Assay for and enzymatic formation of an ethylene precursor, 1-aminocyclopropane-1-carboxylic acid

A simple and sensitive chemical assay was developed for 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor of ethylene. The assay is based on the liberation of ethylene from ACC at pH 11.5 in the presence of pyridoxal phosphate, MnCl₂ and H₂O₂. This assay was used to detect ACC in extracts of tomato fruits (Lycopersicon esculentum Mill.) and to measure the activity of a soluble enzyme from tomato fruit that converted S-adenosylmethionine (SAM) to ACC. The enzyme had a K_m of 13 M for SAM, and conversion of SAM to ACC was competitively and reversibly inhibited by aminoethoxyvinylglycine (AVG), an analog of rhizobitoxine. The K_i value for AVG was 0.2 M. The level of the ACC-forming enzyme activity was positively correlated with the content of ACC and the rate of ethylene formation in wild-type tomatoes of different developmental stages. Mature fruits of the rin (non-ripening) mutant of tomato, which only produce low levels of ethylene, contained much lower levels of ACC and of the ACC-forming enzyme activity than wild-type tomato fruits of comparable age.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG ammoethoxyvinylglycine, the aminoethoxy analog of rhizobitoxine L-2-amino-4-(2-aminoethoxy)-trans-3-butenoic acid - SAM S-adenosyl-L-methionine Michigan Agricultural Experiment Station No. 8876     

Photoaffinity labelling of juvenile hormone binding proteins in the cockroach Leucophaeamaderae

The synthesis and testing of several diazocarbonyl JH analogs (diazo JHA) which act as photoaffinity labels for insect juvenile hormone binding proteins are described. The best competitor, 10,11-epoxyfarnesyl diazoacetate, has been shown to irreversibly reduce [³H]-JH III binding to both ovarian and hemolymph JHBP from Leucophaeamaderae after irradiation at 254 nm for 20 seconds. No loss of activity was observed after incubation of JHBP and diazo JHA without irradiation. Protection from photoinactivation by diazo JHA II was achieved by the presence of an equimolar amount of JH III during the photolysis. Photoaffinity labeled proteins show loss of binding capacity without alteration of the binding affinity. This is the first example of the use of a photoaffinity label in the study of JH action on a molecular level, and may become a valuable tool in the elucidation of JH-receptor-chromatin interactions.     

Modification of sialic acid metabolism of murine erythroleukemia cells by analogs of N-acetylmannosamine

Two analogs of N-acetylmannosamine, $\text{2-}\text{acetamido}\text{-1,3,4,6-}\text{tetra}\text{-O-}\text{acetyl}\text{-2-}\text{deoxy-}\text{d}\text{-mannopyranose}$ (Ac₄-NAcMan) and the 2-trifluoroacetamido derivative (Ac₄F₃-NAcMan), were synthesized as potential inhibitors of the formation of sialic acid-containing glycoconjugates and were examined for their ability to modify the incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into cellular glycoconjugates of Friend murine erythroleukemia cells. Ac₄F₃-NAcMan and Ac₄-NAcMan inhibited cellular replication in suspension culture at concentrations of 0.02 and 0.08 mM, respectively. The cytotoxicity of Ac₄-NAcMan was relatively reversible, whereas that produced by Ac₄F₃-NAcMan was not, as judged by measurement of the cloning efficiencies of cells exposed to these agents. The analogs inhibited incorporation of $\text{N-[}{}^3\text{H]acetylmannosamine}$ into ethanol-soluble and -insoluble materials. Separation of ethanol-soluble metabolites by HPLC demonstrated that Ac₄F₃-NAcMan caused accumulation of radioactivity from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in CMP-N-acetylneuraminic acid (CMP-NeuNAc) equal to the decrease in macromolecular-bound ³H caused by this agent. In contrast, similar exposure to Ac₄-NAcMan produced a large increase in the amount of radioactivity in ethanol-soluble N-acetylneuraminic acid while decreasing the amount of label from $\text{N-[}{}^3\text{H]acetylmannosamine}$ in cellular CMP-NeuNAc, suggesting that the analogs differ in their biochemical sites of action. Treatment of cells with either analog increased the amount of neuraminidase-hydrolyzable sialic acid-like material on the cell surface; this appeared to be due to the incorporation of the analogs into cellular glycoconjugates, since incubation of cells with ³H-labeled analogs resulted in the appearance of radioactivity in cellular ethanol-insoluble and neuraminidase-hydrolyzable material. Incubation of cells with Ac₄-NAcMan labeled with ¹⁴C in the 4-O-acetyl group further demonstrated that incorporation occurred with approx. 50% retention of this substituent. Thus, both the amount and the nature of the surface sialic acid constituents of treated cells were altered, suggesting that these or similar analogs could potentially be used to modify cellular membrane function.     

Uridine transport in basolateral plasma membrane vesicles from rat liver

Summary The characteristics of uridine transport were studied in basolateral plasma membrane vesicles isolated from rat liver. Uridine was not metabolized under transport measurement conditions and was taken up into an osmotically active space with no significant binding of uridine to the membrane vesicles. Uridine uptake was sodium dependent, showing no significant stimulation by other monovalent cations. Kinetic analysis of the sodium-dependent component showed a single system with Michaelis-Menten kinetics. Parameter values were K _M 8.9 m and V _max 0.57 pmol/mg prot/sec. Uridine transport proved to be electrogenic, since, firstly, the Hill plot of the kinetic data suggested a 1 uridine: 1 Na⁺ stoichiometry, secondly, valinomycin enhanced basal uridine uptake rates and, thirdly, the permeant nature of the Na⁺ counterions determined uridine transport rates (SCN^– > NO ₃ ^– > Cl^– > SO ₄ ^2– ). Other purines and pyrimidines cis-inhibited and trans-stimulated uridine uptake.This work has been partially supported by grant PM90-0162 from D.G.I.C.Y.T. (Ministerio de Educación y Ciencia, Spain). B.R.-M. is a research fellow supported by the Nestlé Nutrition Research Grant Programme.     

