Effect of zinc on the biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin in etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell)

The effect of Zn²⁺ ions (in the form of ZnCl₂) in the ceoncentration range 10^?3 to 10^?6 M on the content and biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin has been studied on etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell). In the “long-term” experiment zine ions influenced the seedlings during eight days of germination, whereas in the “short-term” experiment zinc ions acted only 72 h on seven days old intact seedlings. The biosynthesis of indole glucosinolates has been followed by the incorporation of³⁵S from Na₂ ³⁵SO₄ into both glucosinolates in experiments with, hypocotyl segments of the rape seedlings. Zinc ions at chronic “long-term” application increased the glucobrassicin and neoglucobrassicin level in the seedlings. The neoglucobrassicin content especially was increased. A “short-term” application of zinc ions increased the level of both glucosinolates at higher and lower concentrations, whereas medium concentrations (10^?4 and 10^?5 M) lowered their level. Zn²⁺ ions lowered absorption of³⁵SO₄ ^?2 ions by hypocotyl segments and simultaneously lowered the incorporation of³⁵S into glucobrassicin. On the contrary, the incorporation of³⁵S into neoglucobrassicin and proteins was stimulated. Zinc ions do exhibit a specific effect on neoglucobrassicin biosynthesis, on membrane permeability as against sulphate ions and on the incorporation of sulphur into proteins.     

Effect of copper on the content and the biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin in etiolated rape seedlings (Brassica napus var.arvensis (Lam.) Thell.)

The influence of Cu²⁺ ions (in the form of CuCl₂) in the concentration range 10^?3 to 10^?6 M on the content and biosynthesis of indole glucosinolates glucobrassicin and neoglucobrassicin has been studied on etiolated seedlings of rape (Brassica napus var.arvensis (Lam.) Thell.). Ions Cu²⁺ acted on the seedlings either chronically from the beginning of the germination or acutely, during 3 to 72 h, on seven days old seedlings. The biosynthesis of both glucosinolates was followed by the incorporation of³⁵S from Na₂ ³⁵SO₄ into them in hypocotyl segments from seven days old intact etiolated seedlings. After the entry of small amounts of Cu²⁺ ions into the plants, stimulation of the glucosinolates formation occurs, as was found after three h action of Cu²⁺ ions. After the entry of a greater amount of Cu²⁺ ions into the plant, harmful effects appear, as was found after chronic two days action or after 24 and 48 hours acute action of Cu²⁺ ions. Later further stimulation of glucosinolate formation occurs, probably due to enhanced metabolism during reparation processes, as was manifested after chronic action of Cu²⁺ ions lasting four and eight days. The optimal effect of copper was found mainly in the concentration range 5×10^?4 M to 10^?5 M. Ions Cu²⁺ in higher concentration increased the uptake of sulphate ions by hypocotyl segments, and in lower concentrations increased the incorporation of³⁵S from³⁵SO₄ ^2? into the proteins.     

Initiation of protein synthesis in vivo in poliovirus-infected HeLa cells

Initiation of protein synthesis in vivo in poliovirus-infected HeLa cells has been studied. When these cells are synchronized for initiation by fluoride treatment and then double labeled with [³⁵S]methionine and either tritiated proline, phenylalanine, or valine for short pulses, the percentage of N-terminal methionine incorporated in the nascent peptides compared to total incorporation is significantly higher than that of the tritiated amino acids tested. The data indicate that methionine is the initiator amino acid for the synthesis of poliovirus-specific proteins.     

Heparan sulfate biosynthesis by embryonic tissues and primary fibroblast populations.

Fibroblasts from cornea, heart, and skin of day 14 embryonic chicks demonstrate the ability to make heparan sulfate-like polysaccharide when examined during the 10 hr period immediately following their removal from the embryo. Both the whole tissues from which these fibroblasts are isolated and the fibroblasts grown for 2–5 weeks in vitro also synthesize heparan sulfate. During their first few days in vitro, the three fibroblast populations display increasing rates of [³⁵S]-sulfate and d-[1-³H]-Glucosamine incorporation into glycosaminoglycans and sharp fluctuations of those rates, yet the percentage of total [³⁵S]-sulfate incorporated into heparan sulfate-like polysaccharide and the distribution of this polysaccharide between cells and nutrient medium do not change significantly. During their first 48 hr in vitro, skin fibroblasts, but not those from cornea or heart, show steadily decreasing discrepancies between the proportions of [³⁵S]-sulfate and d-[1-³H]-Glucosamine incorporated into heparan sulfate, suggesting a sharp decline in the synthesis of nonsulfated glycosaminoglycans. These data support the hypothesis of Kraemer than many cell-types in vivo may normally make heparan sulfate. The data largely eliminate the hypothesis that the biosynthesis of this polysaccharide is selectively stimulated as embryonic cells adapt to growth in vitro.     

