A simple and specific assay of glycosyltransferase and glycosidase activities by an enzyme-linked immunosorbent assay method, and its application to assay of galactosyltransferase activity in sera from patients with cancer.

A simple, sensitive, and specific assay method for glycosyltransferase and glycosidase activities has been established by means of an enzyme-linked immunosorbent assay (ELISA) using monoclonal antibody, H-11 directed to lactoneotetraosylceramide (nLc4Cer). Enzyme activity was determined by assaying the amount of reaction product, nLc4Cer with the ELISA method. For the assay of galactosyltransferase activity, lactotriaosylceramide (Lc3Cer) immobilized on a 96-well microtiter plate was incubated with bovine milk galactosyltransferase in cacodylate buffer (pH 6.8) containing Triton CF-54, Mn2+, and UDP-galactose. Optimum incubation conditions for the enzyme were determined. Glycosidase activity was also assayed by the ELISA method by using Clostridium perfringens sialidase and neolacto-series gangliosides as substrates, and the substrate specificities towards the gangliosides were examined. By this method, 3-100 pmol of reaction product could be determined. The assay method has several advantages as follows: 1, the method is simple; 2, separation of the reaction product is not required; 3, quantification and identification of the reaction product were done simultaneously; 4, naturally occurring substrates are available (especially for glycosidase); 5, many samples can be assayed in one microplate; 6, sensitivity is very high. The present method was applied for the detection of galactosyltransferase in human sera. Significant elevations of the galactosyltransferase levels were observed in the sera from cancer patients. The formation of nLc4Cer was confirmed by employing the TLC-immunostaining method for bands of Lc3Cer after incubation of the bands with serum and cofactors on an HPTLC plate.     

The metal ion activation of the alkaline β-glycerophosphatase of rabbit small intestine

1. The preparation of gram quantities of isolated epithelial-cell ;ghosts' from mucosal scrapings of rat small intestine is described. The method involves dispersing the tissue by gentle homogenization in 6% dextran in Krebs-Ringer phosphate, pH7.4, followed by filtration through nylon cloth and sedimentation by low-speed centrifuging. 2. The isolated epithelial-cell ;ghosts' contained all of the DNA, but only 52% of the protein and 53-57% of the RNA of the original homogenate. They contained most of the activity of the following enzymes found in the homogenate: aminopeptidase (71%); alkaline beta-glycerophosphatase (82%); invertase (92%); adenosine triphosphatase (93-116%); acid beta-glycerophosphatase (83%); nonspecific esterase (76%); succinate dehydrogenase (96%). Only small proportions of the total lactate-dehydrogenase (10%) and phosphoglucose-isomerase (2%) activities found in the homogenate were recovered in the isolated cell ;ghosts'. 3. The epithelial-cell ;ghost' preparation did not respire unless cofactors and substrates were added, and did not consume glucose or produce lactic acid from glucose. 4. The effect of varying the composition of the homogenization medium was studied. Concentrations of dextran (mol.wt. 15x10(4)) from 1 to 12%, solutions of dextrans (all at 6%) with mol.wt. varying between 3.6x10(4) and 2x10(6), and a solution of 8% polyethylene glycol (mol.wt. 4000) served equally well for the production of epithelial-cell ;ghosts'. Two of these solutions, however, 12% dextran (mol.wt.15x10(4)) and 6% dextran (mol.wt. 2x10(6)), were too viscous to allow the complete sedimentation of the cell ;ghosts' at low relative centrifugal forces. Omission of either Krebs-Ringer phosphate or dextran from the medium resulted in almost complete cell breakage during the homogenization. 5. The isolated cell ;ghosts' were used as a starting material for subcellular fractionation of rat intestinal mucosa by differential centrifugation. The distributions of protein and succinate-dehydrogenase activity among the fractions were compared with corresponding values in fractions isolated by differential centrifugation of mucosa homogenized in 0.3m-sucrose-5mm-EDTA, pH7.4. The method in which cell ;ghosts' were used as starting material gave a better separation and cleaner fractions than the method in which untreated mucosal scrapings were used.     

