Crithidia guilhermei: gelatin- and haemoglobin-degrading extracellular metalloproteinases

The extracellular metalloproteinases of the insect trypanosomatid Crithidia guilhermei were characterized through the incorporation of different protein substrates (gelatin, casein, haemoglobin, and bovine serum albumin) into SDS-PAGE. Two gelatinases (60 and 80 kDa) showed ability to degrade casein as well and a 67-kDa enzyme presented the broadest specificity since it was also able to degrade casein and haemoglobin. Besides the 67-kDa extracellular proteinases detected on haemoglobin-SDS-PAGE, a 43-kDa haemoglobinase was only observed with this substrate. All C. guilhermei proteinases were incapable of using bovine serum albumin. C. guilhermei was also grown in four different culture media and the best proteinase production was reached using yeast extract-peptone medium containing glucose as the major carbon source. The results point to the importance of the use of distinct culture media and proteinaceous substrates on the characterization of extracellular proteolytic activities in trypanosomatids, since alterations in growth conditions and methods of detection could lead to distinct proteolytic profiles.     

Identification and partial characterization of plasma membrane polypeptides of Crithidia guilhermei,crithidia deanei and Crithidia oncopelti

• 1.1. Components of the cell surface of Crithidia guilhermei, Crithidia deanei and Crithidia oncopelti were radioiodinated by the iodogen technique. The distribution of proteins in the detergent-poor (DPP) and detergent-enriched phase (DRP) were studied using a phase separation technique in Triton X-114 and one- and two-dimensional polyacrylamide gel electrophoresis in sodium dodecyl sulphate (1D and 2D SDS-PAGE).
• 2.2. Significant differences were noted in the proteins present in the DRP when the three species were compared.
• 3.3. Two major bands with mol. wt 28,000 and 56,000 and motility in the pH gradient of 7.4 and 6.3, respectively, were observed in C. guilhermei, but not discernible in C. deanei and C. oncopelti.
• 4.4. One polypeptide with mol. wt 50,000 and p1 4.9 was identified in the DRP of C. deanei.
• 5.5. A broad band with mol. wt 68,000–140,000 and pI 4.7–5.5 was clearly observed in the DRP of C. deanei and one or two polypeptides only present in the DPP were observed in the three Crithidia species analyzed.
• 6.6. Our observations show that C. guilhermei has characteristic surface polypeptides not found in C. deanei and C. oncopelti.
• 7.7. Our results, in association with those reported by others, show that the phase separation using Triton X-114 offers a simple approach to the separation and further analysis of a select group of proteins from the bulk of the cellular proteins.

     

Production, purification and characterization of a 50-kDa extracellular metalloprotease from Serratia marcescens

The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation, acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein, bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature optimum of 42° C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 μg/ml) and not inhibited by antipain dihydrochloride (120 μg/ml), aprotinin (4 μg/ml), bestatin (80 μg/ml), chymostatin (50 μg/ml), E-64 (20 μg/ml), leupeptin (4 μg/ml), Pefabloc SC (2000 μg/ml), pepstatin (4 μg/ml), phosphoramidon (660 μg/ml), or phenylmethylsulfonyl fluoride (400 μg/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5% v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma) produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties. Received: 22 November 1996 / Received revision: 27 February 1997 / Accepted: 7 March 1997     

Isolation, purification and partial characterization of a 30-kDa chlorophyll-a/b-binding protein from spinach

A 30-kDa chlorophyll-a/b-binding protein was purified from photosystem II membrane fragments using Ca(2+)-chelating Sepharose 6B chromatography. The protein binds approximately four chlorophyll a molecules, one chlorophyll b molecule and carotenoids. Its 77-K fluorescence-emission spectrum exhibits a maximum at 680 +/- 1 nm. The protein has a high tendency to form a dimer in the presence of Ca2+.Ca2+ binding affects the low-temperature fluorescence-emission maximum, leading to a decrease in its intensity and a blue shift of 1 nm. Similar spectral changes were obtained in the presence of Mg2+, possibly indicating a common binding domain for both cations. We interpret these observations as cation-induced conformational changes of the protein, which were reversible upon subsequent incubation in EDTA. Evidence is presented for the involvement of carboxyl groups in the coordination sphere of the bivalent cations. The possible structural and functional role of the protein is discussed.     