Distribution and Variation of Indole Glucosinolates in Woad (Isatis tinctoria L.)

The exceptionally high levels in woad (Isatis tinctoria L.) of three indolic goitrogens, namely glucobrassicin, neoglucobrassicin, and glucobrassicin-1-sulfonate, permit the facile study of their distribution in the plant and their changes during its development. Woad seeds contain as much as 0.23% fresh weight of glucobrassicin but no other indole glucosinolate, while 1-week-old seedlings also contain substantial amounts of neoglucobrassicin and glucobrassicin-1-sulfonate in their shoots whether grown in the light or dark. The sulfonate is not found in roots, and light depresses neoglucobrassicin levels in shoots. Sterile root cultures synthesize glucobrassicin and neoglucobrassicin, and significant quantities of these were even found to be excreted by the roots of intact sterile seedlings in culture. This may explain the long known deleterious effect of woad and other cruciferous crops on subsequent plantings and the observation could be of ecological importance. Long term changes in levels of all three substances in the plant are similar and are compatible with earlier suggestions that the compounds could be auxin precursors at the time of flower stem elongation. Since sterile seedlings readily incorporate ³⁵SO₄²⁻ into indole glucosinolates and relative specific radioactivities suggest that glucobrassicin is the precursor of the other two compounds, pathways of goitrogen biosynthesis should be relatively easily determined in this material.     

Glucosinolates of egyptian Capparis species

Capparis ovata var. palaestina Zoh., C. spinosa var. aegyptia Boiss. and C. spinosa var. deserti Zoh., were investigated for glucosinolates. Glucoiberin, glucocapparin, sinigrin, glucocleomin, glucocapangulin, glucobrassicin and neoglucobrassicin, in addition to two others, were isolated. Four of these viz. glucoiberin, sinigrin, glucobrassicin and neoglucobrassicin were detected for the first time in Capparis species. Comparative chromatographic analyses of the glucosinolates of the plants examined revealed qualitative differences.     

Early effects of vitamin A on protein synthesis in the epidermis of embryonic chick skin cultured in serum-containing medium

A quantitative study of the incorporation of ³H-leucine and ³⁵S-cystine by the 12-day embryonic chick epidermis during 2 days in culture in serum-containing medium, with and without added vitamin A has been made. This has shown that very soon after explantation, protein synthesis, particularly the synthesis of cyst(e)ine-rich proteins is enhanced dramatically in the epidermis cultured in the control, serum-containing medium alone. This effect is completely absent in the medium containing added vitamin A (5 IU/ml). Under these conditions ³H-leucine and ³⁵S-cystine incorporation remain relatively unchanged during the 2-day culture period.     

The origin of the sulfur atom in thiamine

The mode of biosynthesis of the thiazole moiety of thiamine, 4-methyl-5β-hydroxyethyl thiazole (MHET) was studied using Salmonella typhimurium as test organism. It was shown by isotope incorporation experiments, that the sulfur atom, but not carbon-3, of cysteine is incorporated into MHET, indicating a separation of the sulfur atom of cysteine from the carbon chain during incorporation. Isotope competition experiments revealed that the incorporation of [³⁵S]cysteine is not significantly diluted by the presence of methionine, homocysteine, and glutathione. No incorporation of label from [¹⁴C]glutamate and [¹⁴C]formate was observed, leaving the origin of the five-carbon unit still in doubt.     

