Characterization of a Neutral Aminoacyl-Peptide Hydrolase from Naegleria fowleri1

An intracellular alpha-aminoacyl-peptide hydrolase (EC 3.4.11.-) from Naegleria fowteri nN68 (ATCC 30894) has been characterized. The enzyme preparation hydrolyzed phenylalanyl-, tyrosyl-, leucyl-, arginyl-, alanyl-, tryptophanyl-, histidyl-, methionyl-, and lysyl-naphthylamide but not benzoylleucyl-, leucylglycyl-, glycylprolylleucyl-, glycyl-, threonyl-, aspartyl-, or glutamyl-naphthylamide. The aminopeptidase activity was inhibited by the cysteine-protease inhibitors—hydroxymercuribenzoate, chloromercurisulfate, and iodoacetate- by the aminopeptidase inhibitors-bestatin and trans-epoxysuccinyl-leucyl-agmatine- by an inhibitor of soluble alanyl aminopeptidase EC 3.4.11.14, puromycin, and by the metalloprotease inhibitor, o-phenanthroline. The exopeptidase activity was not inhibited by the chelator, ethylenediaminetetraacetate, or the serine-protease inhibitor, phenylmethylsulfonylfluoride. The pH optimum of the exopeptidase was between 7.0 and 8.0. Enzyme activity was stable at 55°C for 30 min, but all activity was lost after 15 min at 80°C. Enzyme activity was inhibited by 100 μM HgCI₂ and CdCl₂ but not by 1 mM CoCl₂, CuCl₂, MnCl₂, NiCl₂, FeCl₂, or ZnCl₂. Enzyme activity was inhibited by 0.1% sodium dodecyl sulfate but not by 0.2% Brij 35, Tween 20, Tween 80, or Triton X-100.     

Extraction and biochemical characterization of a nuclear deoxyribonucleic acid polymerase activity in bull spermatozoa.

Bull spermatozoa heads were separated from cytoplasmic contaminants, especially mitochondria-rich middle pieces, by centrifugation through 2.4M-sucrose. DNA polymerase activity was demonstrated by incubating nuclear heads for 1 h at 37 degrees C or for 20 h at room temperature in a medium containing detergent and dithiothreitol or 2-mercaptoethanol. Optimal DNA polymerase activity was detected after extraction in a medium containing 50 mM-borate, pH9, 1 mg of soya-bean trypsin inhibitor/ml and supplemented with either 20 mM-dithiothreitol and 4% Tween 80 or 100mM-2-mercaptoethanol and 10% Tween 80. The DNA polymerase reaction was Mg2+-dependent; Mn2+ or Ca2+ could not replace Mg2+ and all four deoxynucleoside triphosphates were required for optimal activity. The polymerase activity was pH-dependent (optimum between 8.2 and 10.5) and was a function of buffer composition and also of pH values. Optimal activity was obtained with 50 mM-Na+ or 150mM-K+ and was partially lowered by N-ethylmaleimide; it was inhibited by spermidine and by salmon protamines, but was greatly stimulated by calf thymus histones. It was also resistant to actinomycin D, netropsin and ethidium bromide. The present results suggest that bull spermatozoa heads contain a beta-type DNA polymerase activity.     

Isolation and characterization of an intracellular aminopeptidase from the extreme thermophilic archaebacterium Sulfolobus solfataricus

An intracellular aminopeptidase (EC 3.4.11.-) was purified from the extreme thermophilic archaebacterium, Sulfolobus solfataricus. The molecular weight of the native enzyme was about 320,000, as calculated by gel-filtration studies, and a subunit Mr of 80,000 was estimated by SDS-polyacrylamide gel electrophoresis. The temperature optimum of the enzyme was at 75 degrees C and the pH optimum was found to be 6.5. The aminopeptidase was highly active against the chromogenic substrates L-Leu-p-NA and L-Ala-p-NA. The enzyme was inhibited by EDTA, but the activity could be partially restored by removal of the EDTA and incubation with Co2+ or Mn2+. Bestatin, a typical inhibitor of aminopeptidase, fully inhibited the enzyme activity, but inhibitors of serine proteinases had no effect. Beside a high thermostability, the enzyme showed a remarkable stability against 6 M urea, organic solvents and acetonitrile.     

