Purification and spectroscopic characterization of a recombinant chloroplastic hemoglobin from the green unicellular alga Chlamydomonas eugametos

Hemoglobins (Hb), which have the important task of delivering molecular oxygen by facilitating its reversible binding to the heme, are now thought to have evolved in all groups of organisms including prokaryotes, fungi, plants and animals. Our recent finding of a light-inducible chloroplastic Hb in the green unicellular alga Chlamydomonas eugametos has further extend this idea, while raising questions about the function that an Hb could play in a high oxygen environment such as in the chloroplast. In order to understand the role played by this new Hb, we have undertaken its biochemical characterization. To facilitate the characterization of Chlamydomonas Hb, which represents less than 0.01% of the soluble protein in the green alga, the protein has been expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant protein possesses a non-covalently bound iron-protoporphyrin IX heme. The oxy form of the recombinant Hb. purified directly from bacterial cells, is very stable, with a measured half-life of 7 days at pH 8 and has an ultraviolet/visible spectrum similar to those of the related cytoplasmic Hbs of the ciliated protozoa Paramecium and Tetrahymena and of the cyanobacterium Nostoc commune. In contrast to what has been reported for oxymyoglobins and oxyhemoglobins, the dioxygen molecule bound to the L1637 Hb can be reduced by the electron-transfer mediator phenazine methosulfate in the presence of NADPH, indicating that the heme pocket of Chlamydomonas Hb may be more accessible to small molecules. With regard to this we found that when the small reducing agent sodium dithionite is used to reduce the met form, it must be removed anaerobically from the Hb prior to oxygenation of the protein to stably produce the oxy form. Otherwise, the oxy form is obtained readily from the met form under an oxygenic atmosphere when ferredoxin and ferredoxin NADP+ reductase are used to enzymically reduce the Hb. Finally, the spectra of the deoxy and met forms were unusual, the heme being partly low-spin at physiological pH. These results confirm the existence of a reversible oxygen-binding protein in the chloroplast of C. eugametos. The unusual spectral and biochemical properties of the protein may reflect a specialized function for this Hb.     

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH.     

Purification and characterization of an isoaspartyl dipeptidase from Escherichia coli

We have identified a gene (iadA) in Escherichia coli encoding a 41-kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-beta-aspartyl, dipeptides. We demonstrate at least a 3000-fold purification of the enzyme to homogeneity from crude cytosol. From the amino-terminal amino acid sequence obtained from this preparation, we designed an oligonucleotide that allowed us to map the gene to the 98-min region of the chromosome and to clone and obtain the DNA sequence of the gene. Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases, while a marked similarity was found with several dihydroorotases and imidases, reflecting the similarity in the structures of the substrates for these enzymes. Using an E. coli strain containing a plasmid overexpressing this gene, we were able to purify sufficient amounts of the dipeptidase to characterize its substrate specificity. We also examined the phenotype of two E. coli strains where this isoaspartyl dipeptidase gene was deleted. We inserted a chloramphenicol cassette into the disrupted coding region of iadA in both a parent strain (MC1000) and a derivative strain (CL1010) lacking pcm, the gene encoding the L-isoaspartyl methyltransferase involved in the repair of isomerized proteins. We found that the iadA deletion does not result in reduced stationary phase or heat shock survival. Analysis of isoaspartyl dipeptidase activity in the deletion strain revealed a second activity of lower native molecular weight that accounts for approximately 31% of the total activity in the parent strain MC1000. The presence of this second activity may account for the absence of an observable phenotype in the iadA mutant cells.     

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.     

Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum

A 3,4-dihydroxybenzoate decarboxylase (EC 4.1.1.63) from Clostridium hydroxybenzoicum JW/Z-1T was purified and partially characterized. The estimated molecular mass of the enzyme was 270 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single band of 57 kDa, suggesting that the enzyme consists of five identical subunits. The temperature and pH optima were 50 degrees C and pH 7.0, respectively. The Arrhenius energy for decarboxylation of 3,4-dihydroxybenzoate was 32.5 kJ . mol(-1) for the temperature range from 22 to 50 degrees C. The Km and kcat for 3,4-dihydroxybenzoate were 0.6 mM and 5.4 x 10(3) min(-1), respectively, at pH 7.0 and 25 degrees C. The enzyme optimally catalyzed the reverse reaction, that is, the carboxylation of catechol to 3,4-dihydroxybenzoate, at pH 7.0. The enzyme did not decarboxylate 2-hydroxybenzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 2,3,4-trihydroxybenzoate, 3,4,5-trihydroxybenzoate, 3-F-4-hydroxybenzoate, or vanillate. The decarboxylase activity was inhibited by 25 and 20%, respectively, by 2,3,4- and 3,4,5-trihydroxybenzoate. Thiamine PPi and pyridoxal 5'-phosphate did not stimulate and hydroxylamine and sodium borohydride did not inhibit the enzyme activity, indicating that the 3,4-dihydroxybenzoate decarboxylase is not a thiamine PPi-, pyridoxal 5'-phosphate-, or pyruvoyl-dependent enzyme.     

Purification and partial characterization of an acid proteinase from Dirofilaria immitis

Tumour necrosis factor-alpha (TNF-alpha) is a pluripotent cytokine with its receptors distributed throughout many different cell types. Because of the diverse effects of the cytokine, it is difficult to clearly define its role in infection and immunity, and appreciate its clinical therapeutic value. We have identified peptides derived from the primary amino acid sequence of human TNF-alpha that have neutrophil-stimulating activity, as measured by enhanced chemiluminescence and superoxide production, and peptides which are both directly cytotoxic for tumour cells (WEHI-164) in vitro and also prevent TNF binding to tumour cells. However, only one of these neutrophil-stimulating peptides was toxic for tumour cells in vitro. Our results indicate that the region of amino acids 54-94 of human TNF-alpha has previously undescribed human neutrophil-stimulatory activity, while peptides encompassing the regions 43-68 and 132-150, which are in close proximity, as indicated in the recently determined three-dimensional structure of human TNF-alpha, have in vitro anti-tumour activity. These peptides also slowed tumour growth or induced tumour regression in WEHI-164 tumour-bearing mice. The peptide 73-94, which activated neutrophils but which was not cytotoxic for tumour cells in vitro, also caused in vivo tumour regression, presumably by activating neutrophils with the consequent release of free radicals at the tumour site. Peptide 63-83, which was able to activate neutrophils in vitro, did not possess tumour regression activity in vivo. The TNF peptides described in this report did not elicit procoagulant activity in cultured bovine aortic endothelial cells and as such are devoid of at least one of the potentially lethal side-effects of elevated TNF levels in vivo.     

Purification and characterization of an endo-1,3-beta-glucanase from Aspergillus fumigatus

An endo-1,3-beta-glucanase was purified from a cell wall autolysate of Aspergillus fumigatus. This beta-glucanase activity was associated with a glycosylated 74-kDa protein. Using a sensitive colorimetric assay and a high-performance anion-exchange chromatography with a pulsed electrochemical detector for product analysis, it was shown that the endoglucanase hydrolysed exclusively linear 1,3-beta-glucan chains, had an optimum pH of 7.0 and an optimum temperature of 60 degrees C. A substrate kinetic study gave a Km value of 0.3 mg/ml for soluble (laminarin and laminari-oligosaccharides) and 1.18 mg/ml for insoluble (curdlan) 1,3-beta-glucan. Laminari-oligosaccharide degradation, analysed by HPLC, showed that the endoglucanase bind to the subtrate at several positions and suggested that the active site of the enzyme recognized five glucose units linked by a 1,3-beta bond. The association of the present endo-1,3-beta-glucanase with the cell wall of A. fumigatus suggests a putative role for this enzyme during cell-wall morphogenesis.     

