Biosynthesis of heparin. Effects of n-butyrate on cultured mast cells

Murine mastocytoma cells were incubated in vitro with inorganic [35S]sulfate, in the absence or presence of 2.5 mM n-butyrate, and labeled heparin was isolated. The polysaccharide produced in the presence of butyrate showed a lower charge density on anion exchange chromatography than did the control material and a 3-fold increased proportion (54 versus 17% for the control) of components with high affinity for antithrombin. Structural analysis of heparin labeled with [3H] glucosamine in the presence of butyrate showed that approximately 35% of the glucosamine units were N-acetylated, as compared to approximately 10% in the control material; the nonacetylated glucosamine residues were N-sulfated. The presence of butyrate thus leads to an inhibition of the N-deacetylation/N-sulfation process in heparin biosynthesis, along with an augmented formation of molecules with high affinity for antithrombin. Preincubation of the mastocytoma cells with butyrate was required for manifestation of either effect; when the preincubation period was reduced from 24 to 10 h the effects of butyrate were no longer observed. Assays for microsomal N-acetylheparosan deacetylase activity failed to show any significant inhibition of the enzyme at butyrate concentrations well above those found to affect heparin biosynthesis in intact mastocytoma cells. Moreover, a polysaccharide formed on incubating mastocytoma microsomal fraction with UDP-[3H]glucuronic acid, UDP-N-acetylglucosamine, and 3'-phosphoadenylylsulfate in the presence of 5 mM butyrate showed the same N-acetyl/N-sulfate ratio as did the corresponding control polysaccharide, produced in the absence of butyrate. These findings suggest that the effect of butyrate on heparin biosynthesis depends on the integrity of the cell.     

Biosynthesis of heparin. Modulation of polysaccharide chain length in a cell-free system.

The formation of heparin-precursor polysaccharide (N-acetylheparosan) was studied with a mouse mastocytoma microsomal fraction. Incubation of this fraction with UDP-[3H]GlcA and UDP-GlcNAc yielded labelled macromolecules that could be depolymerized, apparently to single polysaccharide chains, by alkali treatment, and thus were assumed to be proteoglycans. Label from UDP-[3H]GlcA (approx. 3 microM) is transiently incorporated into microsomal polysaccharide even in the absence of added UDP-GlcNAc, probably owing to the presence of endogenous sugar nucleotide. When the concentration of exogenous UDP-GlcNAc was increased to 25 microM the rate of incorporation of 3H increased and proteoglycans carrying polysaccharide chains with an Mr of approx. 110,000 were produced. Increasing the UDP-GlcNAc concentration to 5 mM led to an approx. 4-fold decrease in the rate of 3H incorporation and a decrease in the Mr of the resulting polysaccharide chains to approx. 6000 (predominant component). When both UDP-GlcA and UDP-GlcNAc were present at high concentrations (5 mM) the rate of polymerization and the polysaccharide chain size were again increased. The results suggest that the inhibition of polymerization observed at grossly different concentrations of the two sugar nucleotides, UDP-GlcA and UDP-GlcNAc, may be due either to interference with the transport of one of these precursors across the Golgi membrane or to competitive inhibition of one of the glycosyltransferases. The maximal rate of chain elongation obtained, under the conditions employed, was about 40 disaccharide units/min. The final length of the polysaccharide chains was directly related to the rate of the polymerization reaction.     

A single-vial biphasic liquid extraction assay for choline acetyltransferase using [3H]choline

A single-vial liquid extraction assay for choline acetyltransferase that uses [³H]choline as the labeled substrate has been devised. [³H]Choline is incubated with an excess of acetyl-CoA in a small reaction vial which also serves as a scintillation vial. After a suitable reaction period, unreacted [³H]choline is quickly and quantitatively converted to phosphoryl-[³H]choline by the addition of an excess of choline kinase. This treatment is followed by the addition of scintillation fluid containing sodium tetraphenylboron after which the vial is capped, shaken, and counted. A two-phase system is produced in which product [³H]acetylcholine is selectively extracted into the scintillation fluid, where it is counted. Phosphoryl-[³H]choline remains in the aqueous phase and is not counted. This assay is rapid, simple, and quite sensitive. In comparison to assays using acetyl-CoA as the labeled substrate, it is less sensitive to interference by other enzymes and thus more suitable for measuring choline acetyltransferase in crude extracts and in the initial stages of purification. Similar single-vial radiometric assays are described for choline kinase and acetyl-CoA hydrolases.     

