Purification and characterization of human microsomal dipeptidase

Human microsomal dipeptidase (MDP, formerly referred to as dehydropeptidase-I or renal dipeptidase) [EC 3.4.13.11] was solubilized from the membrane fraction of kidney by treatment with octyl-beta-D-glucoside and purified by a procedure including ion exchange chromatography and affinity chromatography on cilastatin-immobilized Sepharose. The purified human MDP was found to be homogeneous on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The apparent molecular weight (Mr) was estimated by SDS-polyacrylamide gel electrophoresis under non-reducing conditions to be 130 kDa, comprising a homodimer of two subunits. After treatment with endoglycosidase F, human MDP showed a single band with an apparent Mr of 42 kDa on SDS-polyacrylamide gel electrophoresis. Human MDP was found to bind to Con A-Sepharose and the activity was eluted with methyl-alpha-D-mannopyranoside, suggesting that human MDP is a glycoprotein. We also examined the substrate specificity of human MDP and found that human MDP catalyzed the hydrolysis of S(substituent)-L-cysteinyl-glycine adducts such as L-cystinyl-bis(glycine) and S-N-ethylmaleimide-L-cysteinyl-glycine, as well as the conversion of leukotriene D4 to leukotriene E4. These results suggest that MDP might play an important role in the metabolism of glutathione and leukotriene.     

Human high molecular weight kininogen as a thiol proteinase inhibitor: presence of the entire inhibition capacity in the native form of heavy chain

High molecular weight (HMW) kininogen was purified from fresh human plasma by two successive column chromatographies on DEAE-Sephadex A-50 and Zn-chelate Sepharose 4B. The purified HMW kininogen appeared to be a single band on sodium dodecyl sulfate (SDS)-polyacrylamide disc gel electrophoresis in both the presence and absence of beta-mercaptoethanol. However, it gave two bands on nonreduced SDS-polyacrylamide slab gel electrophoresis, a major band of dimeric form (Mr 200 000, ca. 95%) and a minor band of monomeric form (Mr 105 000, ca. 5%). Under reduced conditions, the dimeric form was converted stoichiometrically to a monomeric form (Mr 110 000), and the monomeric form observed under nonreduced conditions (Mr 105 000) was converted to a heavy chain (Mr 60 000) and a light chain (Mr 50 000). The formation of a dimer of HMW kininogen was also confirmed by an immunoblotting experiment. This unique property of intact HMW kininogen to form a dimer was further utilized in studies on the kininogens and their derivatives as thiol proteinase inhibitors. The purified HMW kininogen strongly inhibited the caseinolytic activities of calpain I, calpain II, and papain but not those of trypsin, chymotrypsin, and thermolysin, indicating that it was a group-specific inhibitor for thiol proteinases. When HMW kininogen was reduced with 0.14 or 1.4 M beta-mercaptoethanol, its inhibitory activity was partially or mostly inactivated, but on subsequent air oxidation its activity was almost completely recovered. In addition, kinin-free and fragment 1,2 free HMW kininogen showed higher inhibitory activity than the intact HMW kininogen.(ABSTRACT TRUNCATED AT 250 WORDS)     

The extraction and reconstitution of the alpha-cyanocinnamate-sensitive pyruvate transporter from castor bean mitochondria

The pyruvate carrier from castor bean mitochondria has been solubilized with Triton X-114 and partially purified using hydroxyapatite column chromatography. SDS-polyacrylamide gel electrophoresis of the hydroxyapatite-eluate showed that there were 6 major protein bands of Mr, 74kDa, 66kDa, 34kDa, 32kDa, 30kDa 12kDa. When the eluate was reconstituted into liposomes it was shown to catalyze a pyruvate exchange reaction which was sensitive to N-ethyl maleimide and a series of analogues of alpha-cyanocinnamate. The characteristics of this pyruvate exchange activity are similar to that found in intact mitochondria, and it is concluded that one or more proteins in the hydroxyapatite-eluate correspond to the pyruvate carrier.     

