and the hydrolysis of 5.8 micronmoles/min of BAEE by hog pancreatic kallikrein [EC 3.4.21.8]. The inhibitor also inhibited other glandular and plasma kallikreins, trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1], etc. The optimal pH of the inhibitor was 7.5-8. The inhibitor was unstable below pH 5, and was destroyed by heating at temperature above 60 degrees. The isoelectric point of the inhibitor was determined by Ampholine focusing to be 4.4, and its molecular weight was estimated to be 73,000 by Sephadex G-100 and G-150 filtrations. Several experimental results suggested that this inhibitor differed from alpha1-antitrypsin.     

Purification of a High Molecular Weight Kininogen from Rat Plasma

A high molecular weight kininogen has been isolated from rat plasma and purified. At each preparative step the kininogen concentration and purity were monitored by assay on the perfused isolated rat uterus in terms of bradykinin equivalents formed per mg protein following incubation of the plasma fractions with rodent acid protease for 24 hours at 37 and pH 4.0. Kinin formation by crystalline trypsin and human pancreatic kallikrein also was compared. Citrated rat plasma first was precipitated with 43% ammonium sulfate. The kininogen fractions then were subjected to a series of gel filtration ion exchange chromatographic columns that included G-200 Sephadex, G-200: G-100 Sephadex interconnected columns, DEAE-A50 Sephadex, and hydroxylapatite. The kininogen fractions finally were subjected to preparative polyacrylamide gel electrophoresis, resulting in a final purification of 92.9-fold compared to the initial rat plasma. A single major kininogen protein band and a minor band of protein impurity were obtained on disc gel electrophoresis. Only the pancreatic kallikrein did not form kinin from this purified kininogen. The apparent molecular weight was estimated by SDS polyacrylamide gel technique to be 110,000.     

Direct angiotensin II formation by rat submandibular gland kallikrein

Submandibular gland kallikrein [EC 3.4.21.8] of male Sprague-Dawley rats was purified by chromatography on soybean trypsin inhibitor (SBTI)-CH-Sepharose 4B, DEAE-Sephadex A-50, aprotinin-CH-Sepharose 4B and Sephadex G-100 columns and preparative isoelectrofocusing. The molecular weight of the kallikrein was estimated to be 30,000 by Sephadex G-100 gel filtration and 29,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric points ranged from pH 3.55 to 4.30. The kinin formed at pH 8 by this kallikrein from bovine low molecular weight (LMW) kininogen showed the same behavior as lysyl-bradykinin on HPLC in a solution of ammonium biphosphate containing acetonitrile. At physiological pH, this kallikrein also generated angiotensin II, a potent vasopressor, from human plasma protein. Rat submandibular gland kallikrein differs from tonin in the isoelectric point, the optimal pH for angiotensin II formation and the type of kinin formed. The tissue kallikrein might play a role in the regulation of local blood flow in view of its ability to form both vasoconstrictive and vasodilatory peptides.     

Isolation and Properties of a New Kallikrein Inhibitor from Tityus serrulatus Venom

The kallikrein inhibitor-peptide content of Tityus serrulatus scorpion crude venom was purified by Sephadex G-50 and Sephadex G-25 fine gel filtration chromatographies, followed by two steps of reverse-phase column on HPLC. The isolated inhibitor peptide was homogeneous in its N-terminal and partial amino acid sequence, showing a molecular weight of 4.489 Da by mass spectrometry and amino acid analysis. The peptide was tested with rat plasma and urine kallikrein, which resulting in an inhibition with similar afinity to both enzymes, showing an IC₅₀ of 14.3 μM after 13 and 8 min, respectively, using kininogen as substrate on the isolated guinea-pig ileum bioassay. The porcine pancreatic kallikrein showed after 10 min an IC₅₀ value of 12.6 μM with H-D-Val-Leu-Arg-pNA HCl as substrate. In addition, the isolated peptide significantly inhibited porcine pancreatic kallikrein with values in the range of apparent or absolute calculated peptide K _i = 2.5 μM. The inhibitor was heat resistant and stable at pH values less than 5.     

