Purification and characterization of purple acid phosphatase PAP1 from dry powder of sweet potato

Purple acid phosphatase (PAP) was purified from sweet potato dry powder, which is used as a food additive. Spectrometric and enzymatic analyses, and analysis of the amino-terminal sequence indicated that the purified purple acid phosphatase was PAP1. High activity in neutral and acidic conditions, broad substrate specificity, and good thermal stability of PAP1 suggest the possibility of practical applications of PAP1.     

Effects of polyethylene glycol on bovine intestine alkaline phosphatase activity and stability

In this study, we evaluated the effects of polyethylene glycol (PEG) on bovine intestine alkaline phosphatase (BIALP) activity and stability. In the hydrolysis of p-nitrophenylphosphate (pNPP) at pH 9.8 at 20 °C, the k(cat)/K(m) values of BIALP plus 5-15% w/v free PEG with molecular masses of 1, 2, 6, and 20 kDa (PEG1000, PEG2000, PEG6000, and PEG20000 respectively) were 120-140%, 180-300%, 130-170%, and 110-140% respectively of that of BIALP without free PEG (1.8 μM(-1) s(-1)), indicating that activation by PEG2000 was the highest. Unmodified BIALP plus 5% PEG2000 and BIALP pegylated with 2,4-bis(O-methoxypolyethylene glycol)-6-chloro-s-triazine exhibited 1.3-fold higher activity on average than that of BIALP without free PEG under various conditions, including pH 7.0-10.0 and 20-65 °C. The temperatures reducing initial activity by 50% in 30-min incubation of unmodified BIALP plus 5% PEG2000 and pegylated BIALP were 51 and 47 °C respectively, similar to that of BIALP without free PEG (49 °C). These results indicate that the addition of PEG2000 and pegylation increase BIALP activity without affecting its stability, suggesting that they can be used in enzyme immunoassay with BIALP to increase sensitivity and rapidity.     

Rat cytochrome P450-mediated transformation of dichlorodibenzo-p-dioxins by recombinant white-rot basidiomycete Coriolus hirsutus

Rat cytochrome P450, CYP1A1, has been reported to play an important role in the metabolism of mono-trichlorodibenzo-p-dioxins (M-TriCDDs). To breed lignin (and M-TetraCDDs)-degrading basidiomycete Coriolus hirsutus strains producing rat CYP1A1, an expression cassette [C. hirsutus gpd promoter-C. hirsutus gpd 5′ portion (224-bp of 1st exon–8th base of 4th exon)-rat cyp1a1 cDNA-Lentinula edodes priA terminator] was constructed and inserted into pUCR1 carrying the C. hirsutus arg1 gene. The resulting recombinant plasmid, MIp5-(cyp1a1 + arg1) was introduced into protoplasts of C. hirsutus monokaryotic strain OJ1078 (Arg⁻, Leu⁻), obtaining three good Arg⁺ transformants. These transformants [ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), and ChTF5-6(CYP1A1)] were estimated to carry nine, six, and seven copies of the expression cassette on their chromosomes, respectively. Immunoblot analysis revealed that the three transformants produce similar amounts of rat CYP1A1 enzyme. ChTF5-2(CYP1A1), ChTF5-4(CYP1A1), ChTF5-6(CYP1A1) and recipient OJ1078 were cultivated in a liquid medium containing 2,7/2,8(at a ratio of 1:1)-dichlorodibenzo-p-dioxins (2,7/2,8-DCDDs) and the amount of intra- and extracellular 2,7/2,8-DCDDs remaining was measured. The results showed that all three transformants efficiently transform 2,7/2,8-DCDDs through the action of the recombinant rat CYP1A1 enzyme.     

Characteristics of Omega-conotoxin GVI A and MVIIC Binding to Ca<Subscript>v</Subscript> 2.1 and Ca<Subscript>v</Subscript> 2.2 Channels Captured by Anti-Ca<Superscript>2+</Superscript> Channel Peptide Antibodies

A New Binding Method (NBM) was used to investigate the characteristics of the specific binding of ¹²⁵I-omega-conotoxin (ω-CTX) GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels captured from chick brain membranes by antibodies against B1Nt (a peptide sequence in Ca_v2.1 and Ca_v2.2 channels). The results for the NBM were as follows. (1) The ED₅₀ values for specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels were about 68 and 60 pM, respectively, and very similar to those (87 and 35 pM, respectively) to crude membranes from chick brain. (2) The specific ¹²⁵I-ω-CTX GVIA (100 pM) binding was inhibited by ω-CTX GVIA (0.5 nM), dynorphine A (Dyn), gentamicin (Gen), neomycin (Neo) and tobramicin (Tob) (100 μM each), but not by ω-agaconotoxin (Aga) IVA, calciseptine, ω-CTX SVIB, ω-CTX MVIIC (0.5 nM each), PN200-110 (PN), diltiazem (Dil) or verapamil (Ver) (100 μM each). Calmodulin (CaM) inhibited the specific binding in a dose-dependent manner (IC₅₀ value of about 100 μg protein/ml). (3) The specific ¹²⁵I-ω-CTX MVIIC (60 pM) binding was inhibited by ω-CTX MVIIC, ω-CTX GVIA, ω-CTX SVIB (0.5 nM each), Dyn, Neo and Tob (100 μM, each), but not by ω-Aga IVA, calciseptine (0.5 nM each), PN, Dil, Ver (100 μM each) or 100 μg protein/ml CaM. These results suggested that the characteristics of the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to Ca_v2.1 and Ca_v2.2 channels in the NBM were very similar to those to crude membranes from chick brain, although the IC₅₀ values for CaM and free Ca²⁺ of CaM were about 33- and 5000-fold higher, respectively, than those for the specific binding of ¹²⁵I-ω-CTX GVIA and ¹²⁵I-ω-CTX MVIIC to crude membranes.     