The use of [3H]aniline to identify the essential carboxyl group in the bovine mitochondrial F1-ATPase that reacts with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline

The inactivation of the bovine heart mitochondrial F1-ATPase with 1-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) in the presence of [3H]aniline at pH 7.0 led to the covalent incorporation of 3H into the enzyme. When the ATPase was inactivated by 94% with 0.9 mM EEDQ in the presence of 3.6 mM [3H]aniline in a large-scale experiment in which the protein concentration was 21 mg/ml, 4.2 mol [3H]anilide were formed per mol enzyme, of which 0.35 mol was incorporated per mol of the alpha subunit and 1.0 mol was incorporated per mol of the beta subunit. Examination of a tryptic digest of the isolated alpha subunit revealed that the majority of the 3H was contained in a single tryptic peptide, which, when purified, was shown to contain the [3H]anilide of a glutamic acid residue which corresponds to alpha-Glu-402 of the Escherichia coli F1-ATPase. This residue was labeled to the extent of about 1.0 mol/mol enzyme. Analysis of tryptic peptides purified from the isolated beta subunit showed that 0.8 and 1.5 mol, respectively, of the [3H]anilides of beta-Glu-341 and beta-Glu-199 were formed per mol MF1 during the inactivation of the enzyme at 21 mg/ml. When the ATPase was inactivated by 90% at a protein concentration of 1.7 mg/ml by 0.9 mM EEDQ in the presence of 1.7 mM [3H]aniline, 3.1 mol [3H]anilide were formed per mol enzyme. From the analysis of the radioactive peptides purified from a tryptic digest of the labeled ATPase from this experiment it was estimated that 0.7 mol of the [3H]anilide of alpha-Glu-402, 0.3 mol of the [3H]anilide of beta-Glu-341, and 1.5 mol of the [3H]anilide of beta-Glu-199 were formed per mol F1-ATPase. Since beta-Glu-199 is labeled to the same extent in the two experiments while alpha-Glu-402 and beta-Glu-341 were not, suggests that the modification of beta-Glu-199 is responsible for inactivation of the enzyme by EEDQ.     

Radiometric assay for carboxypeptidase H (EC 3.4.17.10) and other carboxypeptidase B-like enzymes

Carboxypeptidase H, EC 3.4.17.10, also known as enkephalin convertase, carboxypeptidase E, and crino carboxypeptidase B, is an important enzyme involved in the biosynthesis of bioactive peptides. To assay the enzyme, tissues are homogenized in at least 20 vol (ml/g) of 0.025 M Tris-HCl buffer, pH 8, with 5 mg/ml of bovine serum albumin. After centrifugation, the supernatant is brought to pH 5.6 and centrifuged again. Following a 20-min preincubation in 2 mM CoCl2, the supernatant is incubated with 0.1 mM (final concentration) of the radioactive substrate [3H]benzoyl-Phe-Ala-Arg. The 100-microliters assay is stopped by the addition of 680 microliters of acetonitrile/0.25 M HCl (0.7/1). The 1.5-ml tube is transferred into a scintillation vial and is flushed with 4 ml of Econofluor, a water-immiscible scintillation fluid. The product, [3H]benzoyl-Phe-Ala, recovered in the organic phase, is counted directly with no interference from the substrate remaining in the aqueous phase. The blank is below 1%. Expressed in nanomoles per minute per milligram of tissue, the activity of the soluble enzyme in rat is 0.34 for striatum, 21.0 for pancreatic islet, 16.6 for anterior pituitary, 46.0 for intermediate pituitary, and 10.9 for neural pituitary. In every case 25 microM guanidinoethylmercaptosuccinic acid, an active site-directed inhibitor of carboxypeptidase H, completely inhibits the activity.     