Production of extracellular lipases by Penicillium cyclopium purification and characterization of a partial acylglycerol lipase

Penicillium cyclopium, grown in stationary culture, produces a type I lipase specific for triacylglycerols while, in shaken culture, it produces a type II lipase only active on partial acylglycerols. Lipase II has been purified by ammonium sulfate precipitation and chromatographies on Sephadex G-75 and DEAE-Sephadex. The enzyme exists in several glycosylated forms of 40-43 kDa, which can be converted to a single protein of 37 kDa by enzymatic deglycosylation. Activity of lipase II is maximal at pH 7.0 and 40 degrees C. The enzyme is stable from pH 4.5 to 7.0. Activity is rapidly lost at temperatures above 50 degrees C. The enzyme specifically hydrolyzes monoacylglycerols and diacylglycerols, especially of medium chain fatty acids. The sequence of the 20 first amino acid residues is similar to the N-terminal region of P. camembertii lipase and partially similar to lipases from Humicola lanuginosa and Aspergillus oryzae, but is different from Penicillium cyclopium lipase I. However, it can be observed that residues of valine and serine at positions 2 and 5 in Penicillium cyclopium lipase II are conserved in Penicillium expansum lipase, of which 16 out of the 20 first amino acid residues are similar to Penicillium cyclopium lipase I.     

Polysaccharides of Crithidia fasciculata: Identification and partial characterization of a cell surface constituent

A carbohydrate-containing fraction was extracted from the trypanosomatid Crithidia fasciculata by a phenol-water procedure. Ion-exchange chromatography separated this fraction into three components: a polysaccharide which was not retained on the column; RNA which eluted upon addition of salt; and, another polysaccharide which eluted upon addition of detergent. The unretained fraction was shown to be composed solely of d-mannose. The mannan, which was heterodisperse on Sephadex G-100, had an average molecular weight of approx. 14 000 as based on analysis of reducing groups. The detergent-eluted material yielded arabinose and galactose upon acid hydrolysis. The arabinogalactan was excluded from Sephadex G-100 and Sephacryl S-200 molecular sieve columns, suggesting a molecular weight ≥ 200 000. Cell fractionation studies showed the bulk of extractable polysaccharide was associated with a particulate fraction. Further determination of the cellular localization of the polysaccharide was accomplished by employing a specific antiserum prepared from rabbits immunized with the polysaccharide extract. The cell surface localization of the arabinogalactan was demonstrated by cell agglutination studies as well as immunocytochemical techniques using fluorescein and ferritin conjugated antibodies.     

Human placental sialidase: partial purification and characterization

A sialidase [EC 3.2.1.18] has been partially purified from human placenta by means of procedures comprising Con A-Sepharose adsorption, ammonium sulfate precipitation, sucrose density gradient centrifugation, and high-pressure liquid chromatography on a Shim pack Diol 300 column. On high-pressure liquid chromatography, most of the beta-galactosidase that comigrated with the sialidase on sucrose density gradient centrifugation was removed. The sialidase was purified 3,600-fold from the preparation obtained by Con A-Sepharose adsorption. The enzyme liberated the sialic acid residues from (alpha 2-3) and (alpha 2-6) sialyllactose, colomic acid, fetuin, and transferrin, but not from bovine submaxillary mucin. The enzyme also hydrolyzed gangliosides GM3, GD1a, and GD1b in the presence of sodium cholate as a detergent, but GM1 and GM2 were less susceptible to the enzyme. The optimum pHs for 4-methylumbelliferyl-N-acetylneuraminate, sialyllactose, fetuin, and GM3 lay between 4.0 and 5.0.     