Purification and characterization of a cytotoxin from Enterobacter cloacae

OBJECTIVE: To document our evolving surgical management of colonoscopic perforation and examine factors crucial to the improvement of patient care. DESIGN: We conducted a computer-based retrospective analysis of medical records (1980 through 1995). MATERIAL AND METHODS: Among 57,028 colonoscopic procedures performed, 43 patients (0.075%, or 1 perforation in 1,333 procedures) had a colonic perforation. Two additional patients were treated after colonoscopy performed elsewhere. The outcomes analyzed included surgical morbidity and mortality. RESULTS: Twenty-six women and 19 men who ranged in age from 28 to 85 years (median, 69) were treated for colonic perforation. More than 80% of perforations occurred during the latter half of the study period because of the increased volume of colonoscopic procedures (8 perforations among 12,581 examinations from 1980 through 1987 versus 35 perforations among 44,447 colonoscopies from 1988 through 1995). Emergency laparotomy was performed in 42 patients (93%). Perforations occurred throughout the colon: right side = 10; transverse = 9; and left side = 23. Three patients without evidence of peritoneal irritation fared well with nonoperative management. Most patients underwent primary repair or limited resection in conjunction with end-to-end anastomosis. In 14 patients (33%), an ostomy was created. One patient underwent laparotomy without further treatment. Intra-abdominal contamination ranged from none (31%) to local soiling (48%) to diffusely feculent (21%). Postoperative complications occurred in 12 patients and were associated with older age (P = 0.01), large perforations (P = 0.03), and prior hospitalization (P = 0.04). No postoperative deaths occurred. CONCLUSION: Despite a consistently low risk of colonic perforation, the increasing use of colonoscopy in our practice has resulted in an increased number of iatrogenic colonic perforations. In order to minimize morbidity and mortality, prompt operative intervention is the best strategy in most patients. Non-operative management is warranted in carefully selected patients without peritoneal irritation.     

Purification and characterization of a prolidase from Aureobacterium esteraromaticum

An EDTA-insensitive prolidase (proline dipeptidase, EC 3.4.13.9) was isolated from a cell-free extract of Aureobacterium esteraromaticum IFO 3752. The enzyme was purified almost to homogeneity using acetone precipitation, hydrophobic chromatography, ion-exchange chromatography, and gel-permeation chromatography. The enzyme has a molecular weight of about 440,000 by gel permeation chromatography, and about 40,000 by SDS polyacrylamide gel electrophoresis. The isoelectric point was 4.6. The enzyme hydrolyzed aminoacylprolines such as Ser-Pro. Thr-Pro, Gly-Pro, Ala-Pro, Ile-Pro, Leu-Pro, and Pro-Pro. It also hydrolyzed Gly-Hyp and Pro-Hyp. The rate of hydrolysis for Pro-Hyp was the highest among the substrates tested. Optimum pH for hydrolyzing Pro-Hyp was 9.0 and the enzyme was stable in the pH range from 5 to 10. The optimum temperature was estimated to be 45 degrees C using 10 min of reaction. At least 90% of the initial activity remained after 30 min of incubation at 60 degrees C. p-Chloromercuribenzoic acid and o-phenanthrolin inhibited the enzyme's activity while EDTA did not. Addition of Mn2+ ion did not stimulate activity. These results suggest either that the metal ion in the enzyme may be tightly bound to the polypeptide chain, or that the enzyme is not a metallo-enzyme but a thiol-enzyme.     

Purification, characterization, and localization of an ADP-ribosylactin hydrolase that uses ADP-ribosylated actin from rat brains as a substrate