A di-N-acetylchitobiase activity is involved in the lysosomal catabolism of asparagine-linked glycoproteins in rat liver

A perfused rat liver was used to study the effects of 5-diazo-4-oxo-L-norvaline on lysosomal glycoprotein catabolism. Addition of this compound (1.0 mM) to the perfusate reduced activity of beta-aspartyl-N-acetylglucosylamine amidohydrolase by 99% in 1 h. Treated livers were unable to completely degrade endocytosed N-acetyl[14C]glucosamine-labeled asialo-alpha 1-acid glycoprotein as evidenced by a 50% reduction in radiolabeled serum glycoprotein secretion compared to controls. This decreased degradation was matched by a lysosomal accumulation of glycopeptides with the structure: GlcNAc beta(1-4)GlcNAc-Asn. The result suggested the presence of an unrecognized glycosidase in rat liver lysosomes, since this remnant was extended by one more GlcNAc residue than would have been expected after specific inactivation of the amidohydrolase. Such a novel enzyme would therefore catalyze cleavage of the N-acetylglucosamine residue at the reducing end of alpha 1-acid glycoprotein oligosaccharides only following removal of the linking Asn. The activity was then detected in lysosomal extracts by using intact asialo-biantennary oligosaccharides labeled with [3H] galactose or N-acetyl[14C]glucosamine residues as a substrate. To prevent simultaneous digestion of the material from its nonreducing end, beta-D-galactosidase in the enzyme extract was first inactivated with the irreversible active site-directed inhibitor, beta-D-galactopyranosylmethyl-p-nitrophenyltriazene. The observed di-N-acetylchitobiose cleaving activity worked optimally at pH 3.4 and was uniquely associated with the lysosomal fraction of the liver homogenate. The enzyme also cleaved triantennary chains and di-N-acetylchitobiose, but failed to hydrolyze substrates that had been reduced with NaBH4. The new glycosidase was well separated from N-acetyl-beta-D-glucosaminidase (assayed with p-nitrophenyl-beta-D-glucosaminide) by gel filtration chromatography and had an apparent molecular weight of 37,000. A similar enzyme that hydrolyzes di-N-acetylchitobiose had previously been found in extracts of human liver (Stirling, J. L. (1974) FEBS Lett. 39, 171-175).     

Variety and variability of glycosidase activities in an Oenococcus oeni strain collection tested with synthetic and natural substrates

Aims: To evaluate the capacity of Oenococcus oeni strains to release aroma compounds from glycosylated precursors by measuring glycosidase activities with both synthetic and natural substrates. Methods and Results: Five glycosidase activities were investigated in 47 O. oeni strains using synthetic substrates. This screening revealed that activity levels vary considerably, not only for each strain (depending on the substrate tested), but also between strains. Fifteen strains exhibiting different activity profiles were further analysed using natural substrates extracted from both untoasted and toasted oak. In the latter, various amounts of aromatic compounds were measured, thus confirming the specific potentials of the selected strains, but the results were different from those obtained using synthetic substrates. In addition, the use of toasted wood extracts significantly increased the release of wood aromas, which minimized differences between strains. Conclusions: The capability of O. oeni to hydrolysate glycoconjugate aroma precursors is strain‐dependent and variable, depending on the substrate. Significance and Impact of the Study: Instead of synthetic substrates, natural aroma precursors should be used for an adequate evaluation of the glycosidase potential of O. oeni.     

Subcellular distribution of glycosidases in human polymorphonuclear leucocytes.

The subcellular distribution of nine glycosidases were studied in fractions of homogenized human polymorphonuclear leucocytes (neutrophils) obtained by isopycnic centrifugation through linear sucrose density gradients. The substrates were 4-methylumbelliferyl glycosides. All nine glycosides were hydrolysed by enzymes in neutrophil cytosol fractions, and by enzymes in at least one granule population. alpha-Glucosidase activity sedimented in sucrose density gradients to a point (p = 1.180 g/ml) just above the specific granules, possibly the 'tertiary' granule population. The peak corresponding to alpha-glucosidase did not co-sediment with, but considerably overlapped, the peak corresponding to lactoferrin, a marker for specific granules (p = 1.187 g/ml). alpha-Galactosidase activity was found primarily in heavy azurophil granules (p = 1.222 g/ml). alpha-Mannosidase activity was found primarily in light azurophil granules (p = 1.206 g/ml), following the distribution of myeloperoxidase, the commonly used azurophil granule marker. beta-Glucosidase activity was concentrated in mitochondrial fractions (p = 1.160 g/ml). All other glycosidases presented complex distributions, with activities not restricted to one granule class. Granule-associated glycosidase activities were increased 2--38 times when measured in the presence of 0.05% Triton X-100, indicating latency of the enzymes within granules.     

