A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids β-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [l-(4-benzoyl)phenylalanyl-β-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (k_cat/K_m) of 3.8 ± 0.5 × 10⁴ M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors.     

Purifications and Enzymatic Properties of β-Amylase and Pullulanase from Bacillus cereus var. mycoides

A β-amylase and a pullulanase produced by Bacillus cereus var. mycoides were purified by means of ammonium sulfate fractionation, adsorption on starch and celite and Sephadex G–100 column chromatography. The purified enzymes were homogeneous in disc electrophoresis.

The β-amylase released only maltose from amylose, amylopectin, starch and glycogen, and the released maltose was in β-form. The pullulanase released maltose, maltotriose and maltotetraose from β-limit dextrin and maltotriose from pullulan, but not amylose-like substance from amylopectin.

The optimum pHs of β-amylase and pullulanase were about 7 and 6~6.5, respectively. The optimum temperatures of the enzymes were about 50°C. The enzymes were inhibited by the sulfhydryl reagents such as mercuric chloride and p-chloromercuribenzoate, and the inhibitions with p-chloromercuribenzoate were restored by the addition of cysteine. The molecular weights of β-amylase and pullulanase were estimated to be 35,000±5,000 and 110,000±20,000, respectively.     

13C-NMR Spectra and Streochemistry of Isoechinulins A,B and C

¹³C-NMR spectra of isoechinulins A, B and C, metabolites from Aspergillus ruber, were fully assigned on the basis of chemical shifts and multiplicities and comparison with their analogues. Taking advantage of the symmetrical structure of the diketopiperazine ring, the stereochemistry of the trisubstituted carbon-carbon double bond in a dehydrotryptophyl moiety was determined as Z (cis) by measuring the coupling constants, , in the proton nondecoupled spectrum of isoechinulin B.     

Enzymatic Assay of d-Xylose Isomerase with d-Sorbitol Dehydrogenase

Previously a cyclic pathway for the partial oxidation of propionyl-CoA to pyruvate has been proposed. Enzymatic evidence for the presence of the key reactions involved in this pathway is described and discussed herein. The condensation of propionyl-CoA with oxaloacetate into methylcitrate is shown to be catalyzed by an enzyme contained in cell-free extracts of Candida lipolytica; the enzyme seems to differ from the usual citrate synthase. Methylcitrate is easily convertible to a mixture of C₇-acids by the action of cell-free extract of the mutant strain. On the other hand, a similar mixture is changed into pyruvate and succinate by the action of cell-free extract of the parent strain. Evidence is given that methylisocitrate, one of the products of the conversion, is mainly cleaved by the action of an additional enzyme other than the usual isocitrate lyase. The accumulation of methylisocitrate in a large amount from odd-carbon n-alkanes by the mutant strain can be safely ascribed to the absence or a low level of this enzyme in the mutant strain.     

Hydrolytic Activity of α-Mannosidase against Deoxy Derivatives of p-Nitrophenyl α-d-Mannopyranoside

Deoxy derivatives of p-nitrophenyl (PNP) α-d-mannopyranoside, PNP 2-deoxy-α-d-arabino-hexopyranoside, 3-deoxy-α-d-arabino-hexopyranoside, 4-deoxy-α-d-lyxo-hexopyranoside, and α-d-rhamnopyranoside, were synthesized and hydrolytic activities of jack bean and almond α-mannosidases against them were investigated. These α-mannosidases scarcely acted on the 2-, 3-, and 4-deoxy derivatives, while the 6-deoxy one was hydrolyzed by the enzymes as fast as PNP α-d-mannopyranoside, which is a common substrate for α-mannosidase. These results indicate that the hydroxyl groups at C-2, 3, and 4 of the mannopyranoside are necessary to be recognized as a substrate by these enzymes, while that at C-6 does not have so a crucial role in substrate discrimination. Values of K_m and V_max of the enzymes on the hydrolysis of PNP α-d-rhamnopyranoside were obtained from kinetic studies.     

Purification and Properties of α-Glucosidase and Glucoamylase from Lentinus edodes (Berk.) Sing.

An α-glucosidase and a glucoamylase have been isolated from fruit bodies of Lentinus edodes (Berk.) Sing., by a procedure including fractionation with ammonium sulfate, DEAE-cellulose column chromatography, and preparative gel electrofocusing. Both of them were homogeneous on gel electrofocusing and ultracentrifugation. The molecular weight of α-glucosidase and glucoamylase was 51,000 and 55,000, respectively. The α-glucosidase hydrolyzed maltose, maltotriose, phenyl α-maltoside, amylose, and soluble starch, but did not act on sucrose. The glucoamylase hydrolyzed maltose, maltotriose, phenyl α-maltoside, soluble starch, amylose, amylopectin, and glycogen, glucose being the sole product formed in the digests of these substrates. Both enzymes hydrolyzed phenyl a-maltoside into glucose and phenyl α-glucoside. The glucoamylase hydrolyzed soluble starch, amylose, amylopectin, and glycogen, converting them almost completely into glucose. It was found that β-glucose was liberated from amylose by the action of glucoamylase, while α-glucose was produced by the α-glucosidase.