Conformation and structure of 2-amino-8-(β-d-ribofuranosyl)imidazo [1,2-a]-s-triazin-4-one (5-aza-7-deaza guanosine), a potent antiviral nucleoside

The crystal and molecular structure of one imidazo[1,2-a]-s-triazine nucleoside and its antiviral activity are described. The crystal structure of 2-amino-8-(β-d-ribofuranosyl)imidazo-[1,2-a]-s-triazin-4-one monohydrate (C₁₀H₁₃N₅O₅·H₂O) was solved by X-ray counter data. The compound crystallizes in the monoclinic space group P2₁ with cell dimensions a = 7.353 (1), b = 6.465 (1), c = 13.701 (1) Å, β = 104.64 (1)°. The structure was solved by direct methods and refined by full matrix least-squares technique to a final value of the conventional R-factor of 0.049 using 1998 observed intensities. The orientation of the base relative to the sugar ring defined in terms of rotation about the C(1′)-N(8) glycosyl bond is anti (47.8°). The ribose moiety exhibits C(2′)-endo, ²E conformation. The conformation around C(4′)-C(5′) is gauche^?. Molecular packing is dominated by hydrogen bonds. Base stacking occurs long the b axis. 5-Aza-7-deazaguanosine has shown a marked antiviral activity in vitro against herpes simplex virus despite the fact that N(3) is effective as the hydrogen acceptor only.     

An analog of ACTH/MSH (4–9), ORG-2766, reduces cerebral uptake of morphine

The uptake of morphine was significantly reduced in most regions of the brains of conscious, unrestrained rats within 10 minutes after treatment with an analog of ACTH/MSH (4–9), ORG-2766. The effect was most obvious in regions with significant densities of enkephalin receptors, namely basal ganglia, hippocampus and cortex. The results explain, in part, how some fragments and analogs of ACTH/MSH may antagonize behavioral actions of morphine, even though some of these peptides lack significant opiate receptor binding properties. We believe that this effect of ORG-2766 is related to an action on the permeability characteristics of the brain microvasculature. The underlying mechanism is unknown.     

Facilitated transport of 6-mercaptopurine and 6-thioguanine and non-mediated permeation of 8-azaguanine in novikoff rat hepatoma cells and relationship to intracellular phosphoribosylation

6-Mercaptopurine and 6-thioguanine strongly inhibited the zero-trans entry of hypoxanthine into Novikoff rat hepatoma cells which lacked hypoxanthine/guanine phosphoribosyltransferase, whereas 8-azaguanine had no significant effect. 6-Mercaptopurine was transported by the hypoxanthine carrier with about the same efficiency as its natural substrates (Michaelis-Menten constant = 372 ± 23 μM; maximum velocity = 30 ± 0.7 pmol/μl cell H₂O per s). 8-Azaguanine entry into the cells, on the other hand, showed no sign of saturability and was not significantly affected by substrates of the hypoxanthine/guanine carrier. The rate of entry of 8-azaguanine at 10–100 μM amounted to only about 5% of that of hypoxanthine transport and was related to its lipid solubility in the same manner as observed for various substances whose permeation through the plasma membrane is believed to be non-mediated. Only the non-ionized form of 8-azaguanine (pK_a = 6.6) permeated the cell membrane.Studies with wild type Novikoff cells showed that permeation into the cell was the main rate-determining step in the conversion of extracellular 8-azaguanine to intracellular aza-GTP and its incorporation into nucleic acids. In contrast, 6-mercaptopurine was rapidly transported into cells and phosphoribosylated; the main rate-determining step in its incorporation into nucleic acids was the further conversion of 6-mercaptopurine riboside 5'-monophosphate.     

Entamoeba histolytica: localization and characterization of ca2+-dependent nucleotidases

An activity of Ca²⁺-dependent nucleotidase was detected in axenically-cultivated trophozoites of Entamoeba histolytica. The enzyme was concentrated by differential and sucrose density gradient centrifugation and catalyzed hydrolysis of nucleoside tri- and diphosphates and also thiamine pyrophosphate. Hydrolysis of nucleoside mono-phosphates was not affected by Ca²⁺. Among substrates tested, ATP was most active. Addition of Zn²⁺ or heat treatment almost abolished the enzyme activity. The enzyme exhibited almost the identical activity at acid and neutral pH. Among 6 bands isolated by polyacrylamide gel electrophoresis, 4 were stained with ATP, UTP, CTP and ADP, whereas the other 2 were stained only with ATP, UTP and CTP. The concentrated enzyme preparation, primarily composed of membrane fragments, also had activities of acid phosphatase, acid inorganic pyrophosphatase, 5'-nucleotidase and Mg²⁺-dependent ATPase. These observations suggest that E. histolytica has 2 Ca²⁺-dependent nucleotidases, i.e. one Ca²⁺-dependent ATPase and the other Ca²⁺-dependent nucleoside diphosphatase or an apyrase-like enzyme, and that these nucleotidases are at least partially associated with the plasma membrane or an organelle of lysosomal nature in this parasite.