Detection of an indole oxidizing system in maize leaves

An enzyme activity which brings about a rapid indole disappearance has been detected in cell free extracts of maize ($\text{Zea}\text{mays}$ L.) leaves. The indole utilization by this enzyme system is not dependent on L-serine and pyridoxal phosphate. It does not result in incorporation of (5-³H) indole or (1-¹⁴C) serine into tryptophan. There was no net tryptophan synthesis concomittant with indole disappearance. The enzyme activity is strongly inhibited by dithionite and diethyl-dithiocarbamate. The inhibition by the latter could be specifically removed by Cu²⁺. The activity of dialyzed enzyme could be restored by addition of Cu²⁺ and FAD. The products of indole oxidation were characterized as anthranilic acid and anthranil (2,1-benzisooxazole). The activity of the indole oxidizing system was 2 to 3 times higher in normal maize varieties (Ganga-2 and Ganga-5) than in Opaque-2.     

Evidence for the incorporation of a long-chain 1,2-alkanediol into diol phospholipids by mammalian brain

[2-¹⁴C,2-³H]1,2-Heptadecanediol administered intracerebrally to 18-day-old rats was found to be incorporated as such into phospholipid Chemical and enzymic degradation of the choline phosphatide fraction showed that an appreciable amount of radioactivity was associated with 2-$\text{O}$-acyl heptadecanediol phosphoryl choline.     

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.     

The metabolic fate of [porphyrin-14C] cytochrome c in dogs.

[Porphyrin-¹⁴C] cytochrome $\text{c}$ (isolated from tissues of dogs injected with [¹⁴C] ALA) was given intravenously to one normal and one bile fistula dog. Essentially all of the injected ¹⁴C was excreted in the urine during the first six days. No (unchanged) cytochrome $\text{c}$ was detectable in the urine. Analysis of ¹⁴C and of light absorption at 400 nm in the successive eluates from Florisil columns showed that all ¹⁴C peaks coincided with pigment peaks, but some pigment peaks has no increase in ¹⁴C. The relative distribution of ¹⁴C in these pigment peaks changed markedly between the first and third days. Delayed excretion of some ¹⁴C suggested cellular uptake of cytochrome $\text{c}$ prior to the urinary excretion of its endogenous metabolites.     

The biosynthesis of lubimin from [1-14C]isopentenyl pyrophosphate by cell-free extracts of potato tuber tissue inoculated with an elicitor preparation from Phytophthora infestans

A cell-free enzyme system, which catalyses the incorporation of radiolabel from [$\text{1}\text{2}$-¹⁴C]isopentenyl pyrophosphate into the sesquiterpenoid phytoalexin lubimin, has been prepared from tuber tissue of Solanum tuberosum inoculated with an elicitor preparation from Phytophthora infestans. Biosynthesis of lubimin is optimum at pH 7.$\text{3}\text{2}$-7.5 and is dependent upon Mg²⁺ and NADPH. Lubimin labelling by cell-free enzyme system prepared from tissue 48 hr after treatment with elicitor rises rapidly to a maximum over the first 30 min of incubation and does not decline for a further 150 min. The biosynthetic capacity for lubimin in cell free extracts can be observed as early as 3 hr after inoculation of tuber tissue, and rises to a maximum at about 48 hr after treatment, declining thereafter. Lubimin labelling is inhibited by iodoacetamide, the effect of which is reversed by 3,3-dimethylallylpyrophosphate. Preliminary observations on the cell-free system show that it will also catalyse the incorporation of [2-¹⁴C]mevalonic acid into lubimin in the presence ofan ATP-generating system.     

Effect of the oral hypoglycemic agent 2-tetradecylglycidic acid on fatty acid oxidation in suckling rats in vivo and in perfused liver

The hypoglycemic agent, 2-tetradecylglycidic acid (TDGA), administered in vivo lowered the concentration of plasma glucose and ketone bodies but raised the concentration of liver and plasma triglycerides in 10-day-old suckling rats. Phospholipid and cholesterol content of the plasma and liver were unaffected by drug treatment. TDGA inhibited the in vivo oxidation of [1-¹⁴C]palmitate but not that of [1-¹⁴C]decanoate. In suckling rat liver perfusion, TDGA totally inhibited ketone body formation from palmitate and depressed ketone body production from decanoate by 20%. Liver ATP and ADP content in the presence of TDGA decreased although this was probably a reflection of the increased triglyceride content of the liver since the $\text{ATP}\text{ADP}$ was the same as control livers. The results are discussed in relation to the diet and to the inhibition of carnitine acyl transferase in suckling rats.     

