Purification and partial characterization of glyoxalase I from a higher plant Brassica juncea.

The glyoxalase I was purified from Brassica juncea by affinity chromatography on S-hexyl GSH sepharose 4B. Homogeneity of the protein was confirmed electrophoretically by a silver stained gel. Activity staining on a native starch gel also showed a single band. The effect of glutathione, methylglyoxal, and pH on enzyme kinetics was studied. Magnesium was found to stimulate the enzyme activity.

Stability of mutagenic nitrosated products of indole compounds occurring in vegetables.

Levels of indolylglucosinolates in Brassica vegetables correlated significantly with the amounts of N-nitroso compounds formed in these vegetables after nitrite treatment. Nitrosation of indole-3-carbinol, indole-3-acetonitrile and indole, hydrolysis products of an indolylglucosinolate, resulted in formation of nitrosated products, which were directly mutagenic to Salmonella typhimurium TA100. The nitrosated products were unstable at pH 2 but stable at pH 8. Experiments to elucidate the mechanisms behind these differences in stability showed an equilibrium between the nitrosated indole compound and the free compound plus nitrite.

Complete amino acid sequence of a subunit from rapeseed high molecular weight protein.

A subunit (Mr 15,600) from the high molecular weight protein from rapeseed was separated and isolated; its purity and homogeneity were ascertained. The subunit was cleaved with cyanogen bromide, trypsin, chymotrypsin, and Staphylococcus aureus V8 protease. The fragments were separated and isolated by polyacrylamide gel electrophoresis, gel filtration, column chromatography on Dowex 1 x 2, and paper electrophoresis. The amino acid compositions of the intact subunit and different fragments obtained from enzymatic and chemical cleavages were determined. The subunit and its fragments were sequenced by manual Edman method. The phenylthiohydantoin amino acids obtained after each step were identified by thin-layer chromatography and ultraviolet spectroscopy. The complete amino acid sequence of the subunit consisting of 125 amino acid residues has been established by the overlapping method.

N-nitroso compounds and their precursors in Brassica oleracea.

In the present study, Brassica a dried green vegetable from Kashmir, which is a major constituent of the local diet, was analysed for nitrosatable aliphatic amines, N-nitrosamines prior to and after nitrosation) and alkylating activity due to N-nitrosamides following nitrosation. The cooked vegetable contained 11 micrograms/kg nitrosodimethylamine and 21 micrograms/kg nitrosopyrrolidine. Nitrosation under chemical conditions yielded 1200 micrograms/kg N-methylnitrosourea.

The occurrence of aflatoxins in mustard and mustard products.

Natural occurrence of aflatoxins at concentrations of 750, 87 and 1420 micrograms/kg was recorded in 44, 33 and 80 samples out of 100 samples each of seeds, oil and cakes of mustard respectively. Out of 17 species of fungi isolated from mustard seeds, percentage incidence of the Aspergillus flavus group was maximum during monsoon and summer. Twenty-five per cent of isolates (out of 1143 isolates screened) of the A. flavus group were toxigenic, producing different components of aflatoxins in varying concentrations.

A rapid immunoprecipitation assay for neomycin phosphotransferase II expression in transformed bacteria and plant tissues.

Anti-kanamycin antibodies produced in rabbits, following coupling of the antibiotic to bovine serum albumin, were used to immunoprecipitate radioactively labelled phosphorylated kanamycin from transformed bacterial or plant extracts in a novel assay system, for the detection of neomycin phosphotransferase II (NPTII) activity. Radioactive counts in the immunoprecipitated pellet give a semiquantitative measure of the kanamycin phosphorylation and hence the amount of NPTII activity. This assay is sensitive, uses very small amounts of radioactivity, and is very rapid, allowing many samples to be processed within a few hours. Immunoprecipitated counts from reactions with bacteria carrying a kanamycin resistance gene or from tobacco and Brassica napus plants transformed with NPTII gene-containing vectors were consistently higher than counts from nontransformed controls. Results obtained with this assay correlate well with those from the previously described gel overlay and dot-blot assays, but can be obtained in an appreciably shorter time frame.

Cadmium deposition and hepatic microsomal activity in mice fed Swiss chard grown on municipal incinerator refuse ash.

