Distribution and characterization of diglycosyldiacylglycerol isomers with different anomeric configuration in higher plants.

The novel diglycosyldiacylglycerol (DGDG), galactosyl(beta 1----6)-galactosyl(beta 1----3')-diacylglycerol (B isomer), present in Adzuki beans was found to be distributed together with the well-known galactosyl(alpha 1----6)-galactosyl(beta 1----3')-diacylglycerol (A isomer), in all (10) of the higher plants examined. The highest levels were found in leguminous seeds were the amounts were always less than 33% of the total DGDG of mature seeds. The highest proportion of the B isomer was found in Adzuki bean seed DGDG (26-33%), with the lowest in pea seed DGDG (2%). The amounts of the B isomer in DGDG of Adzuki and kidney beans cotyledons were almost equal to those in mature seeds. Immature seeds and hypocotyls of three kinds of beans also contained the B isomer in small amounts compared with the mature beans, while only trace amounts of the isomer was found in other organs such as leaves, stems, pods, roots and generative organs of plants, except for root from kidney beans. The molecular species composition of the principal diacylglycerol moieties in the A and B isomers of DGDG were found to be significantly different among several plant seeds, although the component diacylglycerol species were qualitatively similar to each other.     

The effects of Penicillium digitatum and Fusarium oxysporum rots on nutritional content of pawpaw (Carica papaya L.)

Investigations were carried out on the effects of Penicillium digitatum and Fusarium oxysporum on the nutritional value of pawpaw (Carica papaya). Decreases were observed in ash content, phosphorus, sodium, reducing sugars and ascorbic acid levels of fruits infected with P. digitatum, but increases in calcium and potassium content. In fruits infected by F. oxysporium, there were decreases in phosphorus, calcium, sodium ascorbic acid and reducing sugar levels; but the levels of ash content increased. The total protein level increased in the fruits infected with both fungi. These results revealed a reduction in fruit quality.     

Dot-blot assays and their use as a direct antigen-binding method to screen monoclonal antibodies to 1,4-beta- and 1,3-beta-glucan synthases

A rapid method has been developed to assay beta-glucan synthases spotted on a nitrocellulose sheet. The sensitivity of this method allows screening of hybridoma-making monoclonal antibodies in a direct antigen-binding assay by measurement of the activity of the enzymes retained by the antibodies previously fixed on nitrocellulose.     

The differential functional stability of various forms of bovine liver rhodanese

Bovine liver rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) was prepared in dilute solutions and subjected to conditions that led to a time-dependent loss of enzyme activity. The rate of this activity loss was found to be dependent upon the sulfur substitution state of the enzyme, and the presence or absence of the substrates, thiosulfate and cyanide. In the absence of excess substrates, free enzyme (E), and the covalent intermediate form of the enzyme bearing a divalent sulfur atom in the active site (ES), are of approximately equal functional stability. In comparison, E, in the presence of excess cyanide, was markedly more labile, while ES, supported by 10-50 mM thiosulfate, showed no significant loss of activity under any of the conditions tested. All the enzyme solutions were shown to be losing assayable protein from solution. However, it was demonstrated that, for rhodanese in the E form, the amount of protein lost was insufficient to account for the activity lost, and a marked decline in specific activity was observed. Enzyme in the ES form, whether supported by additional thiosulfate or not, did not decline in the specific activity, though comparable protein loss did occur from these solutions. Intrinsic fluorescence measurements of rhodanese in the ES form, before and after removal of the persulfide sulfur through the addition of cyanide, indicated that loss of enzymic activity was not accompanied by loss of the bound sulfur atom. Therefore, the stabilizing effect observed with thiosulfate could not be explained simply by its ability to maintain enzyme in the sulfur-substituted state. Since the concentration of thiosulfate employed in these experiments was insufficient to maintain all the enzyme in ES.S2O3 form, thiosulfate was acting as a chemical reagent rather than a substrate in stabilizing enzyme activity.     

Inhibition of activity and quenching of intrinsic fluorescence of transglutaminase by acrylamide are independent events

Addition of low concentrations of acrylamide to the assay mixture of erythrocyte transglutaminase leads to a strong inhibition of the enzyme with a mechanism consistent with a non-competitive inhibition against both substrates. For the quenching of the intrinsic fluorescence of the protein, much higher concentrations of acrylamide are required so that both phenomena appear independent of each other. In this particular case, therefore, acrylamide can be employed to obtain information on ligand-triggered conformational changes.     

Three distinct H-2Ks molecules differing at the carboxy terminus are expressed on a tumor from SJL/J mice

A dimethylbenzanthracene-induced leukemia of H-2s origin expressed at least two class I molecules on the cell surface that were precipitated by anti-H-2.19, an alloantiserum prepared against the private H-2Ks specificity. Mapping studies in recombinant inbred strains along with comparisons of tryptic peptide maps and N-terminal sequences indicated that the proteins were virtually identical and probably encoded by the same class I gene. When cells were labeled in the presence of tunicamycin, the proteins precipitated by anti-H-2.19 were further resolved into three distinct peptides. Experiments were performed to determine which of these various proteins were phosphorylated and which were recognized by an anti-synthetic peptide serum directed against the ultimate C-terminus of H-2K class I molecules. The results indicate that a single class I gene from the H-2Ks region may encode three class I molecules that differ only at the C-terminus due to alternative splicing of pre-mRNA.