John M. Lawrence

Effects of sodium dodecyl sulfate and other dissociating reagents on the globulins of peas

Abstract Pea seed globulins were investigated in NaCl solution by DEAE-cellulose chromatography, polyacrylamide-gel electrophoresis, and ultracentrifugal analysis. These criteria confirmed the presence of two components named vicilin and legumin by earlier investigators. At pH 10.0 vicilin underwent a reversible dissociation. Legumin was unaffected up to pH 10.5. Urea or formamide solutions caused the appearance of six protein bands in a gel, four arising from legumin and two from vicilin. Such apparent dissociation was also accompanied by association phenomena. Sodium dodecyl sulfate (SDS) treatment resulted in twelve well-defined bands; four of these arose from vicilin, six others from legumin, and two were of uncertain origin. Ultracentrifugal analysis with added SDS revealed one peak with a sedimentation coefficient of 3.24, which is much lower than the native proteins. Protein fractions resulting from SDS treatment were recovered from preparative gels. Amino acid compositions of all these fractions were similar. All fractions, like the native proteins, had two or more different N-terminal amino acids. Difference in end-group composition occurred among the fractions, but some N-terminal amino acids were common to several fractions. Incomplete and somewhat random dissociation of the peptide chains which constitute the complex native globulin molecules is postulated.

Disk electropheresis of wheat flour proteins with a modified apparatus utilizing gels of rectangular cross section

Abstract A modified disk electrophoretic apparatus has been constructed out of Plexiglas. This apparatus is simple in design and has several advantages over the standard apparatus. In particular, gels of rectangular cross section rather than circular cross section were used and this facilitated normal photography and densitometry of the patterns. The pH 2.3 gel formula was modified in order to obtain reproducible polymerization. Wheat albumin, globulin, and gliadin fractions of known purity were investigated in this apparatus, both at the pH of 9.5 and at the pH of 2.3. Several extracts of wheat flour were also investigated. The data indicated that, while the patterns were complex, certain sets of bands could be associated with the various purified fractions. Contrary to previous observations, it was demonstrated that the addition of urea to the extracts resulted in different electrophoretic patterns, possibly due to dissociation. Some of the basic differences between starch gel electrophoresis and acrylamide disk electrophoresis have been discussed and the differences observed for wheat flour proteins in the two systems explained on this basis.

Media for in vivo nitrate reductase assay of plant tissues

Abstract The in vivo nitrate reductase (NR) activities of leaf sections of lambs-quarters ( Chenopodium album ), tomato, wheat, pea, maize and pigweed were greatly stimulated by the inclusion of various surfactants in the assay medium. 1-Propanol caused only slight stimulation. The corresponding activities of root tissue were greatly enhanced by 1-propanol, and slightly inhibited by surfactants. Studies of the time course of root NR assays showed that, in the absence of exogenous nitrate, propanol addition resulted in an abrupt increase in reaction rate from a previous low level; and that, at suitable concentrations of propanol, peaks of activity were found at two successive times. These results suggest that the stimulating effect of propanol is due to its promotion of the transfer of nitrate substrate from a vacuolar storage pool to an active cytoplasmic pool accessible to the enzyme. Stimulation of activity by Tergitol NPX in lambs-quarters leaf disc assays was more gradual, and two peaks could not be shown, but the mechanism of stimulation may well be the same. If these explanations are correct, it would follow that there are significant differences in the properties of roof and leaf vacuolar membranes.

Simple methods for determination of methionine and cystine in legume seeds

Abstract Convenient and reliable methods have been developed for the routine determination of the sulfur-containing amino acids in legume seeds. These procedures allow assessment of the content of the nutritionally critical amino acids which should be considered in breeding programs. The method for the determination of methionine in pea, bean, and lentil seeds is based on thin-layer chromatographic separation of the methionine sulfone produced by performic acid treatment of the sample. The separated spots are treated with ninhydrin and the color read by reflectance densitometry. Each 1 μl spot represents 40 μg of seed material containing about 0.4 to 0.8 nmole of methionine. Interfering spots due to the product of ninhydrin with pipecolic acid are decolorized by light treatment. An adaptation and clarification of the method of Goa (6) is suitable for routine determination of cystine plus cysteine. It is based on hydrazinolysis of the samples, producing H 2 S from the cystine. The H 2 S is converted to bismuth sulfide and read in the colorimeter. Sample size was 40 mg of seed material containing 0.3 to 0.5 μmole of cystine.

Incorporation of lysine-C14 into the developing grain of wheat

Uniformly labeled l -lysine-C14 (5 μc. total, 6.6×106 μc. per mole) was injected into the top internodes of wheat stems. Injection 20–24 days after flowering (early dough stage) resulted in ripened wheat in which 46–49% of the radioactivity was recovered. About a quarter of the injected tracer was found in the flour fraction. A little over half the total seed C14 was contributed by lysine. About half the total flour activity and about 60% of the activity in the bran fraction was in lysine. Flour lysine and bran lysine had specific activities of about 40,000 and 50,000 μc. per mole, respectively. Injection at a late dough stage of development of the grain resulted in much lower deposition of radioactivity. Quantitative analyses for the free amino acids of total wheat stem juices from the top internode at three times during wheat development showed the presence of considerable amounts of lysine along with other expected amino acids, but no diaminopimelic acid. This finding, together with the relatively high specific activity of wheat lysine following lysine-C14 injection, suggests that lysine in wheat grain may derive from preformed lysine delivered to the head by way of the stem.

Homoserine in seedlings of the tribe vicieae of the leguminosae

Abstract Young seedlings of 10 subspecies and varieties of Pisum all accumulated high levels of homoserine, as did also Lathyrus latifolius seedlings. There was less in L. odoratus , and little or none in Cicer arietinum , Lens Culinaris , Vicia faba , and V. sativa seedlings.

The effect of terramycin on the growth of rabbits

Abstract The results obtained by feeding graded levels of terramycin to weanling rabbits failed to show any effect of the antibiotic on body weight gains during a 6-week experimental period.