Characterization of two uterine proteases and their actions on the estrogen receptor

We have characterized two previously undetected proteases from the calf uterine cytosol and measured their actions on the estrogen receptor. One is an exopeptidase, purified 60-fold, that hydrolyzed amino acid (lysine-, and alanine-, or leucine-) p-nitroanilide substrates and leucylglycylglycine, did not hydrolyze [14C]methemoglobin, was completely inhibited by 1 mM bestatin or puromycin (specific inhibitors of leucine aminopeptidase like enzymes), and was unable to influence the sedimentation of the 8S form of the estrogen receptor in sucrose gradients containing dilute Tris buffer. A commercial porcine leucine aminopeptidase, like the calf uterine aminopeptidase, did not convert the 8S estrogen receptor to a 4S form. Evidently, removal of the N-terminal amino acid(s) from the estrogen receptor by exopeptidase action cannot alter the sedimentation of the 8S form of the receptor, or the N-terminal amino acid(s) of the receptor is (are) unaccessible or resistant to exopeptidase activity. The second, a receptor-active protease, is an endopeptidase that did not hydrolyze any of the synthetic amide or peptide substrates tested but did possess [14C]methemoglobin-degrading activity and the ability to convert the 8S estrogen receptor to a modified 4S form in sucrose gradients containing dilute Tris buffer. The modified 4S receptor was separable from the native receptor by DEAE-cellulose chromatography. The endopeptidase did not require Ca2+ for activity, and its chromatographic properties were distinctly different from a previously isolated Ca2+-activated protease. It was inhibited by leupeptin or dipyridyl disulfide, suggesting the presence of a thiol group that is essential for its activity. These data indicate that a decrease in the sedimentation rate of the estrogen receptor in sucrose gradients with low salt or a change in the receptor's elution on DEAE-cellulose chromatography is not related to receptor activation but is produced by the receptor-active protease or other proteases.     

Classification of sheep abomasal mucosal mast cell proteinase as a serine endopeptidase (EC 3.4.21)

1. Diisopropylphosphofluoridate (Dip-F) and phenylmethanesulphonylfluoride (Pms-F) are inhibitors of "serine" proteinases, and L-trans-epoxysuccinylleucylamido-(4-guanido)-butane (E-64) is an inhibitor of "thiol" proteinases. The effects of these inhibitors on sheep mast cell proteinase (SMCP) were examined. 2. Enzyme activity was completely inhibited by 5 mM Dip-F following a 4-hr preincubation period at either 4 degrees C or 30 degrees C but was unusually resistant to the action of 1 mM Dip-F. 3. SMCP activity was inhibited by 1 mM Pms-F at both 4 degrees C and 30 degrees C. Inhibition was reversed by dithiothreitol (DTT), but this effect was virtually eliminated following preincubation with Pms-F at 30 degrees C for 12 hr. 4. SMCP activity was unaffected by E-64. 5. These properties are consistent with the classification of SMCP as a "serine" endopeptidase (EC 3.4.21).     

A novel alkaline protease from wild edible mushroom Termitomyces albuminosus

A protease with a molecular mass of 30 kDa and the N-terminal sequence of GLQTNAPWGLARSS, was isolated from fresh fruiting bodies of the wild edible mushroom Termitomyces albuminosus. The purification protocol included ion exchange chromatography on DEAE-cellulose, Q-Sepharose, SP-Sepharose and FPLC-gel filtration on Superdex 75. The protein was unadsorbed on DEAE-cellulose and Q-Sepharose, but adsorbed on SP-Sepharose. The optimal pH and temperature of the purified enzyme were 10.6 and 60 °C, respectively. The enzyme was stable in the presence of 2 % (v/v) Tween 80 and 4 M urea. More than 80 % of the enzyme activity was retained in 2 % (v/v) Triton X 100, 54 % in 10 mM EDTA and 31 % in 2 % (w/v) SDS. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), but not inhibited by dithiothreitol (DTT), pepstatin or lima bean trypsin inhibitor suggesting that it was a serine protease but not a trypsin-like one. The protease was inhibited by Hg(2+), Cu(2+), and Fe(3+) ions. The K(m) and V(max) values of the purified enzyme for casein were 8.26 mg ? ml(-1) and 0.668 mg ? ml(-1) ? min(-1), respectively.     