Hepatic glutathione S-transferases in rainbow trout and their interaction with 2,4-dichlorophenoxyacetic acid and 1,4-benzoquinone

Glutathione S-transferase in the cytosol of rainbow trout liver was partially purified by affinity chromatography on a column with glutathione coupled to epoxy-activated Sepharose 6B, which retained 94% of the total activity. Chromatofocussing on a Polybuffer exchanger 118 column separated the glutathione S-transferase into six major cationic isoenzymes (K1-K6), and some minor fractions. SDS-polyacrylamide slab gel electrophoresis showed K1-K3 to be heterodimers with subunits of Mr 25,000 and 26,500, and K4-K6 to be homodimers with subunits of Mr 25,000. The glutathione S-transferase isoenzymes were partially characterized by different biochemical parameters. The hepatic rainbow trout glutathione S-transferases were inhibited by the organic water pollutants, 1,4-benzoquinone and 2,4-dichlorophenoxyacetic acid. The same kinetic inhibition patterns were observed with these inhibitors as for rat liver glutathione S-transferases. It is concluded that rainbow trout glutathione S-transferases can play a key role in the detoxication of organic micropollutants in the aquatic environment.     

Purification and characterization of a new metalloproteinase with elastolytic activity from the catfish pancreas

An elastolytic enzyme was purified to homogeneity from the pancreas of the catfish Parasilurus asotus by chromatography on CM-cellulose column and affinity chromatography on (Ala)3-CH-Sepharose 4B column. The molecular weight of the enzyme was estimated to be 24 000 by SDS-polyacrylamide gel electrophoresis. The enzyme had elastolytic activity as well as activity toward Suc-(Ala)3-NA. The enzyme was inhibited by EDTA, EGTA, o-phenanthroline, dithiothreitol, and cysteine, but not by the serine proteinase inhibitors iPr2P-F and PhCH2SO2F. In addition, the enzyme was found to require Zn2+ for activity. These results indicate that the catfish enzyme belongs to the group of metalloproteinases and that it is a new type of pancreatic enzyme, unlike the pancreatic elastases which are serine proteinases.     

柞蚕卵组织蛋白酶B纯化及性质

昆虫卵内蛋白酶在胚胎发育中水解卵黄蛋白,为胚胎发育提供氨基酸,昆虫中已报道过几类卵蛋白酶,如家蚕中半胱氨酸蛋白酶和丝氨酸蛋白酶等。但是,目前尚不清楚这些蛋白酶是否存在于其他鳞翅目昆虫。了解这些蛋白酶的作用机理可以为我们提供害虫防治的新方法。并且,由于蛋白水解在许多生理过程中具有重要作用,如蛋白质的成熟和转运、受精、萌芽、肿瘤转移和其他形态发生等。因此,阐明这些蛋白酶的生物功能具有重要意义。由于（ｉ蚕卵粒大产卵量也很大,因此被选作研究鳞翅目昆虫卵蛋白酶的材料,我们希望通过对数种昆虫卵内蛋白酶的研究、找出卵黄蛋白水解的一般规律。在我们前一篇文章中报道了（ｉ蚕组织蛋白酶Ｂ的鉴定,该蛋白酶属于半胱氨酸蛋白酶类的组织蛋或最适ｐＨ为３．５,可被Ｅ－６４抑制。本文报道蛋白酶的纯化和性质。经过５步纯化过程,从（ｉ蚕卵母细胞中纯化出组织蛋白酶Ｂ,用ＳＤＳ聚丙烯酰胺凝胶电泳测得蛋白酶的亚基分子量在４７ｋＤａ左右。纯化的蛋白酶活性可被Ｅ－６４和Ｌｅｕｐｅｐｔｉｎ抑制。因此,该蛋白酶属于半胱氨酸蛋白酶。天冬氨酸蛋白酶特异性抑制剂ｐｅｐｓｔａｔｉｎ不抑制其活性。其活性可被ＤＦＰ和ＰＭＳＦ部分抑制。这两种抑制剂通常抑制丝氨酸蛋白酶活性     