Maltotriose was the main α-glucosyltransfer product formed from maltose by the α-glucosidase.     

Application of Axial-haloketone Rule to Lactones

Antifungal activities were examined and compared for some 40 kinds of aliphatic and aromatic aldehydes, alcohols, phenolic compounds, ether compounds and hydrocarbons from essential oils and for some related compounds, using seven fungi.     

Supplemental Effects of Arginine and Methionine on Growth,and on Formations of Urea and Creatine of Adrenalectomized Rats Fed High Glycine Diets

The effects of adrenalectomy on growth, some enzyme activities in the liver and kidney, and urinary excretion of urea, creatinine and creatine were investigated in rats fed the 10% casein diets containing 7% glycine with or without l-arginine and l-methionine (10C, 10C7G and 10C7ArgMet).

Body weight gains of the intact 10C and 10C7GArgMet groups were almost same as the corresponding adrenalectomized groups. The body weight of the adrenalectomized 10C7G group was extremely decreased though that of the intact 10C7G group was maintained almost constant; but the decrease was recovered by the administration of hydrocortisone. The activities of liver arginase and carbamylphosphate synthetase were not affected by those diets. Liver serine dehydratase and ornithine δ-aminotransferase activities were increased in the intact 10C7G and 10C7GArgMet groups, but these increases were depressed by adrenalectomy. Glutamate-pyruvate transminase activities in the liver of intact 10C7G and 10C7GArgMet groups were also enhanced, but were extremely decreased in the corresponding adrenalectomized groups. Kidney transamidinase activity was not affected by adrenalectomy. The amount of urinary excreted urea was almost unchanged by adrenalectomy, but was increased by hydrocortisone administration. The amounts of excreted creatine of the adrenalectomized groups were generally larger than the corresponding intact groups, but slightly decreased by the administration of hydrocortisone. The amount of excreted creatinine was not generally affected by adrenalectomy.     

Production of Lysine by Pyruvate Dehydrogenase Mutants of Brevibacterium flavum

Mutants with low pyruvate dehydrogenase (PD) activities were derived from a pyruvate kinase-deficient lysine-producing mutant of Brevibacterium flavum, No. 22. They were selected as prototrophic revertants of the acetate auxotrophs of strain No. 22. Among them strain KD-11 produced 55g/liter of lysine as its HCI salt when cultured for 72 hr in a medium containing lOOg/liter of glucose, soybean-meal hydrolysate and methionine. The lysine yield of strain KD-11 was the highest ever reported (55%). The mutant required a higher concentration of methionine for maximum production and gave a smaller amount of cell mass in cultivation than its parent. PD activity of strain No. 22 was stimulated by cysteine, stabilized by glycerol, and gave apparent Kms of 89, 22, 380, 83 μM for pyruvate, coenzyme A, 3-acetylpyridine adenine dinucleotide, and NAD, respectively, under standard conditions. The apparent Km for NAD of PD from strain KD-11 was 10-times higher than that from No. 22. When the concentration of NAD was low, the cell extracts of strain KD-11 showed low PD activity. The specific activity of phosphoenolpyruvate carboxylase of strain KD-11 was slightly higher than that of strain No. 22, while the inhibition by aspartate of the former enzyme was weaker than that of the latter.     

Substrate Shape Preference of Escherichia coli Ribonuclease P Ribozyme and Holo Enzyme Using Bottom-Half Part-Shifting Variants of Pre-tRNA

We showed previously that the bacterial ribonuclease P (RNase P) ribozyme has substrate shape preference depending on the concentrations of catalytically important magnesium ions. The ribozyme discriminates a canonical cloverleaf precursor tRNA from a hairpin RNA with a CCA-tag sequence at low concentrations of magnesium ions. By detailed analysis of the shape preference using the bottom-half part-shifting variants of a tRNA precursor, we showed that the RNAs in a T-shape structure can be substrates for the ribozyme reactions even at low concentrations of magnesium ions, and that the RNA in a natural L-shape is the best substrate for both the ribozyme and the holo enzyme. The results also showed that the position of the bottom-half part did not affect the cleavage site selection of a substrate by the enzyme. Our results are the first kinetic evidence to show the importance of the bottom-half part of tRNA molecule, and our result also showed that the holo enzyme can discriminate substrate shape as well as the ribozyme at low concentrations of metal ions.