Studies on the biosynthesis of N-(γ-L-glutamyl)-4-hydroxyaniline] in Agaricus bisporus: Identification of the position in shikimic acid at which the amination occurs

Labelled shikimic acid was efficiently incorporated into the aniline moiety of $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$, a characteristic aromatic compound of the common mushroom, Agaricus bisporus. Incubations with [3-³H]- and [1,6-¹⁴C]shikimic acid clearly proved that the amination of shikimic acid occurs at its 4-position during the biosynthesis of $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$.     

In vivo biosynthesis of -linolenic acid in plants

[1-¹⁴C]acetate was readily incorporated into unsaturated fatty acids by leaf slices of spinach, barley and whole cells of $\text{Chlorella}\text{pyrenoidosa}$ and $\text{Candida}\text{bogoriensis}$. In these systems the [¹⁴C] label in newly synthesized oleate and linoleate was approximately equally distributed in the C_1–9 and the C_10–18 fragments obtained by reductive ozonolysis of these acids, whereas in a-linolenic acid over 90% of the total [¹⁴C] was localized in the C_1–9 fragment. While [1-¹⁴C]oleic acid was converted by whole cells of $\text{Chlorella}$ to [1-¹⁴C]linoleic and [1-¹⁴C]linolenic acids, [U-¹⁴C]oleic acid yielded [U-¹⁴C]linoleic acid but a-linolenic acid was labeled only in the carboxyl terminal carbon atoms. When spinach leaf slices were supplied with carboxyl labeled octanoic, decanoic, dodecanoic, tetradecanoic and octadecanoic acids, only the first three acids were converted to a-linolenic acids while the last two acids were ineffective. Thus we suggest that (a) linoleic acid is not the precursor of a-linolenic acid and (b) 12:3(3, 6, 9) is the earliest permissible trienoic acid which is then elongated to a-linolenic acid.     

Biosynthesis of N-methyl-l-glucosamine from d-glucose by Streptomyces griseus

Biosynthesis of $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ moiety of streptomycin from d-glucose by Streptomyces griseus was studied. A mixture of d-[1-¹⁴C]glucose and d-[6-³H]glucose was given to the culture of S. griseus. The ³H/¹⁴C ratio found in $\text{N-}\text{methyl}\text{-}\text{d}\text{-}\text{glucosamine}$ further supports a mechanism that the conversion of d-glucose to l-hexose is carried out without scission of carbon skeleton. When d-[1-¹⁴C]glucose and d-[3-³H]glucose were used, the fall of ³H/¹⁴C ratio in $\text{N-}\text{methyl}\text{-}\text{l}\text{-}\text{glucosamine}$ showed that the hydrogen atom at C-3 plays a rôle in such a transformation.     

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.     

Quantitation of the sulfated disaccharides of heparin by high performance liquid chromatography

Heparin was converted by treatment with nitrous acid primarily into sulfated disaccharides. The mixture of disaccharides was reduced with sodium boro[³H]hydride and the disaccharides were purified by preparative paper electrophoresis and paper chromatography. Four disaccharides were obtained. On the basis of their paper electrophoretic mobilities and the products formed at intermediate stages of their acid hydrolysis, the disaccharides were identified as 4-O-(2-O-sulfo-α-l-idopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol, 4-O-(2-O-sulfo-α-l-idopyranosyluronic acid)-2,5-anhydro-d-mannitol, 4-O-(α-l-idopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol, and 4-O-(β-d-glucopyranosyluronic acid)-6-O-sulfo-2,5-anhydro-d-mannitol. The purified disaccharides were used as standards in the development of a high-performance liquid chromatography procedure for their separation and quantitation on a Partisil-10 SAX anion-exchange column. The three monosulfated disaccharides were resolved by isocratic elution with 40 mm KH₂PO₄. The KH₂PO₄ concentration was tehn increased to 400 mm to elute the disulfated disaccharide. Column effluents were collected in $\text{1}\text{2}\text{-}\text{ml}$ fractions, and the recovery of each ³H-labeled product was determined by scintillation counting. When sodium boro-[³H]hydride with a specific activity of 315 mCi/mmol was used in the reduction of the heparin deamination products, the disaccharides gave 28,500 cpm/nmol in the effluent peaks. Quantitative recoveries of the ³H-disaccharides were obtained. It was demonstrated that the method developed using the purified disaccharides gave reproducible and quantitative results in direct assays of aliquots of boro[³H]hydride-reduced heparin deamination mixtures.     