A highly sensitive, mixed-phase assay for chloramphenicol acetyltransferase activity in transfected cells

We describe a simple, rapid yet extremely sensitive assay for chloramphenicol acetyltransferase (CAT) activity in extracts from transfected eukaryotic cells. Using our modified reaction conditions and the mixed-phase assay, less than 0.000010 unit of CAT activity in transfected cells can be reliably detected. The mixed-phase assay is based on the inability of the polar [3H]-acetyl-Coenzyme A (CoA) substrate to partition out of a urea containing aqueous phase into the nonpolar scintillation fluor, while the [3H]chloramphenicol reaction products partition into the toluene scintillation fluor and are quantitated by scintillation counting. The increased sensitivity of this assay is due to the optimization of the acetyl-CoA concentration, to a urea-containing aqueous phase which lowers the assay background, and to the use of extract blanks. The mixed-phase assay is simpler, is quantitative, uses less costly substrates, and is far more sensitive than the most widely used CAT assays, which require solvent extraction followed by thin-layer chromatography to separate the unreacted substrate from product.     

Sialidase in Cerebellar Granule Cells Differentiating in Culture

The optimal conditions for the assay of sialidase in cerebellar granule cells cultivated in vitro, established using [3H]GD1a and 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid (MUB-NeuNAc) as substrates, were the following: pH optimum for both substrates, 3.9; optimal molarity of sodium acetate/acetic acid buffer, 0.05 M with [3H]GD1a and 0.1 M for MUB-NeuNAc; substrate concentration for apparent maximal activity, 0.5 mM for MUB-NeuNAc and 0.1 mM for [3H]GD1a; enzyme activity linear with time up to 30 min with MUB-NeuNAc and up to 90 min with [3H]GD1a; and enzyme activity linear with enzyme protein content up to 80 micrograms with MUB-NeuNAc and up to 20 micrograms with [3H]GD1a. The assay with [3H]GD1a required the presence of Triton X-100 in a molar ratio to GD1a of 15:1. Poly-L-lysine, which was used for plating the cells, was capable of decreasing sialidase activity against [3H]GD1a/Triton X-100 when added to the incubation mixture. However, it had no effect on the enzyme working on MUB-NeuNAc. Using no more than 20 micrograms of cellular protein, the contamination, if any, by poly-L-lysine released from the dish was below the concentration limit exhibiting inhibition. Using the above optimal conditions, sialidase activity was measured during cerebellar granule cell differentiation in culture. From day 0 to day 7-8 in culture, the enzyme activity rose from 20 to 130 nmol of product released/h/mg of protein with MUB-NeuNAc and from 1 to 100 nmol of product released/h/mg of protein with [3H]GD1a.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of a dolichol phosphokinase activity associated with rat liver microsomes

Rat liver microsomes show a capacity to synthesize [1-3H]dolichyl phosphate from [1-3H]-dolichol. Formation of [1-3H]dolichyl phosphate increased continuously over 15 min although the reaction rate was never completely linear. Product formation was directly proportional to microsomal protein concentration between 1.1 mg/mL and the highest concentration tested, 5.5 mg/mL. The reaction rate was linear with respect to the dolichol content of the assay mixture to a saturation point (120 microM). An apparent Km of 50 microM was established for dolichol. The normal phosphate donor for the reaction is CTP and not ATP. The optimum concentration of CTP was 10 mM, and an apparent Km of 4 mM was calculated for this nucleoside triphosphate. The reaction was totally dependent on divalent metal ion, magnesium being more effective than calcium. The optimum concentration of magnesium ion and CTP were the same (10 mM), suggesting that MgCTP2- is utilized as the normal enzyme substrate. Activity measured in the absence of Triton X-100 was only 5% of the activity observed at the optimum (0.5% w/v) detergent concentration. The measurable levels of dolichol phosphokinase could be doubled by the inclusion of 10-15 mM NaF as phosphatase inhibitor. Optimal enzymatic activity was obtained between pH 7.0 and pH 7.5 and could be inhibited by EDTA. The sulfhydryl reagent DTT was slightly stimulatory while the product of the reaction, dolichyl phosphate, was noninhibitory at the highest concentration tested (13.8 microM). The second reaction product (CDP) inhibits the enzymatic phosphorylation of dolichol.     