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.     

Isolation of 4α-methyl-5α-Ergosta-8, 24 (28)-dien-3β-ol from ester fractions of nutritionally deprived neurospora crassa

A method of isolating pure fractions of 4α-methyl-5α-ergosta-8, 24 (28)-dien-3β-ol for sterol intermediate studies is described. Starvation cultures of $\text{Neurospora crassa}$ readily incorporate exogenous mevalonic acid into the sterol ester fraction. Isolation involves a simple solvent extraction and two chromatograms. Only the ester fraction yielded the required purity. Radioactive 4α-methyl-5α-ergosta-8, 24 (28)-dien-3β-ol is readily produced from DL-[2-¹⁴C] mevalonic acid.     

Synthetic polynucleotides as model substrates for ribosomal RNA processing

A nuclear exoribonuclease from Novikoff ascites cells was used to study the hydrolysis of single-stranded heteropolymers containing [¹⁴C]adenylic acid and either uridylic acid or cytidylic acid and heteropolymers of [¹⁴C]adenylic acid and one of the corresponding 2′-$\text{O}$-methylated nucleotides. The results of these studies indicate that both the rate and extent of hydrolysis are greatly inhibited by the presence of 2′-$\text{O}$-methylated nucleotides. Restriction of exonuclease activity by 2′-$\text{O}$-methylated nucleotides provides a possible mechanism for rRNA processing.     

Biosynthesis of 3-methylpentacosane in the cockroach Periplaneta americana.

The biosynthesis of 3-methylalkanes was investigated in the cockroach $\text{Periplaneta americana}$. Between 0.2 and 0.3 percent of the labelled acetate and propionate injected into the insect was incorporated into the cuticular hydrocarbons, compared to 0.01 percent for labelled isoleucine. Twenty-three ± four percent of the [2-¹⁴C]acetate, 42 ± 3 and 44 ± 4 percent of the [2-¹⁴C] and [3-¹⁴C]propionate, and 75 ± 5 percent of the [1-¹⁴C]propionate incorporated into the cuticular hydrocarbons was found in 3-methylpentacosane. These results indicate that propionate serves as the source of the branching methyl group, suggesting a pathway in which this precursor is incorporated during the penultimate step in 3-methylalkane biosynthesis in insects.     

Juvenile hormone production by corpora allta of Tenebrio molitor in vitro

$\text{In vitro}$ organ cultures of corpora allata or corpora cardiaca-corpora allata complexes from $\text{Tenebrio molitor}$ were found to produce methyl-(2E, 6E)-(10R)-10, 11- epoxy-3, 7, 11 trimethyl-2, 6-dodecadienoate (JH III). No detectable JH I or II was produced. The hormone was identified by derivation, chromatography and mass spectral analysis. A ¹⁴C radiolabel was incorporated into the methyl carbon of the ester function from precursor L-[$\text{methyl}$-¹⁴C]-methionine added to the culture medium.     

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 $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$.     

Studies on compartmentation of S-adenosyl-L-methionine in Saccharomyces cerevisiae and isolated rat hapatocytes

The existence of metabolically distinct pools of $\text{S-}\text{adenosyl-L-methionine}$ in Saccharomyces cerevisiae and isolated rat hepatocytes was investigated. Utilizing a relatively long labeling period with [methyl-¹⁴C]methionine, a metabolically ‘stable’ pool was labeled. A subsequent short labeling with [methyl-³H]methionine selectively labeled a putative metabolically ‘labile’ pool. The existence of these distinguishable pools was ascertained by following the ³H and ¹⁴C label disappearance in $\text{S-}\text{adenosyl-L-methionine}$ during the chase-period in label-free media containing cycloleycine to prevent futher synthesis of $\text{S-}\text{adenosyl-L-methionine}$. In both yeast and hepatocytes, the ${}^3\text{H}{}^{14}\text{C}$ ratio in $\text{S-}\text{adenosyl-L-methionine}$ decreased sharply. The individual ³H and ¹⁴C decrease in $\text{S-}\text{adenosyl-L-methionine}$ showed $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 3 and 8 min for yeast and 4 and 18 min for hepatocytes. The results strongly indicate that at least two metabolically distinct $\text{S-}\text{adenosyl-L-methionine}$ pools actually do exist in both systems. Subcellular fractionation revealed that the ‘labile’ pool exist in the cytosol for both yeast and hepatocytes while the ‘stable’ pool exists in the vacuolar and the mitochondrial fraction for the yeast and hepatocytes respectively. The $\text{S-}\text{adenosyl-L-methionine}$ pools were also studied in normal yeast under anaerobic chase condition and petite mutant yeast. Sharply contrasting with aerobically chased normal yeast, both showed closely parallel ³H and ¹⁴C decreases in $\text{S-}\text{adenosyl-L-methionine}$.     