Alcalase rapeseed inhibitors: purification and partial characterization

Extensive rapeseed protein hydrolysate obtained sequentially with Alcalase and Flavourzyme showed inhibitory activity towards Alcalase. Inhibitory activity decreased as the hydrolytic process progressed probably by heat denaturation and/or partial protease degradation. Alcalase rapeseed inhibitors were purified by gel filtration and subsequent ion exchange chromatography. They are composed of peptides of 8.4 and 6.1 kDa linked by interchain disulphide bonds, as observed by reducing SDS-PAGE, with a native molecular weight of 18 kDa. Aminoacid composition of the inhibitors was characterized by the high proportion of methionine (4.2%) and cysteine (4.6%). Alcalase inhibitors were partially resistant to heat treatment; after heating at 70 degrees C for 45 minutes more than 50% of the original inhibitory activity remained in the purified protein but after heating at 90 degrees C for 5 minutes, inhibitory activity decreased very fast to a basal level. The possible relation of these protease inhibitors with the 2S albumin storage proteins is discussed.     

Zebra chorionic gonadotropin: partial purification and characterization

Six samples of pregnant zebra (z) serum from the first and second trimesters of pregnancy were analyzed by RIA and shown to have chorionic gonadotropin levels comparable to that of the mare (0.9-5.3 micrograms/ml); first trimester levels in most cases were higher than second trimester levels. A pool of the sera (10 ml) was fractionated by methods previously employed for the purification of equine (e) and donkey (d) chorionic gonadotropin to achieve a concentration of the zebra chorionic gonadotropin (zCG). A yield of 1.0 mg of glycoprotein was obtained. HPLC analysis of the material indicated the content of zCG to be about 7%. Its molecular size as judged by Ve/Vo values is smaller than eCG, greater than ovine LH, and about the same as equine LH. The zCG was tested in RIAs for LH and eCG, radioreceptor assays (RRA) for LH and FSH, and the rat testis Leydig cell assay for LH. Comparisons were made with equine and donkey chorionic gonadotropin, and equine and zebra LH. The results, preliminary because the preparation is not of high purity, showed that zCG is bioactive as an LH; immunologically similar to eCG, eLH, dCG, and zLH; and competes in RRAs for LH but not FSH receptors. It differs, therefore, from eCG and eLH--which have high levels of intrinsic FSH activity, and is more like dCG, dLH, and zLH--all of which have minimal if any FSH activity.     

Protein glycosylation in Haloferax volcanii: partial characterization of a 98-kDa glycoprotein

Eubacterium ramulus, a flavonoid-degrading anaerobic bacterium from the human gastrointestinal tract, was tested for its ability to transform the isoflavonoids genistein-7-O-glucoside (genistin), genistein and daidzein. Genistein was completely degraded by E. ramulus via 6'-hydroxy-O-desmethylangolensin to 2-(4-hydroxyphenyl)-propionic acid. Dihydrogenistein was neither observed as an intermediate in this transformation nor converted itself by growing cells or cell-free extracts of E. ramulus. Genistein-7-O-glucoside was partially transformed by way of genistein to the product 2-(4-hydroxyphenyl)-propionic acid. Daidzein was in part degraded to O-desmethylangolensin, the corresponding metabolite to 6'-hydroxy-O-desmethylangolensin. The hydroxyl group in position 6' of O-desmethylangolensin is crucial for further degradation.     