Incineration of municipal solid waste results in the production of millions of tons of ash that may be typically high in heavy metals such as cadmium. Disposal of such ash in landfills capped with soil could lead to absorption of such metals by plants and deposition in foraging animal tissues. In this study, weanling, male mice were fed swiss chard that was grown on soil amended with 10% w/w municipal incinerator refuse ash. Cadmium was taken up by the swiss chard (8.15 ppm, dry wt). The mice fed diets containing 25% of ash-grown chard showed mean kidney and liver concentrations of cadmium (ppm, dry wt), respectively, of 2.80 +/- 0.30 and 0.45 +/- 0.03. Control mice fed soil-grown chard showed significantly lower kidney and liver concentrations of cadmium, i.e. 0.39 +/- 0.02 and 0.05 +/- 0.00 ppm. Since refuse incinerator ashes may contain various organic toxicants that can be hepatic microsomal inducers, the relative liver weights and hepatic microsomal aminopyrine N-demethylase and p-nitroanisole O-demethylase activities of mice fed control or ash-grown chard were measured. No consistent increases in these latter parameters were found in the ash-grown chard fed mice as compared with the control animals.

Structural studies on native and chemically modified storage proteins from rapeseed (Brassica napus L.) and related plant proteins.

Recent data on the structure and chemical modification of the two main storage proteins of rapeseed, the high-molecular mass 12 S globulin and the low-molecular mass 2 S protein (napin) are summarized and compared with those of related seed proteins. The 12 S globulin is built up of six subunits forming a quaternary structure which can be approximated by the model of a trigonal antiprism. The subunits, composed of a larger and a smaller polypeptide chain each, have a two-domain structure which is typical for all related plant proteins. These are characterized by a sedimentation coefficient of 11-13 S, a molecular mass of 300,000-360,000 g/mol and a high percentage of beta-sheet conformation. Increasing succinylation results in a step-by-step dissociation up to the subunits and to an unfolding of the latter at a critical level of modification amounting to 60-70%. These structural changes affect the functional properties remarkably. The napin fraction comprises a group of closely related and highly basic proteins with molecular masses of 12,000-14,000 g/mol, a high content of sulphur-containing amino acids and rich in helical conformation. They are built up of a larger and a smaller disulphide bridged polypeptide chain. Acylation does not abolish the secondary or tertiary structure which are stabilized by inter- and intrachain disulphide bonds. Acylation results, however, in a stabilization of the protein against heat-induced aggregation.

Gas chromatographic determination of diquat and paraquat in crops.

A sensitive and reproducible gas chromatographic procedure for the determination of diquat and paraquat in potatoes and rapeseed was developed. The volatilization of analytes was carried out via their hydrogenation with sodium borohydride-nickel(II) chloride. After their isolation from the reaction mixture, the derivatives of bipiperidine were separated on a column packed with Apiezon L plus potassium hydroxide. Comparable detection limits (0.005 mg/kg) were achieved with a nitrogen-phosphorus detector and by mass fragmentography, however, the latter method was preferred for analyses of rapeseed extracts owing to its higher selectivity.

Hydrogen ion titration of 12 S rape seed protein and partial N-terminal sequence of one of it's subunits.

The high molecular weight 12 S protein from rape seed was isolated in a homogeneous form and characterized. Six subunits were isolated by PAGE in the presence of SDS and 0.2 M 2-mercaptoethanol. These subunits (s1 to s6) were found in the protein in the weight ratio of 1.32:1.2:1.15:1.0:1.21:1.11. The molecular weights and first two N-terminal amino acids of the isolated subunits were 64,800 and phenylalanine, alanine (s1), 50,650 and valine, tyrosine (s2), 42,500 and phenylalanine, leucine (s3), 28,800 and threonine, glutamic acid (s4), 19,100 and cystine, isoleucine (s5) and 15,600 and alanine, phenylalanine (s6). The number of side chain carboxyl, imidazole and epsilon-amino groups were calculated from the hydrogen ion titrations, which were in agreement with the amino acid assay. Besides, the N-terminal amino acid sequence upto 43 residues for one subunit (s6) is reported using Edman degradation.

Oil content and fatty acid composition of promising Indian Brassica campestris L. (Toria) genotypes.

Seeds of sixty four genotypes of Brassica campestris L. (Toria) were analyzed for oil content and fatty acid composition. Oil content varied from 38.9% to 44.6%. Major fatty acids viz. oleic, linoleic, linolenic, eicosenoic and erucic acids exhibited ranges from 10.1% to 17.3%, 5.9% to 14.5%, 5.2% to 15.0%, 7.7% to 13.7% and 39.6% to 59.9%, respectively. Compared to the standard cultivar ITSA, four genotypes contained 5 to 8% lower content of erucic acid; and six genotypes contained significantly higher content of linoleic acid. Oil was positively correlated with erucic acid. The observed inverse relationship between linoleic and erucic acid contents might be utilized in breeding nutritionally better Toria cultivars with both high linoleic and low erucic acid contents.

Comparison of the ion exclusion chromatographic method with the Monier-Williams method for determination of total sulfite in foods.

