A simple radioassay for dihydroorotate dehydrogenase

A simple radioassay for dihydroorotate dehydrogenase (DHO-DHase; EC 1.3.3.1) has been developed. l-[carboxy-¹⁴C]Dihydroorotate was prepared from [carboxy-¹⁴C]orotic acid using DHO-DHase derived from Zymobacterium oroticum and was purified by elution from DEAE-Sephadex A-25 with 0.2 m ammonium formate, pH 7. DHO-DHase activity in human spleen mitochondria was determined by the release of ¹⁴CO₂ from the carboxy-¹⁴C-labeled l-dihydroorotate, the reaction being coupled with added orotate phosphoribosyltransferase and orotidylate decarboxylase. An apparent K_m value of ～5 μm for l-dihydroorotate was established using the radioassay. This value correlated well with results from other methods.     

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.     

A hexokinase-coupled radioassay for pyruvate kinase

A procedure for the assay of low activities of pyruvate kinase (0.01 to 4 mIU) is described. The method consists of coupling the formation of ATP by the pyruvate kinase reaction to hexokinase in the presence of uniformly labeled [¹⁴C]glucose. The labeled glucose 6-phosphate thus formed is easily separated from the unreacted glucose using small columns of Dowex 1-X8 formate and detected by liquid scintillation spectrometry. Chromatographic patterns of pyruvate kinases from 25 mg of rat liver, 3.5 mg of frog oocyte, and 0.5 mg of the whole body of the fruit fly Drosophila melanogaster are presented as illustrations of the sensitivity of the radioassay.     

A novel method for the detection of receptors and membrane proteins by scintillation proximity radioassay

A rapid and convenient binding assay for receptors and membrane proteins has been developed. It is based on the binding of 125I-labeled ligands to membrane proteins adsorbed to polyvinyltoluene plastic scintillation microspheres. Membranes or isolated membrane proteins adsorb to the beads upon mixing, and addition of 125I-labeled ligand induces photon emission which is proportional to the amount of added receptor or membrane protein. The interaction of acetylcholine receptor with 125I-labeled alpha-bungarotoxin and antigens with 125I-labeled antibodies or protein A were used as models to test the system. As little as 1 ng of acetylcholine receptor is detected by the assay and a linear relationship with receptor concentration is observed up to 50 ng of receptor per 250 microliter reaction medium. The effects of detergents, salts, soluble proteins, and neutral membranes were studied. Inclusion of bovine serum albumin up to 1 mg/ml, sodium chloride up to 0.5 M, and membranes up to 10 micrograms/ml cause little or no effect on the assay. Detergents at 10-fold below their critical micelle concentrations had little or no effect on the assay. The pharmacological effects of agonists such as acetylcholine were conveniently studied by following the displacement of the 125I-labeled ligand. Similarly, the amount of toxin in crude snake venom can be assayed by measuring competition with the labeled toxin. Only a few seconds are required to perform each binding assay.     

A Radioiodinated Azo Dye with Affinity for Amyloid: A Preliminary Report

Investigation was undertaken to produce a radioactive material with affinity for amyloid tissue. Studies of a number of major dyes using paraffin histological sections of an amyloid spleen as test material showed that the only compounds with marked affinity for amyloid were watersoluble disazo dyes of the type E←D→E, where D is usually benzidine, o-tolidine or o-dianisidine, and E is a naphthol or aminonaphthol sulfonic acid. Substitution of an amino or a hydroxyl group in position 1 of the naphthalene nucleus caused loss of staining affinity. It was found that trypan blue could be radioiodinated at position 8 using the Sandmeyer reaction, and the labelled dye showed excellent staining and radioautographs of the test amyloid section. Metabolism of the tagged dye using 10-200 μg./kg. in animals showed minimal organ uptake, except for the liver which accumulated 30-40% of the injected dose by the end of four days. Use of this material in the diagnosis of human amyloid disease is being explored.     