Patterns of incorporation of tritiated thymidine by the dorsal polytene foot-pad nuclei of Sarcophaga bullata (Sarcophagidae: Diptera)

The incorporation of tritiated thymidine into the DNA of the dorsal foot-pad nuclei of Sarcophaga bullata, during pharate adult development, was studied by scintillation counting and autoradiography. Incorporation was maximal on day 4 and showed a progressive temporal decline on days 5 to 8. Autoradiographs of chromosomal arms A¹, A² and D¹, from late stages of the prolonged S period, showed discontinuous incorporation. On days 7 and 8 loci which incorporated the radio-labeled precursor were almost exclusively associated with DNA granules. DNA granules were associated with 8 specific loci in chromosomal arm A¹, 10 in A², and 8 in D¹. The relationship between patterns of incorporation of tritiated thymidine and DNA granules is discussed.Deceased June 15, 1975     

Glucosinolate profiling of Brassica rapa cultivars after infection by Leptosphaeria maculans and Fusarium oxysporum

The glucosinolate contents of two different cultivars of Brassica rapa (Herfstraap and Oleifera) infected with Leptosphaeria maculans and Fusarium oxysporum were determined. Infection triggered the accumulation of aliphatic glucosinolates (gluconapin, progoitrin, glucobrassicanapin and gluconapoleiferin) and indole glucosinolate (4-hydroxy-glucobrassicin) in Herfstraap and of two indole glucosinolates (glucobrassicin and 4-hydroxy-glucobrassicin) in Oleifera. While total and aliphatic glucosinolates decreased significantly in Oleifera, a large increase was observed in Herfstraap after fungal infection. The indole glucosinolate glucobrassicin accumulated in Oleifera at a higher rate than Herfstraap especially after infection with F. oxysporum. Apparently the interaction between fungus and B. rapa is cultivar and fungal species specific.     

Role of Polysulfides in Reduction of Elemental Sulfur by the Hyperthermophilic Archaebacterium Pyrococcus furiosus

Polysulfides formed through the breakdown of elemental sulfur or other sulfur compounds were found to be reduced to H₂S by the hyperthermophilic archaebacterium Pyrococcus furiosus during growth. Metabolism of polysulfides by the organism was dissimilatory, as no incorporation of ³⁵S-labeled elemental sulfur was detected. However, [³⁵S]cysteine and [³⁵S]methionine were incorporated into cellular protein. Contact between the organism and elemental sulfur is not necessary for metabolism. The sulfide generated from metabolic reduction of polysulfides dissociates to a strong nucleophile, HS⁻, which in turn opens up the S₈ elemental sulfur ring. In addition to H₂S, P. furiosus cultures produced methyl mercaptan in a growth-associated fashion.     

Studies of the Biochemical Background to Differences in Glucosinolate Content in Brassica napus L. II. Administration of Some Sulphur-35 and Carbon-14 Compounds and Localization of Metabolic Blocks

The aim of the investigation was to study the differences in the metabolism of substances that are utilized in the synthesis of glucosinolates between the Brassica napus cv. Bronowski, which is very low in glucosinolate content, and a cultivar (cv. Regina II) that contains approximately average amounts of these compounds. By experiments in which the plants were grown in nutrient solutions supplied with sulphate-³⁵S, it was shown that the rate of sulphate uptake was similar in the two cultivars. No accumulation of intermediate metabolites could be demonstrated by autoradiography in Bronowski. Sulphate-³⁵S, methionine-³⁵S, methionine-2-¹⁴C, 2-amino-6-(methylthio)caproic acid-2-¹⁴C, and S-(β-d -glucopyranosyl)-4-pentenethiohydroximic acid (desulpho-3-butenylglucosinolate) (glucose-U-¹⁴C or ³⁵S) were fed to shoots of the two cultivars. After incubation, the plant material was extracted with methanol. The extracts were separated into various fractions, and in some experiments glucosinolates or derivatives of their degradation products were isolated. When measuring the radioactivity of the various fractions or isolated products, the incorporation of radioactivity into glucosinolates was found to be poor in Bronowski from sulphate, methionine, and 2-amino-6-(methylthio)caproic acid. Desulpho-3-butenyl-glucosinolate was an efficient precursor of 3-butenylglucosinolate in Bronowski, but a poor precursor of 2-hydroxy-3-bute-nylglucosinolate, which suggests a metabolic block at the hydroxylation step in this cultivar. In Regina II desulpho-3-butenylglucosinolate was a good precursor of both 3-butenylglucosinolate and of 2-hydroxy-3-butenylglucosinolate, which demonstrates that these glucosinolates may be synthesized without prior formation of the corresponding co-methylthioalkyl glucosinolates and that the hydroxylation can take place after the formation of desulpho-3-butenylglucosinolate. The results indicate that the low glucosinolate content of Bronowski is caused by block(s) in the separate pathway leading to the biosynthesis of glucosinolates.     