Experimental data comparing the alkali extraction/ion exclusion chromatographic method with the Monier-Williams method for determination of total sulfite are presented in (a) enzymatic and nonenzymatic browning systems, (b) vegetables containing naturally occurring sulfite, and (c) a carbohydrate-type food additive, erythorbic acid. Excellent agreement, with a linear correlation coefficient of 0.99, was observed in fresh potato samples homogenized with sulfite and allowed to react for different time intervals (enzymatic browning system). A good overall correlation was observed in dehydrated, sulfited apple samples heated for different times (nonenzymatic browning system); however, as heating time increased, higher results were obtained by the Monier-Williams method than by the alkali extraction/ion exclusion chromatographic method. The results of determining sulfite in the alkali trapping solution following acid distillation or acid treatment without heat suggested that this deviation was due to a fraction of sulfite bound to the browning reaction products in such a way that it was released by acid distillation but not by alkali extraction or acid treatment without heat. Similar behavior was demonstrated in cabbage with naturally occurring sulfite, which was released by acid distillation but not by alkali extraction or acid treatment without heat. The ion exclusion chromatographic method could overcome interference by the volatile caramelization reaction products in the Monier-Williams determination of erythorbic acid.

[Changes in the levels of nitrates and nitrites in vegetables and vegetable products and vitamin C in white sauerkraut]. / Zmiany zawartosci azotanów i azotynow w warzywach i przetworach warzywnych oraz witaminy C w bialej kapuscie kiszonej.

The authors demonstrated that during souring nitrates disappear from the white sauerkraut as a mean rate of 42% after 7 days and 23% after 30 days (souring in spring 1987) and by 29% and 77% of the initial level after 7, 30 and 90 days (souring in winter-spring period 1988). After 7 days the level of l-ascorbic acid rose from a mean value of 52 mg/kg to 300 mg/kg, and after 90 days this level fell to one-half of the initial value. No significant changes were noted in the content of nitrites. A decrease of nitrates and increase of nitrite content was found in freshly obtained carrot juice (with a juice extractor). The effect of time and temperature of juice storage on the concent of nitrates and nitrites was studied.

[Effect of the variety and size of fresh and frozen brussels sprouts on the vitamin C level]. / Wplyw odmiany i wielkosci glówek na zawartosc witaminy C w swiezej i mrozonej kapuscie brukselskiej.

The content of vitamin C was determined in the following varieties of Brussels sprouts: Maczuga, Citadel F1, Lancelot F1 and Valiant F1 with diameter 15, 20, 25, 30 and 35 mm. The raw vegetable, the blanched raw sprouts, frozen sprouts immediately after freezing and after 2, 4 and 6 months of frozen storage, and sprouts kept frozen for 6 months and boiled then. The content of vitamin C in the raw vegetable, in the frozen semiproduct, and in the product ready for consumption depended on the variety and on the size of the vegetable, and with rising size the content of vitamin C was decreasing. Immediately after harvesting the sprouts contained from 109 to 198 mg of vitamin C in 100 g of fresh mass. Higher levels of vitamin C were present in heterotic varieties, lower in the stabilized variety Maczuga. Depending on the variety blanching led to a decrease of vitamin C content by 15 to 27%, freezing reduced it by 1% to 8%, during 6 months of storage of frozen vegetables this reduction was 14 to 32%, and boiling of frozen sprouts decreased it by another 30-39%. Consequently, the final product contained from 33% to 48% of vitamin C amount in the raw vegetable, which was, on the average, 73 mg/100 g of fresh mass in the Citadel variety, 60 mg in the Valiant variety, 59 mg in the Lancelot variety, and 52 mg in the Maczuga variety. The boiled sprouts of 15 mm diameter contained, on average, 59% vitamin C more than the sprouts with 35 mm diameter.

Primary structure of the major allergen of yellow mustard (Sinapis alba L.) seed, Sin a I.

Sin a I, a 2-S albumin from the seeds of yellow mustard, is herein described as the major allergen of these seeds. This protein is composed of two disulfide-linked polypeptide chains of 39 and 88 amino acids, whose primary structures are reported. The Sin a I allergen is found to be related to other low-molecular-mass albumins, such as those isolated from rapeseed, castor bean and Brazil nut. Additional structural similarity has also been found between the glutamine-rich large chain of Sin a I and a proline-rich zein, a gliadin, and trypsin and alpha-amylase inhibitors isolated from the seeds of several monocotyledons. Internal amino acid sequence similarity has been detected at both termini of the small and large chains of Sin a I and involves the location of proline and glycine residues at similar positions in relation to the processing cleavage sites. Prediction of secondary structure, based on the amino acid sequences of the mature chains of the mustard allergen, indicates that the precursor polypeptide is cleaved at regions showing a high beta-turn probability. This is also observed with the amino acid sequence deduced from the rapeseed napin gene nucleotide sequence.