Identification of P-57, a serine proteinase, from human erythrocyte membranes, which cleaves both chains of human third component (C3) of complement

A proteinase, which cleaves human third component of complement, was solubilized from erythrocyte membranes then purified by gel filtration chromatography, fluid phase electrophoresis, and hydroxylapatite chromatography. Labeling of the purified material by 125I or 3H-DFP and measurement of proteolytic activity subsequently isolated by SDS-polyacrylamide gel electrophoresis allowed to identify a 57 kDa single band, in non reducing conditions. Inhibition of this activity by PMSF supports covalent modification of an active serine residue. This membrane serine proteinase cleaved alpha and beta chains of human third component of complement, suggesting that p-57 is distinct from plasma serine proteinases.     

Purification of a high-molecular-weight inhibitor of the calcium-activated proteinase

An inhibitor of the muscle calcium-activated proteinases has been purified from porcine skeletal muscle by using DEAE-cellulose column chromatography, thermal treatment, Sephacryl S-400 column chromatography in 6 M urea and Sephacryl S-300 column chromatography in 6 M urea. Sodium dodecyl sulfate polyacrylamide slab gel electrophoresis shows that the purified inhibitor is homogeneous and has a subunit molecular weight of 172 000. The inhibitor inactivates both the low- and high-calcium-requiring forms of the calcium-activated proteinase but does not inhibit other proteinases against which it has been tried. It thus appears that the inhibitor is specific for the calcium-activated proteinase. Studies using homogeneous inhibitor and high-calcium-requiring proteinase show that one molecule of the inhibitor can inactivate up to eight molecules of the calcium-activated proteinase. Inactivation of the calcium-activated proteinase by the inhibitor cannot be reversed by calcium concentrations as high as 25 mM, thus eliminating the possibility that the inhibitor functions by chelating calcium. The inhibitory peptide appears to be extremely susceptible to proteolysis during its isolation. Even in the presence of synthetic proteinase inhibitors different inhibitor preparations yield homogeneous inhibitory peptides ranging in molecular weight from 145 000 to 172 000. Preparative electrophoresis and column chromatography have been used to isolate putative proteolytic breakdown products of the 172 kDa peptide at 145, 114, 41 and 29 kDa.     

Purification of protein phosphatase from hen oviduct

Two protein phosphatases of 103 and 29 kDa as determined by gel filtration, were purified from hen oviducts. The 103 -kDa phosphatase was purified 7300-fold to near homogeneity and dissociated into two polypeptides in the presence of SDS. Molecular masses of these polypeptides were estimated to be 60 and 38 kDa by SDS-polyacrylamide slab gel electrophoresis using the buffer system of Laemmli, but 68 and 35 kDa using the buffer system of Weber and Osborn. The stoichiometry of these polypeptides was approx 1:1 according to the densitometric analysis of gels at 550 nm. The 29 -kDa phosphatase was purified 2900-fold. Both phosphatases dephosphorylated the alpha-subunit of phosphorylase kinase more rapidly than the beta-subunit.     

Molecular mass analysis of murine immunosuppressive immunoglobulin G-binding factors (IgG-BFs) produced by T-cell hybrids

Induced and constitutive murine IgG-binding factors (IgG-BFs) have been purified by affinity chromatography from supernatants of T-cells preincubated with or without murine monoclonal IgG1 and IgG2b, respectively. IgG-BF Mr values have been studied by SDS-polyacrylamide gel electrophoresis (PAGE) after treatment with SDS under conditions which do not noticeably alter their immunosuppressive activities on the secondary in vitro IgG antibody response. Suppression was recovered at Mr values of 80000, 40000 and 20000. When induced IgG-BF was tested, the isotype-specific suppressive activity was found only at 40 kDa. The 20-kDa moiety appeared to derive from the 40-kDa component and the material found at 80 kDa exerted non-specific immunosuppressive effects. We conclude therefore that isotype-specific IgG-BF has an apparent Mr of 40000.     