Effects of ATP on Phosphatidylinositol-Phospholipase C and Inositol 1-Phosphate Accumulation in Rat Brain Synaptosomes

Incubation of rat brain synaptosomes prelabeled with [2-3H]inositol resulted in a time-dependent release of labeled inositol 1-phosphate. This process was Ca2+ dependent, and ATP (1 mM) enhanced the inositol 1-phosphate formation three- to fivefold. Using [1-14C]arachidonoyl-phosphatidylinositol which was introduced into saponin-permeabilized synaptosomes, ATP (1 mM) and free Ca2+ (approximately 20 microM) enhanced the phospholipase C hydrolysis of this substrate to form labeled diacylglycerol. When the same permeabilized synaptosomal preparation was incubated with [2-3H]inositol-phosphatidylinositol, ATP not only enhanced the formation of labeled inositol 1-phosphate, but also inhibited the conversion of inositol 1-phosphate to inositol. Furthermore, ATP appeared to reduce the Ca2+ requirement of the phosphatidylinositol-phospholipase C. Inhibition of the conversion of inositol 1-phosphate to inositol could not be overcome by increasing the Mg2+ concentration in the incubation medium. Although the ATP effect is not viewed as a receptor-mediated event, it is possible that such an event may occur in synaptosomes under conditions in which intrasynaptic Ca2+ concentration becomes elevated.     

A simple quantitative assay for chloramphenicol acetyltransferase by direct extraction of the labeled product into scintillation cocktail.

A simple, rapid, sensitive, quantitative, and inexpensive assay for chloramphenicol acetyltransferase (CAT) is described. The assay is based on the direct extraction of the products of the reaction into toluene-based liquid scintillation cocktail. The assay is carried out in 7-ml scintillation vials using 1 mM chloramphenicol and either 100 microM acetyl-CoA and 0.1 microCi of [3H]acetyl-CoA or 1 mM acetyl-CoA and 0.5 microCi of [3H]acetyl-CoA. After incubation, the reaction is terminated with 0.5 ml of 0.1 M sodium borate-5 M NaC, pH 9. The acetylchloramphenicols are extracted with 5 ml of 0.4% 2,5-diphenyloxazole-0.005% 1,4-bis(5-phenyloxazol-2-yl)benzene in toluene by a 30-s shaking. After a short centrifugation to clarify the layers, the vials are counted in a liquid scintillation counter. Extracted products are stable in the organic layer. Under these conditions, nearly 100% extraction of acetylchloramphenicols is shown using nonlabeled compounds and spectrophotometric methods. Using pure enzyme in the assay, linearity of activity with enzyme concentration, time, and temperature of incubation is demonstrated. Assays may even be carried out at 60 degrees C, where the enzyme activity is 3.4-fold higher than that at 23 degrees C. The increase in enzyme activity with increasing temperature is due to the increased formation of predominantly 3-acetyl and 1-acetylchloramphenicols and not to 1,3-diacetylchloramphenicol. The present assay compared very well with the standard assay using [14C]chloramphenicol and TLC. Using this assay, we measured quantitatively the CAT activity in extracts of pSV2-CAT-transfected CV-1 cells in 10 min and NIH 3T3 cell extracts in 60 min at 60 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new tritium-release assay for 25-hydroxyvitamin D-1 alpha-hydroxylase

