Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

Evidence for a proteolytic modification of liver pyruvate kinase in fasted rats.

Liver pyruvate kinase was purified to homogeneity from rats fed a high carbohydrate, low protein diet (LPK-C) and from rats fasted for 84 h (LPK-F). Although the enzymes have similar electrophoretic mobilities in 7% polyacrylamide disc gels, the specific activity of LPK-C was two to three times the value of the specific activity of LPK-F. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of LPK-C yields a single protein band of 56,000 daltons. In contrast, LPK-F yields two bands of protein. Approximately one-third of the LPK-F has an electrophoretic mobility similar to the 56,000-dalton LPK-C peptide. The remaining two-thirds of the LPK-F protein migrates as a 51,000-dalton peptide. Cyanogen bromide was used to cleave LPK-C and LPK-F. Similar peptide patterns were obtained from LPK-C and LPK-F when the cyanogen bromide fragments were resolved by 12% polyacrylamide gel electrophoresis in 7.5 m urea containing 6 mm Triton X-100 and 5% acetic acid. Separation of the two peptides from LPK-F was accomplished by selective immunologie absorption of the 56,000-dalton peptide with anti-LPK-C gammaglobulin immobilized on Sepharose 4B. Tryptic digests of LPK-C, LPK-F and the 51,000-dalton peptide yield similar peptide patterns when analyzed via sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. These results suggest that the 51,000-dalton peptide could be derived by a proteolytic cleavage or limited digestion of the 56,000-dalton subunit. Phosphorylation of LPK-C and LPK-F by [γ-³²P]ATP in vitro with cyclic AMP-activated protein kinase results in covalent incorporation of ${}^{32}$P into only the 56,000-dalton subunit. These results suggest that anin vivo proteolytic modification that yields the 51,000-dalton subunit.     

Selective photoaffinity labeling of acetylcholine receptor using a cholinergic analogue

A bisazido derivative was synthesized from bis(3-aminopyridinium)-1,10-decane diiodide and it was shown that it was bound (KD congruent to 2.2 muM) specifically to purified acetylcholine receptor and fulfilled the requirements for a photoaffinity label. Like the parent compound the derivative could transform membrane-bound receptor from a low ligand affinity conformation(s) to a high ligand affinity form (s), a transition which is thought to resemble desensitization processes observed in vivo. Photolysis of 3H-labeled bisazido reagent was carried out in the presence of the receptor. After dodecyl sulfate-polyacrylamide gel electrophoresis of labeled purified receptor two of the four subunits (mol wt 40 000 and 60 000) contained 90% of the bound radioactivity while for membrane-bound receptor the subunits of mol wt 40 000 and 50 000 were labeled. The results favor the assumption that the specific ligand binding sites are located on mol wt 40 000 subunits and labeling of the other subunits reflects (a) their proximity to the ligand-binding site and (b) alterations in subunit topography between membrane-bound and solubilized states.     

Trophic interactions in soils as they affect energy and nutrient dynamics. IV. Flows of metabolic and biomass carbon

Flows of biomass and respiratory carbon were studied in a series of propylene-oxide sterilized soil microcosms. One-half of the microcosms received three pulsed additions of 200 ppm glucose-carbon to mimic rhizosphere carbon inputs. Biotic variables were: bacteria (Pseudomonas) alone, or amoebae (Acanthamoeba) and nematodes (Mesodiplogaster) singly, or both combined in the presence of bacteria.Over the 24-day experiment, respiration was significantly higher in the microcosms containing the bacterial grazers. Biomass accumulation by amoebae was significantly higher than that by nematodes. The nematodes respired up to 30-fold more CO₂ per unit biomass than did amoebae. Similar amounts of carbon flowed into both respiratory and biomass carbon in microcosms with fauna, compared with the bacteria-alone microcosms. However, partitioning of available carbon by the microfauna varied considerably, with little biomass production and relatively more CO₂-C produced in the nematode-containing microcosms. The amoebae, in contrast, allocated more carbon to tissue production (about 40% assimilation efficiency) and correspondingly less to CO₂.     

Alpha-Glycerophosphate dehydrogenase (insoluble) and lactic dehydrogenase activities in the skeletal muscles of two insects.

The flight muscle preparations of the dragonfly Pantala flavescens and the aquatic beetle Cybister confusus showed extremely low levels of lactic dehydrogenase activity and high levels of alpha-glycerophosphate dehydrogenase (insoluble) activity. The activities of these two enzymes in the leg muscle of the beetle were approximately the same (1:1), but lactic dehydrogenase activity was several times higher than that in the flight muscles of both Insects. These results have been interpreted as indicating the high energy-yielding demands of the flight muscles during continuous sustained activity, while the leg muscles of the beetle which are involved in swimming activity derive their energy predominantly through anaerobic glycolysis.     

Endocrine cells of the mucous membrane of the fundus region of the stomach of hibernating rodents]

The gastric mucous membrane was studied in the hibernating animal Citellus erythrogenys in different seasons (during 7-8 months a year the animal is in hibernation and its digestive tract is not functioning). During hibernation the general amount of enterochromaffin-like cells decreases and their composition changes: the number of active cells diminishes and that of less active cells grows. Such dynamics of cells suggests the participation of enterochromaffin-like cells of Citellus erythrogenys in the regulation of gastric secretion (in rats and mice these cells contain histamine stimulating the mucous membrane glands). The amount of true enterochromaffin cells is not sufficiently changed and they seem to be not directly related with digestion but to be producers of serotonin which (together with serotonin of the brain) takes part in sustaining the mechanisms of appearance and supporting of winter hibernation.     

FURTHER STUDIES ON THE BREAKDOWN OF 2:4-DICHLOROPHENOXYACETIC ACID BY A SOIL BACTERIUM

Bacterial growth rates and the kinetics of detoxication have been followed during the course of breakdown of 2:4-dichlorophenoxyacetic acid in a liquid mineral salt medium by cultures of Bacterium globiforme isolated from garden soil. It was found that toxicity persisted apparently unchanged long after bacterial growth had ceased, and finally disappeared suddenly after a lag period comparable in duration with those obtained in soil perfusion experiments. These results can best be explained by the production of highly phytotoxic intermediates in the first stages of 2:4-D breakdown. Preliminary experiments involving paper partition chromatography indicate that there may be at least two such intermediates.