Screening of Lactobacillus spp. and Pediococcus spp. for glycosidase activities that are important in oenology

AIMS: To assess glycosidase activities from a range of Lactobacillus and Pediococcus species and characterize these activities under conditions pertinent to the wine industry. METHODS AND RESULTS: Lactic acid bacteria were cultured in MRS broth supplemented with apple juice before being harvested, washed and assayed for glycosidase activity using p-nitrophenol-linked substrates. All strains exhibited a detectable capacity for the hydrolysis of the beta- and alpha-d-glucopyranosides. The magnitude of these activities and their response to the physico-chemical parameters investigated varied in a strain-dependent manner. The use of an assay buffer with a pH below 4 generally resulted in a reduced hydrolysis of both substrates while temperature optima ranged between 35 and 45 degrees C. The effect of the inclusion of ethanol in the assay buffer (up to 12%, v/v) ranged from near complete inhibition to increases in activity approaching 80%. With the clear exception of a single strain, glucose and fructose (0.1-20 g l(-1)) acted as inhibitors. An assessment of glycosidase activity during simultaneous exposure to glucose and ethanol at a pH of 3.5 suggested that ethanol decreased loss of activity under these wine-like conditions. CONCLUSIONS: Lactobacillus spp. and Pediococcus spp. possess varying degrees of beta- and alpha-d-glucopyranosidase activities, which in turn are influenced differently by exposure to ethanol and/or sugars, temperature and pH. Several strains appeared suited for further evaluation under winemaking conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This work highlights the fact that strains of Lactobacillus and Pediococcus have the potential to influence the glycoside composition of wine. Tailoring of wine may therefore be possible through selective application of strains or enzymatic extracts thereof.     

The effect of the carbohydrate growth substrate on the glycosidase activity of hemicellulose-degrading rumen bacterial isolates

Thirteen strains of hemicellulose-degrading bacteria isolated from the rumen were grown on ten different monosaccharide, disaccharide and hemicellulosic poly-saccharide substrates in vitro , and the range and specific activities of the glycosidase enzymes formed were determined. Alkaline phosphatase activities were also measured. The specific activities were affected by the carbohydrate source supplied in the growth medium and were usually reduced in glucose grown cells, and less frequently following growth on cellobiose; the responses to other substrates were both enzyme and organism dependent. In several of the strains examined many of the enzymes were found following growth on all of the substrates tested, although there were wide variations in the specific activities. The activities of several of the glycosidases were increased after growth on hemicellulosic polysaccharides.     

Activation of multiple protein kinases during the burst in protein phosphorylation that precedes the first meiotic cell division in Xenopus oocytes

A number of different protein and peptide substrates were used to identify and characterize stimulated kinase activities in Xenopus oocyte extracts prepared during the major burst in protein phosphorylation that precedes meiotic cell division. While total cAMP-dependent protein kinase activity in the cytosol was not stimulated, this kinase was the major kinase phosphorylating a number of the substrates and consequently had to be inhibited to prevent its masking cAMP-independent protein kinase activities. Sizable stimulations of kinase activities were then observed in extracts from progesterone-treated oocytes as compared to controls when the following substrates were utilized: Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) (8-fold); the synthetic peptide, Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala, the sequence of which is based on that of a phosphorylation site in ribosomal protein S6 (8-fold); ribosomal protein S6 (8-fold); histone H1 (5-fold); skeletal muscle glycogen synthase (3-fold); and myelin basic protein (30-fold). When these substrates were used to assay extracts fractionated on DEAE-Sephacel, at least three distinct peaks of stimulated kinase activity were detected, eluting at 0.12, 0.17, and 0.21 M NaCl. These peaks were tentatively designated M-phase Activated Kinases(s), MAK-H, MAK-S, and MAK-M, respectively. Using histone H1 as a selective probe for MAK-H and S6 peptide or Kemptide as probes for MAK-S, the kinase activities comprising these peaks were found to cycle with the meiotic cell cycle.     