Characterization and purification of a kallikrein from human pancreatic juice and immunological comparison with other kallikreins.

A prekallikrein has been demonstrated in human pancreatic juice and the active enzyme has been purified from this material. The purification procedure included filtration on Sephadex G-100, chromatography on DEAE-cellulose and affinity chromatography on trypsin-inhibitor Sepharose. The purified kallikrein appeared to be homogeneous by polyacrylamide gel electrophoresis at pH 8.3 and by immunoelectrophoresis. Human pancreatic kallikrein is immunologically different from human plasma kallikrein and from pancreatic kallikreins of other species (hog, cat, rat and dog). Human pancreatic kallikrein has common antigenic determinants with human urinary and submandibular kallikreins but probably not with parotid kallikrein.     

Purification and characterization of rat plasma kallikrein

Kallikrein enzyme initially was isolated from rat plasma by passage of citrated plasma through a DEAE-Sephadex column at pH 7.2. The active fraction was purified to electrophoretic apparent homogeneity by precipitation to 60% ammonium sulfate saturation, sequential passage through DE-52 cellulose, Sephadex G-200 and SP-Sephadex columns and finally by chromatofocusing on a PBE-94 column. The kallikrein content of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME) and by its ability to form kinin from heat-treated rat plasma. The molecular weight was estimated by gel filtration to be 50,000 and by SDS-gel electrophoresis 41,000. Multiple isozymic forms were obtained with pI values ranging from 4.2 to 5.0. The enzyme has a pH optimum of 8.3. The Km and Vmax values for TAME, Bz-pro-phe-arg-pNA and H-D-val-leu-lys-pNA were 1.6, 0.16 and 1.7 mM and 3.09, 0.96 and 0.25 microM/mg/min respectively. The enzyme was inhibited by soybean trypsin inhibitor but not by lima bean trypsin inhibitor.     

A cytocidal tissue kallikrein isolated from mouse submandibular glands

A cytocidal factor against mouse thymocytes was purified from the submandibular glands of female BALB/c mice using Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. SDS-PAGE and amino acid sequence analysis revealed that the cytocidal factor was mouse glandular kallikrein (mGK)-6. mGK-6 showed an optimal enzyme activity at pH 10 and a cytocidal activity against thymocytes in a dose-dependent manner.     

Purification of cat submaxillary kallikrein.

The cat submaxillary gland contains 1,000--2,400 kallikrein units (KU)/g of tissue. The submaxillary kallikrein was purified to homogeneity by acetone fractionation, DEAE-Sephadex A-50 chromatography, Sephadex G-75 gel filtration, and Ampholine isoelectric focusing. The kallikrein was separated by isoelectric focusing into 6--7 forms with pI's between 4.2 and 5.1. One mg of the purified kallikrein contained 930--1,260 KU in the dog vasodilator assay, and hydrolyzed 15--25 and 9--12 mumol of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-alpha-toluenesulfonyl-L-arginine methyl ester (TAME), respectively, in 1 min at 25 degrees C and pH 8.0. The Km's of the purified kallikrein with BAEE and TAME were 0.67 and 0.34 mM, respectively. Hydrolysis of N-alpha-benzoyl-L-tyrosine ethyl ester (BTEE), N-alpha-benzoylarginine-p-nitroanilide (BApNA), and casein was small or negligible. The apparent molecular weight of the kallikrein was estimated to be 5 X 10(4) by Sephadex G-100 gel filtration and 4.7 X 10(4) by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS). The kallikrein was found to contain 18.5% carbohydrate by weight. Trasylol and soybean trypsin inhibitor were not specific inhibitors of this kallikrein.     

Dog renal kallikrein: purification and some properties.

