Biochemical and histochemical studies on p-nitrophenyl phosphate and beta-glycerophosphate hydrolysis at acid pH by rabbit parotid tissue.

Synopsis A biochemical and histochemical study has been made of the characteristics and distribution of acid phosphatases in the parotid gland of the rabbit. The substratesp-nitrophenyl phosphate and -glycerophosphate have been used. Homogenate studies showed that substantial hydrolysis ofp-nitrophenyl phosphate occurred in the presence of tartrate and fluoride ions but that -glycerophosphate hydrolysis was almost abolished by these additives. The pH optima usingp-nitrophenyl phosphate and -glycerophosphate were found to be around pH 5.0 and pH 4.0 respectively. Studies on subcellular fractions revealed that maximalp-nitrophenyl phosphate hydrolysis occurred in the microsomal fraction whereas maximal hydrolysis of -glycerophosphate occurred in the mitochondrial fraction. The histochemical staining patterns produced by the two substrates differed in several respects. Whenp-nitrophenyl phosphate was used heavy plasmalemmal staining occurred, while reactions using -glycerophosphate as substrate produced heavy staining of the acinar cell cytoplasm including many of the zymogen granules. Both substrates resulted in the staining of lysosomes and certain vesicles associated with the Golgi complex. Formaldehyde fixation resulted in an overall reduction in staining intensity and glutaraldehyde fixation used in electron microscopic studies heavily inhibited much of the extralysosomal activity. The conclusion is reached that significant extralysosomal acid hydrolase activity is present in the rabbit parotid gland, much of which is located on smooth microsomes. The data is discussed in the context of the distribution of extralysosomal acid hydrolases and the staining patterns are considered in the light of the limitations inherent in the techniques employed.     

Autoradiographic localization of 3H-GABA uptake in the thyroid gland of the rat

Summary The uptake of -aminobutyric acid (GABA) in the thyroid gland of the rat was studied autoradiographically following in vitro incubation.High-affinity GABA uptake was localized in follicle cells, whereas C cells (parafollicular cells) in general did not accumulate GABA by high-affinity transport. The follicle cells were also the main sites of low-affinity GABA uptake. Additionally, some nerve fibres were found to accumulate GABA.The predominant localization of GABA uptake in follicle cells is discussed in view of a presumed role of GABA in thyroid function.     

Histochemistry of non-specific phosphatases: the use of p-nitrophenyl phosphate and -glycerophosphate as substrates

Synopsis It has been found that the acid and alkaline phosphatases in homogenates of respectively intestine and hypodermis ofAscaris suum hydrolyse sodiump-nitrophenyl phosphate at about twice the rate of sodium -glycerophosphate. This difference was also observed histochemically. Thus, when sections of intestine were incubated for acid phosphatase withp-nitrophenyl phosphate as substrate, the intensity of staining was about twice as great as that obtained after incubation in -glycerophosphate. Further, alkaline phosphatase was evident in sections of hypodermis after only 2 hr incubation inp-nitrophenyl phosphate but was not apparent after 10 hr incubation with -glycerophosphate. Hence biochemical assays and histochemical studies both indicate thatp-nitrophenyl phosphate is a superior substrate to -glycerophosphate for the visualization of acid and alkaline phosphatases in tissues.This paper was presented in part at the 1969 Aberdeen meeting of the British Society for Parasitology.     