Mammalian ADP-ribosylation is poorly understood. An ADP-ribosylprotein hydrolase that acted on ADP-ribosylated actin was purified from rat brain. The molecular weight of this enzyme was 62, 000 as determined by SDS-polyacrylamide gel electrophoresis and gel filtration. Enzyme activity with ADP-ribosylated actin as a substrate was inhibited by NAD, ATP, ADP, and ADP-ribose, but not by AMP. Mg2+ increased Vmax. Purified ADP-ribosylactin hydrolase catalyzed the hydrolysis of ADP-ribosylated subunits Gsalpha, Gialpha, and Goalpha and elongation factor-2. After de-ADP-ribosylation by the purified ADP-ribosylactin hydrolase, the proteins were re-ADP-ribosylated by brain mono-ADP-ribosyltransferases and bacterial toxins. The actin that was de-modified by ADP-ribosylactin hydrolase could form actin filaments. Two kinds of monoclonal antibodies against ADP-ribosylactin hydrolase were prepared and characterized. In an immunohistochemical study, the plasma membranes and cytoplasmic regions of the nerve cells in the rat brain were immunoreactive. In subcellular fractionation of the brains, most of the ADP-ribosylactin hydrolase activity was found in the cytosol and synaptosome fractions. When the synaptosomes were treated with a hypotonic solution, ADP-ribosylactin hydrolase activity was found in the supernatant. Our findings suggest that brain ADP-ribosylactin hydrolase has the important function of polymerizing actin for signal transduction in the cytosol of nerve cells and synaptosomes.     

Purification and characterization of an NAD-malic enzyme from Bradyrhizobium japonicum A1017

An NAD-malic enzyme was purified to homogeneity from Bradyrhizobium japonicum A1017, and its molecular characteristics were surveyed. The enzyme exhibited native and subunit molecular masses of 388 and 85 kDa, respectively, suggesting that it exists as a homotetramer, and was activated by metabolic intermediates in glycolysis. The role of the enzyme in bacteroids' carbon metabolism is discussed.     

Purification and characterization of cysteine proteinase from a baculovirus gene

To analyze the degradation of product proteins at the late stage of virus infection in the baculovirus expression system, a cysteine proteinase was purified from hemolymph of Bombyx mori infected with wild-type B. mori nuclear polyhedorosis virus (BmNPV). The purified cysteine proteinase preparation had two protein bands (major 35-kDa active protein and 28-kDa inactive protein) on SDS-PAGE. Based on the N-terminal amino acid sequences of them, it was found that both proteins originated in the cysteine proteinase gene of BmNPV. The purified cysteine proteinase had an optimum pH at 4.0, and also had activities at neutral pHs. When recombinant luciferase was used as a natural substrate, it was degraded rapidly by the cysteine proteinase at the physiological pH of hemolymph. These results suggest that the cysteine proteinase from a BmNPV gene participates in the degradation of foreign protein expressed by the baculovirus system.     

Purification and characterization of a cephalosporin esterase from Rhodosporidium toruloides

A novel cephalosporin esterase (EC 3.1.1.41) from Rhodosporidium toruloides was purified to gel electrophoretic homogeneity. The enzyme is a glycoprotein with a molecular mass of 80 kDa. Upon deglycosylation, several forms of the enzyme were observed with a molecular mass range between 60 and 66 kDa. The isoelectric point of the enzyme is approximately 5.6, with the pH optimum for activity occurring at 6.0. The optimal activity of the enzyme occurred at 25 degrees C, with the enzyme rapidly losing activity at temperatures above 25 degrees C. The enzyme deacetylated a variety of cephalosporin derivatives, including cephalosporin C; the Km for this substrate is 51.8 mM, and the Vmax is 7.9 mumol/min/mg. In addition to cephalosporins, the enzyme hydrolyzed short-chain p-nitrophenyl esters, with the activity decreasing with increasing ester chain length. The enzyme also has the ability to acetylate desacetyl cephalosporins in high yields under mild conditions in the presence of various acetyl donors. A comparison of the physical properties of the esterase with those of other well-characterized cephalosporin esterases indicates that the enzyme is unique in this class.     

