Phosphodiesterase in Microsomes from Bovine Milk

Phosphodiesterase was solubilized from bovine milk microsomes and partially purified. The purified enzyme showed 20-fold specific activity compared with that of microsomes, and 1,500-fold with that of the original milk.

The properties of the enzyme were investigated by using NpT. The pH optimum was at 9.5. The enzyme was inhibited with EDT A and reactivated with the addition of magnesium or calcium ions. This enzyme was strongly inhibited with reducing reagents. K_m, value was 7.4 x 10^-4 M for NpT at pH 9.5.

RNA was hydrolyzed completely to 5′-mononucleotides, and this enzyme may be considered to show the exonucleolytic action for RNA.     

Affinity chromatographic purification of beta-glucosidase of Candida gulliermondii.

A beta-glucosidase was isolated from Candida guilliermondii, a yeast capable of growth on cellobiose. The enzyme was partially purified by treatment with polyethyleneimine and ammonium sulfate precipitation. Further purification was achieved by affinity chromatography using a Sepharose 4B matrix to which oxidized salicin was coupled through adipic dihydrazide. The final product was a 12.5-fold purification of the crude extract with a recovery of 27% of the initial enzyme activity. Polyacrylamide disc electrophoresis of the purified enzyme gave a single band. A km of 1.25 x 10(-4)M was obtained using p-nitrophenyl beta-D-glucopyranoside as the substrate. The optimum pH for enzyme activity was 6.8. Maximum activity was observed at temperature of 37 degrees C. Enzyme activity was completely inhibited by Hg++, Pb++, and Zn++ ions. The molecular weight of the enzyme is 48,000 as estimated by sucrose density gradient centrifugation.     

Peroxidase-mediated toxicity to schistosomula of Schistosoma mansoni

Guinea pig eosinophil peroxidase (EPO) was capable of killing schistosomula of Schistosoma mansoni in vitro when combined with hydrogen peroxide and a halide. Killing was measured by 51Cr release, by microscopic evaluation of viability, and by reinfection experiments in mice. Parasite killing was dependent on each component of the EPO-H2O2-halide system, was completely inhibited by catalase and azide, and was partially inhibited by cyanide. The EPO-mediated system required 10(-4) M H2O2 and 10(-4) M iodide at pH 7.0, and the schistosomula were killed with exposure to this system of less than 30 min at 37 degrees C. At pH 6.0, the EPO-mediated system showed significant cidal activity with 10(-6) M iodide. Canine neutrophil peroxidase (myeloperoxidase [MPO]) was also able to kill schistosomula in vitro in the presence of 10(-4) M H2O2 and 10(-4) iodide at pH 7.0 and pH 6.0. Physiologic concentrations of chloride (0.1 M) could substitute for iodide at pH 7.0 and pH 6.0 as the halide cofactor; however, at pH 7.0, a higher concentration of enzyme was required. These findings with isolated enzyme systems are compatible with a role for peroxidase in the host defense against schistosomula.     

Purfication and properties of a specific isoflavone 7-O-glucoside-6'-malonate malonyestrase from roots of chickpea (Cicer arietinum L.)

Protein extracts from roots of chickpea (Cicer arietinum L.) plants contained high esterase activity hydrolyzing malonate hemiesters of isoflavone 7-O-glucosides. Using 5,7-dihydroxy-4'-methoxyisoflavone (biochanin A) 7-O-glucoside-6"-malonate as a substrate, a specific malonylesterase was purified about 700-fold to near homogeneity. The purified enzyme possesses an extremely low enzyme activity with synthetic esterase substrates. Various putative nonspecific esterases, as tested with alpha-naphthylacetate, were removed during enzyme purification. The malonylesterase demonstrated a very high molecular mass in gel chromatography and in sedimentation analyses with sucrose gradients (greater than or equal to 2 X 10(6)). Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis pointed to a single subunit of 32,000. The catalyzed reaction showed a pH optimum at 7.5 and a temperature optimum between 30 and 35 degrees C. The apparent Km for biochanin A 7-O-glucoside-6"-malonate was (4.2 +/- 1.2) X 10(-4) M. The malonylesterase was insensitive to the esterase inhibitors eserine and neostigmine (10(-3) M) as well as phenylmethylsulfonyl fluoride, paraoxon, and diisopropylfluorophosphate (10(-4) M). On the other hand enzyme activity was totally inhibited by Hg2+ ions (10(-5) M) and p-hydroxymercuribenzoate (10(-4) M), whereas iodoacetamide (10(-6)-10(-4) M) inhibited only partially. Di- and tricarboxylic acids strongly stimulated enzyme activity at 10(-2) M. These properties indicate that the malonylesterase from chickpea roots greatly differs from other known esterases. The possible biological function of the specific malonylesterase is discussed in relation to isoflavone conjugate metabolism in chickpea.     

