Time-resolved fluoroimmunoassay for equol in plasma and urine

We present a method for the determination of the isoflavan equol in plasma and urine. This estrogenic isoflavan, which is formed by the action of the intestinal microflora, may have higher biological activity than its precursor daidzein. High urinary excretion of equol has been suggested to be associated with a reduction in breast cancer risk. The method is based on time-resolved fluoroimmunoassay, using a europium chelate as a label. After synthesis of 4′-O-carboxymethylequol the compound is coupled to bovine serum albumin (BSA), then used as antigen to immunize rabbits. The tracer with the europium chelate is synthesized using the same 4′-O-derivative of equol. After enzymatic hydrolysis (urine) or enzymatic hydrolysis and ether extraction (plasma) the immunoassay is carried out. The antiserum cross-reacted to variable extent with some isoflavonoids. For the plasma method the cross-reactivity does not seem to influence the results, which were highly specific. The overestimation of the values using the urine method (164%) compared to the results obtained by a gas chromatography–mass spectrometry (GC–MS) method is probably due to some influence of the matrix on the signal, and interference of structurally related compounds. It is suggested that plasma assays are used but if urine samples are measured a formula has to be used to correct the values making them comparable to the GC–MS results. The correlation coefficients between the time-resolved fluoroimmunoassay (TR-FIA) methods and GC–MS methods were high; r-values for the plasma and urine method, were 0.98 and 0.91, respectively. The intra-assay coefficient of variation (CV%) for the TR-FIA plasma and urine results at three different concentrations vary between 5.5–6.5 and 3.4–6.9, respectively. The inter-assay CV% varies between 5.4–9.7 and 7.4–7.7, respectively. The working ranges of the plasma and urine assay are 1.27–512 and 1.9–512 nmol/l, respectively.     

Rapid analysis of phytoestrogens in human urine by time-resolved fluoroimmunoassay

A time-resolved fluoroimmunoassay (TR-FIA), with europium labeled phytoestrogens as tracers, was developed for the quantitative determination of enterolactone, genistein and daidzein in human urine. The aim was to create a method for the screening of large populations in order to assess the possible correlations between the urinary levels and the risk of Western diseases.

After the synthesis of the 5′-carboxymethoxy derivative of enterolactone and 4′-O-carboxymethyl derivatives of daidzein and genistein, the respective compound was coupled to bovine serum albumin and then used as an antigen in the immunization of rabbits. The same derivatives of the phytoestrogen were used in preparing the europium tracers. After the enzymatic hydrolysis, the TR-FIA was carried out using the Victor 1420 multilabel counter. The method has sufficient sensitivity to measure the phytoestrogens at concentrations even below 5 nmol/l. The intra- and inter-assay coefficients of variation, at three different concentrations, varied from 1.9 to 5.3 and from 2.4 to 9.7, respectively.

We measured urinary enterolactone, genistein and daidzein in 215 samples from Finnish healthy women and found that more than 50% of the values ranged between 1 and 7, <0.1 and 0.6 and below 0.6 μmol/24 h, respectively. The TR-FIA method including only a hydrolysis step gave higher values than those measured by gas chromatography–mass spectrometry (GC–MS). However, the assay results by the present method showed strong correlation with those obtained by GC–MS. It is concluded that the TR-FIA is suitable for population screening of urinary phytoestrogens.     

Apparent transinhibition of peptide uptake in the scutellum of barley grain

The uptake of glycylsarcosine (Gly-Sar) into scutella separated from germinating grains of barley ( Hordeum vulgare L. cv. Himalaya) is inhibited by other peptides; in most cases the inhibition is not purely competitive but of a mixed type (simultaneous increase in the apparent K_m and decrease in V_max) (Sopanen, T. 1979. FEBS Lett. 108: 447–450). The aim of the present experiments was to elucidate the mechanism of the mixed inhibition by studying how peptides already taken up into the cells affect the uptake of Gly-Sar.
When scutella were preincubated in the presence of various peptides, 11 of the 13 peptides tested inhibited the subsequent uptake of Gly-Sar by 10 to 45%. The inhibition, studied in detail with leucylleucine and prolylproline, was due to a decrease in V_max. The two peptides having no effect were glycylglycine and D-alanyl-L-alanine which are the only peptides known to date acting as purely competitive inhibitors when present together with the substrate Gly-Sar.
Preincubation with leucine, proline and alanine was not inhibitory, although preincubation with the corresponding dipeptides was. This result, together with the demonstration of intact leucylleucine in the scutella after preincubation with leucylleucine, indicates that the inhibition was caused by the intact peptides.
The results support the notion that in the mixed type inhibition the increase in the apparent K_m is due to competition for the carrier at the outside of the membrane, while the decrease in V_max is due to peptides taken up and binding to the carrier at the inside of the membrane.     