Novel metabolism of 1 alpha,25-dihydroxyvitamin D3 with C24-C25 bond cleavage catalyzed by human CYP24A1

Our previous study revealed that human CYP24A1 catalyzes a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways that used both 25(OH)D(3) and 1alpha,25(OH)(2)D(3) as substrates, while rat CYP24A1 showed extreme predominance of the C-24 over C-23 hydroxylation pathway [Sakaki, T., Sawada, N., Komai, K., Shiozawa, S., Yamada, S., Yamamoto, K., Ohyama, Y. and Inouye, K. (2000) Eur. J. Biochem. 267, 6158-6165]. In this study, by using the Escherichia coli expression system for human CYP24A1, we identified 25,26,27-trinor-23-ene-D(3) and 25,26,27-trinor-23-ene-1alpha(OH)D(3) as novel metabolites of 25(OH)D(3) and 1alpha,25(OH)(2)D(3), respectively. These metabolites appear to be closely related to the C-23 hydroxylation pathway, because human CYP24A1 produces much more of these metabolites than does rat CYP24A1. We propose that the C(24)-C(25) bond cleavage occurs by a unique reaction mechanism including radical rearrangement. Namely, after hydrogen abstraction of the C-23 position of 1alpha,25(OH)(2)D(3), part of the substrate-radical intermediate is converted into 25,26,27-trinor-23-ene-1alpha(OH)D(3), while a major part of them is converted into 1alpha,23,25(OH)(3)D(3). Because the C(24)-C(25) bond cleavage abolishes the binding affinity of 1alpha,25(OH)D(3) for the vitamin D receptor, this reaction is quite effective for inactivation of 1alpha,25(OH)D(3).     

Inhibitory effect of 0.19 alpha-amylase inhibitor from wheat kernel on the activity of porcine pancreas alpha-amylase and its thermal stability

The inhibitory effect of 0.19 alpha-amylase inhibitor (0.19 AI) from wheat kernel on the porcine pancreas alpha-amylase (PPA)-catalyzed hydrolysis of p-nitrophenyl-alpha-D-maltoside (pNP-G2) was examined. 0.19 AI is a homodimer of 26.6 kDa with 13.3-kDa subunits under the conditions used. The elution behaviors in gel filtration HPLC of PPA and 0.19 AI indicated that a PPA molecule bound with a 0.19 AI molecule (homodimer) at a molar ratio of 1:1. 0.19 AI inhibited PPA activity in a competitive manner with an inhibitor constant, K(i), of 57.3 nM at pH 6.9, 30 degrees C, and the binding between them was found to be endothermic and entropy-driven. The activation energy for the thermal inactivation of 0.19 AI was determined to be 87.0 kJ/mol, and the temperature, T(50), giving 50% inactivation in a 30-min incubation at pH 6.9 was 88.1 degrees C. The high inhibitory activity of 0.19 AI against PPA and its high thermal stability suggest its potential for use in the prevention and therapy of obesity and diabetes.     

Metabolism of A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 by CYP24A1

The metabolism of 1alpha,25(OH)(2)D(3) (1alpha,3beta) and its A-ring diastereomers, 1beta,25(OH)(2)D(3) (1beta,3beta), 1alpha,25(OH)(2)-3-epi-D(3) (1alpha,3alpha), and 1beta,25(OH)(2)-3-epi-D(3) (1beta,3alpha), was examined to compare the substrate specificity and reaction specificity of CYP24A1 between humans and rats. The ratio between C-23 and C-24 oxidation pathways in human CYP24A1-dependent metabolism of (1alpha,3alpha) and (1beta,3alpha) was 1:1, although the ratio for (1alpha,3beta) and (1beta,3beta) was 1:4. These results indicate that the orientation of the hydroxyl group at the C-3 position determines the ratio between C-23 and C-24 oxidation pathways. A remarkable increase of metabolites in the C-23 oxidation pathway was also observed in rat CYP24A1-dependent metabolism. The binding affinity of human CYP24A1 for A-ring diastereomers was (1alpha,3beta)>(1alpha,3alpha)>(1beta,3beta)>(1beta,3alpha), indicating that both hydroxyl groups at C-1 and C-3 positions significantly affect substrate-binding. The information obtained in this study is quite useful for understanding substrate recognition of CYP24A1 and designing new vitamin D analogs.     

Extracellular production of recombinant thermolysin expressed in Escherichia coli, and its purification and enzymatic characterization

Thermolysin is a representative zinc metalloproteinase derived from Bacillus thermoproteolyticus and a target in protein engineering to understand the catalytic mechanism and thermostability. Extracellular production of thermolysin has been achieved in Bacillus, but not in Escherichia coli, although it is the most widely used as a host for the production of recombinant proteins. In this study, we expressed thermolysin as a single polypeptide pre-proenzyme in E. coli under the original promoter sequences in the npr gene, the gene from B. thermoproteolyticus, which encodes thermolysin. Active mature thermolysin (34.6 kDa) was secreted into the culture medium. The recombinant thermolysin was purified to homogeneity by sequential column chromatography procedures of the supernatant with hydrophobic-interaction chromatography followed by affinity chromatography. The purified recombinant product is indistinguishable from natural thermolysin from B. thermoproteolyticus as assessed by hydrolysis of N-[3-(2-furyl)acryloyl]-glycyl-L-leucine amide and N-carbobenzoxy-L-asparatyl-L-phenylalanine methyl ester. The results demonstrate that our expression system should be useful for structural and functional analysis of thermolysin.