Purification and properties of ap-nitrobenzyl esterase fromBacillus subtilis

A procedure for purifying to homogeneity a microbially produced biocatalyst useful for deblocking intermediates in the manufacture of beta-lactam antibiotics is reported. In aqueous solution the purifiedp-nitrobenzyl (PNB) carboxy-esterase was soluble, monomeric (molecular weight: 54 000 by SDS-PAGE or by gel filtration) and exhibited an acidic pl, 4.1. The PNB carboxy-esterase catalyzed rapid ester hydrolysis for simple organic esters such as PNB-acetate, benzyl acetate and -naphthyl acetate and catalyzed deblocking (ester hydrolysis) of beta-lactam antibiotic PNB esters such as cephalexin-PNB and loracarbef-PNB. TheN-terminal amino acid sequence and the amino acid composition are reported. A serine residue is involved in ester hydrolysis: the PNB carboxy esterase was inhibited by phenylmethylsulfonyl fluoride and diethylp-nitrophenyl phosphate; one mole of diisopropyl fluorophosphate titration was required per mole of PNB carboxy-esterase for complete inhibition. When the [³H]-diisopropyl fluorophosphate-treated biocatalyst was digested with Lys C and the resulting peptides separated by HPLC, a single [³H]-labeled peptide was obtained; its amino acid sequence is reported. Inhibition of the PNB carboxy esterase by diethyl pyrocarbonate suggests that a histidinyl residue (or residues) is (are) also involved in the catalytic site of the esterase.Abbreviations used -ME -mercaptoethanol - Cf cefaclor - Cf nucleus-PNB - (6R, 7R) 7-amino-3-chloro-8-oxo-5-thia-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Cp cephalexin - Cp-PNB p-nitrobenzyl carboxy-ester of cephalexin - DEPC diethyl, pyrocarbonate - DFP diisopropyl fluorophosphate - DMSO dimethyl sulfoxide - DNP diethylp-nitrophenyl phosphate - EDTA ethylenediaminetetraacetic acid - EGTA ethylene, glycol-bis(aminoethyl ether) - N,N,NN tetracetic acid - Lc loracarbef - Lc-PNB p-nitrobenzyl carboxy-ester of loracarbef - Lc nucleus-PNB - (6R, 7S) 7-amino-3-chloro-8-oxo-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylic acid, (4-nitrophenyl)methyl ester - Lys C an endoproteinase specifically cleaving at C terminal lysine residues - MW_r relative molecular weight - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - PNB p-nitrobenzyl - PNBCE p-nitrobenzyl carboxy-esterase - SDS sodium dodecyl sulfate     

A quantitative study of peroxidase activity in unfixed tissue sections of the guinea-pig thyroid gland

Summary A technique for the cytochemical demonstration of peroxidase activity in unfixed guinea-pig thyroid tissue is described in this paper. The substrate 3,3-diaminobenzidine tetrahydrochloride (DAB) is oxidized by the peroxidase to form an insoluble reaction product. Optimal results were obtained after 20 min incubation at 37° C in reaction medium containing 1.4mm DAB (in 0.1m Tris-HCl) and 0.15mm hydrogen peroxide at pH 8.0. Peroxidase activity was seen in the thyroid follicle cells as a diffuse brown reaction product (which was more dense and granular in erythrocytes). The enzyme activity was quantified using a scanning-integrating microdensitometer, and the effects of two specific peroxidase inhibitors were evaluated. Both 3-amino-1,2,4-triazole and methimazole inhibited peroxidase activity in the follicle cells (enzyme activity was still seen in the erythrocytes), maximal inhibition occurring at 10mm. Stimulation of peroxidase in the thyroid was observedin vivo (1 I.U. TSH administered every 8 h for two days), with the maximal stimulation occurring after 1 day.     

On the Fermenting Ability of Sand Cultures of Acetone-Butanol Organisms Stored for Long Years

β-N-Acetyl-D-hexosaminidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purifted by making an acetone-dried preparation of the mid-gut gland, extracting with 50 mM citrate-phosphate buffer (pH 4.0) (about 13% of the extracted proteins was β-N-acetyl-D-hexosaminidase), ammonium sulfate fractionation, and column chromatographies on CM-Sepharose and DEAE-Sepharose. The purifted β-N-acetyl-D-hexosaminidase was homogeneous on SDS–PAGE, and sufficiently free from other exo-type glycosidases. The molecular weight was 56,000 by SDS–PAGE. The enzyme hydrolyzed both p-nitrophenyl β-N-acetyl-D-glucosaminide and p-nitrophenyl β-N-acetyl-D-galactosaminide. For p-nitrophenyl β-N-acetyl-D-glucosaminide, the pH optimum was 3.7, the optimum temperature was 45°C, and the K_m was 0.24 mM. For p-nitrophenyl β-N-acetyl-D-galactosaminide, these were pH 3.4, 45°C, and 0.15 mM, respectively. The enzyme liberated non-reducing terminal β-Iinked N-acetyl-D-glucosamine or N-acetyl-D-galactosamine from various 2-aminopyridyl derivatives of oligosaccharides of N-glycan or glycolipid type except of G_M2-tetrasaccharide. As the enzyme was stable around pH 3.5–5.5, it may be useful for long time reactions around the optimum pH.     