The contents of kallikrein [EC 3.4.21.8] in the kidneys of various animals were estimated and the activity was found to be most potent in dogs. The dog renal kallikrein (DRK) was located mainly in the kidney cortex. Following the activation of a dog kidney cortex homogenate with acetone, kallikrein was purified about 2,000-fold with an overall yield of 18% by diethylaminoethyl (DEAE)-cellulose adsorption, acetone fractionation, and chromatography on Sephadex G-75 and DEAE-Sephadex A-50. The final purified preparation of dog renal kallikrein had a vasodilator activity of 65.5 KU per A280, and appeared to be homogeneous both in disc electrophoresis and ultracentrifugal analysis. Its molecular weight was estimated to be approximately 3.8 X 10(4) from the sedimentation coefficient obtained by ultracentrifugation, and by Sephadex gel filtration. However, isoelectric fractionation of the purified DRK preparation gave three isoelectric point, 3.9, 4.1, and 4.3. The DRK had an optimum pH of about 8.6 and was stable at pH 8. This enzyme was hardly inhibited by Trasylol, soybean trypsin inhibitor, ovomucoid trypsin inhibitor or potato kallikrein inhibitors. These properties were compared with those of kallikrein from other sources; DRK appeared to be similar to urinary kallikrein.     

Purification to apparent homogeneity of inactive kallikrein from rat urine

Inactive kallikrein was purified from rat urine by a procedure including ammonium sulfate fractionation, DEAE cellulose chromatography, phenyl-Sepharose CL-4B chromatography, and gel filtration on Sephadex G-100 and Sephadex G-75 columns. The resulting preparation was essentially homogeneous, as assessed by polyacrylamide gel electrophoresis. This preparation migrated as a single protein band on a SDS-polyacrylamide gel and the molecular weight was 41000. The purified material underwent marked activation by trypsin, but not by deoxycholate, Triton X-100, SDS or acidification. These results indicate that the purified inactive kallikrein is the precursor rather than a complex with a substance binding to the active form of kallikrein.     

A kallikrein-specific inhibitor in rat kidney tubules.

A kallikrein inhibitor was found in tubules of the rat kidney and purified by chromatography on Sephadex G-100. The molecular weight of the inhibitor, estimated by gel filtration and dodecylsulfate electrophoresis, is about 4700. It inhibits the following kallikreins: porcine submanidbular and pancreatic kallikrein, rat kidney and urine kallikrein, and human urine and plasma kallikrein. An inhibition of bovine trypsin was not observed.     

Purification of glandular kallikrein in maxillary mucosa from humans suffering from chronic inflammation

Glandular kallikrein was purified from human maxillary mucosa with chronic inflammation and its biochemical properties were characterized. The purification procedure consisted of extraction with 3 mol/l KCl, saturation of ammonium sulfate (66%), DEAE-Sepharose, arginine-Sepharose and Sephadex G-150 chromatographies. Maxillary mucosa with chronic inflammation contains considerable activity of glandular kallikrein, which is a serine protease with limited proteolytic activity and its biochemical properties resemble those of pancreatic kallikrein.     

Isolation and Properties of a New Kallikrein Inhibitor from Tityus serrulatus Venom

The kallikrein inhibitor-peptide content of Tityus serrulatus scorpion crude venom was purified by Sephadex G-50 and Sephadex G-25 fine gel filtration chromatographies, followed by two steps of reverse-phase column on HPLC. The isolated inhibitor peptide was homogeneous in its N-terminal and partial amino acid sequence, showing a molecular weight of 4.489 Da by mass spectrometry and amino acid analysis. The peptide was tested with rat plasma and urine kallikrein, which resulting in an inhibition with similar afinity to both enzymes, showing an IC₅₀ of 14.3 M after 13 and 8 min, respectively, using kininogen as substrate on the isolated guinea-pig ileum bioassay. The porcine pancreatic kallikrein showed after 10 min an IC₅₀ value of 12.6 M with H-D-Val-Leu-Arg-pNA HCl as substrate. In addition, the isolated peptide significantly inhibited porcine pancreatic kallikrein with values in the range of apparent or absolute calculated peptide K _i = 2.5 M. The inhibitor was heat resistant and stable at pH values less than 5.     

Studies on urinary kallikreins. I. Purification and characterization of human urinary kallikreins.