Effects of microbivore species composition and basal resource enrichment on trophic-level biomasses in an experimental microbial-based soil food web

Previous theoretical and empirical evidence suggests that species composition within trophic levels may profoundly affect the response of trophic-level biomasses to enhanced basal resources. To test whether species composition of microbivorous nematodes has such an effect in microbial-based soil food webs, I created three microcosm food webs, consisting of bacteria, fungi, bacterial-feeding nematodes (Acrobeloides tricornus, Caenorhabditis elegans), fungal-feeding nematodes (Aphelenchus avenae, Aphelenchoides sp.) and a predatory nematode (Prionchulus punctatus). The food webs differed in species composition at the second trophic level: food web A included A. tricornus and Aph. avenae, food web B included C. elegans and Aphelenchoides sp., and food web AB included all four species. I increased basal resources by adding glucose to half of the replicates of each food web, and sampled microcosms destructively four times during a 22-week experiment to estimate the biomass of organisms at each trophic level. Microbivore species composition significantly affected bacterivore and fungivore biomass but not bacterial, fungal or predator biomass. Greatest bacterivore and fungivore biomass was found in food web A, intermediate biomass in food web AB, and smallest biomass in food web B. Basal resource addition increased the biomass of microbes and microbivores but did not affect predator biomass. Importantly, microbivore species composition did not significantly modify the effect of additional resources on trophic-level biomasses. The presence of a competitor reduced the biomass of A. tricornus and Aph. avenae, in that the biomass of these species was less in food web AB than in food web A, whereas the biomass of C. elegans and Aphelenchoides sp. was not affected by their potential competitors. The biomass of Aph. avenae increased with additional resources in the absence of the competitor only, while the biomass of A. tricornus and Aphelenchoides sp. increased also in the presence of their competitors. The results imply that microbivore species composition may determine the second-level biomass in simple microbe-nematode food webs, but may not significantly affect biomass at other levels or modify the response of trophic-level biomasses to enhanced basal resources. The study also shows that even if the role of predation in a food web is diminished, the positive response of organisms to increased resource availability may still be hindered by competition. Received: 22 June 1998 / Accepted: 28 August 1998     

Fed-batch fermentor synthesis of 3-dehydroshikimic acid using recombinant Escherichia coli.

3-Dehydroshikimic acid (DHS), in addition to being a potent antioxidant, is the key hydroaromatic intermediate in the biocatalytic conversion of glucose into aromatic bioproducts and a variety of industrial chemicals. Microbial synthesis of DHS, like other intermediates in the common pathway of aromatic amino acid biosynthesis, has previously been examined only under shake flask conditions. In this account, synthesis of DHS using recombinant Escherichia coli constructs is examined in a fed-batch fermentor where glucose availability, oxygenation levels, and solution pH are controlled. DHS yields and titers are also determined by the activity of 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) synthase. This enzyme's expression levels, sensitivity to feedback inhibition, and the availability of its substrates, phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P), dictate its in vivo activity. By combining fed-batch fermentor control with amplified expression of a feedback-insensitive isozyme of DAHP synthase and amplified expression of transketolase, DHS titers of 69 g/L were synthesized in 30% yield (mol/mol) from D-glucose. Significant concentrations of 3-dehydroquinic acid (6.8 g/L) and gallic acid (6.6 g/L) were synthesized in addition to DHS. The pronounced impact of transketolase overexpression, which increases E4P availability, on DHS titers and yields indicates that PEP availability is not a limiting factor under the fed-batch fermentor conditions employed.     

Acid Carboxypeptidases in Grains and Leaves of Wheat, Triticum aestivum L

Extracts of resting and germinating (3 days at 20°C) wheat (Triticum aestivum L. cv Ruso) grains rapidly hydrolyzed various benzyloxycarbonyldipeptides (Z-dipeptides) at pH 4 to 6. Similar activities were present in extracts of mature flag leaves. Fractionation by chromatography on CM-cellulose and on Sephadex G-200 showed that the activities in germinating grains were due to five acid carboxypeptidases with different and complementary substrate specificities. The wheat enzymes appeared to correspond to the five acid carboxypeptidases present in germinating barley (L Mikola 1983 Biochim Biophys Acta 747: 241-252). The enzymes were designated wheat carboxypeptidases I to V and their best or most characteristic substrates and approximate molecular weights were: I, Z-Phe-Ala, 120,000; II, Z-Ala-Arg, 120,000; III, Z-Ala-Phe, 40,000; IV, Z-Pro-Ala, 165,000; and V, Z-Pro-Ala, 150,000. Resting grains contained carboxypeptidase II as a series of three isoenzymes and low activities of carboxypeptidases IV and V. During germination the activity of carboxypeptidase II decreased, those of carboxypeptidases IV and V increased, and high activities of carboxypeptidases I and III appeared. The flag leaves contained high activity of carboxypeptidase I and lower activities of carboxypeptidases II, IV, and V, whereas carboxypeptidase III was absent.     

