The Biosynthesis of Some Isothiocyanates and Oxazolidinethiones in Rape (Brassica campestris L.)

The incorporation of the radioactivity from acetate-1-¹⁴C, acetate-2-¹⁴C, dl-methionine-1-¹⁴C, dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, dl-allyl-glycine-2-¹⁴C, and dl-2-amino-5-hydroxyvalerate-2-¹⁴C into the aglycones of progoitrin, gluconapin, and glucobrassicanapin of maturing rape plants (Brassica campestris L.) was investigated. Radioactivity from dl-methionine-2-¹⁴C, dl-methionine-3,4-¹⁴C, dl-homomethionine-2-¹⁴C, and acetate-2-¹⁴C were incorporated into the 3 major thioglucosides. The other organic compounds were poorly incorporated except for dl-allylglycine-2-¹⁴C into glucobrassicanapin. The results obtained suggest that the rape plant can synthesize amino acids by the condensation of acetate (as acetyl CoA) to α-keto acids to yield a homologue of the original amino acid. These newly formed amino acids are then employed to synthesize the 3 major thioglucosides.     

Inhibition of Ornithine Decarboxylase and Growth of the Fungus Helminthosporium maydis

α-dl-Difluoromethylornithine (DFMO), a specific enzyme-activated inhibitor of ornithine decarboxylase, at 0.5 to 2.0 millimolar significantly inhibited mycelial growth and especially sporulation of Helminthosporium maydis in the dark; its inhibitory effect on sporulation was greatly increased under light conditions. Putrescine at 0.25 millimolar fully prevented the inhibitory effects of DFMO; the inhibition caused by the latter could not be prevented by cadaverine or CaCl₂. α-dl-Difluoromethylarginine, a specific enzyme-activated inhibitor of arginine decarboxylase, at 0.1 to 2.0 millimolar had a weak inhibitory effect on the fungus. The effect was not dependent on the inhibitor concentration and there was no detectable arginine decarboxylase activity in the fungus.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Enzymatic Esterification of Indole-3-acetic Acid to myo-Inositol and Glucose

Incubation of mature sweet corn kernels of Zea mays in dilute solutions of ¹⁴C-labeled indole-3-acetic acid leads to the formation of ¹⁴C-labeled esters of myo-inositol, glucose, and glucans. Utilizing this knowledge it was found that an enzyme preparation from immature sweet corn kernels of Zea mays catalyzed the CoA- and ATP-dependent esterification of indole-3-acetic acid to myo-inositol and glucose. The esters formed were 2-O-(indole-3-acetyl)-myo-inositol, 1-dl-1-O-(indole-3-acetyl)-myo-inositol, di-O-(indole-3-acetyl)-myo-inositol, tri-O-(indole-3-acetyl)-myo-inositol, 2-O-(indole-3-acetyl)-d-glucopyranose, 4-O-(indole-3-acetyl)-d-glucopyranose and 6-O-(indole-3-acetyl)-d-glycopyranose. An assay system was developed for measuring esterification of ¹⁴C-labeled indole-3-acetic acid by ammonolysis of the esters followed by isolation and counting the radioactive indole-3-acetamide.     

Polyamines, hydroxycinnamoylputrescines, and root formation in leaf explants of tobacco cultivated in vitro: effects of the suicide inhibitors of putrescine synthesis

In vitro formation of roots is obtained directly, without intermediate growth of callus, from foliar explants of a tobacco (Nicotiana tabacum) plant cultured on Murashige and Skoog medium containing IAA. Auxin-induced root formation was accompanied by significant changes in hydroxycinnamoylputrescine levels. Increasing levels were found in leaf explants during the first 14 days in culture; this was followed by a sharp decline after 20 days. Early changes in putrescine conjugates were detected in leaf explants before the visible appearance of roots. An early and transitory accumulation of hydroxycinnamoylputrescines was observed in the roots. Free polyamines (putrescine, spermidine, and spermine) in leaf explants and roots were always at a low level and only small changes in their concentrations were observed, α-dl-difluoromethylarginine and α-dl-difluoromethylornithine, specific, irreversible inhibitors of arginine decarboxylase and ornithine decarboxylase, respectively, inhibited putrescine accumulation and root initiation and reduced the fresh and dry weights of leaf explants. These effects were reversed by free putrescine or hydroxycinnamoylputrescines. The results reported here suggest that hydroxycinnamoylputrescines are associated with root formation. The relationship among free polyamines, hydroxycinnamoylputrescines, cell division, and root formation is discussed.     