An intramolecular C-2 → C-1 hydrogen transfer during the acid-catalyzed conversion of d-xylose to d-threo-pentulose (d-xylulose)

Aldose-ketose isomerases are known to catalyze a partial and sometimes complete intramolecular hydrogen transfer between C-1 of the ketose and C-2 of the aldose. It was recently shown (Feather, M. S., and Harris, D. W. (1975) J. Amer. Chem. Soc.97, 178–181) that the same type of transfer occurs during the acid-catalyzed interconversion of d-fructose, d-glucose, and d-mannose. A similar transfer is demonstrated herein for the conversion of d-xylose to d-xylulose in acid solution. d-[2-³H]xylose was isomerized in aqueous sulfuric acid and the resulting d-[³H]xylulose was isolated in 6% yield. The ketose had 18.3% the activity of the starting aldose. Chemical degradation showed that all the carbon-bound tritium of the d-[³H]xylulose was located at C-1, thus indicating a C-2 → C-1 intramolecular hydrogen transfer. During the reaction, less than 1.2% of the total radiochemical activity was found in the solvent, and, the unreacted d-[2-³H]xylose was recovered, having an activity nearly the same as the starting material. The differences in activity, therefore, of the d-[2-³H]xylose and the d-[1-³H]xylulose are due to an isotope effect ($\text{K}{}_{\mathrm{<mtext>H</mtext>}}\text{K}{}_{\mathrm{<mtext>T</mtext>}}$) which is indicated to be 5.4. The data are discussed in terms of currently accepted models for isomerase mechanisms.     

Compartmentation of cytosolic dehydrogenases studied by transfer of tritium from labelled substrates into lactate in rat hepatocytes

This paper describes the transfer of tritium from [2-³H]xylitol or (1R)-[1-³H]ethanol into lactate in cells from fed rats either untreated or triiodothyronine-treated. The labelling pattern of lactate during the metabolism of [2-³H]xylitol or (1R)-[1-³H]ethanol follows the equation $\text{L = K(1?e}{}^{\mathrm{<mtext>?</mtext><mtext>t</mtext><mtext>\tau </mtext>}}\text{)}$ (μmol tritium/μmol lactate). The yield in lactate together with the minimum value of the total flux of reducing equivalents are used to estimate the specific radioactivity of NADH. We have calculated the lactate dehydrogenase-catalysed oxidation rate of NADH from the experimental values of lactate labelling and the specific radioactivity of NADH. We found the calculated flux of reducing equivalents from NADH to pyruvate to be of the same order of magnitude whether labelled ethanol or labelled xylitol was metabolized. We found the flux to be only a few percent of the maximal activity of lactate dehydrogenase. The results obtained suggest that the cytoplasm can be regarded as one compartment, containing a single pool of NAD(H).     

Biosynthesis of -linolenic acid by disrupted spinach chloroplasts

A disrupted spinach chloroplast preparation readily synthesized $\text{[}{}^{14}\text{C]α-linolenate}$ from [2-¹⁴C]acetate under anaerobic conditions. It can be shown by degradation data that [¹⁴C]oleate is not a precursor of [¹⁴C]linolenate and that $\text{cis}$ 7,10,13-hexadecatrienoic acid is the probable immediate precursor of the [¹⁴C]linolenate.     

Isolation and identification of a naturally occurring 7, 8-didemethyl-8-hydroxy-5-deazariboflavin derivative from Mycobacterium avium

A yellow pigment (C₂₄H₂₆N₄PO₁₅Na₃) was isolated from Mycobacterium avium. On the acid hydrolysis the pigment (1 mol) gave L-glutamic acid (1 mol), lactic acid (1 mol) and 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5′-phosphoric acid. The structure of $\text{N-[O-[[5-(8-}\text{hydroxypyrimidol[4,5-b]}\text{quinoline}\text{-10-}\text{yl}\text{-2,4(3H, 10H)-}\text{dione}\text{)-2,3,4-}\text{trihydroxypentyloxy]hydroxyphosphoryl]-}\text{L}\text{-lactyl]-}\text{L}\text{-glutamic acid}$ was proposed for this compound. The compound isolated functions presumably as a new cofactor in a redox system of the bacteria.     