A novel fibrinolytic enzyme from <Emphasis Type="Italic">Cordyceps militaris</Emphasis>, a Chinese traditional medicinal mushroom

A novel fibrinolytic enzyme from Cordyceps militaris was purified and partially characterized for the first time, which was designated C. militaris fibrinolytic enzyme (CMase). This extracellular enzyme from C. militaris was isolated by ammonium sulphate fraction, and purified to electrophoretic homogeneity using gel filtration chromatography. The apparent molecular mass of the purified enzyme was estimated to be 27.3 kDa by SDS-PAGE. The optimum pH and temperature for the enzyme activity were pH 6.0 and 25 °C, respectively. In the presence of metal ions such as Mg²⁺ and Fe²⁺ ions the activity of the enzyme increased, whereas EDTA and Cu²⁺ ion inhibited the enzyme activity. Interestingly the N-terminal amino acid sequences of the enzyme is extremely similar to those of the trypsin proteinases from insects, and has no significant homology with those of the fibrinolytic enzyme from other medicinal mushroom. In conclusion, C. militaris produces a strong fibrinolytic enzyme CMase and may be considered as a new source for thrombolytic agents.     

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

L-asparaginase activity in Aeromonas sp. isolated from freshwater mussel

Aeromonas sp. from Lamellidens marginalis produced L-asparaginase when grown at 37 degrees C. The optimum enzyme activity was at pH 9 when temperature was 45 degrees C. Half-life of partially purified enzyme at 50 degrees C and 55 degrees C was 35 and 20 min, respectively. Activation and deactivation energies of partially purified enzyme were 17.48 and 24.86 kcal mol-1 respectively. The enzyme exhibited a Km (L-asparagine) value of 4.9 x 10(-6) mol l-1 and a Vmax of 9.803 IU ml-1. Three metal ions inhibited the enzyme activity at 10-20 mumol l-1 concentrations. Catalytic activity was also inhibited by EDTA, iodoacetic acid, parachloromercuribenzoic acid and phenylmethylsulphonyl fluoride at 0.1 mumol l-1.     

Purification and properties of phospholipase A2 from the venom of scorpion, (Heterometrus fulvipes)

Phospholipase A2 was purified about 78 fold from the venom of scorpion Heterometrus fulvipes. The molecular weight of the enzyme was found to be 16,000 and it was optimally active at pH 7.4 and at 50 degrees C. The Km value of the enzyme was 1.8 x 10(-3) M. Calcium, Magnesium and Zinc ions stimulated whereas Mercury ion and EDTA inhibited the enzyme activity. This enzyme exhibited fluorescence emission maximum between 310-320 nm.     

Carbonic anhydrase of blue-green alga Spirulina platensis]

Carboanhydrase (carbonate-hydroliase EC 4.2.1.1.) is found in the extract of Spirulina platensis cells. A linear dependency of the enzyme activity on the protein concentration; pH optimum is found to be 8.0. Specific activity of carboanhydrase is 3 muM/min-mg of protein under the concentration of CO2 of 4-10(-3) M, appearing Michelis constant being 4.9-10(-3) M. The enzyme was stabilized with 10 mM of cisteine, its activity was inhibited by 50% with sulphanylamide (1-10(-5) M), acetazolamide (8--10(-7) M) and Cl- ions (5-10(-2) M). The activity of carboanhydrase, as well as the rate of NaH14CO3 fixation, depended on the pH value of cultural medium.     

Development and properties of fructose 1,6-bisphosphatase in the endosperm of castor-bean seedlings.