Some observations on the biosynthesis of the plant sulpholipid by Euglena gracilis

1. dl-Cysteine decreases the uptake of ³⁵SO₄²⁻ by Euglena gracilis but does not decrease the relative incorporation of the isotope into sulpholipid; cysteic acid, on the other hand, does not affect the uptake of ³⁵SO₄²⁻ but does dilute out its incorporation into the sulpholipid. 2. Both l-[³⁵S]cysteic acid and dl-+meso-[3-¹⁴C]cysteic acid appear almost exclusively in 6-sulphoquinovose. 3. Molybdate inhibits the incorporation of ³⁵SO₄²⁻ into sulpholipid but not its uptake into the cells; this suggests that adenosine 3′-phosphate 5′-sulphatophosphate may be concerned with the biosynthesis of sulpholipid, and it was shown to be formed by chloroplast fragments. 4. An outline scheme for sulpholipid biosynthesis based on these observations is discussed.     

Arginine Metabolism in Developing Soybean Cotyledons: III. Utilization

Tracerkinetic experiments were performed using l-[guanidino-¹⁴C]arginine, l-[U-¹⁴C]arginine, l-[ureido-¹⁴C]citrulline, and l-[1-¹⁴C]ornithine to investigate arginine utilization in developing cotyledons of Glycine max (L.) Merrill. Excised cotyledons were injected with carrier-free ¹⁴C compounds and incubated in sealed vials containing a CO₂ trap. The free and protein amino acids were analyzed using high performance liquid chromatography and arginine-specific enzyme-linked assays. After 4 hours, 75% and 90% of the ¹⁴C metabolized from [guanidino-¹⁴C]arginine and [U-¹⁴C]arginine, respectively, was in protein arginine. The net protein arginine accumulation rate, calculated from the depletion of nitrogenous solutes in the cotyledon during incubation, was 17 nanomoles per cotyledon per hour. The data indicated that arginine was also catabolized by the arginase-urease reactions at a rate of 5.5 nanomoles per cotyledon per hour. Between 2 and 4 hours ¹⁴CO₂ was also evolved from carbons other than C-6 of arginine at a rate of 11.0 nanomoles per cotyledon per hour. It is suggested that this extra ¹⁴CO₂ was evolved during the catabolism of ornithine-derived glutamate; ¹⁴C-ornithine was a product of the arginase reaction. A model for the estimated fluxes associated with arginine utilization in developing soybean cotyledons is presented.     

Feruloylputrescine and Caffeoylputrescine Are Not Involved in Growth and Floral Bud Formation of Stem Explants from Nicotiana tabacum L. var Xanthi nc

The role of feruloylputrescine (FP) and of caffeoylputrescine (CP) was investigated in an explant system of stem explants from day-neutral Nicotiana tabacum L. var Xanthi nc. Previously, a correlation between cortical callus formation and increase in FP content, as well as between in vitro flower formation and increase in CP content had been shown. During the explant growth in vitro, the increase of both FP and CP was inhibited by 4-fluor-(1-amino-2-phenylethyl)phosphonic acid and 2-amino-indene-2-phosphonic acid, both inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5). dl-α-difluoromethylarginine, an inhibitor of arginine decarboxylase (ADC, EC 4.1.1.19), prevented only the increase in FP, while dl-α-difluoromethylornithine, an inhibitor of ornithine decarboxylase (EC 4.1.1.17), reduced only that of CP. Increase in dry weight and the formation of cortical callus and of floral buds of explants were not affected by any of the inhibitors. We conclude, in contrast to earlier hypotheses, that FP and CP do not trigger growth and differentiation in the explants. It seems more likely that FP and CP increase in response to auxin and cytokinin in the media.     

Selective Inhibition of the Demethylation at C-14 in Ergosterol Biosynthesis by the Fungicide,Denmert (S-1358)

In cell-free homogenates of Saccharomyces cerevisiae, Denmert (S-1358) inhibited the incorporation of radioactivity from dl-mevalonate-2-¹⁴C into 14-desmethyl-lanosterol, 4α-methyl-cholesta-8,24-dien-3-one, 4α-methyl-zymosterol and 4-desmethyl sterols (zymosterol and episterol) at a concentration of 10^?4 m. Concomitantly, a large accumulation of radioactivity was observed in the Ianosterol fraction.

In good agreement with the results described above, Denmert inhibited the conversion of ¹⁴C-labeled lanosterol to 4-desmethyl sterols, while the conversion of ¹⁴C-labeled 14-desmethyl-lanosterol to 4-desmethyl sterols was hardly affected by the fungicide. It is therefore evident that Denmert is a potent selective inhibitor of the demethylation at the C–14 position in ergosterol biosynthesis.