A comparison of aroclor 1254-induced and uninduced rat liver microsomes to human liver microsomes in phenytoin O-deethylation, coumarin 7-hydroxylation, tolbutamide 4-hydroxylation, S-mephenytoin 4'-hydroxylation, chloroxazone 6-hydroxylation and testosterone 6beta-hydroxylation

Aroclor 1254-induced rat liver homogenate supernatant (liver S-9) is routinely used as an exogenous metabolic activation system for the evaluation of mutagenicity of xenobiotics. The purpose of this study is to evaluate whether results obtained with Aroclor 1254-induced liver microsomes would be relevant to human. Aroclor 1254-induced and uninduced rat liver microsomes were compared to human liver microsomes in the metabolism of substrates which are known to be selectively metabolized by the major human cytochrome P450 (CYP) isoforms. The activities studied and the major CYP isoforms involved were as follows: phenacetin O-deethylation (CYP1A2); coumarin 7-hydroxylation, (CYP2A6); tolbutamide 4-hydroxylation (CYP2C9), S-mephenytoin 4'-hydroxylation (CYP2C19); dextromethorphan O-demethylation (CYP2D6); chloroxazone 6-hydroxylation (CYP2E1); and testosterone 6beta-hydroxylation (CYP3A4). We found that both induced and uninduced rat liver microsomes were active in all the pathways studied with the exception of coumarin 7-hydroxylation. Coumarin 7-hydroxylation was observed with human liver microsomes but not the rat liver microsomes. Aroclor-1254 was found to induce all activities measured, with the exception of coumarin 7-hydroxylation. Dextromethorphan O-deethylation activity was higher in the rat liver microsomes than the human liver microsomes. Testosterone 6beta-hydroxylation activity was found to be similar between the human liver microsomes and the induced rat liver microsomes. Our results suggest that experimental data obtained with Aroclor 1254-induced rat liver microsomes may not always be relevant to human.     

Beta-galactosidase, phospho-beta-galactosidase and phospho-beta-glucosidase activities in lactobacilli strains isolated from human faeces

AIMS: Lactose intolerance, a serious health problem for Asians, can be solved using probiotic bacteria having high lactose hydrolysis activities. We determined the distribution of beta-galactosidase (beta-gal), phospho-beta-galactosidase (P-betagal) and phospho-beta-glucosidase (P-beta-glc) activities in species of lactic acid bacteria (LAB) isolated from human faeces to select strains for potential use in fermented dairy products, e.g. yogurt. METHODS AND RESULTS: The sugar substrates, o-nitrophenyl-beta-D- galactopyranoside 6-phosphate and o-nitrophenyl-beta-D-glucopyranoside 6-phosphate, were synthesized and used to measure respectively P-beta-gal and P-beta-glc activities. Sixty-five toluene-treated strains were examined for three lactase enzyme activities. Lactobacillus mucosae OLL2848 showed the highest beta-gal activity (107.09 U mg(-1) of protein) among the Lactobacillus strains from human faeces. Lactobacillus gasseri OLL2836 and OLL 2948 showed the highest P-beta-gal (46.58 U) and P-beta-glc (50.19 U)activity, respectively, with no beta-gal activity. CONCLUSIONS: The expression of P-beta-glc induced by lactose was characteristic of Lact. gasseri. Because this LAB is a major inhabitant of the human intestine. This enzyme is a key glycosidase involved in lactose utilization. SIGNIFICANCE AND IMPACT OF STUDY: This is the first report describing the distribution of three glycosidase activities used in lactose metabolism in LAB isolated from human faeces for possible use in functional foods.     

Glycosidases of rat Kupffer cells, hepatocytes and peritoneal macrophages.

