Purification and characterization of an alkaline elastase from Myxococcus xanthus

An extracellular elastase, termed Myxococcus xanthus alkaline protease 1 (MAP1), has been purified from M. xanthus DK1622 culture supernatants by a combination of ion-exchange and affinity chromatographies. It consists of a single peptide chain of 39 kDa. The elastolytic activity was totally suppressed by 10 mM 1,10-phenanthroline and the enzyme may then be classified as a metalloprotease. Its pH optimum was estimated to be 8.2 with both elastin-orcein and succinyl-Ala3 p-nitroanilide as substrates. Despite its low pI (5.2), MAP1 was adsorbed on elastin at 80%, a result which privileges hydrophobic interactions between MAP1 and elastin rather than salt bridges, as for known basic elastases. About 80% of the original amidasic and elastolytic activities were conserved after a 30-min prior incubation of the enzyme at 40 degrees C; however, 70% of the amidasic activity is measured, instead of 15% for the elastolytic activity, after 30 min at 50 degrees C. Thermal denaturation at this temperature may prevent adsorption of the enzyme on elastin without any important change of the elastase structure. MAP1 readily hydrolyzes the Gly23-Phe24 bond in the oxidized insulin B chain; the peptide bonds Ala14-Leu15, Leu15-Tyr16, Phe24-Phe25, Phe25-Tyr26 are also cleaved, suggesting a primary specificity of the enzyme for hydrophobic or aromatic residues at the first amino acid towards the C-terminus from the cleavage site (P'1 position) [Schechter, I. & Berger, A. (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. This hypothesis is consistent with the fact that Ala2-Phe-Ala and Ala3-Phe-Ala are hydrolyzed even though tri-alanine to hexa-alanine oligomers are not. The evidence of an elastase with the same molecular mass and pI as MAP1 is given during fruiting body development in submerged culture of M. xanthus. The fact that aromatic amino acids have been found to be the most representative of A-signal [Kuspa, A., Plamann, L. & Kaiser, D. (1992) J. Bacteriol. 174, 3319-3326] is consistent with the hypothesis that, regarding its specificity, MAP1 is likely to play a role in development of myxobacteria.     

Human pancreatic alpha-amylase. I. Purification and characterization

alpha-Amylase was extracted from human pancreas and purified by using ammonium sulfate fractionation, Sephadex G-100 and DEAE-Sephadex A-50 column chromatography. The enzyme was shown to be homogenous by three different criteria: polyacrylamide disc gel electrophoresis, SDS polyacrylamide gel electrophoresis and analytical ultracentrifugation. The values of SO20,w, D20,w, v, and frictional ration of the enzyme were calculated to be 5.01S, 7.56D, 0.718 ml g-1 and 1.10, respectively. The molecular weight of the alpha-amylase was determined by three different methods: sedimentation velocity-diffusion, conventional sedimentation equilibrium and SDS polyacrylamide gel electrophoresis and was found to be 57,850; 50,100 and 53,200 g mole-1, respectively (average value 53,700). The amino acid composition of the enzyme was determined and compared with those of alpha-amylases from various other sources.     

Isolation and partial characterization of elastase from dog granulocytes

Hemoglobin Hope (beta(H14)136gly leads to asp), a mildly unstable variant, was found to have decreased oxygen affinity, a normal Bohr effect and diminished cooperativity. Decreased oxygen affinity of hemoglobin Hope may explain the previous failure to find an appropriate response to hemolysis in individuals studied who were heterozygous for both hemoglobin Hope and sickle hemoglobin. Salt bridge formation between NA1 valine and H14 aspartic acid may stabilize the beta Hope subunit in its deoxy form thus producing intrinsically low oxygen affinity and reduced cooperativity.     