1. The activity of fructose 1,6-bisphosphatase (EC 3.1.3.11) in the fatty endosperm of castor bean (Ricinus communis) increases 25-fold during germination and then declines. The developmental pattern follows that of catalase, a marker enzyme for gluconeogenesis in this tissue. 2. The enzyme at its peak of development was partially purified, and its properties were studied. It has an optimal activity at neutral pH (7.0-8.0). The apparent Km value for fructose 1,6-bisphosphate is 3.8 X 10(-5) M. The activity is inhibited by AMP allosterically with an apparent Ki value of 2.2 X 10(-4) M. The enzyme hydrolyses fructose 1,6-bisphosphate and not ribulose 1,5-bisphosphate or sedoehptulose 1,7-bisphosphate. 3. Treatment of the partially purified enzyme with acid leads to an 80% decrease in activity. The remaining activity is insensitive to AMP and has optimal activity at pH 6.7 and a high apparent Km value (2.5 X 10(-4) M) for fructose 1.6-bisphosphate. Enzyme extracted from the tissue with water instead of buffer has a similar modification. The effect of acid explains the discrepancies between this report and previous ones on the properties of the enzyme in this tissue. 4. The storage tissues of various fatty seedlings all contain a 'neutral' fructose 1,6-bisphosphatase. The activities of the enzyme from some of the tissues are inhibited by AMP. 5. The properties of the enzyme in fatty seedlings and in green leaves are discussed in comparison with that in animal tissues.     

Further Characterization of a Myelin-Associated Neuraminidase: Properties and Substrate Specificity

A neuraminidase activity in myelin isolated from adult rat brains was examined. The enzyme activity in myelin was first compared with that in microsomes using N-acetylneuramin(alpha 2----3)lactitol (NL) as a substrate. In contrast to the microsomal neuraminidase which exhibited a sharp pH dependency for its activity, the myelin enzyme gave a very shallow pH activity curve over a range between 3.6 and 5.9. The myelin enzyme was more stable to heat denaturation (65 degrees C) than the microsomal enzyme. Inhibition studies with a competitive inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, showed the Ki value for the myelin neuraminidase to be about one-fifth of that for the microsomal enzyme (1.3 X 10(-6) M versus 6.3 X 10(-6) M). The apparent Km values for the myelin and the microsomal enzyme were 1.3 X 10(-4) M and 4.3 X 10(-4) M, respectively. An enzyme preparation that was practically devoid of myelin lipids was then prepared and its substrate specificity examined. The "delipidated enzyme" could hydrolyze fetuin, NL, and ganglioside substrates, including GM1 and GM2. When the delipidated enzyme was exposed to high temperature (55 degrees C) or low pH (pH 2.54), the neuraminidase activities toward NL and GM3 decreased at nearly the same rate. Both fetuin and 2,3-dehydro-2-deoxy-N-acetylneuraminic acid inhibited NL and GM3 hydrolysis. With 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, inhibition of NL was greater than that of GM3; however, the Ki values for each substrate were almost identical. GM3 and GM1 also competitively inhibited the hydrolysis of NL and NL similarly inhibited GM3 hydrolysis by the enzyme. These results indicate that rat brain myelin has intrinsic neuraminidase activities toward nonganglioside as well as ganglioside substrates, and that these two enzyme activities are likely catalyzed by a single enzyme entity.     

Reactivation and aging of diphenyl phosphoryl acetylcholinesterase.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) is readily in hibited by 10(-5) M diphenylphosphorochloridate even though the inhibitor hydrolyzes in a few seconds. The fluoridate is a much weaker inhibitor. The inhibited enzyme, diphenyl phosphoryl enzyme spontaneously recovers only about 50% of its activity with a half time of about 17 min at pH 7.0 and 6 min at pH 8.0. The fact that only 50% of the original activity returns is due to aging. The rates of reactivation and aging can be very greatly increased by a few percent of an organic solvent. Depending on the solvent even 1% may increase the rates by a factor of 5 or 6. The highest increase in rate was 70-fold. Quaternary NH+4 also increases the rates. Organic solvents and NH+4 also accelerate the reactivation of the much more stable diethyl phosphoryl enzyme derivative.     

5'-methylthioadenosine phosphorylase from Caldariella acidophila. 1. Purification and partial characterization]

5'-Methylthioadenosine phosphorylase has been isolated from C.acidophila, a thermophilic bacterium living in acid hot springs at temperatures ranging from 63 to 89 degrees C. The enzyme has been purified to homogeneity in 32% yield. The enzyme shows a high degree of thermophilicity, its temperature optimum being 93 degrees C in the in vitro assay. The enzyme is exceptionally stable; no loss of activity was observable after exposure for 1 h at 100 degrees C. The optimum pH is about 7,2, with one-half of the maximal activity occurring at pH 6 and 9. The apparent Km for the substrates are: 8,3 x 10(-5) M for MTA and 4,3 x 10(-4) M for phosphate ions.     