The fungicide, triarimol, was also found to exhibit the same effect on sterol biosynthesis as Denmert.     

Lipogenesis in the brain of suckling rats. Studies on the mechanism of mitochondrial–cytosolic carbon transfer

1. Studies on the incorporation of [3-¹⁴C]pyruvate and d-3-hydroxy[3-¹⁴C]butyrate into the brain lipid fraction by brain homogenates of the suckling (7-day-old) rat have been carried out. 2. Whereas approximately twice as much CO₂ was evolved from pyruvate compared with 3-hydroxybutyrate metabolism, similar amounts of the radioactivity of these two precursors were incorporated into the lipid fraction. Furthermore, in both cases the incorporation into lipid was almost tripled when glucose (10mm) or NADPH (2.5mm) was added to the incubation media. 3. If 5mm-(—)-hydroxycitrate, an ATP–citrate lyase inhibitor, was added to the incubation the incorporation of carbon from pyruvate was inhibited to 39% of the control and from 3-hydroxybutyrate to 73% of the control, whereas CO₂ production from both precursors was not affected. 4. The incorporation from pyruvate or 3-hydroxybutyrate into lipids was not affected by the presence of 10mm-glutamate in the medium (to encourage N-acetylaspartate production). However, incorporation from pyruvate was inhibited by 21% in the presence of 5mm-amino-oxyacetate (a transaminase inhibitor) and by 83% in the presence of both hydroxycitrate (5mm) and amino-oxyacetate. 5. Incorporation from 3-hydroxybutyrate into brain lipids was inhibited by 20% by amino-oxyacetate alone, but by 55% in the presence of both hydroxycitrate and amino-oxyacetate. 6. It is concluded that the mechanism of carbon transfer from pyruvate into lipids across the mitochondrial membrane in the suckling rat brain is mainly via citrate and N-acetylaspartate. 3-Hydroxybutyrate, in addition to using these routes, may also be incorporated via acetoacetate formation and transport to the cytosol.     

Inositol Metabolism in Plants. V. Conversion of Myo-inositol to Uronic Acid and Pentose Units of Acidic Polysaccharides in Root-tips of Zea mays

The metabolism of myo-inositol-2-¹⁴C, d-glucuronate-1-¹⁴C, d-glucuronate-6-¹⁴C, and l-methionine-methyl-¹⁴C to cell wall polysaccharides was investigated in excised root-tips of 3 day old Zea mays seedlings. From myo-inositol, about one-half of incorporated label was recovered in ethanol insoluble residues. Of this label, about 90% was solubilized by treatment, first with a preparation of pectinase-EDTA, then with dilute hydrochloric acid. The only labeled constituents in these hydrolyzates were d-galacturonic acid, d-glucuronic acid, 4-O-methyl-d-glucuronic acid, d-xylose, and l-arabinose, or larger oligosaccharide fragments containing these units. Medium external to excised root-tips grown under sterile conditions in myo-inositol-2-¹⁴C contained labeled polysaccharide.     

d-Glucosone and l-Sorbosone, Putative Intermediates of l-Ascorbic Acid Biosynthesis in Detached Bean and Spinach Leaves

d-[6-¹⁴C]Glucosone that had been prepared enzymically from d-[6-¹⁴C]glucose was used to compare relative efficiencies of these two sugars for l-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, ¹⁴C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from [6-¹⁴C]glucose underwent considerable redistribution during AA formation, whereas ¹⁴C from [6-¹⁴C]glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, l-[U-¹⁴C]sorbosone was found to be equivalent to [6-¹⁴C]glucose as a source of ¹⁴C for AA. In the presence of 0.1% d-glucosone, conversion of [6-¹⁴C] glucose into labeled AA was greatly repressed. In a comparable experiment with l-sorbosone replacing d-glucosone, the effect was much less. The experiments described here give substance to the proposal that d-glucosone and l-sorbosone are putative intermediates in the conversion of d-glucose to AA in higher plants.     

Degradation of phenylalanine and tyrosine by Sporobolomyces roseus

Ammonia-lyase activity for l-phenylalanine, m-hydroxyphenylalanine and l-tyrosine was demonstrated in cell-free extracts of Sporobolomyces roseus. Cultures of this organism converted dl-[ring-¹⁴C]phenylalanine and l-[U-¹⁴C]tyrosine into the corresponding cinnamic acid. Tracer studies showed that these compounds were further metabolized to [¹⁴C]protocatechuic acid. Benzoic acid and p-hydroxybenzoic acid were intermediates in this pathway. Washed cells of the organism readily utilized cinnamic acid, p-coumaric acid, caffeic acid, benzoic acid and p-hydroxybenzoic acid. Protocatechuic acid was the terminal aromatic compound formed during the metabolism of these compounds. The cells of S. roseus were able to convert m-coumaric acid into m-hydroxybenzoic acid, but the latter compound, which accumulated in the medium, was not further metabolized. 4-Hydroxycoumarin was identified as the product of o-coumaric acid metabolism by this organism.     

Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon

1. Human uterine cervical stroma was found to contain a Ca²⁺-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×10⁵ by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.     

Biosynthesis of (+)-Tartaric Acid from l-[4-C]Ascorbic Acid in Grape and Geranium

The metabolic fate of l-[4-¹⁴C]ascorbic acid has been examined in the grape (Vitis labrusca L.) and lemon geranium (Pelargonium crispum L. L'Hér. cv. Prince Rupert) under conditions comparable to data from l-[1-¹⁴C]ascorbic acid and l-[6-¹⁴C]ascorbic acid experiments. In detached grape leaves and immature berries, l-[4-¹⁴C]ascorbic acid and l-[1-¹⁴C]ascorbic acid were equivalent precursors to carboxyl labeled (+)-tartaric acid. In geranium apices, l-[4-¹⁴C]ascorbic acid yielded internal labeled (+)-tartaric acid while l-[6-¹⁴C]ascorbic acid gave an equivalent conversion to carboxyl labeled (+)-tartaric acid. These findings clearly show that two distinct processes for the synthesis of (+)-tartaric acid from l-ascorbic acid exist in plants identified as (+)-tartaric acid accumulators. In grape leaves and immature berries, (+)-tartaric acid synthesis proceeds via preservation of a four-carbon fragment derived from carbons 1 through 4 of l-ascorbic acid while carbons 3 through 6 yield (+)-tartaric acid in geranium apices.     

Polyamine synthesis in maize cell lines

Uptake of [¹⁴C]putrescine, [¹⁴C]arginine, and [¹⁴C]ornithine was measured in five separate callus cell lines of Zea mays. Each precursor was rapidly taken into the intracellular pool in each culture where, on the average, 25 to 50% of the total putrescine was found in a conjugated form, detected after acid hydrolysis. Half-maximal labeling of each culture was achieved in less than 1 minute. Within this time frame of precursor incorporation, only putrescine derived from arginine was conjugated, indicating that putrescine pools derived from arginine may initially be sequestered from ornithine-derived putrescine. The decarboxylase activities were measured in each culture after addition of exogenous polyamine to the growth medium to assess differential regulation of the decarboxylases. Arginine and ornithine decarboxylase activities were augmented by added polyamine, the effect on arginine decarboxylase being eightfold greater than on ornithine decarboxylase. Levels of extractable ornithine decarboxylase were consistently 15- to 100-fold higher than arginine decarboxylase, depending on the titer of extracellular polyamine. Taken as whole the results support the idea that there are distinct populations of polyamine that are initially sequestered after the decarboxylase reactions and that give rise to separate end products and possibly have separate functions.     

Effects of the suicide inhibitors of arginine and ornithine decarboxylase activities on organogenesis, growth, free polyamine and hydroxycinnamoyl putrescine levels in leaf explants of Nicotiana xanthi N.C. Cultivated in vitro in a medium producing callus formation

We studied the effects of dl-α-difluoromethylarginine (DFMA) and dl-α-difluoromethylornithine (DFMO), specific, irreversible inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), respectively, on organogenesis growth and titers of free polyamines and conjugated putrescines (hydroxycinnamoyl putrescines) in tobacco (Nicotiana tabacum cv Xanthi n.c.) calli. These results suggest that ADC and ODC regulate putrescine biosynthesis during early and later stages of tobacco callus development, respectively. ADC appears active in biosynthesis of large levels of free amines (agmatine and putrescine) while ODC appears active only in biosynthesis of large levels of putrescine conjugates (hydroxycinnamoyl putrescines). DFMA inhibits the fresh and dry weight increases of tobacco calli, whereas DFMO even promoted the fresh and dry weight increases, thus supporting the view that ADC is important for cell division and callus induction. Inhibition of ODC activity by DFMO resulting in an amide deficiency after 4 weeks of culture facilates the expression of differentiated cell functions. Formation of buds is associated with a significant decrease of hydroxycinnamoyl putrescines.     