Characterization of an alpha-glucan from chick embryo sternum whose synthesis is stimulated by L-3,5,3'-triiodothyronine and human serum.

Incorporation of [³H]glucose into macromolecular components of 12-day chick embryo sternum incubated in vitro was stimulated by both human serum and l-3,5,3′-triiodothyronine. Under all conditions, 65–70% of the radioactivity was incorporated into glycosaminoglycans. About 10% of the radioactivity was incorporated into a fraction separable by ion-exchange chromatography which was stimulated two- to sixfold by addition of 2–10 nm triiodothyronine and 5–20% ($\text{v}\text{v}$) human serum. Further characterization of this fraction by paper electrophoresis at pH 3.5 showed the presence of two components, one apparently anionic and one neutral. All of the increase in incorporation of [³H]glucose was into the former species. Acid hydrolysis of this material showed that it contained only glucose. Treatment with α-amylase released 78% of the label as maltotriose and maltose; digestion with crystalline β-amylase released 75% as maltose; and treatment with glucoamylase and α-amylase released 93% as glucose. There was no incorporation of any amino acid into this fraction, nor could any incorporation of [³²P]phosphate, [³⁵S]sulfate, [³H]uridine, or [³H]acetate be demonstrated. Mild acid hydrolysis (0.1 N HC1, 100 °C, 10–20 min) converted the material to a neutral species with a much lower molecular weight. The results indicate that chick embryo sternum contains a species of glycogen whose synthesis is stimulated by thyroid hormones and other serum factors.     

A stereochemical study on the metabolism of isobutyrate in Pseudomonas putida

Ammonium[2-³H,1-¹⁴C]isobutyrate was converted by Pseudomonas putida ATCC 21244 into S(+)-β-hydroxyisobutyric acid (β-HIBA) with loss of the α-tritium atom. The recovered isobutyrate had the same ${}^3\text{H}{}^{14}\text{C}$ as the starting material. Ammonium (2S)-[3-¹³C]isobutyrate was synthesized and converted by P. putida into β-HIBA. The ¹³C-nmr of the corresponding methyl ester benzoate showed ¹³C enrichment in the hydroxymethyl carbon atom. The results therefore indicate that isobutyrate metabolism in this organism proceeds via an unsaturated intermediate (probably methacrylyl-CoA) formed by dehydrogenation of the 2-pro-S-methyl group of the precursor (isobutyryl-CoA). Hydration of the intermediate proceeds with addition of a proton at C-2 from the same side as the hydrogen removed in the dehydrogenation.     

A 3H-labelled trisaccharide from heparin as substrate for acetyl:CoA: 2-amino-2-deoxy-α-d-glucoside N-acetyltransferase

The tetrasaccharide fraction obtained by gel chromatography after treatment of commercially available heparin with nitrous acid was reduced with NaB³H₄ and then hydrolysed with 2m trifluoracetic acid at 70° for 3 days. By gel chromatography and electrophoresis, the ³H-labelled trisaccharide 1 bearing an unsubstituted 2-amino-2-deoxy-d-glucosyl group in the non-reducing position was obtained (18% from the ³H-labelled tetrasaccharide). By sequential, enzymic degradation, the structure $\text{α-}\text{d}\text{-GlcN}\text{-(1→4)-β-}\text{d}\text{-GlcA}\text{-(1→4)-[1-}{}^3\text{H]aManol}$ was obtained for 1, which is a substrate for acetyl-CoA: 2-amino-2-deoxy-α-d-glucoside N-acetyltransferase, an enzyme that is deficient in the Sanfilippo C syndrome. In human-skin fibroblasts, the pH optimum of acetyl transfer onto 1 was between pH 5.5 and 7.0, and dependent on the buffer. An apparent K_m for 1 of 0.14mM was found.     