An acidic protease from the grass carp intestine (Ctenopharyngodon idellus)

The acidic Protease was extracted from the intestine of the grass carp (Ctenopharyngodon idellus) by 0.1 M sodium phosphate buffer, pH 7.0 at 4 degrees C after neat intestine was defatted with acetone, and partially purified by ammonium sulfate precipitation, gel filtration chromatography and ionic exchange chromatography. SDS-PAGE electrophoresis showed that the enzyme was homogeneous with a relative molecular mass of 28,500. Substrate-PAGE at pH7.0 showed that the purified acidic protease has only an active component. Specificity and inhibiting assays showed that it should be a cathepsin D. The optimal pH and optimal temperature of the enzyme were pH2.5 and 37 degrees C, respectively. It retained only 20% of its initial activity after incubating at 50 degrees C for 30 min. The enzyme lost 81% of its activity after incubation with pepstatin A at room temperature, but was not inhibited by soybean trypsin inhibitor or phenylmethylsulfonyl fluoride (PMSF). Its V(max) and K(m) values were determined to be 3.57 mg/mL and 0.75 min(-1), respectively.     

Purification and characterization of fibrinolytic enzyme from cultured mycelia of Armillaria mellea

A fibrinolytic enzyme was purified from the cultured mycelia of Armillaria mellea by ion-exchange chromatography followed by gel filtration, and was designated A. mellea metalloprotease (AMMP). The purification protocol resulted in a 627-fold purification of the enzyme, with a final yield of 6.05%. The apparent molecular mass of the purified enzyme was estimated to be 21kDa by SDS-PAGE, fibrin-zymography and gel filtration chromatography, which revealed a monomeric form of the enzyme. The optimal reaction pH value and temperature were, pH 6.0, and 33 degrees C, respectively. This protease effectively hydrolyzed fibrinogen, preferentially digesting the Aalpha-chain over the Bbeta- and r-chains. Enzyme activity was inhibited by Cu(2+) and Co(2+), but enhanced by the addition of Ca(2+) and Mg(2+) ions. Furthermore, AMMP activity was potently inhibited by EDTA, and was found to exhibit a higher specificity for the substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 24 amino acid residues of the N-terminal sequence were MFSLSSRFFLYTLCL SAVAVSAAP, which is extremely similar to the 24 amino acid residues of the N-terminal sequence of the fruiting body of A. mellea. These data suggest that the fibrinolytic enzyme AMMP, obtained from the A. mellea exhibits a profound fibrinolytic activity. The mycelia of A. mellea may thus represent a potential source of new therapeutic agents to treat thrombosis.     

Purification and characterization of a carboxylesterase from rabbit liver lysosomes

Carboxylesterase [EC 3.1.1.1] was purified from rabbit liver lysosomes by means of detergent solubilization, and by hydroxyapatite, phenyl-Sepharose and chromatofocusing column chromatographies. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 58,000. This enzyme was eluted at an isoelectric point of approximately 5.8 by chromatofocusing, and exhibited a broad pH optimum of between 6.0 and 9.0. The enzyme hydrolyzed 4-methylumbelliferyl esters of saturated fatty acids (C2-C12), and it also hydrolyzed p-nitrophenylacetate, methyl butyrate, and tributyrin, but not acetanilide. Its activity was completely inhibited by diisopropyl-fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) at 10(-4) M, but was not affected by eserine, or by alpha- or beta-naphthyl acetate at 10(-3) M. Various metal ions (Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Zn2+, Ni2+) at 10(-3) M also had no effect on the enzyme activity.     

Interaction of thiolsubtilisin with Streptomyces subtilisin inhibitor, SSI.

Subtilisin BPN' was chemically converted to thiolsubtilisin and the interaction of this modified enzyme with Streptomyces subtilisin inhibitor (SSI) was examined. SSI competitively inhibited the esterolytic activity of thiolsubtilisin toward p-nitrophenyl acetate with a K1 value of 1.3 X 10(-5) M at pH 7.5 Spectrophotometric analysis of the interaction between SSI and the modified enzyme yielded a Kd value of 4 X 10(-5) M at pH 9.7. These values are about 10(5)-fold greater than the Kd value (less than 10(-9) M at pH 7.5) for the native enzyme. This indicates that the small change in the active site structure of subtilisin (Ser221 to Cys221) leads to a considerable decrease in the binding affinity (by about 6-7 kcal/mol) to SSI.     