Studies on the nature of the Li locus in Trifolium repens L. II. The effect of genotype on enzyme activity and properties

Various reagents which prevent enzyme inhibition by phenolic compounds were tested in attempts to improve the medium used to extract the Li-controlled enzyme activities from white clover leaves. The addition of 50 mm diethyldithiocarbamate to the extraction medium gave a fivefold increase in the enzyme activity of LiLi white clover extracts against p-nitrophenyl -d-glucoside, linamarin-lotaustralin, and p-nitrophenyl -d-galactoside. These three substrates were used in tests on the effect of genotype on enzyme activity. An absence of dominance at the Li locus was demonstrated, with a dosage effect of Li alleles on enzyme activity. A new Li allele was identified in the Lili clone, C11, which had low levels of enzyme activity. In crosses with two lili clones, Li(C11)li progeny were produced with activity levels similar to those of the C11 parent. Inhibition and heat-inactivation tests suggest that the lili clone, D4, produces an altered form of -glucosidase which may also be present in LiLi plants. The nature of the Li locus is discussed.     

A study of the strength and stability of gas vesicles isolated from a blue-green alga

Summary Acid phosphatase was studied by means of electron microscope cytochemistry in glutaraldehyde-fixed myxamoebae of Dictyostelium discoideum grown on dead bacteria. The enzyme activity was localized to the digestive vacuoles in vegetative as well as in aggregating cells. Biochemical experiments showed that the enzyme was not inactivated by fixation in 2% purified glutaraldehyde.Abbreviations used NPP p-nitrophenyl phosphate - NP p-nitrophenol - GP -glycerophosphate - glc-6-P glucose-6-phosphate - Pi orthophosphate     

Systematic Synthesis of Sulfur-containing p-Nitrophenyl α-Maltopentaoside Derivatives for a Differential Assay of Human α-Amylases

For use in a differential assay of human α-amylases, a variety of 6⁵-S-substituted p-nitrophenyl α-maltopentaoside derivatives (6-54) were systematically synthesized via the key intermediate, p-nitrophenyl O-(2,3-di-O-acetyl-6-S-acetyl-4-O-benzoyl-6-thio-α-D-glucopyranosyl)-(1 →4)-tris[O-(2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-(1→4)]-2,3,6-tri-O-acetyl-α-D-glucopyranoside (4), which was easily prepared from p-nitrophenyl α-maltopentaoside (G5P) in four steps. The sulfoxide and sulfone derivatives were prepared by oxidizing the corresponding sulfides with m-chloroperbenzoic acid.     

Purification and Properties of Acid β-d-Galactosidase from Corticium rolfsii

An acid β-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SP-Sephadex C-50 chromatography. The maximum activity of the enzyme towards p-nitrophenyl β-D-galactopyranoside was found to be at pH 2.0 to 2.5 and the enzyme was fairly active at pH 1.5 to l.8. The enzyme was quite stable over a pH range 2.0 to 8.0 at 2°C for 72 hr. The enzymic activity was clearly inhibited by Hg²⁺. Km value was determined to be 3.84 × 10^?4 m, and V_max was calculated to be 6.9 μ moles per min per mg for p-nitrophenyl β-d-galactopyranoside. Contrary to high activity on the synthetic galactoside, reaction velocity was small when the enzyme acted on lactose.     

Subcellular distributions and properties of rat testicular acid phosphatases

Synopsis Acid phosphatase activities againstp-nitrophenyl phosphate, -naphthyl phosphate -naphthyl phosphate, naphthol AS-BI phosphate, -glycerophosphate and -glycerophosphate were studied in whole homogenates and subcellular fractions of rat testes. The mitochondrial-lysosomal fraction (the 9000 g sediment) was the most active against all these substrates, but a high specific activity was also present in the soluble fraction (105,000 g supernatant). The effect of various inhibitors and activators onp-nitrophenyl phosphate hydrolysis was different in these fractions. The soluble enzyme was markedly stimulated by Co²⁺, Mg²⁺, Mn²⁺, Ni²⁺ and Zn²⁺. The particulate enzyme was inhibited by sodium fluoride, sodium tartrate and sodium molybdate, whereas the soluble activity was sensitive to Cd²⁺, Cu²⁺ and Sn²⁺ as well as to formaldehyde and glutaraldehyde. The soluble acid phosphatase is mainly localized within the seminiferous tubules. The high sensitivity of the soluble activity to the commonly used fixatives may interfere in the histochemical demonstration of the enzyme.     