Maize leaf adenylate kinase : purification and partial characterization

Adenylate kinase (EC 2.7.4.3) from leaves of maize (Zea mays) was purified to homogeneity using (NH₄)₂SO₄ fractionation, followed by chromatography on DEAE-cellulose, hydroxyapatite, Sephadex G-75SF, and Green A dye-ligand columns. The purified enzyme had specific activity of about 1,550 micromoles ADP produced per minute per milligram protein, and the ratio of velocities of the reverse (utilization of ATP) to forward (formation of ATP) reaction was about 1.5. The M_r value of adenylate kinase, determined by electrophoresis in dissociating conditions and by gel filtration, was 29,000 and 31,000 respectively, suggesting monomeric nature of the enzyme. Purified preparations were stable for at least 1 month at 0 to 4°C. Magnesium ions were essential for activity of adenylate kinase in both directions of the reaction. Optimal rates in the forward direction were observed at the magnesium to ADP ratio of about 0.6 to 0.8. For the reverse reaction, ATP served as a substrate only when complexed with magnesium, while AMP reacted as a free species. The enzyme preferentially utilized adenine ribonucleotides in both directions of the reaction. The nucleoside triphosphate-binding site of adenylate kinase was fairly nonspecific with regard to nucleotide species. On the other hand, the primary amino group of either adenine and cytosine moieties was essential for effective binding to the nucleoside monophosphate site of the enzyme.     

Rat kidney histamine N-methyltransferase: purification and partial characterization

Histamine N-methyltransferase (HMT, EC 2.1.1.8) was purified 8,420-fold in 44% yield from rat kidney. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE cellulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous as determined by gel electrophoresis and was stable for at least five months at -80 degrees C. The apparent molecular weight of the enzyme was found to be 31,500 as determined by gel filtration through Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point of the enzyme was determined to be 5.4. The Km's for histamine and S-adenosyl-L-methionine were 12.4 +/- 1.3 microM and 10.2 +/- 0.5 microM, respectively. When S-adenosyl-L-methionine was the variable substrate, the Ki's for S-adenosyl-L-homocysteine and S-adenosyl-D-homocysteine were 31.9 +/- 3.4 microM and 32.0 +/- 3.5 microM, respectively. When histamine was the variable substrate, the Ki for S-adenosyl-L-homocysteine was 11.8 +/- 0.6 microM. Comparison of physico-chemical and catalytic properties of the rat kidney and the guinea pig enzymes suggest that these proteins have similar structural and catalytic characteristics.     

Tritrichomonas foetus: characterization of isolates and partial purification of a secreted cytotoxin

Putative virulence factors including extracellular proteases, hemagglutinin, hemolysins, and soluble cytotoxins may play significant roles in the pathogenesis of trichomoniasis. The cytotoxicity, hemagglutinating, and hemolytic activity of Tritrichomonas foetus isolate ATCC #30003 and several field isolates were compared. All isolates were hemolytic toward mouse and bovine erythrocytes but not other tested species. The isolates varied significantly in hemagglutinating ability and cytotoxin production. A 40,000 Da soluble cytotoxin was partially purified and characterized. Chromatography separated cytotoxic activity from hemagglutinating and hemolytic activity but not from protease activity. However, protease assays indicated that protease activity was inversely correlated with cytotoxic activity. Characterization studies indicated that cytotoxic activity was destroyed by heat and acidic conditions but repeated freeze/thawing did not diminish activity. Target cell specificity assays showed Henle cells were twice as sensitive to the effects of the cytotoxin as Vero cells. These results suggest that T. foetus isolates vary in the production of virulence factors and produce a soluble relatively stable non-protease cytotoxic protein capable of killing cultured mammalian cells in vitro.     