Specificity and reactivity of human leukocyte elastase, porcine pancreatic elastase, human granulocyte cathepsin G, and bovine pancreatic chymotrypsin with arylsulfonyl fluorides. Discovery of a new series of potent and specific irreversible elastase inhibitors

The reactivity and specificity of a series of substituted benzenesulfonyl fluorides with human leukocyte (HL) elastase, cathepsin G, porcine pancreatic (PP) elastase, and bovine chymotrypsin A alpha are reported. Benzenesulfonyl fluorides with 2-fluoroacyl substituents were found to be potent and specific inhibitors of elastase. HL elastase was inhibited most rapidly by 2--(CF3CF2CONH)--C6H4SO4F (kobs/[I] = 1700 M-1 s-1) which is slightly better than our best peptide chloromethyl ketone inhibitor of this enzyme. PP elastase was most rapidly inhibited by 2--(CF3CONH)--C6H4-SO2F (kobs/[I] = 2300 M-1S-1). The 2--(CF3CF2CF2CONH) and 2--(CF3SNH) derivatives were quite selective for HL elastase and inhibited PP elastase, cathepsin G, and chymotrypsin A alpha quite slowly. A specific and potent chymotrypsin inhibitor (2--(Z--Gly--NH)--C6H4SO2F) was also discovered. A model for the elastase inhibition reaction is proposed which involves interaction of the fluroacyl group of the inhibitor with the primary substrate recognition site S1 of the enzyme. Hydrogen bonding also occurs between the inhibitor NH and a backbone peptide carbonyl group, probably from residue 214. The 2-fluoroacyl group plays the dual role of binding in the hydrophobic S1 pocket and through electronic effects, increasing the strength f the hydrogen bond. The results of this study demonstrate that it is possible to construct simple organic molecules which are specific inhibitors of HL elastase, PP elastase, or chymotrypsin.     

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid L-arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K(m) for L-arginine 2.8, 2.0 and 2.3 microM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.     

Differing susceptibility of young and adult hamster lungs to injury with pancreatic elastase

A study was undertaken to determine whether there are differences in the initial response of the lungs of young (28 days) and adult (105 days) hamsters to pancreatic elastase treatment. The elastase was given intratracheally (0.1 mg/100 g body weight), and the animals were studied 24 h later. The mean linear intercept of the lungs of young animals increased less than that of adult animals (p less than 0.05). Adult body weights decreased significantly with elastase treatment, whereas those of young elastase-treated animals did not. The lungs of young animals gained proportionately less weight than those of adult animals, suggesting they experienced less hemorrhage and edema. When the volumes, at given transpulmonary pressures, of fluid-filled lungs were expressed as percent predicted, the values for young animals were significantly less than those for adult animals. The most pronounced differences were at low transpulmonary pressures (1 to 3 cm H2O). Volume-pressure hysteresis was not altered by elastase treatment in either the young or adult animals. We conclude that the lungs of adult hamsters are more susceptible to elastase injury than the lungs of young hamsters.     

Resistance of purified cholera toxin to enzymatic treatment with pancreatic elastase and papain

Treatment of Cholera toxin with pancreatic elastase and papain in vitro showed a high resistance of the toxin molecule to these enzymes, under non-denaturing conditions or in the presence of 2 M urea. These experiments support the hypothesis of a particularly stable molecular structure of the toxin, as an explanation of its activity in the intestinal lumen where the pancreatic proteases are active.     

Structure of a specific acyl-enzyme complex formed between beta-casomorphin-7 and porcine pancreatic elastase

BACKGROUND: An accurate determination of the extent of Barrett's metaplasia is critical to the study of its natural history and response to therapy. Our hypothesis is that area calculations offer advantages over length estimates of Barrett's. METHODS: Changes in both measures and estimates of progression or regression between two endoscopies in 17 patients were compared. Area was calculated using a computer image analysis technique. RESULTS: Although there was no significant difference in length correlation versus area correlation between endoscopies (r = 0.90 vs 0.99), the mean change in absolute length (1.4 +/- 0.2 cm) was greater than the change in area (4.5 +/- 1.4 cm2, equivalent to a length of 0.67 +/- 0.2 cm, p = 0.001). The percent change in absolute length (26.9%) was greater than the change in area (16%, p = 0.001). Discordance of estimates of progression or regression between area and length was found in nine patients. The image technique detected no change in the area of squamous islands. CONCLUSIONS: Imaging analysis can precisely measure the extent of Barrett's including squamous islands. Area showed little change, whereas measures of length were more varied. Computer based image analysis provides a more precise estimate of interval change of Barrett's.     

