A new method for rapid assignment of S-S bridges in proteins

A new method for complementing existing protein chemical techniques for the assignment of S-S bridge positions in amino-acid sequences is described. The principle of the method is the direct examination of the masses of protein fragments, obtained by chemical or enzymatic degradation. Proteins are digested under conditions known to minimise disulphide reduction and reshuffling, and the unfractionated digest is examined directly by high field magnet (or other high mass) fast atom bombardment or Californium mass spectrometry. Disulphide linked peptides are identified from their unique masses, and by comparison with the spectrum of digested and reduced samples in which the signal corresponding to the S-S linked peptide(s) is replaced by two signals corresponding to the respective thiol peptide components, if INTER-bridged, or shifted by two mass units (dithiol) if INTRA-bridged. This rapid procedure has considerable potential in assisting with studies on the primary structure of proteins, in crystallographic studies and the monitoring of denaturation/renaturation of recombinant proteins.     

Superoxide perturbation of the organization of vascular endothelial cell membranes

Cultured porcine thoracic aorta endothelial cells were covalently labeled with 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl. Electron paramagnetic resonance spectrometry revealed two major binding environments representing strongly and weakly immobilized species. The disorder parameter of weak/strong, determined from the respective peak amplitudes, was irreversibly elevated following incubation of endothelial cells with a superoxide-generating system, indicating increased membrane fluidity. The rate of increase in membrane disorder was dependent upon superoxide generation rates. Incorporation of the spin-label at concentrations less than 250 microM had no effect on cell viability. The cellular proteins reacting with the spin-label were predominantly membrane proteins, characterized by immunoblotting using a rabbit anti-4-maleimido-2,2,6,6-tetramethylpiperidinooxyl IgG, following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophorectic transfer to nitrocellulose.     

Induced maternal behavior in the domestic hen. Influence of partial or total separation on the maintenance of maternal responsiveness

The influence of direct physical contact on maintaining maternal responsiveness in induced adult hens was investigated using a separation procedure. Partial separation from chicks causes a significant decline of the clucking rate in hens, this response however does not disappear as in the case of total separation. The possibility for physical contact between hen and chicks contributes largely, but not uniquely, to maintain maternal responsiveness in the domestic hen.     

Growth-dependent expression of a cell surface glycoprotein

The expression of the hepatocellular membrane receptor for desialylated galactose-termining glycoproteins was studied during different proliferative stages of a human hepatoma cell line. Rapidly growing cells exhibited a reduced endocytotic rate of desialylated orsomucoid as compared to non-growing cells. This reduction was shown to be the consequence of a lower concentration of active cell-surface associated receptor protein in the dividing cells.