Glucosinolate biosynthesis in oilseed rape (Brassicanapus L.): studies with 35SO2-4 and glucosinolate precursors using oilseed rape pods and seeds

³⁵SO^2–₄ and glucosinolate precursors were administeredto investigate the capacity of oilseed rape intact whole pods,isolated pod walls and seeds to carry out reactions of the glucosinolatebiosynthetic pathway. Pod walls incorporated ³⁵S-label from³⁵SO^2–₄ into both the thio-glucose and sulphonate-sulphurof glucosinolates. Pulse-labelling experiments showed that podwalls were the predominant source of the glucosinolates accumulatedby the seeds. Isolated immatureseeds were capable of reductiveassimilation of ³⁵SO^2–₄ and incorporated ³⁵S into sulphur-containingamino acids and the sulphonate moiety of glucosinolates, butwere not able to incorporate ³⁵S into the thio-glucose of glucosinolates. Key words: Biosynthesis, glucosinolates, oilseed rape, pod walls, seeds     

Assimilatory Sulfur Metabolism in Marine Microorganisms: Considerations for the Application of Sulfate Incorporation into Protein as a Measurement of Natural Population Protein Synthesis

The sulfur content of residue protein was determined for pure cultures of Nitrosococcus oceanus, Desulfovibrio salexigens, 4 mixed populations of fermentative bacteria, 22 samples from mixed natural population enrichments, and 11 nutritionally and morphologically distinct isolates from enrichments of Sargasso Sea water. The average 1.09 ± 0.14% (by weight) S in protein for 13 pure cultures agrees with the 1.1% calculated from average protein composition. An operational value encompassing all mixed population and pure culture measurements has a coefficient of variation of only 15.1% (n = 41). Short-term [³⁵S]sulfate incorporation kinetics by Pseudomonas halodurans and Alteromonas luteoviolaceus demonstrated a rapid appearance of ³⁵S in the residue protein fraction which was well modelled by a simple exponential uptake equation. This indicates that little error in protein synthesis determination results from isotope dilution by endogenous pools of sulfur-containing compounds. Methionine effectively competed with sulfate for protein synthesis in P. halodurans at high concentrations (10 μM), but had much less influence at 1 μM. Cystine competed less effectively with sulfate, and glutathione did not detectably reduce sulfate-S incorporation into protein. [³⁵S]sulfate incorporation was compared with [¹⁴C]glucose assimilation in a eutrophic brackish-water environment. Both tracers yielded similar results for the first 8 h of incubation, but a secondary growth phase was observed only with ³⁵S. Redistribution of ¹⁴C from low-molecular-weight materials into residue protein indicated additional protein synthesis. [³⁵S]sulfate incorporation into residue protein by marine bacteria can be used to quantitatively measure bacterial protein synthesis in unenriched mixed populations of marine bacteria.     

Sulfation of fucoidan in Fucus embryos. I. Possible role in localization

Zygotes of the brown alga Fucus distichus L. Powell divide into two cells which are structurally and biochemically different from each other. Cytochemical staining and autoradiography indicate that a sulfated polysaccharide is localized in only one of the two cells. Up to 10 hr after fertilization, no localization of sulfated polysaccharides is detectable in zygotes, and little ³⁵S (Na₂³⁵SO₄) is incorporated into an acid-soluble carbohydrate fraction. Between 10 and 16 hr, during rhizoid initiation and several hours before the first cell division, there is a large increase in the amount of ³⁵S incorporated into this fraction. The label is found associated with the sulfated fucose polymer fucoidan. Various extraction techniques and labeling experiments demonstrate that fucoidan is unsulfated at fertilization and undergoes little metabolic activity or turnover during the first 24 hr. Thus, the incorporation of sulfate into this carbohydrate fraction appears to involve a sulfation of a preexisting, unsulfated fucan polymer. The degree of sulfation achieved at this time in vivo is sufficient for migration of fucoidan through an electric field in agarose or acrylamide gels. The possible role of sulfation as a mechanism for the localization of fucoidan in the rhizoid cell by means of an intracellular electrical gradient is discussed.     