Loss of Hydrogen from Carbon 5 of d-Glucose during Conversion of d-[5-H,6-C]Glucose to l-Ascorbic Acid in Pelargonium crispum (L.) L'Hér

Conversion of d-[5-³H,6-¹⁴C]glucose to l-ascorbic acid in detached apices of Pelargonium crispum (L.) L'Hér cv Prince Rupert (lemon geranium) was accompanied by complete loss of tritium in the product. Chemical degradation of d-glucose which was recovered from the labeled apices yielded d-glyceric acid (corresponding to carbons 4, 5, and 6 of glucose) with a ³H:¹⁴C ratio of 4 to be compared with 9, the ratio in d-[5-³H,6-¹⁴C]glucose initially. Conversion of d-[6-³H,6-¹⁴C]glucose in the same tissue was accompanied by retention of tritium in l-ascorbic acid with a ³H:¹⁴C ratio comparable to that of compounds from the hexose pool. Results indicate that during l-ascorbic acid biosynthesis from glucose in Pelargonium crispum hydrogen at carbon 5 undergoes exchange with the medium, suggesting an epimerization at this carbon atom.     

Effect of tunicamycin on epidermal glycoprotein and glycosaminoglycan synthesis in vitro

1. When pig ear skin slices were cultured for 18h in the presence of 1μg of tunicamycin/ml the incorporation of d-[³H]glucosamine into the epidermis, solubilized with 8m-urea/5% (w/v) sodium dodecyl sulphate, was inhibited by 45–55%. This degree of inhibition was not increased by using up to 5μg of tunicamycin/ml or by treating the skin slices with tunicamycin for up to 8 days. The incorporation of (U-¹⁴C)-labelled l-amino acids under these conditions was not affected by tunicamycin. Polyacrylamide-gel electrophoresis indicated that the labelling of the major glycosaminoglycan peak with d-[³H]glucosamine was unaffected, whereas that of the faster migrating glycoprotein components was considerably decreased in the presence of tunicamycin. 2. Subcellular fractionation indicated that tunicamycin specifically inhibited the incorporation of d-[³H]glucosamine but not of (U-¹⁴C)-labelled l-amino acids into particulate (mainly plasma-membrane) glycoproteins by about 70%. The labelling of soluble glycoproteins was hardly affected. Polyacrylamide-gel electrophoresis of the plasma-membrane fraction showed decreased d-[³H]glucosamine incorporation into all glycoprotein components, indicating that the plasma-membrane glycoproteins contained mainly N-asparagine-linked oligosaccharides. 3. Cellulose acetate electrophoresis of both cellular and extracellular glycosaminoglycans showed that tunicamycin had no significant effect on the synthesis of the major component, hyaluronic acid. However, the incorporation of both d-[³H]glucosamine and ³⁵SO₄²⁻ into sulphated glycosaminoglycans was inhibited by about 50%. This inhibition was partially overcome, at least in the cellular fraction, by 2mm-p-nitrophenyl β-d-xyloside indicating that tunicamycin-treated epidermis retained the ability to synthesize sulphated glycosaminoglycan chains. Tunicamycin may affect the synthesis and/or degradation of proteoglycan core proteins or the xylosyltransferase. 4. Electron-microscopic examination of epidermis treated with tunicamycin for up to 4 days revealed no significant changes in cell-surface morphology or in epidermal-cell adhesion. Either N-asparagine-linked carbohydrates play little role in epidermal-cell adhesion or more probably there is little turnover of these components in epidermal adhesive structures such as desmosomes and hemidesmosomes during organ culture.     

Use of N-Bromoacetyl-l-ornithine to Study l-Ornithine and l-Arginine Biosynthesis in Soybean (Glycine max L.) Cell Cultures

The effect of the inhibitor N²-bromoacetyl-l-ornithine (NBAO) on the biosynthesis of ornithine in higher plants, was investigated using soybean cells (Glycine max L. var Mandarin), grown in suspension culture. The NBAO was found to reduce the specific activity of the enzyme N²-acetyl-l-ornithine: l-glutamate N-acetyltransferase (EC 2.3.1.35). In contrast, the specific activity of the enzyme acetyl coenzyme A:L-glutamate N-acetyltransferase (EC 2.3.1.1), which is also involved in N-acetylglutamate biosynthesis, was not significantly changed. Estimation of the concentrations of free amino acids in the soluble fraction of the cells showed that while ornithine levels were decreased, glutamic acid levels were increased in the presence of NBAO. While arginine levels initially increased in the presence of NBAO, they finally decreased near the end of the growth period. Evidence was obtained that the initial increase in arginine levels was due to the inhibition of arginase (EC 3.5.3.1) by N²-bromoacetyl l-ornithine. We conclude that the reaction catalyzed by N²-acetyl-l-ornithine:l-glutamate N-acetyl transferase is a rate limiting reaction in vivo.