A new, rapid assay for 1 alpha-hydroxylase has been developed using 25-hydroxy-[1 alpha-3H]vitamin D3 as the substrate. Using the solubilized and reconstituted chick 1 alpha-hydroxylase, conversion of this substrate to 1,25-dihydroxyvitamin D3 causes the release of tritium into the aqueous medium. This 3H2O can be easily separated from the labeled substrate by passing the reaction mixture through a reverse-phase silica cartridge. The release of tritium is stereospecific as evidenced by the lack of 3H2O formed when 25-hydroxy-[1 beta-3H]vitamin D3 is used as the substrate. In parallel reactions containing the 25-hydroxy-[26,27-3H]vitamin D3 substrate, production of labeled 1,25-dihydroxyvitamin D3 was assessed by extraction and high-performance liquid chromatography and found to agree very closely with the amount of 3H2O produced from 25-hydroxy-[1 alpha-3H]vitamin D3, validating the accuracy of the new assay. Finally, a major advantage of the tritium-release assay for 1 alpha-hydroxylase is that the results are not affected by further metabolism of the 1,25-dihydroxyvitamin D formed in the incubations.     

Radioenzymatic determination of phenylpropanolamine in plasma

We modified a norepinephrine radioenzymatic method (C. R. Lake, M. G. Ziegler, and I. J. Kopin, 1976, Life Sci. 18, 1315-1326) for determination of plasma phenylpropanolamine (PPA) concentrations. PPA is converted to N[methyl-3H]ephedrine ([3H]EPD) by the enzyme phenylethanolamine N-methyltransferase (PNMT) and S-[methyl-3H]adenosyl-L-methionine ([3H]AdoMet). The product, [3H]EPD, is isolated from unreacted [3H]AdoMet and labeled side products by an organic extraction and a TLC procedure. In addition, a preincubation organic extraction procedure is included to remove inhibitors of PNMT from plasma and to concentrate the sample for enhanced enzymatic conversion. In order to accurately quantitate PPA across the wide range of possible concentrations, the assay is conducted at two plasma volumes. PPA concentrations between 0.3 and 50 micrograms/liter can be detected with 1 ml of plasma, while concentrations between 4 and 1500 micrograms/liter can be detected with 0.1 ml of plasma. The intra-assay coefficients of variation (CVs) are 9.3 and 5.7% at 0.5 and 1500 micrograms/liter, respectively, while the mean interassay CV is 13.8%.     

Assay and properties of N-acetylglucosamine-6-phosphate deacetylase from rat liver

A single-vial assay has been developed for N-acetylglucosamine-6-phosphate deacetylase, in which [3H]acetate released from 3H-acetyl-labeled substrate is measured in a biphasic liquid scintillation counting system after acidification of the reaction mixture. The deacetylase was partially purified from rat liver, and some of its properties were determined. Chromatography on a calibrated Sepharose CL-6B column indicated a molecular weight of 345,000. The Km for the substrate at pH 8.0 was 0.3 mM. Glucosamine 6-phosphate and glucose 6-phosphate inhibited the enzyme, whereas N-acetylgalactosamine, N-acetylglucosamine, N-acetylglucosamine 1-phosphate, and glucosamine 1-phosphate were without effect. The effects of several divalent cations were also examined. Under the conditions tested, Ca2+, Mg2+, and Ba2+ had essentially no effect, whereas Mn2+, Ni2+, and Cu2+ were inhibitory and Co2+ stimulated activity at low concentrations but inhibited above 5 mM. An increase in the ionic strength of the reaction mixture to 0.3 M decreased the activity by 40%.     

A radiochemical assay for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase extracted from mitochondrial membrane of rat intestinal epithelial cells