Purification and characterization of a lectin from Wistaria floribunda seeds

A lectin from Wistaria floribunda seeds which specifically binds to N-acetyl-D-galactosamine was purified to homogeneity as judged by polyacrylamide gel electrophoresis. Its molecular weight was estimated to be 68,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It dissociated into subunits on reduction with 2-mercaptoethanol with concomitant loss of hemagglutinating activity. On oxidation in air, the subunits reassociated into the lectin molecules with hemagglutinating activity. Carboxymethylation of the subunits with iodoacetic acid prevented their reassociation on oxidation in air. The molecular weight of the subunits was 32,000, which is about one-half that of the native lectin, suggesting that the lectin consists of two subunits. The results total, NH2-terminal, and COOH-terminal amino acid analyses, and mapping of the tryptic digest of the lectin indicated that these two subunits were indistinguishable and were probably identical, and that they were linked together covalently through a single disulfide bond. Equilibrium dialysis experiments show that the lectin and its subunit molecules are divalent and monovalent, respectively, with respect to sugar binding. The lectin is a glycoprotein, containing 3.2% carbohydrate. The carbohydrate moiety is composed of mannose, galactose, and glucosamine is a molar ratio of 1:2:1 and these sugars seem to be linked as a single oligosaccharide chain to each subunit of the protein.     

Purification and characterization of a DNA helicase from Saccharomyces cerevisiae

A novel DNA helicase, scHelI, has been purified from whole cell extracts of Saccharomyces cerevisiae using biochemical assays to monitor the fractionation. The enzyme unwinds partial duplex DNA substrates, as long as 343 base pairs in length, in a reaction that is dependent on either ATP or dATP hydrolysis. scHelI also catalyzes a single-stranded DNA-dependent ATP hydrolysis reaction; the apparent Km for ATP is 325 microM. The unwinding reaction on circular partial duplex substrates is biphasic, with a fast component occurring within 5 min of the initiation of the reaction and a slow component continuing to 60 min. This is in contrast to the ATP hydrolysis reaction, which exhibits linear kinetics for 60 min. The direction of the unwinding reaction is 5' to 3' with respect to the strand of DNA on which the enzyme is bound. The unwinding reaction is strongly stimulated by the addition of Escherichia coli single-stranded DNA-binding protein when long partial duplex substrates are used. The enzymatic activity of scHelI copurifies with a polypeptide of 135 kDa as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The polypeptide sediments as a monomer in a glycerol gradient in the presence of 0.2 M NaCl.     

Purification and characterization of a novel ceramidase from Pseudomonas aeruginosa

We report here a novel type of ceramidase of Pseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis. The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17. After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed quite wide specificity for various ceramides, i.e. it hydrolyzed ceramides containing C12:0-C18:0 fatty acids and 7-nitrobenz-2-oxa-1, 3-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:1) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base. However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from a Pseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K. (1995) J. Biol. Chem. 270, 24370-24374). This bacterial ceramidase had a pH optimum of 8.0-9.0, an apparent Km of 139 microM, and a Vmax of 5.3 micromol/min/mg using N-palmitoylsphingosine as the substrate. The enzyme appears to require Ca2+ for expression of the activity. Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. Our study demonstrated that ceramidase is widely distributed from bacteria to mammals.     

Purification and characterization of beta-glucosidase from Bacteroides JY-6, a human intestinal bacterium

A beta-glucosidase (EC 3.2.1.21.) was purified 2500-fold from Bacteroides JY-6, an intestinal anaerobic bacterium of human. The specific activity of the homogeneously purified enzyme was 210 mumol/min/mg protein. The enzyme (M(r) 75kDa) was an monomer whose pI and optimal pH values were 4.6 and 5.5-6, respectively. The best substrates were p-nitrophenyl beta-D-glucopyranoside and natural beta-bound glucosides, such as prunin and poncirenin. Puerarin, which is a C-glycoside, was weakly effective. However, cellobiose, alpha-bound glycosides and rhamnoglucosides were not effective. The apparent Kms for prunin and p-nitrophenyl-beta-D-glucopyranoside were determined to be 0.08 and 0.19 mM, respectively. The enzyme was strongly inhibited by p-chloromercuriphenylsulfonic acid and reaction products such as p-nitrophenol and glucose.