A novel stimulating protein of mammalian DNA polymerase alpha

A DNA polymerase alpha-primase complex, which had been purified by means of immunoaffinity column chromatography, showed little activity in a reaction mixture composed of Tris-HCl buffer, but showed full activity in potassium phosphate buffer. It was found that potassium ion is required for the reaction by the immunoaffinity-purified enzyme. On the other hand, the DNA polymerase alpha purified by the orthodox biochemical method showed full activity in both buffer systems. A protein factor, which could restore the activity of immunoaffinity-purified DNA polymerase alpha-primase complex in the potassium-free reaction mixture, was separated from biochemically purified DNA polymerase alpha. The factor, designated as factor T, was stable to heat up to 70 degrees C, but was sensitive to trypsin. It sedimented at about 4S through a glycerol gradient. SDS-polyacrylamide gel electrophoresis revealed two polypeptide bands at 56 and 54 kDa. By immunoprecipitation, the factor T was shown to be physically associated with DNA polymerase alpha-primase complex. The stimulation was also observed with poly[d(A-T)], primed M13 DNA, and heat-denatured DNA.     

Agglutinins in the horseshoe crab hemolymph: purification of a potent agglutinin of horse erythrocytes from the hemolymph of Tachypleus tridentatus, the Japanese horseshoe crab

Agglutinins from Tachypleus (Tachypleus tridentatus, the Japanese horseshoe crab) hemolymph were isolated by affinity chromatography on BSM-coupled Sepharose 4B. The agglutinins showed multiple species and were composed of eight heterogeneous subunits with molecular weights of 45,000, 42,000, 41,000, 39,000, 33,000, 29,000, 27,000, and 22,000 as determined by SDS-polyacrylamide gel electrophoresis. The affinity-isolated agglutinins were fractionated into four groups by gel filtration on a Fractogel TSK (Toyopearl) HW-65 column, and these were designated as Tachypleus tridentatus agglutinin (TTA)-I, -II, -III, and -IV in the order of elution. These agglutinins were demonstrated to be heterogeneous as judged by their specificity towards horse erythrocytes, subunit structures, and immunological properties. TTA-III showed a potent agglutination activity towards horse erythrocytes and was further purified by gel filtration on a Cellulofine GC-700 column. The purified TTA-III is a highly purified (46,000-fold) protein composed of homogeneous subunits (Mr, 42,000) as judged by SDS-polyacrylamide gel electrophoresis and immunological analysis.     

Mechanism of activation of inactive renin in human plasma by puff adder venom

Venom of the puff adder (Bitis arietans) contains a potent, basic, Mr 24,000 metalloproteinase activity that can destroy all detectable trypsin and chymotrypsin inhibitory activity, when venom is incubated with human plasma. We have found that during such incubation, concomitant activation of inactive renin occurs. In an examination of the mechanism involved we now report the activation, in addition, of plasma prekallikrein and serine proteinase activity, but not plasminogen, when human plasma is incubated with venom. Furthermore, venom was not able to release active trypsin from its complex with alpha 1-proteinase inhibitor and human renin was not inhibited by alpha 1-proteinase inhibitor. The activities in venom and venom/plasma mixtures were analysed using Sephacryl S-200 gel filtration and the effect of 10 mM EDTA and 5 mM phenylmethanesulphonyl fluoride on activities in column fractions was tested. The inactive-renin-activating, plasma prekallikrein-activating and serine proteinase-activating activities could be accounted for to a large extent by a venom metalloproteinase which was estimated to have a Mr of 24,000 by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis. This enzyme activity appeared to complex with alpha 2-macroglobulin when venom was mixed with plasma. Since both EDTA and phenylmethanesulphonyl fluoride could inhibit the activation of inactive renin by this metalloproteinase, it is suggested that the enzyme activates serine proteinase(s), which then activate inactive renin. Plasma kallikrein may have a role in this process. Additional peaks of inactive-renin-activating activity eluted from Sephacryl S-200 at Mr 30,000 and 80,000 (minor) and an additional, minor peak of caseinolytic activity eluted at Mr 60,000. The Mr 24,000 metalloproteinase in venom may have considerable utility in activating inactive renin at physiological pH owing to its ability to destroy plasma proteinase inhibitors at the same time.     