Studies on the metabolism of guanidine compounds in mammals

[¹⁴C]Guanidine was observed in the urine after subcutaneous administration to rats of l-[guanidino-¹⁴C]arginine or l-[guanidino-¹⁴C]canavanine. [${}^{14}\text{C}$]Hydroxyguanidine was additionally detected in the urine after injection of dl-[guanidino-¹⁴C]canavanine. These ${}^{14}\text{C}$ metabolites were characterized by high-voltage electrophoresis and paper chromatography, by enzymatic conversion of [¹⁴C]hydroxyguanidine to [¹⁴C]guanidine, and by repeated recrystallization of isolated urinary [${}^{14}\text{C}$]guanidine as the picrate salt with no significant loss of specific activity. These experiments demonstrate that both l-arginine and l-canavanine can serve as precursors of guanidine in the rat.     

The extrasplanchnic origin of blood dihydrotestosterone in elderly men

The splanchnic extraction and interconversion of testosterone (T) and dihydrotestosterone (DHT) were studied in 5 elderly men undergoing cardiac catheterization using a constant Infusion of [1,2-³H] testosterone and [4-¹⁴C] DHT. Metabolic clearance rate (MCR), splanchnic extraction (SE), splanchnic clearance (SC), extrasplanchnic clearance (ESC), transfer constant In blood ([P]_BB^T-DHT) and transfer constant across the liver ([P]_BB^T-DHT) were calc?ulated. The MCR^T was 675 ± 108 (mean ± SC) L/day and MCR^DHT was 409 ± 68 L/day. SE^T was 45.9 ± 7.0% and SE^DHT was 18.5 ± 5.4%. When these values are compared with those recently reported by us for normal men, there is a $\text{1}\text{3}$ reduction in SE^T and $\text{1}\text{2}$ reduction for SE^DHT in elderly men. The calculated SC^T and ESC^T were 355 ± 72 L/day and 320 ± 86 L/day, respectively. SC^DHT and ESC^DHT were 145 + 48 L/day and 263 ± 77 L/day respectively, suggesting that a major fraction of DHT is metabolized in extrasplanchnic organs. No evidence for a net appearance of DHT by either mass or specific activity analysis in hepatic vein blood was observed indicating that the splanchnic compartment does not contribute DHT into the circulation either by $\text{de novp}$ synthesis or via conversion from testosterone. This work indicates that conversion of testosterone to DHT in elderly men occurs entirely in extrasplanchnic tissue.     

Conversion of sterigmatocystin to aflatoxin B 1 by Aspergillus parasiticus

¹⁴C-Sterigmatocystin isolated from cultures of $\text{Aspergillus}$$\text{versicolor}$ supplemented with (1-¹⁴C)acetate was shown to be efficiently converted to aflatoxin B₁ by the resting mycelium of $\text{A. parasiticus}$. The experimental results may indicate a biosynthetic pathway leading from 5-hydroxysterigmatocystin to sterigmatocystin and then to aflatoxin B₁.     

Effect of dichloroacetate and glucagon on the incorporation of labeled substrates into glucose and on pyruvate dehydrogenase in hepatocytes from fed and starved rats

Dichloroacetate (2 mm) stimulated the conversion of [1-¹⁴C]lactate to glucose in hepatocytes from fed rats. In hepatocytes from rats starved for 24 h, where the mitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio is elevated, dichloroacetate inhibited the conversion of [1-¹⁴C]lactate to glucose. Dichloroacetate stimulated ¹⁴CO₂ production from [1-¹⁴C]lactate in both cases. It also completely activated pyruvate dehydrogenase and increased flux through the enzyme. The addition of β-hydroxybutyrate, which elevates the intramitochondrial $\text{NADH}\text{NAD}{}^{\mathrm{+}}$ ratio, changed the metabolism of [1-¹⁴C]lactate in hepatocytes from fed rats to a pattern similar to that seen in hepatocytes from starved rats. Thus, the effect of dichloroacetate on labeled glucose synthesis from lactate appears to depend on the mitochondrial oxidation-reduction state of the hepatocytes. Glucagon (10 nm) stimulated labeled glucose synthesis from lactate or alanine in hepatocytes from both fed and starved rats and in the absence or presence of dichloroacetate. The hormone had no effect on pyruvate dehydrogenase activity whether or not the enzyme had been activated by dichloroacetate. Thus, it appears that pyruvate dehydrogenase is not involved in the hormonal regulation of gluconeogenesis. Glucagon inhibited the incorporation of 10 mm [1-¹⁴C]pyruvate into glucose in hepatocytes from starved rats. This inhibition has been attributed to an inhibition of pyruvate dehydrogenase by the hormone (Zahlten et al., 1973, Proc. Nat. Acad. Sci. USA70, 3213–3218). However, dichloroacetate did not prevent the inhibition of glucose synthesis. Nor did glucagon alter the activity of pyruvate dehydrogenase in homogenates of cells that had been incubated with 10 mm pyruvate in the absence or presence of dichloroacetate. Thus, the inhibition by glucagon of pyruvate gluconeogenesis does not appear to be due to an inhibition of pyruvate dehydrogenase.     