General Characteristics of the Intracellular Myrosinase from Aspergillus niger

The enzymatic properties of intracellular myrosinase produced by Aspergillus niger AKU 3302 were investigated. Maximum activity occurred at pH 6.2, and the enzyme was stable in a pH range of 7.6 to 8.0 at 5°C for 24 hr. Optimum temperature was about 34°C. Enzyme activity was stimulated by copper (I), (II), manganese (II) and cobalt (II) and was inhibited by mercury (II) and stannous (II) ions. However, metal complexing agents and DFP had little effect, while PCMB was a strong inhibitor. In contrast to plant myrosinase, this enzyme was neither activated nor inhibited by L-ascorbic acid. Glucosides and δ-gluconolactone inhibited enzyme activity but sugars were ineffective. The Km value for sinigrin was 3.3 × 10^?3 M and that for p-nitrophenyl β-glucoside was 1.5 × 10^?3 M. The relation between fungous myrosinases and β-glucosidase is discussed in comparison to plant myrosinase.     

Purification and Properties of α-d-Galactosidase Produced by Corticium rolfsii

An α-d-galactosidase was purified from the culture filtrate of Corticium rolfsii IFO 6146 by a combination of QAE-Sephadex A-50 and SE-Sephadex C-50 chromatography. The purified enzyme was demonstrated to be free of other possibly interfering glycosidases and glycanases. The maximum activity of the enzyme towards p-nitrophenyl α-d-galactopyrano-side was found to be at pH 2.5 to 4.5, and the enzyme was fairly active at pH 1.1 to 2.0. The enzyme was stable over a pH range 4.0 to 7.0 at 5°C for 72 hr and relatively unstable at pH 1.1 to 2.0 as compared with endo-polygalacturonase, α-l-arabinofuranosidase and β-d-galactosidase produced by C. rolfsii. The enzymic activity was completely inhibited by Hg²⁺ and Ag⁺ ions, respectively. Km values were determined to be 0.16 × 10^?3 m for p-nitrophenyl α-d-galactopyranoside and 0.26 × 10^?3m for o-nitrophenyl α-d-galactopyranoside. The values of V_max were also determined to be 26.6 μmoles and 28.6 μmoles per min per mg for p- and o-nitrophenyl α-d-galactopyranoside, respectively.     

Isolation of a Novel Lipase Gene from <Emphasis Type="Italic">Serratia liquefaciens</Emphasis> S33 DB-1, Functional Expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and its Properties

A new lipase gene designated as SlLipA was isolated from Serratia liquefaciens S33 DB-1 by the genomic-walking method. The cloned gene contained an open reading frame (ORF) of 1,845 bp encoding 615 amino acids with a conserved GXSXG motif. Genome sequence analysis showed that an aldo/keto reductase gene closed to the SlLipA gene. The lipase gene was cloned into the expression vector pPICZαA and successfully integrated into the heterologous host, methylotrophic yeast Pichia pastoris GS115. Five transformants could be expressed as secreted recombinant proteins with the high activity on Triglyceride–Agarose plate and as candidates to produce the recombinant enzyme. A C-terminal His tag was used for its purification. The lipase activity of different transformants against substrate para-nitrophenyl laurate (p-NPL) varied from 14 to 16 U ml⁻¹. For the substrates para-nitrophenyl caprate (p-NPC), p-NPL, para-nitrophenyl myristate (p-NPM), para-nitrophenyl palmitate (p-NPP), and para-nitrophenyl stearate (p-NPS), the specific activity was shown to be preferred to long acyl chain length of p-NPS.     

Optimisation of the dehydrogenase assay for measurement of indigenous microbial activity in beach sediments contaminated with petroleum

A dehydrogenase enzyme assay has been developed to measure microbial activity in petroleum-contaminated beach sediments. The assay, using 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyltetrazoliumchloride (INT) as the substrate provides a reliable measurement of overall indigenous microbiological activity in beach sediments containing 7% (w/w) oil. The method has been effectively used in field studies (Mathew et al. 1999) to monitor microbial activity in conjunction with the biodegradation of petroleum hydrocarbons.     