Adhesion-promoting factor from embryonic fibroblasts for normal liver epithelial cells

In the present study we show that adhesion of normal rat liver epithelial cells (RL34) to substratum coated with type I collagen (collagen substratum) is promoted by a factor involved in 80% ammonium sulfate precipitated proteins from serum-free conditioned medium (PCM) of rat embryo fibroblasts. Adhesion of RL34 cells to collagen substratum was promoted dose dependently by whole PCM and the maximum effects on adhesion could be achieved by 200 micrograms/ml whole PCM. Kinetics studies with 100 micrograms/ml whole PCM showed that adhesion proceeded very slowly, taking 16 h to reach a plateau. Adhesion-promoting activity in whole PCM was sensitive to treatments with trypsin, acid, and heat but stable to dithiothreitol treatment. Further purification of whole PCM was performed using a combination of chromatography on blue Sepharose column, gel filtration column and heparin Sepharose column. The partially purified proteins, referred to as heparin PCM, are not bound or only weakly bound to heparin under physiological ion strength and pH, and the apparent molecular weight (Mr) range is estimated to be 40,000 to 60,000 from gel filtration chromatography and SDS-polyacrylamide gel electrophoresis. When whole PCM or heparin PCM was used for coating on plastic or collagen substratum, they no longer exerted the promoting activity.     

Purification and characterization of thermostable alpha-galactosidase from Ganoderma lucidum

Alpha-galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea alpha-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70 degrees C, respectively. The enzyme was fully stable to heating at 70 degrees C for 30 min. It hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km=0.4 mM) but hydrolyzed little o-nitrophenyl-alpha-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses.     

Activation of human platelet adenylate cyclase by a bovine sperm component

The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of Mr 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of Mr 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.     

A serine proteinase inhibitor isolated from Tamarindus indica seeds and its effects on the release of human neutrophil elastase

Proteinaceous inhibitors with high inhibitory activities against human neutrophil elastase (HNE) were found in seeds of the Tamarind tree (Tamarindus indica). A serine proteinase inhibitor denoted PG50 was purified using ammonium sulphate and acetone precipitation followed by Sephacryl S-300 and Sephadex G-50 gel filtration chromatographies. Inhibitor PG50 showed a Mr of 14.9 K on Sephadex G-50 calibrated column and a Mr of 11.6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PG50 had selective activity while cysteine proteinases (papain and bromelain) and serine proteinases (porcine pancreatic elastase and bovine chymotrypsin) were not inhibited, it was strongly effective against serine proteinases such as bovine trypsin and isolated human neutrophil elastase. The IC50 value was determined to be 55.96 microg.mL-1. PG50 showed neither cytotoxic nor haemolytic activity on human blood cells. After pre-incubation of PG50 with cytochalasin B, the exocytosis of elastase was initiated using PAF and fMLP. PG50 exhibited different inhibition on elastase release by PAF, at 44.6% and on release by fMLP, at 28.4%. These results showed that PG50 preferentially affected elastase release by PAF stimuli and this may indicate selective inhibition on PAF receptors.     