Purification and partial characterization of a 31-kDa cysteine endopeptidase from germinated barley

Proteolytic enzymes hydrolyze cereal seed storage proteins into small peptides and amino acids, which are very important for seed germination and the malting process. A cysteine-class endopeptidase was purified from 4-d-germinated barley (Hordeum vulgare L. cv. Morex). Four purification steps were used, carboxymethyl cellulose cation-exchange chromatography, chromatofocusing, size-exclusion chromatography, and electroelution from a polyacrylamide gel. The endopeptidase was most active at pH 4.5. It's isoelectric point (pI) was 4.4, as determined by isoelectric focusing, and it's SDS-PAGE molecular size was 31 kDa. The enzyme specifically hydrolyzed peptide bonds when the S₂ site contained relatively large hydrophobic amino acids. The N-terminal amino acid sequence residues (1–9) of the 31-kDa endopeptidase had high homology to those of the EP-A and EP-B cysteine proteinases reported previously. The 31-kDa endopeptidase had a hydrolytic specificity similar to that of the Morex green malt 30-kDa endopeptidase we characterized previously, and also reacted with the antibody raised against the purified 30-kDa proteinase, but the two had different mobilities on non-denaturing PAGE. The hydrolytic specificities of both 30- and 31-kDa endopeptidases are such that both would very quickly cleave hordein (barley storage) proteins to small glutamine- and proline-rich peptides that could be quickly degraded to amino acids by barley exopeptidases.Abbreviations CMC carboxymethyl cellulose - E-64 transepoxysuccinyl-l-leucylamido-(4-guanidino)butane - EMI N-ethylmaleimide - IEF isoelectricfocusing - Phen 1,10-phenanthroline - PI isoelectric point - PMSF phenylmethylsulfonyl fluoride We thank the American Malting Barley Association for partially funding this work. Germinated barley seeds were kindly prepared by Eddie D. Goplin. Special thanks to Laurie Marinac for her excellent technical assistance.     

Penicillium chrysogenum extracellular acid phosphatase: purification and biochemical characterization

An extracellular acid phosphatase (EC 3.1.3.2) from crude culture filtrate of Penicillium chrysogenum was purified to homogeneity using high-performance ion-exchange chromatography and size-exclusion chromatography. SDS-PAGE of the purified enzyme exhibited a single stained band at an Mr of approx. 57,000. The mobility of the native enzyme indicated the Mr to be 50,000, implying that the active form is a monomer. The isoelectric point of the enzyme was estimated to be 6.2 by isoelectric focusing. Like acid phosphatases from several yeasts and fungi the Penicillium enzyme was a glycoprotein. Removal of carbohydrate resulted in a protein band with an Mr of 50,000 as estimated by SDS-PAGE, suggesting that 12% of the mass of the enzyme was carbohydrate. The enzyme was catalytically active at temperatures ranging from 20 degrees C to 65 degrees C with a maximum activity at 60 degrees C and the pH optimum was at 5.5. The Michaelis constant of the enzyme for p-nitrophenyl phosphate was 0.11 mM and it was inhibited competitively by inorganic phosphate (ki = 0.42 mM).     

Isolation and partial characterization of membrane-associated cyclophilin and a related 22-kDa glycoprotein.

The presence of membrane-associated proteins which stereospecifically bind cyclosporin A and react with anti-cyclophilin antibodies has been documented in rat tissues. Extraction of membranes with 6 M urea or 0.5% Chaps releases cyclosporin-binding activity that is 5-12% of that found in cytosol. Cyclosporin-A-binding proteins are present in most subcellular organelles of liver, but microsomes contain the greatest activity. These proteins can be purified by adsorption onto a cyclosporin-A affinity column and elution with cyclosporin A. Two major fractions are resolved on SDS/PAGE: an 18-kDa fraction is comprised of two isoforms that are similar if not identical to the two major cytosolic isoforms of cyclophilin. In addition, in microsomes an approximately equal quantity of a 22-kDa glycoprotein was detected. Based on partial sequencing (five peptides, 89 amino acids) this protein is similar but not identical to human cyclophilin B. This 22-kDa isoform is poorly recognized by affinity-purified anti-cyclophilin antibodies and comprises several predominant isoforms (pI approximately 9.3-9.6). Selective binding of membrane 22-kDa cyclophilin to peanut lectin suggests the oligosaccharides contain a terminal galactosyl-N-galactosamine residue.