A radiochemical assay has been developed for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase from rat intestinal epithelial cells. The spectrophotometric assay utilized to measure the enzyme in bacterial cell homogenates is not sensitive enough for homogenates from rat mitochondria, which require an assay that can measure as little as 0.5 nmol NADPH formed/min/ml extract. The assay described here is sensitive to 0.1 nmol product formed/min/ml of extract and employs the use of [3H]pyrroline 5-carboxylate which is phosphorylated and oxidized by the enzyme to gamma-[3H]glutamyl phosphate, a product that decomposes to [3H]pyrrolidone 5-carboxylate. The latter product is separated from the substrate by ion-exchange chromatography. In order to correct for any product loss during separation by ion-exchange [14C]pyrrolidone 5-carboxylate is added as an internal standard to the deproteinized assay mixture. Under the assay conditions described mammalian gamma-glutamate semialdehyde dehydrogenase activity is linear with respect to time and protein concentration. Comparison between the kinetic parameters reported for the bacterial enzyme and those reported here for the mammalian enzyme indicate similarities in the pH optima as well as a requirement for phosphate. Kinetic studies on mammalian enzyme yield apparent Km values of 1.8 mM for pyrroline 5-carboxylate, 0.2 mM for NADP+, and 11.3 mM for phosphate.     

Microassay for interferon, using [3H]uridine, microculture plates, and a multiple automated sample harvester.

A microassay for interferon is described which uses target cells grown in microculture wells, [3H]uridine to measure vesicular stomatitis virus replication in target cells, and a multiple automated sample harvester to collect the radioactively labeled viral ribonucleic acid onto glass fiber filter disks. The disks were placed in minivials, and radioactivity was counted in a liquid scintillation spectrophotometer. Interferon activity was calculated as the reciprocal of the highest titer which inhibited the incorporation of [3H]uridine into viral ribonucleic acid by 50%. Interferon titers determined by the microassay were similar to the plaque reduction assay when 100 plaque-forming units of challenge vesicular stomatitis virus was used. However, it was found that the interferon titers decreased approximately 2-fold for each 10-fold increase in the concentration of challenge vesicular stomatitis virus when tested in the range of 10(2) to 10(5) plaque-forming units. Interferon titers determined by the microassay show a high degree of repeatability, and the assay can be used to measure small and large numbers of interferon samples.     

Inhibition of the lignin peroxidase of Phanerochaete chrysosporium by hydroxylamino-dinitrotoluene, an early intermediate in the degradation of 2,4,6-trinitrotoluene.

The ability of the white rot fungus Phanerochaete chrysosporium to mineralize 2,4,6-trinitrotoluene (TNT) was studied in the concentration range of 0.36 to 20.36 mg/liter. The initial rate of 14CO2 formation was 30% in 4 days at 0.36 mg of [14C]TNT per liter and decreased to 5% in 4 days at 20.36 mg of [14C]TNT per liter. Such a pronounced inhibition was not observed when a mixture of [14C]2-amino-4,6-dinitrotoluene and [14C]4-amino-2,6-dinitrotoluene was used as a substrate. 2-Hydroxylamino-4,6-dinitrotoluene and its isomer 4-hydroxylamino-2,6-dinitrotoluene were identified as the first detectable degradation products of TNT. Their transient accumulation correlated with the inhibition of TNT degradation and of the veratryl alcohol oxidase activity of lignin peroxidase. With purified lignin peroxidase H8, it could be shown that the two isomers of hydroxylamino-dinitrotoluene were oxidized by lignin peroxidase. The corresponding nitroso-dinitrotoluenes apparently were formed, as indicated by the formation of azoxy-tetranitrotoluenes.     

Binding between mammalian spermatid-ectoplasmic specialization complexes and microtubules.

Ectoplasmic specializations (ESs) are submembrane specializations that consist of Sertoli cell plasma membrane linked by an ordered array of actin filaments to a cisterna of endoplasmic recticulum (ESER). They are thought to function in the spermatid-Sertoli cell adhesion junction. Microtubules occur adjacent to the cytoplasmic face of the ESER and are oriented parallel to the long axis of the Sertoli cell, the direction of spermatid translocation during spermatogenesis. Our hypothesis that spermatid orientation and translocation in the seminiferous epithelium is microtubule dependent predicts that microtubules bind to ESs. To test for binding between microtubules and ESs, we have developed an in vitro assay in which spermatid-ES complexes were isolated from the seminiferous epithelium and incubated with bovine brain microtubules that were labeled with [3H]GTP and stabilized with taxol. Binding was determined by scintillation counts from gradient fractions enriched for spermatid-ES complexes and depleted of unbound microtubules by differential centrifugation. Our data indicate that microtubules bind to spermatid-ES complexes in a substrate concentration-dependent manner and can be released with 5 mM GTP or 10 mM MgATP. Binding is competitively reduced with excess unlabeled microtubules and is inhibited by 100 microM vanadate and 2 mM N-ethylmaleimide (NEM). The amount of binding is unchanged by 10 microM vanadate, 2 mM erythro-(2-hydroxy-3-nonyl)adenine (EHNA) or 1 mM 5'-adenylylimidodiphosphate (AMP-PNP). Immunofluorescence and autoradiographic data confirm that labeled microtubules bind to ES locations on spermatid-ES complexes. These data are consistent with the hypothesis that spermatid translocation is a microtubule-based transport event.     