Purification and characterization of phosphoenolpyruvate carboxylase from a cyanobacterium.

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified 100-fold from the cyanobacterium Coccochloris peniocystis with a yield of 10%. A single isozyme was found at all stages of purification, and activity of other beta-carboxylase enzymes was not detected. The apparent molecular weight of the native enzyme was 560,000. Optimal activity was observed at pH 8.0 and 40 degrees C, yielding a Vmax of 8.84 mumol/mg of protein per min. The enzyme was not protected from heat inactivation by aspartate, malate, or oxalacetate. Michaelis-Menten reaction kinetics were observed for various concentrations of PEP, Mg2+, and HCO3-, yielding Km values of 0.6, 0.27, and 0.8 mM, respectively. Enzyme activity was inhibited by aspartate and tricarboxylic acid cycle intermediates and noncompetitively inhibited by oxalacetate, while activation by any compound was not observed. However, the enzyme was sensitive to metabolic control at subsaturating substrate concentrations at neutral pH. These data indicate that cyanobacterial PEP carboxylase resembles the enzyme isolated from C3 plants (plants which initially incorporate CO2 into C3 sugars) and suggest that PEP carboxylase functions anapleurotically in cyanobacteria.     

Inhibition of glucose 6-phosphatase by pure and impure C-type phospholipases. Reactivation by phospholipid dispersions and protection by serum albumin.

1. Pure or impure C-type phospholipases hydrolysed rat liver microsomal phosphatides in situ at 5 degrees or 37 degrees C. At 5 degrees C mean hydrolysis of total phospholipids was 90% by Bacillus cereus and 75% by Clostridium perfringens (Clostridium welchii) C-type phospholipases. 2. Four degrees of inhibition of glucose 6-phosphatase (D-glucose 6-phosphate phosphohydrolase; EC 3.1.3.9) resulted. (a) At 37 degrees C inhibition was virtually complete and apparently irreversible. (b) At 5 degrees C phospholipase C inhibited 50-87% of the activity expressed by intact control microsomal fractions. (c) Bovine serum albumin present during delipidation alleviated most of this inhibition: at 5 degrees C phospholipase C plus bovine serum albumin inhibited by 0-35% (mean 18%):simultaneous stimulation by the destruction of its latency seems to offset glucose 6-phosphatase inhibition, sometimes completely. (d) If latency was first destroyed, phospholipase C plus bovine serum albumin inhibited 30-50% of total glucose 6-phosphatase activity at 5 degrees C. Only this inhibition is likely largely to reflect the lower availability of phospholipids, essential for maximal enzyme activity, as it is virtually completely reversed by added phospholipid dispersions. Co-dispersions of phosphatidylserine plus phosphatidylcholine (1:1, w/w) were especially effective but Triton X-100 was unable effectively to restore activity. 3. Considerable glucose 6-phosphatase activity survived 240min of treatment with phospholipase C at 5 degrees C, but in the absence of substrate or at physiological glucose 6-phosphate concentrations the delipidated enzyme was completely inactivated within 10min at 37 degrees C. However, 80mM-glucose 6-phosphate stabilized it and phospholipid dispersions substantially restored thermal stability. 4. It is concluded that glucose 6-phosphatase is at least partly phospholipid-dependent, and complete dependence is not excluded. For reasons discussed it is impossible yet to be certain which phospholipid class(es) the enzyme requires for activity.     