In Vitro Sulfation of Glycoprotein with Sulfotransferase from Dictyosterium discoideum

A screening test for incorporation of [³⁵S]-labeled sulfate into glycoprotein with the sulfotransferase system from Dictyosterium discoideum was done. [³⁵S]-Labeled sulfate was incorporated effectively into the aspartic proteinase of Mucor miehei. The oligosaccharide chain of the aspartic proteinase was about 2 kDa by Endo F digestion and sulfate was incorporated into the oligosaccharide chain of the enzyme.     

Effect of radiation on the biosynthesis of L-Tryptophan from 14C-Anthranilic acid in kohlrabi (Brassica oleracea L., var.gongylodes L.)

The metabolism of¹⁴C-anthranilic acid (¹⁴C-AA) in kohlrabi (Brassica oleracea L. var.gongylodes L.) and the effect of radiation gamma⁶⁰Co on this metabolism was investigated. In hypocotylar segmnents of seven days old etiolated seedlings¹⁴C-AA was metabolised par, tially to its detoxication product¹⁴C-β-glucoside of AA. Simultaneously L-tryptophan was also formed, which in these plants is a precursor of indolic glucosinolates glucobrassicin and neoglucobrassicin. The metabolism of¹⁴C-AA was followed for 97 h. Radiation, applied both to seeds and to seven days old plants did not affect the metabolism of¹⁴C-AA substantially. The intermediary reaction AA → L-tryptophan in the biosynthesis of L-tryptophan is not a radiosensitive part of the synthesis of this amino acid. A not too high radiation sensitivity (max. 45%) was observed in the metabolic pathway leading from L-tryptophan to glucobrassicin.     

Post-translational processing in the synthesis of egg-specific protein in the silkworm,Bombyx mori

The post-translational processing of egg-specific protein (ESP) in developing ovarian follicles of the silkworm, Bombyx mori was analyzed using in vivo and in vitro labeling systems with some radioactive precursors. The labeling with[³⁵S]methionine revealed that ESP is first synthesized as 69 kDa peptide (69K-ESP) which is then converted to 72 kDa peptide (72K-ESP) until 2 h. Some of 72K-ESP molecules were converted to 64 kDa peptide (64K-ESP) after 10h-labeling. [¹⁴C]Mannose was incorporated into 69K-ESP and 72K-ESP. In the presence of tunicamycin, labeling with [³⁵S]methionine brought about a new 67 kDa peptide (67K-ESP) by reducing the incorporation into 69K- and 72K-ESP. [³²P]Ortho-phosphate was incorporated into only 72K-ESP by a 2 h-pulse labeling. Treatment of 72K-ESP with alkaline phosphatase converted it to 69K-ESP. These results along with the available information led to the conclusion that the primary translation product is sequentially processed to 67K-ESP by signal peptide cleavage, to 69K-ESP by glycosylation, and finally to 72K-ESP by phosphorylation as the actual product in the peptide synthesis. The limited conversion of 72K-ESP to 64K-ESP is proposed to be a post-endocytotic event.     

Fasciola hepatica: A light and electron autoradiographic study of shell-protein and glycogen synthesis by vitelline follicles in tissue slices

The incorporation of tritiated amino acids and monosaccharides by the vitelline cells of F. hepatica slices maintained in vitro was studied by light and electron microscope autoradiography.A “pulse-chase” labeling technique was used with tritiated tyrosine, phenylalanine, leucine, and methionine, of which H³-tyrosine was the most readily incorporated into shell-protein globules of immature vitelline cells. The mechanism of protein synthesis appeared to resemble the GER-Golgi mediated mechanism of vertebrates. Young vitelline cells were the most active in protein synthesis, and they matured considerably during the 60 min chase period. Maturing cells, which were carrying out glycogenesis, incorporated no amino acids.An “accumulation” labeling technique was used with H³-galactose and H³-glucose. Both monosaccharides were readily incorporated into glycogen by vitelline cells which had reached the stage of glycogenesis, but mature cells, which were already packed with glycogen, incorporated little monosaccharide. Labeling appeared in the nurse cells of follicles containing many mature vitelline cells. No evidence was found for the involvement of any cell organelle in glycogenesis, but preformed glycogen may have acted as a “template” for further synthesis.