A rapid radioassay for sphingosine kinase

A solvent-extraction-based radioassay for measuring sphingosine kinase (SKase) activity has been developed. The assay utilizes [3H]sphingosine substrate and differentially extracts the [3H]sphingosine-1-phosphate product. The extracted radioactivity is demonstrated to be primarily [3H]sphingosine-1-phosphate with less than 1% contamination by [3H]sphingosine. When assaying SKase activity in the soluble cell fraction, the extraction efficiency of the labeled sphingosine-1-phosphate product is a reproducible 78%, which allows for a simple back calculation to correct for the 22% extraction loss. With minor modification, the assay is also a reproducible procedure for determining SKase activity in subcellular membrane fractions. The assay is far more rapid than thin-layer chromatography and high-performance liquid chromatography methods, which makes it possible to do a large number of assays in a short period of time. The utility of the assay is demonstrated by using it to conduct a complete bisubstrate kinetic analysis of rat heart SKase.     

Measurement of testosterone and dehydroepiandrosterone 16alpha-hydroxylase activities by a tritium exchange method.

A new isotopic method, based upon the stereospecific replacement of a proton (3H) by a hydroxyl group has been developed for the measurement of rat liver testosterone and dehydroepiandrosterone 16alpha-hydroxylase activity. Specifically 16-tritiated substrates were prepared by microbiological (Cylindrocarpon radicicola) transformation of the [16-3H]progesterone and [16-3H]pregnenolone. The incubation medium consists of a phosphate buffer (pH7; 150mM), NADPH (0.1 mM), nicotinamide (10mM) and magnesium chloride (4 mM). Tween 80 (1 mg/ml) is used to solubilize saturating concentrations of [16-3H]testosterone (50 micron) or [16-3H]dehydroepiandrosterone (100 micron). The enzymatically released tritium is recovered in the incubation medium as tritiated water which is distilled under reduced pressure and counted by liquid scintillation. The method is easy to perform, very sensitive (50 pmol of 16alpha-hydroxylated metabolites) and is independent of any further metabolism of the 16alpha-hydroxylated products.     

An anion-exchange radioassay for glucose 6-phosphate phosphatase: use in topological studies with endoplasmic reticulum vesicles.

A simple procedure is presented for the enzymatic preparation of [2-3H]mannose 6-phosphate (Man 6-P) with purified yeast hexokinase and unlabeled ATP. The enzymatically synthesized [2-3H]Man 6-P is utilized as the radiolabeled substrate in a new rapid assay for glucose 6-phosphate (Glc 6-P) phosphatase. The principle of the assay procedure is that the unreacted substrate, [2-3H]Man 6-P, is retained by the anion-exchange resin, AG 1-X8 (acetate), while the enzymatic product, [2-3H]-mannose, is eluted directly into a scintillation counting vial. When Glc 6-P phosphatase activity associated with mouse liver endoplasmic reticulum (ER) vesicles is assayed by the new chromatographic assay, the same characteristic latency and properties are observed, as determined by the commonly used colorimetric assay of inorganic phosphate produced. The anion-exchange radioassay described should be useful for a variety of topological studies on enzymes associated with membrane vesicles derived from liver and kidney ER.