Effect of thermal and chemical denaturants on Thermoanaerobacter ethanolicus secondary-alcohol dehydrogenase stability and activity

Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (2 degrees ADH) was optimally active near 90 degrees C displaying thermostability half-lives of 1.2 days, 1.7 h, 19 min, 9.0 min, and 1.3 min at 80 degrees C, 90 degrees C, 92 degrees C, 95 degrees C, and 99 degrees C, respectively. Enzyme activity loss upon heating (90-100 degrees C) was accompanied by precipitation, but the soluble enzyme remaining after partial inactivation retained complete activity. Enzyme thermoinactivation was modeled by a pseudo-first order rate equation suggesting that the rate determining step was unimolecular with respect to protein and thermoinactivation preceded aggregation. The apparent 2 degrees ADH melting temperature (T(m)) occurred at approximately 115 degrees C, 20 degrees C higher than the temperature for maximal activity, suggesting that it is completely folded in its active temperature range. Thermodynamic calculations indicated that the active folded structure of the 2 degrees ADH is stabilized by a relatively small Gibbs energy (triangle upG(stab.)(double dagger) = 110 kJ mol(-1)). 2 degrees ADH catalytic activities at 37 degrees C to 75 degrees C, were 2-fold enhanced by guanidine hydrochloride (GuHCl) concentrations between 120 mM and 190 mM. These results demonstrate the extreme resistance of this thermophilic 2 degrees ADH to thermal or chemical denaturation; and suggest increased temperature or GuHCl levels seem to enhance protein fixability and activity.     

Lung contains an inhibitor for nicotinatemononucleotide pyrophosphorylase (carboxylating) of NAD biosynthesis

Rat, cow and foal lung extracts contained an inhibitor for the liver NAD biosynthetic-pathway enzyme, nicotinatemononucleotide pyrophosphorylase (carboxylating) [EC 2.4.2.19]. The inhibitor was not dialyzable, was labile at 100 degrees C, was retained by a 30,000 dalton pore size Amicon membrane and, when partially purified by precipitation at 40-100% ammonium sulfate, inhibited the enzyme stoichiometrically. Lung reportedly does not contain nicotinate-mononucleotide pyrophosphorylase or make NAD de novo. However, the inhibitor would mask detection of the enzyme in lung extracts. We detected a low nicotinatemononucleotide pyrophosphorylase-like activity (0.003 +/- 0.001 nanomoles CO2 produced from quinolinic acid per mg of extract protein) in rat lung but none in foal or cow lung.     

Enzyme activities of cell-free extracts from mutant strains of lactic streptococci subjected to sublethal heating or freeze-thawing

Two mutant lactose-negative (Lac-), proteinase-negative (Prt-) strains of lactic streptococci, Streptococcus lactis 25Sp and S. cremoris KHA2, and their parents, S. lactis C2 and S. cremoris KH Lac+ Prt+, were grown in a suitable medium with the pH maintained at 6.5 by addition of NH4OH. Cells were harvested by centrifugation, resuspended, and then heated sublethally at 54 or 69 degrees C for 15 sec. Cells also were frozen and stored for 1 week at -20 or -100 degrees C. Cell-free extracts of cells heated at 54 degrees C had more proteinase and aminopeptidase activities than did a similar extract of cells heated at 69 degrees C. The greatest enzyme activities occurred in the cell-free extracts prepared from cells frozen and stored at -100 degrees C. Specific activities of proteinase and dipeptidase generally decreased in extracts of freeze-shocked cells compared to those in extracts of untreated cells. Enzyme activity of extracts also decreased in the presence of 5% NaCl at pH 5.0. Cell-free extracts at pH values of 5 to 8 were heated at 69 degrees C for 1.5, or 10 min. Heating them for 10 min caused a loss of dipeptidase activity which was most pronounced at pH 5.0 and least pronounced at pH 7.0.     