Human urinary kallikrein [EC 3.4.21.8] (HUK) was purified about 200-fold with an overall yield of 40 percent from crude powder by DEAE-cellulose chromatography, acetone fractionation, Sephadex G-100 gel filtration and DEAE-Sephadex A-50 chromatography. Its activity was 200 kallikrein units (KU) per A280. HUK from active fractions obtained by DEAE-Sephadex A-50 chromatography was separated into three active components showing isoelectric points of 3.9 (HUK-1), 4.0 (HUK-2), and 4.2 (HUK-3) by isoelectric focusing: each HUK component was homogeneous on disc electrophoresis. The approximate molecular weights of HUK-1, -2 and -3 were estimated to be 2.7 X 10(4), 2.7 X 10(4), and 2.9 X 10(4), respectively, by gel filtration on a Sephadex G-100 column. The optimum pH's of HUK-1, -2, and -3 in esterolytic action were found to be 8.0, 8.3, and 7.5, respectively, and they were fairly heat stable in comparison with other glandular kallikreins. The three components of HUK were weakly inhibited by Trasylol, but were not affected by soybean and ovomucoid trypsin inhibitors. They were strongly resistant to treatment with urea and weakly resistant to treatment with guanidine. The activation energies of HUK-1, -2, and -3 were found to by 1.17 X 10(4), 5.1 X 10(3), and 1.45 X 10(4) cal per mole, respectively. The Km values were estimated toward N-alpha-tosyl-L-arginine methyl ester (TAME), N-alpha-benozyl-L-arginine ethyl ester (BAEE), and N-alpha-benozyl-L-arginine methyl ester (BAME).     

A novel anticoagulant protein from Scapharca broughtonii

An anticoagulant protein was purified from the edible portion of a blood ark shell, Scapharca broughtonii, by ammonium sulfate precipitation and column chromatography on DEAE-Sephadex A-50, Sephadex G- 75, DEAE-Sephacel, and Biogel P-100. In vitro assays with human plasma, the anticoagulant from S. broughtonii, prolonged the activated partial thromboplastin time (APTT) and inhibited the factor IX in the intrinsic pathway of the blood coagulation cascade. But, the fibrin plate assay did not show that the anticoagulant is a fibrinolytic protease. The molecular mass of the purified S. broughtonii anticoagulant was measured to be about 26.0 kDa by gel filtration on a Sephadex G-75 column and SDSPAGE under denaturing conditions. The optimum activity in the APTT assay was exhibited at pH 7.0-7.5 and 40-45 degrees C in the presence of Ca(2+).     

Porcine liver succinyltrialanine p-nitroanilide hydrolytic enzyme. Its purification and characterization as a post-proline cleaving enzyme

Succinyltrialanine p-nitroanilide(STANA)-hydrolytic enzyme was purified 5,200-fold from porcine liver soluble fraction with a yield of 75% by ammonium sulfate fractionation and chromatographies on DEAE-Sephacel, Sephadex G-150, and hydroxylapatite columns. The purified enzyme was homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate (SDS). The pI of the enzyme was 4.9 by dis gel electrofocusing and the molecular weight was calculated to be 72,000 by gel filtration on a Sephadex G-150 column and 74,000 by SDS-polyacrylamide gel electrophoresis. Acidic amino acids amounted to 17.2% of the total amino acid residues, and the basic ones, 12.9%. No hexosamine was detected. The STANA-hydrolytic enzyme showed maximal activity at pH 7.4 against succinyltrialanine p-nitroanilide and at pH 6.5 against succinyl-Gly-Pro-4-methylcoumaryl 7-amide (MCA), and was stable between pH 6 and 7 in the presence of dithiothreitol. This enzyme hydrolyzed succinyl-Gly-Pro-Leu-Gly-Pro-MCA, succinyl-Gly-Pro-MCA, succinyl-Ala-Pro-Ala-MCA, and several proline-containing natural peptides in addition to succinyltrialanine p-nitroanilide, but was unable to hydrolyze the substrates of aminopeptidases, dipeptidylaminopeptidase IV, trypsin, and chymotrypsin. Elastatinal and chymostatin were effective inhibitors and their IC50 values were 8.7 micrograms/ml and 18.2 micrograms/ml, respectively. The enzyme was completely inhibited by 10(-7) M p-chloromercuribenzoic acid (pCMB), 10(-7) M p-chloromercuriphenylsulfonic acid (pCMPS), and 10(-4) M diisopropyl phosphofluoridate (DFP), but not by 1 mM E-64, which is known as an inhibitor specific to thiol proteinase. The enzyme was easily inactivated by agitation in a Vortex mixer, and its activity was recovered by the addition of thiol compounds such as dithiothreitol, 2-mercaptoethanol and cysteine. The effects of inhibitors and thiol compounds were substantially identical when the enzyme activity was measured with either succinyltrialanine p-nitroanilide or succinyl-Gly-Pro-MCA as a substrate. These results indicate that the STANA-hydrolytic enzyme in the liver soluble fraction is a post-proline cleaving enzyme [EC 3.4.21.26].     