Purification and characterization of rabbit transcobalamin II

Rabbit transcobalamin II has been purified by labile ligand affinity chromatography and G-200 Sephadex gel filtration. Structural studies indicate Stokes' radii of 2.7 nm and 3.0 nm for transocobalamin II saturated and unsaturated with cobalamin. The amino acid content of the protein is very similar to that of human transcobalamin II (Allen, R. H. (1975) Prog. Hematol. 9, 59-84). The aminoterminal sequence for transcobalamin II is reported for the first time: Glu-Ile-Cys-Gly-Val-Pro-Lys-Val-Asp-Ser-Glu-Leu-Val-Glu-Lys-Leu-Gly-Gln-Arg-Leu-Leu-Pro-(Trp)-Met-Thr). The ultraviolet and circular dichroic spectra of aquo-, hydroxo-, azido- and cyanocobalamin bound to rabbit transcobalamin II are described. On complex formation the molar absorption of the cobalamins increases and the major bands shift to longer wavelengths. The spectra are little affected by change in the fifth ligand, which indicates that the electron density around the cobalt atom is kept fairly constant by the transcobalamin II molecule. This is in contrast to the observations for the same cobalamins attached to human intrinsic factor and human transcobalamin I (Nexo, E. and Olesen, H. (1976) Biochim. Biophys. Acta 446, 143-150).     

Purification and characterization of ceramide-activated protein phosphatases

Ceramide has emerged as a potential regulator of diverse cellular functions, and a few direct targets have been identified for its action including protein kinases and phosphatases. In this study, we have purified the predominant ceramide-activated protein phosphatase (CAPP) from rat brain. Utilizing a novel chromatographic approach, CAPP was purified to near homogeneity using hydrophobic interaction chromatography on phenyl Sepharose followed by anion-exchange chromatography on MonoQ. The purified protein was composed of three major bands on SDS-polyacrylamide gel electrophoresis which comigrated with the three subunits of heterotrimeric PP2A. Immunologic studies further identified CAPP to be composed predominantly of heterotrimeric AB'C and ABalphaC as well as heterodimeric PP2A (AC), where C is the catalytic subunit, and A and B are regulatory subunits. These results were also supported by the coelution of CAPP with trimeric and dimeric PP2A on size-exclusion chromatography. These studies provide a convenient and efficient method for the isolation of trimeric and dimeric PP2A, and they allow the biochemical investigation of CAPP.     

Purification and characterization of a nylon-degrading enzyme

A nylon-degrading enzyme found in the extracellular medium of a ligninolytic culture of the white rot fungus strain IZU-154 was purified by ion-exchange chromatography, gel filtration chromatography, and hydrophobic chromatography. The characteristics of the purified protein (i.e., molecular weight, absorption spectrum, and requirements for 2,6-dimethoxyphenol oxidation) were identical to those of manganese peroxidase, which was previously characterized as a key enzyme in the ligninolytic systems of many white rot fungi, and this result led us to conclude that nylon degradation is catalyzed by manganese peroxidase. However, the reaction mechanism for nylon degradation differed significantly from the reaction mechanism reported for manganese peroxidase. The nylon-degrading activity did not depend on exogenous H2O2 but nevertheless was inhibited by catalase, and superoxide dismutase inhibited the nylon-degrading activity strongly. These features are identical to those of the peroxidase-oxidase reaction catalyzed by horseradish peroxidase. In addition, alpha-hydroxy acids which are known to accelerate the manganese peroxidase reaction inhibited the nylon-degrading activity strongly. Degradation of nylon-6 fiber was also investigated. Drastic and regular erosion in the nylon surface was observed, suggesting that nylon is degraded to soluble oligomers and that nylon is degraded selectively.     