Production, purification, and characterization of a novel thermostable serine protease from soil isolate, Streptomyces tendae

An isolate of Streptomyces tendae produced a extracellular protease which was purified to apparent homogeneity giving a single band on SDS-PAGE with a molecular mass of 21 kDa. Optimum activity was at 70 degrees C and pH 6. It was stable at 55 degrees C for 30 min and between pH 4 and 9. It was resistant to neutral detergents and organic solvents such as Triton X-100, Tween 80, methanol, ethanol, acetone, and 2-propanol at 5% (v/v). The enzyme was completely inhibited by 5 mM PMSF, indicating it to be a serine protease. N-terminal amino acid sequence did not show any homology with other known proteolytic enzymes. The protease may therefore be a novel neutral serine protease, which is stable at high temperature and over a broad range of pH.     

Isolation and fundamental characteristics of a phospholipase B inhibitor from autolyzed Torulaspora delbrueckii.

Phospholipase B inhibitor was found in the autolyzate of yeast cells, Torulaspora delbrueckii. The inhibitor was purified to homogeneity by ethanol precipitation, gel filtration with Sephadex G-10, ion-exchange chromatography with DEAE-Sephacel, and gel filtration with Asahipak GS-320. On thin-layer chromatography the purified inhibitor was detected with the Hanes-Isherwood reagent, which is used to detect phosphorus. The activity of the inhibitor was not affected by heat treatment at 100 degrees C for 1 h. Heating at 100 degrees C for 1 h in 1 M HCl and 1 M NaOH lowered activity to 76 and 80% of the original values, respectively, but heating at 110 degrees C for 24 h in 6 M HCl completely abolished activity. The inhibitor was highly soluble in water, but practically insoluble in alcohol, acetone, ether, and chloroform. The degree of inhibition of enzyme activity was not proportional to the concentration of inhibitor. The inhibitor inhibited both membrane-bound and water-soluble phospholipase B activity from T. delbrueckii at the same level; however, the inhibitor did not inhibit the activity of phospholipase A2 from snake venom (Naja naja).     

Intracellular hydrolases of Aspergillus parasiticus and Aspergillus flavus

When the distribution profile of hydrolases in mycelial homogenates and culture filtrates of A. parasiticus and A. flavus was examined, six hydrolytic enzymes viz. N-acetyl-beta-glucosaminidase, aryl sulfatase, alkaline proteinase, cathepsin B, cathepsin D and aminopeptidase were detected in homogenate. The culture filtrates were devoid of any activity of these enzymes. The enzyme levels varied with the stage of incubation. The most abundant fungal exopeptidase showing preference for basic amino acid naphthylamides seems to be an aminopeptidase B. Incorporation of CEPA, an ethylene generating compound, stimulated the amino peptidase activity in the mycelium but inhibited the enzyme in vitro. The enzyme was also inhibited by different aflatoxins to varying degree. While aminopeptidase B was located intracellularly, a non-dialysable, heat-stable inhibitor of the enzyme was found to be secreted in the culture filtrate. This peptide inhibitor was however ineffective on the other enzymes.     

Characterization of proteolipid protein fatty acylesterase from rat brain myelin.

A protein fatty acylesterase activity that catalyzes the removal of fatty acid from exogenous proteolipid protein (PLP) has been demonstrated in isolated rat brain myelin. Optimum enzyme activity for the deacylation of PLP was obtained in 0.5% Triton X-100, 1 mM dithiothreitol at pH 7.0 and at 37 degrees C. Other detergents (octyl beta-D-glucoside, Nonidet P-40, and Tween 20) have little or no effect, whereas deacylation was completely abolished by 0.1% sodium dodecyl sulfate or boiling the membrane fraction for 5 min prior to incubation. Under optimal conditions, the rate of deacylation was linear up to 20 min, and the apparent Km for bovine [3H]palmitoyl-PLP was 18 microM. The myelin-associated PLP fatty acylesterase has no apparent requirements for divalent cations (Ca2+, Mg2+, Mn2+), and chelators such as EDTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid, and 1,10-phenantroline have little or no effect on enzyme activity. Sulfhydryl and histidine residues are needed for full enzyme activity, whereas the "active serine"-directed inhibitor phenylmethylsulfonyl fluoride has no effect. The myelin-associated protein fatty acylesterase was present throughout brain development and in all myelin subfractions, in agreement with the dynamic metabolism of PLP-bound fatty acids. Enzyme activity was also present in sciatic nerve, brain cortex, and heart whereas liver was devoid of activity. Several esterases, including phospholipase A2, glyoxalase II, and acetylcholinesterase, did not remove fatty acid from PLP. Myelin basic protein, palmitoyl-CoA hydrolase, and myelin-associated nonspecific esterase were also ruled out as the PLP fatty acylesterase. Thus, all data seem to indicate that this enzyme is different from esterases of the lipid metabolism. Finally, stimulation of protein phosphorylation with Ca2+, but not with cyclic-AMP, inhibited PLP deacylation, suggesting that the myelin-associated protein fatty acylesterase activity is regulated by endogenous Ca(2+)-dependent protein kinases.     