Effects of desmethylimipramine and diphenylpropene derivatives on synthesis of rat brain lipids in vivo

The effects of antidepressant compounds on the synthesis of brain lipids from [1-¹⁴C] acetate $\text{in vivo}$ in 15 day old rats have been investigated. Compounds used included the drug desmethylimipramine (DMI), the tetrabenazine antagonist 3-methylamino-1:1-diphenylprop-1-ene (II) and the primary (I) and tertiary (III) amine analogues of (II). Compound (II), the most potent tetrabenazine antagonist in the diphenylpropene series, significantly increased lipogenesis, whereas the remaining compounds did not. The results from fractionation of the lipid extract from rats treated with (II) indicated that the incorporation of radioactivity from [1-¹⁴C] acetate increased proportionally in all neutral lipids and phospholipids. Tetrabenazine also increased brain lipogenesis $\text{in vivo}$ and altered the distribution within lipid classes of radioactivity from [1-¹⁴C] acetate. Using [¹⁴C] labelled compound, the concentration of (II) in the brain under the present experimental conditions has been determined.     

Concerning the specificity of heme oxygenase: the enzymatic oxidation of synthetic hemins.

Hemin XIII $\text{4}\text{～}$, hemin III $\text{5}\text{～}$, and iron $\text{1,4-di(β-hydroxyethyl)porphyrin}\text{6}\text{～}$ were enzymatically oxidized by a microsomal heme oxygenase preparation from rat liver. These are all better substrates of the oxygenase than the natural substrate, hemin IX $\text{1}\text{～}$. The enzymatic oxidation was selective for the α-methine bridge and in every case only the α-biliverdins were obtained. The latter were readily reduced by biliverdin reductase to the corresponding α-bilirubins. The absence of isomers in addition to the α-bilirubins was established by preparing the derived azopigments and by using [α-¹⁴C]$\text{6}\text{～}$ and [α-¹⁴C]$\text{4}\text{～}$ as substrates. The chemical oxidation of $\text{4}\text{～}$, $\text{5}\text{～}$, and $\text{6}\text{～}$ gave the expected mixture of biliverdins. It is concluded that heme oxygenase is not specific for hemin IX. On the other hand, the enzyme is highly selective for the α-methine bridge, defined as the methine opposed to that flanked by the 6,7-propionic acid residues.     

Role of epithelial-mesenchymal interactions in the ontogenesis of intestinal brush-border enzymes

The respective roles of embryonic intestinal mesenchyme and endoderm in the biochemical differentiation of brushborder enzymes have been investigated. As a first step of this study, the prenatal developmental pattern of several enzymes (maltase, sucrase, lactase, alkaline phosphatase), measured in brush-border membranes purified from chick and rat intestine, has been established. Xenoplastic recombinations between the intestinal tissue components of $\text{5-}\text{1}\text{2}\text{-}\text{day-old}$ chick embryos and 14-day-old fetal rats have been performed. After 11 days of intracoelomic graft in 3-day-old chick embryos, the combinations composed of chick mesenchyme and rat endoderm (Cm/Re) showed enzyme activities characteristic of the fetal rat intestine: high lactase activity and traces of sucrase activity. The inverse combinations composed of rat mesenchyme and chick endoderm (Rm/Ce) exhibited a chicken-like pattern: high sucrase activity and traces of lactase activity. In the latter combinations, the specific enzyme activities were similar to those present in the intestine of 15- to 16-day-old chick embryos (theoretical level reached after the grafting period). Conversely, the levels of enzyme activities of the Cm/Re combinations remained lower than those present in the normally developed rat intestine. These results show that the endodermal tissue carries the specific characteristics of its future biochemical differentiation. They also suggest that the important maturation events, which occur shortly before birth in the rat, are dependent upon other factors, presumably hormones.