1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine: A novel source of arachidonic acid in neutrophils stimulated by the calcium ionophore A23187

Rabbit peritoneal neutrophils incorporated [¹⁴C]arachidonic acid into seven molecular species of choline-containing phosphoglycerides. These 2-[¹⁴C]arachidonoyl species differed with respect to the alkyl ether or acyl residue bound at the $\text{sn}$-1 position; four of the seven were ether-linked. Stimulation with calcium ionophore A23187 induced a proportionate release of arachidonate from all seven molecular species: 40% of the released arachidonate came from alkyl ether species. Thus, 1-$\text{O}$-alkyl-2-arachidonoyl-$\text{sn}$-glycero-3-phosphocholine (GPC) is a significant source of metabolizable arachidonic acid. Since 1-$\text{O}$-alkyl-2-lyso-GPC is the metabolic precussor of platelet activating factor, these results further interrelate pathways forming arachidonate metabolites and platelet activating factor; they also supply a rationale for the observation that both classes of stimuli form concomitantly during cell activation.     

Binding of benzo[a]pyrene at the 1,3,6 positions to nucleic acids in vivo on mouse skin and in vitro with rat liver microsomes and nuclei

Loss of tritium from specific positions in [³H,¹⁴C] aromatic hydrocarbons can elucidate their binding site(s) to DNA and RNA and indicate the mechanism of activation. Studies of tritium loss from [6-³H,¹⁴C]benzo[a]pyrene(B[a]P), [1,3-³H,¹⁴C]B[a]P, [1,3,6-³H,¹⁴C]B[a]P, [6,7-³H,¹⁴C]B[a]P, and [7-³H,¹⁴C]B[a]P were conducted in vitro using liver nuclei and microsomes from 3-methylcholanthrene-induced Sprague-Dawley rats and in vivo on the skin of Charles River CD-1 mice. The relative loss of tritium from $\text{[}{}^3\text{H}\text{,}{}^{14}\text{C}\text{]}\text{B}\text{[a]}\text{P}$ was measured after binding to skin DNA and RNA, to nuclear DNA, and to native and denatured calf thymus and rat liver DNA's and poly(G) by microsomal activation. In skin, nuclei, and microsomes plus native DNA, virtually all B[a]P binding occurred at positions 1,3 and 6; while with microsomes plus denatured DNA or poly(G), B[a]P showed no binding at the 6 position and a small amount at the 1 and 3 positions. In vivo and with nuclei, binding at the 6 position predominated. Little loss of tritium from the 7 position was seen; this was expected because binding at this position is not thought to occur. This confirms the interpretation of loss of tritium as an indication of binding at a given position. These results demonstrate that the use of microsomes to activate B[a]P is not a valid model system for delineating the in vivo mechanism of B[a]P activation, and support previous evidence for one-electron oxidation as the mechanism of activation of hydrocarbons in binding to nucleic acids.     

Enhanced activity of tRNA-pseudouridine synthetase in Yoshida ascites sarcoma.

Five days after transplantation of Yoshida ascites sarcoma cells into a rat, specific activity of tRNA-pseudouridine synthetase was extremely high in the supernatant of tumor cells and moderately high in the tumor-bearing rat liver compared with normal rat liver. Enzyme assay was performed at 37°C by determining the release of tritium from heterogeneous [³H] tRNA extracted from $\text{E. coli}$ B grown in the presence of [5,6-³H]-uracil and resulting in the increased ratio of the amount of pseudouridine to uridine residues in [³H] tRNA. Neither [5-³H]-uridine, [5,6-³H]-UTP, nor [5,6-³H]-poly U released tritium in the present assay conditions.     