Isolation and purification of plasminogen activator from Yoshida ascites Sarcoma of rats

The plasminogen activator was purified to the extent of 150-fold from 20,000 x g supernatant of Yoshida ascites Sarcoma by ammonium sulphate precipitation at 33% saturation followed by affinity chromatography on p-aminobenzamidine-Sepharose 4B. The specific activity of the purified activator was 10,260 IU/mg expressed in terms of International units of urokinase, the known activator of plasminogen. The activator was homogeneous by polyacrylamide slab gel electrophoresis with an apparent molecular weight 75 kDa by gel filtration on Sephadex G-100. Analysis by SDS-polyacrylamide gel electrophoresis under reducing conditions, revealed the presence of two subunits of about 48 and 29 kDa. The activator displayed binding preference to fibrin and was immunologically distinguishable from urokinase, indicating that it could be of non-urokinase origin. The preparation further revealed similarity to standard tissue plasminogen activator with respect to fibrin binding and immunological cross reactivity.     

dNTP pool imbalance induced endonuclease: 5-fluorodeoxyuridine induced DNA double strand break in mouse FM3A cells and the mechanism of cell death.

The mechanism of intracellular deoxyribonucleotide triphosphates (dNTP) pool imbalance-induced cell death in mouse FM3A cells was studied. When the cells were treated with 1 microM 5-fluorodeoxyuridine (FdUrd), the imbalance of the cellular dNTP pool was induced. The imbalance was followed by DNA double stranded breaks and subsequent cell death. The endonuclease toward double stranded DNA has been found in a fraction of FdUrd treated cell lysate, and isolated using column chromatography. SDS-polyacrylamide gel electrophoresis showed a major protein species of approximate 45 kDa. The endonuclease was revealed, using electrophoretic separation in SDS-polyacrylamide gels containing DNA, by incubating the gels in buffer to remove SDS and to allow renaturation and enzyme activity.     

The quaternary structure of the plasma membrane b-type cytochrome of human granulocytes

Hydrodynamic, crosslinking and immunoprecipitation studies were performed on detergent solubilized cytochrome b to demonstrate that the two copurifying polypeptides of molecular weight 91,000 (glycosylated) and 22,000 [1,2] formed a molecular complex. The hydrodynamic studies indicated that the cytochrome b/detergent complex had a sedimentation coefficient, partial specific volume and Stokes radius of 5.25 S, 0.82 cm3/g and 6.2 nm in Triton X-100 and 6.05 S, 0.80 cm3/g and 5.6 nm in octylglucoside, respectively. These studies also indicated that the detergent-protein complex has a molecular mass of 202 and 188 kDa in Triton X-100 and octylglucoside, respectively, is asymmetric in shape with a frictional coefficient of 1.3-1.4 and binds significant amounts of detergent. The molecular mass of the protein portion of the detergent-cytochrome complex was estimated to be between 100 and 127 kDa. Crosslinking studies with disuccinimidyl suberate and alkaline cleavable bis[2-(succinimidooxy-carbonyloxy)ethyl]sulfone revealed that the Mr = 91,000 and Mr = 22,000 components of purified cytochrome b are closely associated and can be covalently bound to form a polypeptide which, by SDS-polyacrylamide gel electrophoresis, has Mr values of 110,000-120,000 and 120,000-135,000 on 8% and 11% (w/v) SDS-polyacrylamide gels, respectively. Cleavage of the crosslinked species resulted in the reappearance of the Mr = 91,000 and Mr = 22,000 species. Sedimentation profiles of crosslinked cytochrome b in linear sucrose density gradients made up in H2O were identical to those of non-crosslinked controls. A close association of the two protein species was further confirmed by the ability of antibody specific for the smaller subunit to immunoprecipitate the larger one also. Experiments aimed at identifying the heme-carrying subunit(s) were inconclusive, since dissociation of the complex resulted in loss of cytochrome b spectrum. These results, in combination with our SDS-polyacrylamide gel electrophoresis molecular-weight estimates, provide strong evidence for the cytochrome b being an alpha-beta-type heterodimer composed of a glycosylated Mr = 91,000 and non-glycosylated Mr = 22,000 polypeptide.