A new vasopeptide formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen

A new vasoactive peptide, formed by the action of a Murphy-Sturm lymphosarcoma acid protease on rat plasma kininogen was purified by gel filtration on Sephadex G-50 (fine) and fractions assayed on the isolated rat uterus for smooth muscle stimulating activity. The most active fraction was purified further by CM-cellulose chromatography. High voltage electrophoresis showed the peptide to be one component (Mgly 2.49) with an electrophoretic mobility different from bradykinin, lysyl-bradykinin and methionyl-lysyl-bradykinin. The molecular weight of the peptide was estimated on Sephadex G-25 column to be 1460. The amino acid composition was determined and the carboxyl terminal sequence identified by carboxypeptidase Y treatment to be Pro-Phe-Arg-Leu. Dansyl-Edman procedure yielded an amino terminal sequence of Ile-Ser-Arg-Pro. The peptide produced a dose-dependent contraction of the isolated guinea pig anterior mesenteric vein and relaxed the rabbit superior mesenteric artery contracted by phenylephrine.     

Purification of (Ca2+-Mg2+)-ATPase from rat liver plasma membranes

The Ca2+-stimulated, Mg2+-dependent ATPase from rat liver plasma membranes was solubilized using the detergent polyoxyethylene 9 lauryl ether and purified by column chromatography using Polybuffer Exchanger 94, concanavalin A-Sepharose 4B, and Sephadex G-200. The molecular weight of the enzyme, estimated by gel filtration in the presence of the detergent on a Sephadex G-200 column, was 200,000 +/- 15,000. The enzyme was purified at least 300-fold from rat liver plasma membranes and had a specific activity of 19.7 mumol/mg/min. Polyacrylamide gel electrophoresis under nondenaturing conditions of the purified enzyme indicated that the enzymatic activity correlated with the major protein band. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, one major band in the molecular weight range of 70,000 +/- 5,000 was seen. The isoelectric point of the purified enzyme was 6.9 +/- 0.2 as determined by analytical isoelectric focusing. The enzyme was activated by Ca2+ with an apparent half-saturation constant of 87 +/- 2 nM for Ca2+. Calmodulin and trifluoperazine at the concentration of 1 microgram/ml and 100 microM, respectively, had no effect on the enzymatic activity.     

Purification and characterization of a collagenase from the mackerel,Scomber japonicus

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and 55 degrees, respectively. The K(m) and V(max) of the enzyme for collagen Type I were approximately 1.1mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by Hg2+, Zn2+, PMSF, TLCK, and the soybean-trypsin inhibitor.     

海枣曲霉β-半乳糖苷酶的提纯与性质

 通过过聚乙二醇6000-磷酸钾缓冲液双相分离、Sephadex G-100凝胶过滤、DEAE-Sephadex A-50离子交换层析、羟基磷灰石层析及SephadexG-100凝胶过滤等提纯步骤,从海枣曲霉(Aspergillus phoenicis)麦麸培养物抽提液中提纯得到凝胶电泳均一的β-半乳糖苷酶。该酶的最适pH为3.5—4.0,最适温度为60℃(反应15分钟),在pH5.0—8.5之间及60℃以下稳定。在65℃和70℃保温时失活50％的时间分别为27和2分钟。用SDS凝胶电泳法和梯度凝胶电泳法分别测得该酶的分子量为115,000和118,000。薄层凝胶等电聚焦法测得其等电点为pH4.6。