The acid glycosidase content of rat liver Kupffer cells was compared with that of hepatocytes and resident peritoneal macrophages. Homogenates of all these cells were able to hydrolyze the p-nitrophenyl glycosides of N-acetylglucosamine, N-acetylgalactosamine, glucose, galactose, fucose and mannose, but not xylose. Activity was greatest against the N-acetylglucosaminoside. With Kupffer cell homogenates, most of the glycosidases behaved as if they were lysosomal enzymes. When expressed as rates of hydrolysis per 10(6) cells, activities against a given substrate by homogenates from the three cell types generally agreed within a factor of 2-4. Significant differences between cell types were found, however, when ratios of glycosidase activities were compared. Furthermore, even though the quantity of glycosidase per cell was similar in Kupffer cells and hepatocytes, the glycosidase concentrations were much higher in the former cells, since Kupffer cells are much smaller than hepatocytes.     

Assay system for simultaneous measurement of three steroidogenic enzyme activities in rat and human testis--effect of human chorionic gonadotropin

An assay system that measures the enzymatic activities (17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase) in the delta 4 pathway of testosterone biosynthesis using rat and human testicular homogenate was examined. This system involves the simultaneous separation of the steroid intermediates by a three-step TLC procedure. The observed Rf values were 0.78 for progesterone (P), 0.59 for 17 alpha-hydroxyprogesterone (17 alpha-HP), 0.70 for androstenedione (A), 0.5 for testosterone, 0.64 for dihydrotestosterone, and 0.45 for 3 alpha, 17 beta-androstanediol. The identification of these steroid intermediates was further accomplished by acetylation and rechromatography of the representative samples along with the authentic standards and by recrystallization to constant specific activity until three consecutive crystallizations were within +/- 5% of the mean value. Incubation time up to 30 min and increasing protein concentrations showed a linear relationship with respect to these three enzymatic activities. The optimum temperature for these enzymatic activities varied from 32 to 34 degrees C, with a sharp decline between 37 and 40 degrees C. The Michaelis constants (Km) for the rat testis homogenate samples were 0.17 microM for P, 0.22 microM for 17 alpha-HP, and 2.5 microM for A, while for the human testis the Km values were 1.2, 2.2, and 2.3 microM, respectively, for these substrates. The concentrations of the endogenous steroid substrates present in these homogenate samples did not alter the Km or Vmax values. The effect of human chorionic gonadotropin (hCG) in vitro on these steroidogenic enzyme activities was also studied. In the rat testis, 10 IU of hCG produced a significant rise in all the three enzyme activities whereas in the human testis 10 and 30 IU of hCG showed no significant change in any of these enzymatic activities. However, 100 IU of hCG resulted in a significant increase in 17 alpha-hydroxylase and 17,20-desmolase activities in the human testis. These studies suggest that this assay system for the measurement of these enzymatic activities using a testicular homogenate sample provides consistent and reproducible results. Based on the sensitivities of the measurements and our experience with testicular biopsy technique, we conclude that a routine testicular biopsy in the human should provide sufficient tissue to run these enzymatic assays.     

Ecto- and cytosolic 5'-nucleotidases in normal and AMP deaminase-deficient human skeletal muscle

In skeletal muscle, adenosine monophosphate (AMP) is mainly deaminated by AMP deaminase. However, the C34T mutation in the AMPD1 gene severely reduces AMP deaminase activity. Alternatively, intracellular AMP is dephosphorylated to adenosine via cytosolic AMP 5'-nucleotidase (cN-I). In individuals with a homozygous C34T mutation, cN-I might be a more important pathway for AMP removal. We determined activities of AMP deaminase, cN-I, total cytosolic 5'-nucleotidase (total cN), ecto-5'-nucleotidase (ectoN) and whole homogenate 5'-nucleotidase activity in skeletal muscle biopsies from patients with different AMPD1 genotypes [homozygotes for C34T mutation (TT); heterozygotes for C34T mutation (CT); and homozygotes for wild type (CC): diseased controls CC; and normal controls CC]. AMP deaminase activity showed genotype-dependent differences. Total cN activity in normal controls accounted for 57+/-22% of whole homogenate 5'-nucleotidase activity and was not significantly different from the other groups. A weak inverse correlation was found between AMP deaminase and cN-I activities (r2=0.18, p<0.01). There were no significant differences between different groups in the activities of cN-I, whole homogenate 5'-nucleotidase and ectoN, or in cN-I expression on Western blots. No correlation for age, fibre type distribution and AMPD1 genotype was found for whole homogenate nucleotidase, total cN and cN-I using multiple linear regression analysis. There was no gender-specific difference in the activities of whole homogenate nucleotidase, total cN and cN-I. The results indicate no changes in the relative expression or catalytic behaviour of cN-I in AMP deaminase-deficient human skeletal muscle, but suggest that increased turnover of AMP by cN-I in working skeletal muscle is due to higher substrate availability of AMP.     