Transport-related amino acid metabolism in germinating barley grains

When eight [¹⁴C]-labelled amino acids were separately injected into the endosperm of germinating (4 days at 20°C) barley (Hordeum vulgare L. cv. Himalaya) grains, the label was rapidly taken up by the scutellum and further transported to the shoot and roots. Some of the amino acids (leucine, lysine and asparagine) were transported in an intact form through the scutellum to the seedling, whilst glutamic acid and aspartic acid were largely converted to glutamine in the scutellum. Proline was mainly transported unchanged, but a small part of the label appeared in glutamine. Arginine was mostly broken down in the scutellum, possibly providing ammonia for the synthesis of glutamine. During further transport in the seedling there was a partial transfer of label from glutamine to asparagine, particularly in the shoot. None of the amino acids used supplied carbon for the synthesis of sucrose, glucose or fructose. Glutamine synthetase activity was particularly high in the scutellum during the period of rapid amino acid transport.     

Aspartic proteinase from barley grains is related to mammalian lysosomal cathepsin D

Resting barley (Hordeum vulgare L.) grains contain acid-proteinase activity. The corresponding enzyme was purified from grain extracts by affinity chromatography on a pepstatin-Sepharose column. The pH optimum of the affinity-purified enzyme was between 3.5 and 3.9 as measured by hemoglobin hydrolysis and the enzymatic activity was completely inhibited by pepstatin a specific inhibitor of aspartic proteinases (EC 3.4.23). Further purification on a Mono S column followed by activity measurements and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the affinity-purified enzyme preparation contained two active heterodimeric aspartic proteinases: a larger 48k Da enzyme, consisting of 32-kDa and 16-kDa subunits and a smaller one of 40 kDa, consisting of 29-kDa and 11-kDa subunits. Separation and partial amino acid sequence analysis of each subunit indicate that the 40-kDa enzyme is formed by proteolytic processing of the 48k Da form. Amino-acid sequence alignment and inhibition studies showed that the barley aspartic proteinase resembles mammalian lysosomal cathepsin D (EC 3.4.23.5).     

Localization and activity of various peptidases in germinating barley

Summary Germinating barley grains contain at least eight different peptidases: three carboxypeptidase (pH optima 4.8, 5.2, and 5.7), three aminopeptidases which act on aminoacyl--naphthylamides (pH opitima in the hydrolysis of di- and tripeptides at pH 5.8–6.5), and two peptidases which hydrolyse Ala-Gly and Leu-Tyr optimally at pH 7.8 and 8.6 respectively. We have determined the activities of these enzymes in the different tissues of non-germinated grains and followed the changes in the activities during a 5-day germination at 16°C.The aleurone layers contain high activities of all three groups of peptidases; there are no changes in the activities of the five aminopeptidases on germination, while the carboxypeptidases exhibit a small increase of activity. The starchy endosperms contain high carboxypeptidase activities, which increase during germination, but are totally devoid of the five aminopeptidases.All the peptidases exhibit high activities in the scutella; the carboxypeptidases and the enzymes acting on Ala-Gly and Leu-Tyr increase in activity during germination, while the naphthylamidase activities remain constant.The three peptidase groups occur in the seedling as well, but compared to the other tissues the carboxypeptidase activities are very small and the naphthylamidase activities are very high. The last-named enzymes seem to be characteristic for growing tissues.The starchy endosperm contains about two thirds of the total reserve proteins of the grain. Its internal pH during germination is 5.0–5.2, a value at which all the carboxypeptidases are highly active. As these enzymes are present in high concentrations in this tissue, it is probable that they have a central role in the mobilization of the reserve proteins during germination. The high peptidase activities of the scutellum, on the other hand, suggest that some of the hydrolysis products are absorbed as peptides and these are further hydrolysed to amino acids in this tissue.Abbreviations used DTT dithiothreitol - GA₃ gibberellic acid - -NA -naphthylamide - TNBS 2,4,6-trinitrobenzene sulphonic acid - Z- N-carbobenzoxy     

Purification and partial characterization of barley leucine aminopeptidase

A peptidase acting on Leu-Gly-Gly and Leu-Tyr at pH 8 to 10 was purified about 670-fold from germinated grains of barley (Hordeum vulgare L.). Gel electrophoretic analyses indicated a purity of about 90%. The purified enzyme is remarkably similar to mammalian leucine aminopeptidases (EC 3.4.1.1) both in chemical and in enzymatic properties. It has a sedimentation constant of 12.7S and a molecular weight of about 260,000. The enzyme has a high activity on leucine amide and di- and tripeptides with N-terminal leucine or methionine; leucyl-β-naphthylamide, in contrast, is hydrolyzed very slowly. The enzyme also liberates N-terminal amino acids from the insulin B chain. The pH optima for the hydrolysis of different substrates depend on the buffers used; highest reaction rates are generally obtained at pH 8.5 to 10.5. Mg²⁺ and Mn²⁺ ions stabilize (and probably activate) the enzyme. In contrast to mammalian leucine aminopeptidases, the barley enzyme is inactivated in the absence of reducing sulfydryl compounds.