A direct radioassay for pyruvate kinase activity

Pyruvate kinase catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. A direct radioassay for this enzyme using [14C]PEP as substrate has been developed. The product, [14C]pyruvate, can be separated from the substrate rapidly and easily by applying the mixture to a hydroxyapatite column, and eluting the [14C]pyruvate directly into a scintillation vial. The [14C]PEP is bound to the column which can be regenerated and used indefinitely. The assay is sensitive, rapid, and particularly well suited for the simultaneous assay of large numbers of samples.     

A simple radioassay for angiotensin-converting enzyme.

Angiotensin-converting enzyme can be measured by the rate of release of 3H-labelled hippurate from p-[3H]benzoylglycylglycylglycine. The product is separable from the substrate by extraction of acidified reaction mixtures with ethyl acetate. Assay results for human serum angiotensin-converting enzyme can be obtained within 1.5 h of receipt of serum samples. Within the limits tested, the assay appears to be specific. However, interference by hitherto unrecognized enzymes of abnormal sera must be ruled out.     

A rapid radioassay procedure for plasma lecithin-cholesterol acyltransferase

A rapid and accurate single step procedure is described for the assay of lecithin-cholesterol acyltransferase activity. After incubation, using radiolabeled cholesterol as the substrate, an ethanolic solution of digitonin is added directly to the incubation mixture to extract the lipids. Excess cholesterol is then added, and the labeled cholesterol-digitonide along with denatured proteins are sedimented by low speed centrifugation, leaving the labeled esterified cholesterol in solution. An aliquot of the supernatant is counted in an aqueous scintillation mixture. The method correlates well with the established thin-layer chromatographic procedure using either lecithin-cholesterol vesicles or heat-inactivated plasma as the substrate for lecithin-cholesterol acyltransferase.     

Jejunal brush-border folate hydrolase. A novel enzyme.

Dietary folate, a vitamin required for DNA synthesis and cell regeneration, occurs as pteroylpolyglutamates that are hydrolyzed to pteroylglutamate during the process of intestinal absorption. Studies from our laboratory over the past 15 years have shown that jejunal brush-border folate hydrolase is essential and rate-limiting in folate absorption. Brush-border folate hydrolase activity and pteroylpolyglutamate hydrolysis are inhibited in disease and conditions associated with folate deficiency, including celiac and tropical sprue, the use of sulfasalazine to treat inflammatory bowel disease, and chronic alcoholism. Brush-border folate hydrolase is an exopeptidase located on the jejunal brush-border surface that liberates hydrolytic products of pteroylpolyglutamates in a progressive fashion, with a final release of pteroylglutamate. Subsequent steps in folate absorption include uptake by a brush-border folate-binding-protein receptor and transport across the brush-border membrane into the enterocyte. These steps are probably followed by an intracellular synthesis of pteroylglutamates for folate-dependent reactions and intracellular hydrolysis to pteroylglutamate for transport across the basolateral membrane to the portal circulation. In pigs, the active form of jejunal brush-border folate hydrolase has a molecular weight of 240 kd and is probably a homodimer of the 120-kd protein found after immunoprecipitation with specific antibody. Regulating the synthesis and expression of brush-border folate hydrolase may be critical to the availability of dietary folate.     

A novel cyclic opioid peptide analog showing high preference for mu-receptors

The side-chain to side-chain cyclized opioid peptide analogs H-Tyr-D-Orn-Phe-Asp-NH2 (I) and H-Tyr-D-Lys-Phe-Glu-NH2 (II) were synthesized and tested in the guinea pig ileum and mouse vas deferens assays and in binding assays based on displacement of mu- and delta-opioid receptor-selective radioligands from rat brain membranes. The more rigid cyclic analog I containing a 13-membered ring structure showed very high preference for mu-receptors over delta-receptors, whereas the more flexible cyclic peptide II (15-membered ring) was non-selective. These results indicate that variation in the degree of conformational restriction of opioid peptides can produce drastic shifts in their receptor selectivity profile. Because of its high mu-receptor selectivity and rigidity cyclic analog I will be useful for determining the conformational requirements of mu-opioid receptors.     