Purification and characterization of two forms of urokinase

Affinity chromatography on agmatine-Sepharose was used for the separation of two active forms of urokinase (EC 3.4.99.26) from partially purified human urinary urokinase. The approximate molecular weight of the heavier form was 47 000 and of the lighter 33 400. Both forms were homogeneous by sodium dodecyl sulfate gel electrophoresis and by 3H-labeled diisopropylphosphorofluoridate and 14C-labeled p-nitrophenyl-p'-guanidinobenzoate incorporation studies. The 33 400 mol. wt. form had a single chain, and the 47 000 mol. wt. form had two chains (33 100 and 18 600 mol. wt.) linked by disulfide bonds. The specific activity of the heavier form was 104 000 CTA units/mg protein, compared with 226 000 units/mg for the lighter form but the activities per mmol of active site (molar activities) of the two forms were almost identical (9.6-10(9) and 10.2-10(9) CTA units/mmol). Isoelectric focusing on gels showed that the 47 000 material contained one major subform with a pI of 8.60 and a minor subform with a pI of 8.90, while the 33 400 material had three major subforms with pI values of 8.35, 8.60 and 8.70, respectively, and a minor subform with a pI of 8.05. 3H-labeled diisopropylphosphorofluoridate incorporation studies revealed an active-site serine residue in the heavy chain.     

Chicken oviductal ecto-ATP-diphosphohydrolase. Purification and characterization

An ecto-ATP diphosphohydrolase (ATPDase) was purified to homogeneity from vesiculosomes shed from chicken oviduct. First, the ecto-ATPDase-enriched vesiculosomes were concentrated by filtration, differential centrifugation, and exclusion chromatography. Next, the nonionic detergent, Nonidet P-40, was used to extract the ecto-ATPDase from vesiculosomal membranes, and the solubilized enzyme was further purified by ion exchange (DEAE-Bio-Gel) and lentil-lectin-Sepharose 4B chromatography. In the final stage, immunoaffinity chromatography was utilized to obtain purified ecto-ATPDase. More than 25,000-fold purification was achieved. Specific activity of the purified enzyme was greater than 800 micronol/min/mg of protein with MgATP as the substrate, the highest ever reported for an ATPDase. The enzyme also hydrolyzed other nucleoside triphosphates in the presence of magnesium at similar rates and CaATP and MgADP at lower rates. The molecular mass of the purified glycoprotein was 80 kDa as determined by SDS-polyacrylamide gel electrophoresis and Western blot analysis. Based on its enzymatic properties, the relationship of the chicken oviduct ecto-ATPDase with other reported ATPDases and ecto-ATPases is discussed.     

Purification and characterization of human coagulation factor V

We have purified human coagulation Factor V 6,000-fold to homogeneity from citrated plasma using polyethylene glycol 6000 precipitation, adsorption of Factor V to barium citrate, DEAE-Sepharose chromatography, and gel filtration on Ultrogel AcA 34 (yield 21%). Human Factor V is a single polypeptide chain before and after disulfide bond reduction with an apparent Mr = 335,000 as determined by electrophoresis on 5% acrylamide sodium dodecyl sulfate gels. Human Factor V is a glycoprotein containing 13% of weight carbohydrate and there is a high content of sialic acid (86 residues/mol) compared to the other sugars. When human Factor V is treated with thrombin, coagulation activity increases 25- to 30-fold to a specific activity of 1.7 to 2.0 units/microgram. Thrombin activation is accompanied by the cleavage of three bonds in the Factor V molecule. We have detected activation intermediates with apparent Mr = 295,000 and 248,000 and final products with apparent Mr = 150,000, 121,000, and a doublet at 95,000-91,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final products of thrombin activation of human Factor V and bovine Factor V are similar, yet the intermediates observed are different. This suggests that cleavages are made at similar locations in bovine and human Factor V, but that they occur in a different sequence. When human Factor V is treated with the Factor V activator from Russell's viper venom, it is split into two components with apparent Mr = 303,000 and 95,000-91,000 and is fully activated. The increase in coagulation activity observed upon treatment of human Factor V with thrombin or the Factor V activator from Russell's viper venom seems to correlate with the generation of the doublet Mr = 95,0090-91,000 component.     