A novel thermostable lipase from a thermophilic Bacillus sp.: characterization and esterification studies

An extracellular, thermostable, alkaline lipase was partially purified from a thermophilic Bacillus strain J 33. It was optimally active at pH 8.0 at 60°C, retaining 50% activity at 70°C for 30 min. It had native molecular mass of 45 kDa. The lipase was stable in 90% (v/v) hexane or benzene mixtures in water. It converted 66% oleic acid at 0.25 M with 0.4 M methanol in hexane to methyl oleate at 60°C in 16 h. Activity was stimulated by Mg2 (10 mM) but inhibited by EDTA (10 mM) and PMSF (10 mM). It was stable in Triton X-100, Tween 20 and Tween 80 (0.1% v/v). © Rapid Science Ltd. 1998     

High-level heterologous expression and properties of a novel lipase from Ralstonia sp. M1

The mature lipase LipA and its 56aa-truncated chaperone DeltaLipBhis (with 6xhis-tag) from Ralstonia sp. M1 were over-expressed in Escherichia coli BL21 under the control of T7 promoter with a high level of 70 and 12mg protein per gram of wet cells, respectively. The simply purified lipase LipA was effectively refolded by Ni-NTA purified chaperone DeltaLipBhis in molar ratio 1:1 at 4 degrees C for 24 hours in H2O. The in vitro refolded lipase LipA had an optimal activity in the temperature range of 50-55 degrees C and was stable up to 45 degrees C with more than 84% activity retention. The maximal activity was observed at pH 10.75 for hydrolysis of olive oil and found to be stable over alkaline pH range 8.0-10.5 with more than 52% activity retention. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine (remaining activity 137-334%), but inhibited by 1-butanol and acetonitrile (40-86%). Metal ions Cu2+, Sn2+, Mn2+, Mg2+, and Ca2+ stimulated the lipase slightly with increase in activity by up to 22%, whereas Zn2+ significantly inhibited the enzyme with the residual activity of 30-65% and Fe3+ to a lesser degree (activity retention of 77-86%). Tween 80, Tween 60, and Tween 40 induced the activation of the lipase LipA (222-330%) and 0.2-1% (w/v) of Triton X-100, X-45, and SDS increased the lipase activity by up to 52%. However, 5% (w/v) of Triton X-100, X-45, and SDS inhibited strongly the activity by 31-89%. The inhibitors including DEPC, EDTA, PMSF, and 2-mercaptoethanol (0.1-10mM) inhibited moderately the lipase with remaining activity of 57-105%. The lipase LipA hydrolyzed a wide range of triglycerides, but preferentially short length acyl chains (C4 and C6). In contrast to the triglycerides, medium length acyl chains (C8 and C14) of p-nitrophenyl (p-NP) esters were preferential substrates of this lipase. The enzyme preferentially catalyzed the hydrolysis of cottonseed oil (317%), cornoil (227%), palm oil (222%), and wheatgerm oil (210%) in comparison to olive oil (100%).     