Ribosomes of Brachionus plicatilis (Rotifera) – distribution in homogenate fractions and general properties

(1) The content of DNA, RNA and of proteins of Brachionus plicatilis was estimated and the distribution of RNA and of proteins of different homogenate fractions characterised. (2) Ribosomes were isolated from Brachionus plicatilis homogenates and were characterised by gradient centrifugation. (3) Unlike the RNA content, the yield of ribosomes from different homogenate fractions is strongly dependent on the concentration of Mg²⁺-ions in the buffers. Likewise resuspension of ribosomes is more effective in Mg²⁺- (or Ca²⁺-) free buffers. (4) Dissociation of ribosomes was brought about by centrifugation of ribosomes in gradients containing less than 4 mM Mg²⁺. In this case, beside the peaks of subunits, a peak in the region of 80 S remained which vanished only under conditions destroying ribosomal material altogether. (5) Proteins were isolated from ribosomal subunits and from undissociated ribosomes and were characterised by two-dimensional gel electrophoresis techniques. Patterns of 51 spots were regularly obtained from large subunits and patterns of 41 spots from small subunits. The undissociated ribosomes showed 83 spots, most of which could be attributed to the large or the small subunit. The ribosomal proteins have molecular masses of between 11000 and 56000 Da, while the molecular mass of the total protein content of Brachionus ribosomes was estimated to be 1.8 ±0.5) ×10⁶ Da.     

A distinctive activity of 5'-nucleotidase for dTMP in rat liver mitochondria

The activities of 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5); adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4); AMP deaminase (AMP aminohydrolase, EC 3.5.3.6), and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver 5'-Nucleotidase (5'Nase) and ATP-(Mg2+)-ase activities were compared with similar enzyme activities in the plasma membrane (PM) fraction, obtained from the same biological material. In the regenerating liver, 5'Nase for dTMP diminished its activity by 56% (24 h after partial hepatectomy) and 35 +/- 4% for all substrates in the PM fraction (48 h after operation). In mitochondria, 5'Nase for dTMP manifests sigmoidal substrate activity curve (in contrast with all substrates in the PM fraction and remaining substrates in mitochondria). In vivo 5-azacytidine (a) administered 1 h after partial hepatectomy, prevented changes of 5'Nase activity: (b) administered 24 or 48 h after partial hepatectomy, stabilized low 5'Nase activity (in mitochondria for dTMP, in the PM fraction for all substrates) and decreased ATP-(Mg2+)-ase activity by 51 and 31% in mitochondria and the PM fraction respectively.     

Studies on brain cytosol neuraminadase. I. Isolation and partial characterization of two forms of the enzyme from pig brain.

1. Two forms of cytosol neuraminidase (EC 3.2.1.18) (neuraminidase A and neuraminidase B) were isolated and purified from pig brain homogenate, by proceeding through the following steps: centrifugation of brain homogenate at 105 000 X g (1h); ammonium sulphate fractionation (35-55% saturated fraction); column chromatography on Biogel A 5 m; column chromatography on hydroxy apatite/cellulose gel; affinity chromatography on Affinose-tyrosyl-p-nitrophenyloxamic acid. The separation of the two forms of neuraminidase was provided by chromatography on hydroxylapatite/cellulose gel. Neuraminidase A was purified about 500-fold; neuraminidase B about 400-fold. 2. The pH optima and the maximum activities in various buffers were different for neuraminidase A and B (for instance the pH optimum was in sodium acetate/acetic acid buffer, 4.7 for neuraminidase A and 4.9 for neuraminidase B). Ions affected in a different way the two enzymes: K+ activated neuraminidase A but not neuraminidase B; Na+ and Li+ inhibited neuraminidase A at a higher degree than neuraminidase B. Neuraminidase B seemed to be moderately activated by some bivalent cations (Ca2+; Mg2+; Zn2+); neuraminidase A did not. The Km values for sialyllactose were different: 2.2-10(-3) M for neuramindase A; 0.46-10(-3) M for neuraminidase B.     