Purification and characterization of human topoisomerase I mutants

A system for rapid purification and characterization of eukaryotic topoisomerase-I mutants has been developed. The system utilizes six-histidine tagging of human topoisomerase I expressed in Saccharomyces cerevisiae to enable purification by nickel-affinity chromatography. Virtually homogenous mutant proteins are then tested for their ability to relax supercoiled DNA plasmids and their capacity for binding, cleaving and religating short defined DNA substrates. Relaxation-deficient mutants were obtained by site-directed mutagenesis of selected highly conserved amino acids. The mutants Tyr723Phe (active site mutation), Arg488Gln and Lys532Glu were inert in relaxation of DNA, whereas Lys720Glu showed a 50-fold reduction in specific relaxation activity. Accordingly, only Lys720Glu showed low, but detectable cleavage activity on suicide DNA substrates, uncoupling the cleavage and religation events of topoisomerase I. The relative religation efficiency of Lys720Glu comparable to that of wild-type topoisomerase I, indicating that Lys720 is involved in interactions important for normal DNA cleavage, but not for the religation reaction. All mutants could be cross linked by ultraviolet light to bromo-dUTP-substituted DNA oligonucleotides carrying a topoisomerase-I-binding site, indicating that the deficiency of Tyr723Phe, Arg488Gln and Lys532Glu in DNA relaxation and cleavage is not due to an inability of these mutants to bind DNA non-covalently.     

Purification and characterization of Phaseolus vulgaris alpha-D-galactosidase isozymes

A highly purified preparation of alpha-D-galactosidase [E.C. 3.2.1.22] isozymes was obtained from Phaseolus vulgaris (pinto bean) seeds by extraction, salt precipitation, ion exchange, and affinity chromatography. The final preparation was homogeneous by SDS-PAGE but revealed isozymes of relative mass of 38.3 and 39.6 kDa. The N-terminal sequence for both isozymes was identical, LANGLAKT (one letter code for amino acids). Relative native molecular mass was estimated at 149.3 kDa by Sephacryl S-200 chromatography. Activity was unaffected by ionic strength at high enzyme concentrations, and was specific for alpha-D-galactoside conjugates. No protease or hemagglutinin activity was detected, and activity was stable at 4 degrees C. Studies with soluble oligosaccharides demonstrated high activity against the selected straight and branched-chain substrates. The enzyme was active against terminal alpha 1-3 galactosyl residues on human and rabbit erythrocyte membranes. Because of its activity against membrane glycoconjugates, these isozymes may have potential utility for modifying membrane epitopes on native erythrocytes.     

Purification and characterization of a tumor lipid-mobilizing factor

Cancer patients with weight loss showed urinary excretion of a lipid-mobilizing factor (LMF), determined by the ability to stimulate lipolysis in isolated murine epididymal adipocytes. Such bioactivity was not detectable in the urine of cancer patients without weight loss or in normal subjects. The LMF was purified using a combination of ion exchange, exclusion, and hydrophobic interaction chromatographies to give a single component of apparent Mr 43,000, which showed homology in amino acid sequence with human plasma Zn-alpha2-glycoprotein. Both substances showed the same mobility on denaturing and nondenaturing gels and the same chymotrypsin digestion pattern, both stained heavily for carbohydrate, and they showed similar immunoreactivity. Polyclonal antisera to human plasma Zn-alpha2-glycoprotein was also capable of neutralization of the bioactivity of human LMF in vitro. Using competitive PCR to quantify expression of Zn-alpha2-glycoprotein, we found that only those tumors that were capable of producing a decrease in carcass lipid expressed mRNA for Zn-alpha2-glycoprotein. These results provide strong evidence to suggest that tumor production of Zn-alpha2-glycoprotein is responsible for the lipid catabolism seen in cancer patients.