Isolation and partial characterisation of acid phosphatase isozymes from dormant oilseed of<Emphasis Type="Italic"> Corylus avellana</Emphasis> L.

The acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) complement from dormant hazel (Corylus avellana L.) seeds was found to exhibit significant electrophoretic heterogeneity partially attributable to the presence of distinct molecular forms. In axiferous tissue, total acid phosphatase activity increased in a biphasic fashion during chilling, a treatment necessary to alleviate seed dormancy. Three acid phosphatase isozymes were isolated from cotyledons of dormant hazel seeds by successive ammonium sulphate precipitation, size-exclusion, Concanavalin A affinity, cation- and anion-exchange chromatographies resulting in 75-, 389- and 191-fold purification (APase1, APase2, APase3, respectively). The three glycosylated isoforms were isolated to catalytic homogeneity as determined by electrophoretic, kinetic and heat-inactivation studies. The native acid phosphatase complement of hazel seeds had an apparent M_r of 81.5±3.5 kDa as estimated by size-exclusion chromatography, while the determined pI values were 5.1 (APase1), 6.9 (APase2) and 7.3 (APase3). The optimum pH for p-nitrophenyl phosphate hydrolysis was pH 3 (APase1), pH 5.6 (APase2) and pH 6 (APase3). The hazel isozymes hydrolysed a variety of phosphorylated substrates in a non-specific manner, exhibiting low K_m and the highest specificity constant (V_max/K_m) for pyrophosphate. They were not primary phytases since they could not initiate phytic acid hydrolysis, while APase2 and APase3 had significant phospho-tyrosine phosphatase activity. Inorganic phosphate was a competitive inhibitor, while activity was significantly impaired in the presence of vanadate and fluoride.Abbreviations APase Acid phosphatase (EC 3.1.3.2) - ConA Concanavalin A–Sepharose 4B - CV Column volume - -GP -Glycerophosphate - IEF Isoelectric focusing - IP6 Phytic acid - pNPP p-Nitrophenyl phosphate - PAGE Polyacrylamide gel electrophoresis - PPi Pyrophosphate     

Characterization of Lipoxygenase from Fungi of the Genus Mortierella

The specific activity of lipoxygenase from several strains of the zygomycete Mortierellavaried from 1.02 to 2.02 mol diene per min per mg protein. The enzyme equally used linoleic or arachidonic acid as a substrate. An increase in lipoxygenase activity was observed after adding corn oil to the culture medium. Tests with inhibitors having different chemical structures revealed that the lipoxygenase activity from Mortierellacells was inhibited only by esculetin, ethanol, and nordihydroguaiaretic acid (NDGA). NDGA inhibited the enzyme in vitro(IC₅₀=142 M), but its addition in the exponential phase of growth activated the enzyme.     

A colorimetric method for the determination of lipase activity in soil

A colorimetric method for the determination of lipase activity in soil has been developed. Using p-nitrophenyl butyrate as substrate, soil samples are incubated at 30°C and pH 7.25 for 10 min. After cooling on ice and centrifugation, the released p-nitrophenol is determined at 400 nm. To allow for the adsorption of p-nitrophenol onto soil, a calibration curve is prepared in the presence of soil.     

Ecto-alkaline phosphatase considered as levamisole-sensitive phosphohydrolase at physiological pH range during mineralization in cultured fetal calvaria cells

Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p-nitrophenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6–8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP were close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole was uncompetitive (K_i = 45 μM). Phosphatidylinositol-specific phospholipase C (PI-PLC) produced the release into the medium of a p-nitrophenyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The released activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto-pNPPase activity. After 75 min of PI-PLC treatment the ecto-pNPPase activity remained unchanged despite the 30% decrease in Nonidet P-40-extractable ALP activity. High levels of ⁴⁵Ca incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in accordance with ecto-pNPPase activity variations. These results evidenced the levamisole-sensitive activity of a glycosylphosphatidylinositol-anchored pNPPase consistent with ALP acting as an ecto-enzyme whose functioning under physiological conditions was correlated to ⁴⁵Ca incorporation and permit the prediction of the physiological importance of the enzyme dynamic equilibrium at the cell surface in cultured fetal calvaria cells. © 1996 Wiley-Liss, Inc.