In vivo Responses of Honey Bee Midgut Proteases to Two Protease Inhibitors from Potato

Potato protease inhibitors, POT-1 and POT-2, were fed to newly emerged adult honey bees in cages at different doses in either sugar syrup (0.2 or 0.01% w:v) or pollen food (1 or 0.2% w:w). In vivo activities of three digestive endopeptidases (trypsin, chymotrypsin and elastase) and one exopeptidase (leucine aminopeptidase; LAP) were measured after 3 or 8days' exposure of bees to inhibitor. Enzyme activities were significantly lower at day 8 than at day 3, except for elastase, which did not change. POT-2 significantly reduced the activity of all endopeptidases at both timepoints, regardless of the dose level or the medium in which the inhibitor was administered. POT-1 acted in a similar manner, except that 0.01% POT-1 in syrup had no effect on bees. There was no consistent trend in changes in LAP activity. Bees fed either inhibitor at 1% in pollen or at 0.2% in syrup had significantly reduced lifespans, with the effect of the pollen treatment being greater than the syrup treatment. The survival of bees fed POT-1 or POT-2 at 0.2% in pollen or 0.01% in syrup did not differ from the controls.     

Protease activity of 80 kDa protein secreted from the apicomplexan parasite Toxoplasma gondii

This study describes the characterization of 80 kDa protease showing gelationlytic property among three proteases in the excretory/secretory proteins (ESP) from Toxoplasma gondii. The protease activity was detected in the ESP but not in the somatic extract of RH tachyzoites. This protease was active only in the presence of calcium ion but not other divalent cationic ions such as Cu(2+), Zn(2+), Mg(2+), and Mn(2+), implying that Ca(2+) is critical factor for the activation of the protease. The 80 kDa protease was optimally active at pH 7.5. Its gelatinolytic activity was maximal at 37 degrees C, and significant level of enzyme activity of the protease remained after heat treatment at 56 degrees C for 30 min or 100 degrees C for 10 min. This thermostable enzyme was strongly inhibited by metal chelators, i.e., EDTA, EGTA, and 1,10- phenanthroline. Thus, the 80 kDa protease in the ESP secreted by T. gondii was classified as a calcium dependent neutral metalloprotease.     

Deoxyribonucleic acid synthesis in isolated nuclei from baby-hamster kidney cells (BHK-21/C13). Characterization of the system

A comparative study of some commonly employed laboratory procedures for studying DNA synthesis in isolated nuclei was carried out. Nuclei isolated from baby-hamster kidney (BHK-21/C13) cells synthesize DNA for 30-60min at 37 degrees C in a reaction requiring uni- and bi-valent cations, ATP and all four deoxyribonucleoside 5'-triphosphates. The addition of either ribonucleotides or cytosol from S-phase cells had no effect, but DNA synthesis was stimulated by some dextrans (mol.wt. 5x10(6)). The extent of synthesis was influenced by apparently minor variations in experimental conditions. For example, DNA synthesis by nuclei in Tris/HCl, pH7.5, was only 50% of that observed in Hepes/NaOH, pH7.5; the presence of detergents Triton X-100, Triton N-101, Nonidet P-40, Brij 58 and Tween 80 in the incubation medium altered the amount of synthesis to different extents. Although most detergents inhibited synthesis, a stimulation occurred with Tween 80 (150% of controls). These effects were reversed on washing the nuclei, except that of Brij 58, which inhibited DNA synthesis by 90-95% irreversibly. Anomalous sucrose-density-gradient sedimentation behaviour of the DNA, and of precursor [(3)H]-dTTP, was observed when nuclei were lysed with solutions of sodium dodecyl sulphate/Mg(2+) or with Sarkosyl/Mg(2+), but consistent results, showing that the DNA synthesized in vitro sedimented exclusively at about 4S, were obtained when nuclei were lysed with sodium dodecyl sulphate (without Mg(2+))/EDTA, digested with proteinase K and heated at 100 degrees C with 11% (v/v) formaldehyde to prevent macromolecular association. These results, coupled with density-labelling studies with bromodeoxyuridine and CsCl-density-gradient analysis, showed that DNA synthesis in these nuclei was replicative and was restricted to a covalent extension of Okazaki pieces previously initiated in vivo. No new initiations were observed, and the DNA was not ligated into larger molecules. The cessation of DNA synthesis after about 60 min was due to the complete utilization of available primer/template DNA.