A novel antifungal analog peptide derived from protaetiamycine

Previously, the 9-mer analog peptides, 9Pbw2 and 9Pbw4, were designed based on a defensin-like peptide, protaetiamycine isolated from Protaetia brevitarsis. In this study, antifungal effects of the analog peptides were investigated. The antifungal susceptibility testing exhibited that 9Pbw4 contained more potent antifungal activities than 9Pbw2. A PI influx assay confirmed the effects of the analog peptides and demonstrated that the peptides exerted their activity by a membrane-active mechanism, in an energy-independent manner. As the noteworthy potency of 9Pbw4, the mechanism(s) of 9Pbw4 were further investigated. The membrane studies, using rhodamine-labeled giant unilamellar vesicle (GUV) and fluorescein isothiocyanate (FITC)-dextran loaded liposome, suggested that the membrane-active mechanism of 9Pbw4 could have originated from the poreforming action and the radii of pores was presumed to be anywhere from 1.8 nm to 3.3 nm. These results were confirmed by 3D-flow cytometric contour-plot analysis. The present study suggests a potential of 9Pbw4 as a novel antifungal peptide.     

A rapid and simple radioassay for thymine 7-hydroxylase

This work describes a simple and convenient procedure for measuring the activity of thymine 7-hydroxylase. The principle of the procedure depends upon the conversion of tritiated thymine by the enzyme to 5-hydroxymethyluracil. This reaction simultaneously invokes the loss of a tritium atom and the formation of tritiated water. The quantity of tritiated water formed is stoichiometrically proportional to the amount of 5-hydroxymethyluracil produced.The sensitivity of this procedure was markedly improved when both catalase and BSA were included in the reaction mixture.     

18F]fluoro-deoxy-glucose folate: a novel PET radiotracer with improved in vivo properties for folate receptor targeting

The folate receptor (FR) is upregulated in various cancer types (FR-α isoform) and in activated macrophages (FR-β isoform) which are involved in inflammatory and autoimmune diseases, but its expression in healthy tissues and organs is highly restricted to only a few sites (e.g kidneys). Therefore, the FR is a promising target for imaging and therapy of cancer and inflammation using folate-based radiopharmaceuticals. Herein, we report the synthesis and evaluation of a novel folic acid conjugate with improved properties suitable for positron emission tomography (PET). [(18)F]-fluoro-deoxy-glucose folate ([(18)F]3) was synthesized based on the click chemistry approach using 2-deoxy-2-[(18)F]fluoroglucopyranosyl azide and a folate alkyne derivative. The novel radiotracer [(18)F]3 was produced in good radiochemical yields (25% d.c.) and high specific radioactivity (90 GBq/μmol). Compared to previously published (18)F-folic acid derivatives, an increase in hydrophilicity was achieved by using a glucose entity as a prosthetic group. Biodistribution and PET imaging studies in KB tumor-bearing mice showed a high and specific uptake of the radiotracer in FR-positive tumors (10.03 ± 1.12%ID/g, 60 min p.i.) and kidneys (42.94 ± 2.04%ID/g, 60 min p.i.). FR-unspecific accumulation of radioactivity was only found in the liver (9.49 ± 1.13%ID/g, 60 min p.i.) and gallbladder (17.59 ± 7.22%ID/g, 60 min p.i.). No radiometabolites were detected in blood, urine, and liver tissue up to 30 min after injection of [(18)F]3. [(18)F]-fluoro-deoxy-glucose-folate ([(18)F]3) is thus a promising PET radioligand for imaging FR-positive tumors.     

An ultrasensitive radioassay for hexokinase

A batch chromatographic method for the determination of hexokinase employing trace-labelled glucose and resin chromatography is described. Markedly enhanced sensitivity compared with the standard spectrophotometric assay is shown. Its greater reliability and use for particulate-tissue preparations is discussed.