Cotyledonary Storage Proteins in Pisum sativum. V. Further Studies on Molecular Heterogeneity in the Vicilin Series of Holoproteins

The cotyledonary storage proteins of mature pea seeds were resolved by polyacrylamide gel electrophoresis in an acid buffer system as discrete bands comprising holoproteins referable to either the legumin or vicilin series. Three major and four less abundant vicilin holoproteins were detected in acid-gel separations after subfractionation of storage-protein extracts on the basis of differential solubility at specific pH values and ionic strengths, by electrophoresis in the alkaline pH range or by gel filtration. Acid-gel patterns were qualitatively consistent over a range of genotypes. Up to 12 polypeptides were found in varying proportions in reduced and dissociated eluates from fixed and stained vicilin bands separated on acid gels. Each vicilin holoprotein showed a distinctive quantitative, and in some cases qualitative, polypeptide composition. Genetically determined size or charge variants of certain polypeptides used as markers were found in each of the major vicilin holoproteins. This observation, and the consistent appearance of the vicilin bands on acid gels after exposure to widely different treatments during subfractionation, indicate that these polypeptides are strictly subunits of the vicilins, rather than fortuitously associated components. Amongst the holoproteins of each band, some flexibility of subunit composition is indicated by evidence that the total of the molecular weights of the subunits exceeds the molecular weight of the corresponding holoprotein, and by the apparent relative abundance of certain subunits. The observed complexity of subunit composition of the vicilins is consistent with previous findings of immunological similarities and differences amongst the holoproteins of this series, and with change during development in the subunit composition of these proteins.

Some Characteristics of the Clottable Protein of Limulus Polyphemus Blood Cells

Summary1. The endotoxin-clottable protein of Limulus blood cell extracts has been studied. The concept of the clottable protein as a true cell protein was confirmed.2. 35–55% of total extractable protein was removed from the extracts by clotting. Polyacrylamide disc electrophoresis of the extracts showed 6 to 9 protein bands one of which was reduced in intensity by the clotting. Dimethylf ormamide could be used for fractionated precipitation of the proteins.3. The gel protein was easily soluble in HCl or NaOH and reprecipitated by neutralization. It was also soluble in 0.05 M formate/6.7 M urea pH 4.3 but not in neutral solutions of urea.4. Light absorption spectra of the gel protein in 0.188 N NaOH showed maxima at 283 mμ and 290 mμ, whereas one maximum, at 276 mμ, was observed in 0.189 N HCl. E1 % 1 cm in 0.188 N NaOH was 10.4 and 11.1 at 283 mμ and 290 mμ, respectively, and 9.0 at 276 mμ in 0.189 N HCl.5. Data on the total amino acid composition of the gel protein are given. A mean minimal molecular weight of about 20,000 is calculated from these.6. In starch gel electrophoresis with a discontinuous acid buffer system the gel protein separated into two main zones. Possible relationships between these are discussed in terms of clotting mechanism.7. The data show that Limulus clottable protein differs markedly in its molecular characteristics from those of mammalian fibrinogens.

Analytical Methods for Diethylstilbestrol in Feeds and Premixes

Methods are presented in which diethylstilbestrol is extracted from feeds in the Goldfisch apparatus, transferred into alkaline sodium acetate solution to avoid emulsions, and measured colorimetrically in a sodium acetate-acetic acid buffer system. The procedure is rapid, and results agree closely with those obtained by the official method. Procedures are also presented for determination of diethylstilbestrol in molasses and fat mixtures.

Kinetic behaviour of calf-intestinal alkaline phosphatase with 4-methylumbelliferyl phosphate

1. The effects of varying pH, ionic strength and temperature on the parameters K(m) and V(max.) for a purified alkaline phosphatase from calf intestinal mucosa with a new fluorogenic substrate, 4-methylumbelliferyl phosphate monoester disodium salt, and an ammediol-hydrochloric acid buffer system were determined. 2. It was found that, under varying conditions, a relationship exists between K(m) and V(max.) such that V(max.)=beta/(1+alpha/K(m)), where alpha and beta are constants, temperature- and ionic strength-dependent, but pH-independent. It is shown that this relationship accounts satisfactorily for the well-known effect of varying substrate concentration on optimum pH and velocity. 3. The various results are interpreted in terms of a pH-dependent conformational equilibrium between two forms of the enzyme, E(1) and E(2). Only E(1) combines with substrate, and only E(2) reacts to give inorganic phosphate. 4. To account for the pH-variation of K(m) and V(max.) in terms of this theory, it is postulated that the conformational change is associated with a change in pK of two basic groups in the enzyme.