Androgen hydroxylation catalysed by a cell line (SD1) that stably expresses rat hepatic cytochrome P-450 PB-4 (IIB1).

Androgen hydroxylation catalysed by Chinese hamster fibroblast SD1 cells, which stably express cytochrome P-450 form PB-4, the rat P450IIB1 gene product, was assessed and compared to that catalysed by purified cytochrome P-450 PB-4 isolated from rat liver. SD1 cell homogenates catalysed the NADPH-dependent hydroxylation of androstenedione and testosterone with a regioselectivity very similar to that purified by P-450 PB-4 (16 beta-hydroxylation/16 alpha-hydroxylation = 6.0-6.8 for androstenedione; 16 beta/16 alpha = 0.9 for testosterone). Homogenates prepared from the parental cell line V79, which does not express detectable levels of P-450 PB-4 or any other cytochrome P-450, exhibited no androgen 16 beta- or 16 alpha-hydroxylase activity. The hydroxylase activities catalysed by the SD1 cell homogenate were selectively and quantitatively inhibited (greater than 90%) by a monoclonal antibody to P-450 PB-4 at a level of antibody (40 pmol of antibody binding sites/mg of SD1 homogenate) that closely corresponds to the P-450 PB-4 content of the cells (48 pmol of PB-4/mg of SD1 homogenate). Fractionation of cell homogenates into cytosol and microsomes revealed that the P-450 PB-4-mediated activities are associated with the membrane fraction. Although the P-450 PB-4-specific content of the SD1 microsomes was 15% of that present in phenobarbital-induced rat liver microsomes, the P-450 PB-4-dependent androstenedione 16 beta-hydroxylase activity of the SD1 membrane fraction was only 2-3% of that present in the liver microsomes. This activity could be stimulated several-fold, however, by supplementation of SD1 microsomes with purified rat NADPH P-450 reductase. These studies establish that a single P-450 gene product (IIB1) can account for the hydroxylation of androgen substrates at multiple sites, and suggest that SD1 cells can be used to assess the catalytic specificity of P-450 PB-4 with other substrates as well.     

Comparative xenobiotic metabolism capacities and pesticide sensitivity in adults of Solea solea and Solea senegalensis

The measurement of enzymatic activities involved in xenobiotic biotransformation was carried out in adults of Solea solea and Solea senegalensis. The hepatic enzymes analysed were cytochrome P450 (CYP) related activities using eight fluorometric substrates and carboxylesterases (CbE). The conjugating activities of glutathione S-transferase (GST) and UPD-glucuronosyltransferase (UDPGT) were also assessed. Specific mammalian inhibitors were used as diagnostic tools for related activities of CYP1A (α-naphthoflavone; αNF), CYP2B6 and CYP2C19 (ticlopidine) and CYP3A4 (ketoconazole). The in vitro sensitivity to organophosphorous pesticides (OP) was tested in the S10 homogenate of brain (acetylcholinesterase-AChE) and liver (CbE). Furthermore, the pesticide chlorpyrifos oxon (CLPO) was used to explore the OP sensitivity of CbE of both species in two subcellular fractions (microsomes and cytosol), using two substrates. Overall, only two parameters confirmed species differences: EROD and cytosolic CbE being significantly elevated (p < 0.05) in the common sole, S. solea. A high inhibition of CYP1A related activities using several fluorometric substrates (ER, MR and CEC) after in vitro incubation with αNF confirmed all measure CYP1A1-related activities whereas ketoconazole was more specific for BFCOD (CYP3A4). Pesticide sensitivity was similar for brain AChE but hepatic CbE had a protective role that was species and pesticide dependent.     