Purification and characterization of a fibrinolytic protease from a culture supernatant of Flammulina velutipes mycelia

In this study we purified a fibrinolytic enzyme from the culture supernatant of Flammulina velutipes mycelia by ion exchange and gel filtration chromatographies, it was designated as F. velutipes protease (FVP-I). This purification protocol resulted in 18.52-fold purification of the enzyme at a final yield of 0.69%. The molecular mass of the purified enzyme was estimated to be 37 kDa by SDS-PAGE, fibrin-zymography and size exclusion by FPLC. This protease effectively hydrolyzed fibrin, preferentially digesting alpha-chain over beta-and gamma-gamma chain. Optimal protease activity was found to occur at a pH of 6.0 and a temperature of 20 to 30 degrees C. The protease activity was inhibited by Cu2+, Fe2+ and Fe3+ ions, but was found to be enhanced by Mn2+ and Mg2+ ions. Furthermore, FVP-I activity was potently inhibited by EDTA and EGTA, and it was found to exhibit a higher specificity for chromogenic substrate S-2586 for chymotrypsin, indicating that the enzyme is a chymotrypsin-like metalloprotease. The first 20 amino acid residues of the N-terminal sequence of FVP-I were LTYRVIPITKQAVTEGTELL. They had a high degree of homology with hypothetical protein CC1G_11771, GeneBank Accession no. EAU86463.     

Fructose 1,6-bisphosphate aldolase activity ofRhizobium species

FDP aldolase was found to be present in the cell-free extracts of Rhizobium leguminosarum, Rhizobium phaseoli, Rhizobium trifolii, Rhizobium meliloti, Rhizobium lupini, Rhizobium japonicum and Rhizobium species from Arachis hypogaea and Sesbania cannabina. The enzyme in 3 representative species has optimal activity at pH 8.4 in 0.2M veronal buffer. The enzyme activity was completely lost by treatment at 60 degrees C for 15 min. The Km values were in the range from 2.38 to 4.55 X 10(-6)M FDP. Metal chelating agents inhibited enzyme activity, but monovalent or bivalent metal ions failed to stimulate the activity. Bivalent metal ions in general were rather inhibitory.     

Differentiation of fluorides-stimulated and non-fluoride-stimulated components of beef brain cortex adenylate cyclase cy calcium ions, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid and Triton X-100.

Beef brain cortex adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) activity is 84--88% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the absence of F- but only 50--60% inhibited by 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid in the presence of F-. In either case, further increase in EGTA concentration did not alter the degree of inhibition. The inhibition can be completely reversed in both cases by addition of 3 - 10(-5) M Ca2+, (yielding a [free Ca2+] of approximately 2 - 10(-6) M) and 5 - 10(-5) M Mn2+ or Co2+ and partially by 5 - 10(-5) M Sr2+ but not by addition of 5 - 10(-5) M Ba2+, Zn2+, Ni2+ or Fe2+. A [free Ca2+] of 7.2 - 10(-5) M markedly inhibited cyclase activity in the presence of F-. Solubilization by 1.8% Triton X-100 resulted in an enzyme preparation no longer stimulated by NaF and 100% inhibited by the addition of 5 - 10(-5) M ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid either in the absence or presence of NaF. However, in contrast to ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-TETRAACETIC ACID, EDTA had no measurable effect on adenylate cyclase either in the presence or absence of NaF and ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid did not affect ATPase or phosphodiesterase activities. The data is rationalized by the postulation of two independent enzyme components in brain cortex: one component is about six-fold activated by NaF and the NaF effect is enhanced by low concentrations of Ca2+ and Mg2+. A second component is totally Ca2+ dependent and inhibited by high concentrations of F-. Mn2+, Co2+ and Sr2+ appear to be in vitro Ca2+ substitutes for both enzyme systems. On this basis, Triton X-100 treatment results in about a three-fold increase in specific activity of the Ca2+ dependent cyclase component but a complete abolition of the NaF stimulated component.