The reduction of vinyl side-chains of Mg-protoporphyrin IX monomethyl ester in vitro

Portions of crude homogenates of etiolated wheat seedlings incubated with Mg-protoporphyrin IX and $\text{S-}\text{adenosyl}\text{-}\text{L}\text{-}\text{methionine}$ and then added to other portions of the same crude homogenates that were pretreated with [1-³H]ethanol and yeast alcohol dehydrogenase provided, after a short reaction period, ³H-labeled Mg-protoporphyrin IX monomethyl ester. The ³H-labeled Mg-protoporphyrin IX monomethyl ester thus obtained was shown to contain the ³H in one reduced (to ethyl) vinyl side-chain. Subsequently, ³H-labeled Mg-monoethyl-(monodivinyl)-protoporphyrin IX monomethyl ester was obtained when Mg-protoporphyrin IX monomethyl ester and [³H]NADH were added to dialyzed crude homogenates of etiolated wheat seedlings. Insignificant amounts of ³H were incorporated into poprhyrin substrates when Mg-2,4-divinylpheoporphyrin a₅ or [³H]NADPH were substituted in reaction mixtures for Mg-protoporphyrin IX monomethyl ester or [³H]NADPH, respectively. The results of these and further experiments suggest that an NADPH-dependent enzyme in the crude homogenates of etiolated wheat seedlings was capable of catalyzing the reduction to ethyl of one vinyl side-chain of Mg-protoporphyrin IX monomethyl ester. These findings suggest that the 4-vinyl side-chain reductive reaction likely occurs after the biosynthesis IX monomethyl ester, but before isocyclic ring formation in the pathway to chlorophyll a.     

Acetylcholine stimulates hydrolysis of 32 P-labeled phosphatidic acid in guinea pig synaptosomes

[³H]-inositol or [³H]-arachidonate was injected intracerebrally into guinea pigs. Labeled nerve endings were incubated with Ach¹ or CCh, both of which stimulate labeling of PhA and PhI from ³²P_i by > 100% and 70% respectively. Their addition did not affect the $\text{in}$$\text{vivo}$ labeled phosphatidyl-[³H]-inositol or [³H]-arachidonyl-diglyceride and -PhI. Enhanced hydrolysis of [³H]-inositol-PhiP and -PhIP₂ in the presence of ACh, CCh or choline was not reversed by atropine. In a two-step experiment, PhA was labeled with ³²P_i, and DNP was added to block further $\text{γ-[}{}^{32}\text{P]-ATP}$ formation. Addition of ACh stimulated an atropine-sensitive $\text{decrease}$ in [³²P]-PhA.     

Incorporation of radioactive shikimic acid into N-(γ-Lglutamyl)-4-hydroxyaniline in Agaricus bisporus

Agaricus bisporus contains novel aromatic compounds. By incubation of the mushroom with [G-¹⁴ C] shikimic acid, the radioactivity was incorporated into tyrosine, phenylalanine and several unidentified metabolites. The most radioactive metabolite in the stipe and the cap was identified as $\text{N-(γ-}\text{L}\text{-glutamyl}\text{)-4-}\text{hyrroxyaniline}$. The radioactivity was proved to be localized in the 4-hydroxyaniline moiety of this compound.     

Control of glycolysis and lipogenesis in the liver by glucagon at the phosphofructokinase-fructose 1,6-diphosphatase site

We have examined the effects of glucagon on lipogenesis from fasted-refed rats incubated under two conditions, either without added substrate or with 10 mml-lactate. Net glycolysis (from glycogen) occurs in the absence of glucagon. This glycolysis is inhibited by glucagon under conditions of no added lactate, and reversed by glucagon to a net gluconeogenesis in the presence of 10 mm lactate. Glucagon markedly inhibits fatty acid synthesis (estimated by incorporation of tritium from THO) in hepatocytes incubated without added substrate; but, in the presence of 10 mml-lactate, the inhibition of fatty acid synthesis is only about 10%. The inhibition of lipogenesis from endogenous glycogen is primarily caused by inhibition of glycolysis. Glucagon markedly lowers the $\text{C}\text{-4,5,6}\text{C}\text{-1,2,3}$ ratio in glucose produced from [1-¹⁴C]galactose, indicating a strong inhibition of phosphofructokinase flux. The $\text{C}\text{-1,2,3}\text{C}\text{-4,5,6}$ ratio in glucose from [1-¹⁴C]glycerol is only slightly less than 1, indicating an active fructose diphosphatase flux even under conditions of active net glycolysis. Glucagon increases this ratio only slightly, suggesting that an acute increase of fructose diphosphatase activity by glucagon may occur, but is of much less importance than the decrease of phosphofructokinase.