The isolation and properties of epithelial-cell `ghosts' from rat small intestine

1. The preparation of gram quantities of isolated epithelial-cell `ghosts'' from mucosal scrapings of rat small intestine is described. The method involves dispersing the tissue by gentle homogenization in 6% dextran in Krebs–Ringer phosphate, pH7·4, followed by filtration through nylon cloth and sedimentation by low-speed centrifuging. 2. The isolated epithelial-cell `ghosts'' contained all of the DNA, but only 52% of the protein and 53–57% of the RNA of the original homogenate. They contained most of the activity of the following enzymes found in the homogenate: aminopeptidase (71%); alkaline β-glycerophosphatase (82%); invertase (92%); adenosine triphosphatase (93–116%); acid β-glycerophosphatase (83%); nonspecific esterase (76%); succinate dehydrogenase (96%). Only small proportions of the total lactate-dehydrogenase (10%) and phosphoglucose-isomerase (2%) activities found in the homogenate were recovered in the isolated cell `ghosts''. 3. The epithelial-cell `ghost'' preparation did not respire unless cofactors and substrates were added, and did not consume glucose or produce lactic acid from glucose. 4. The effect of varying the composition of the homogenization medium was studied. Concentrations of dextran (mol.wt. 15×10⁴) from 1 to 12%, solutions of dextrans (all at 6%) with mol.wt. varying between 3·6×10⁴ and 2×10⁶, and a solution of 8% polyethylene glycol (mol.wt. 4000) served equally well for the production of epithelial-cell `ghosts''. Two of these solutions, however, 12% dextran (mol.wt.15×10<sup>4</sup>) and 6% dextran (mol.wt. 2×10<sup>6</sup>), were too viscous to allow the complete sedimentation of the cell `ghosts'' at low relative centrifugal forces. Omission of either Krebs–Ringer phosphate or dextran from the medium resulted in almost complete cell breakage during the homogenization. 5. The isolated cell `ghosts'' were used as a starting material for subcellular fractionation of rat intestinal mucosa by differential centrifugation. The distributions of protein and succinate-dehydrogenase activity among the fractions were compared with corresponding values in fractions isolated by differential centrifugation of mucosa homogenized in 0·3m-sucrose–5mm-EDTA, pH7·4. The method in which cell `ghosts'' were used as starting material gave a better separation and cleaner fractions than the method in which untreated mucosal scrapings were used.     

Seasonal and spatial distribution of extracellular enzymatic activities and microbial incorporation of dissolved organic substrates in marine sediments

Seasonal and spatial distributions of extracellular enzymatic activities and microbial incorporations of dissolved organic substrates were followed in sediments of the brackish water Kiel Bight (Baltic Sea, Federal Republic of Germany). Enzymatic hydrolysis of polymeric organic compounds was determined by means of fluorogenic substrates (4-methylumbelliferyl-beta-d-glucoside, l-leucine-4-methylcoumarinyl-7-amide hydrochloride); incorporation of dissolved organic substrates into microbial biomass was measured by using tritiated substances (acetate, leucine, and thymidine). Based on a recently developed core injection technique, substrates were injected in microliter portions into undisturbed sediment cores. Enzymatic and incorporation activities underwent strong seasonal variations related to the enrichment of organic material in the sediment surface following sedimentation events. The input of the phytoplankton bloom during autumn caused stimulation of both enzymatic hydrolysis of polymeric organic compounds and microbial incorporation of dissolved organic substrates. Following input by spring phytoplankton bloom, mainly incorporation activities were stimulated. In late spring the development of the benthic fauna obviously greatly influenced microbial activities. During summer individual periods of high microbial activities were observed which might be traced back to short-term sedimentation events. The high microbial incorporation of leucine and thymidine during winter demonstrated that the nutrient supply rather than temperature is the dominating factor determining microbial production. Stimulation of microbial activities arose from the sediment surface and spread out relatively quickly into deeper horizons. Generally, the sediments were characterized by distinct patterns of interrelationships between the individual parameters of microbial activities measured.