A non-denaturing gel electrophoresis system for the purification of membrane bound proteins

A new method is described for the purification of a membrane bound glycoprotein, the kappa opioid receptor from human placental tissue. The method uses preparative slab-gel electrophoresis in the presence of the non-denaturing detergent CHAPS. A linear relationship between log molecular weight and SDS PAGE electrophoretic mobility of known molecular weight markers, in the presence of CHAPS, is observed. Using this method, we were able partially to purify an 3H-etorphine binding glycoprotein, from placental villus tissue, with an apparent molecular weight range of 60-70,000. The iodinated glycoprotein migrates in SDS PAGE with an apparent molecular weight of 63,000. This method may be useful for the isolation of membrane bound proteins, especially when an affinity ligand is not available.     

Separation and localization of cell wall layers of a gram-negative bacterium

The cell envelope of a marine pseudomonad as seen in thin section by electron microscopy has the double-membrane structure typical of other gram-negative bacteria. Cells washed with a solution containing Na(+), K(+), and Mg(++) at their concentrations in the growth medium, when suspended briefly in 0.5 m sucrose, lost 13% of their hexosamine in a form nonsedimentable by centrifugation at 73,000 x g. Since the resulting cells in thin section appeared unchanged, it was concluded that the material released was derived from a nonstaining, loosely bound outer layer. This same layer could be removed from the cells by washing with 0.5 m NaCl. A second nonsedimentable fraction was released after successive suspension of the cells in 0.5 m sucrose. Since this material was released only when the outer double-track structure had broken, it was concluded that it arose from a layer immediately underlying the latter layer. The three layers differed in their content of hexosamine and protein. None of the layers released contained muramic or diaminopimelic acid. The cell form remaining was rod shaped and appeared in thin section to be bounded only by its cytoplasmic membrane. This form contained all the muramic and diaminopimelic acid in the cell. Treatment with lysozyme released the muramic and diaminopimelic acid and converted the rod form to a protoplast, indicating that in the rod form (mureinoplast) a thin layer of peptidoglycan is located on the outside surface of the cytoplasmic membrane. Thus, five separate layers have been detected in the cell envelope of this marine pseudomonad.     

Chemical composition of culinary wastes and their potential as a feed for ruminants

Culinary wastes were collected from three different sources, namely, institutional, restaurant and household. As dry matter content of the food wastes increased, protein level (on a dry weight basis) remained relatively constant, whereas fat content markedly increased.Sheep readily adapted to a diet containing 35% of dry matter as food wastes, their daily dry matter intake being 4.5% of body weight; this suggested that palatability was no problem. Digestibility values of 76, 68, 73 and 99% were calculated for dry matter, protein ether extract and acid detergent fibre fractions of garbage, indicating that the material had a high nutritive value for sheep.The culinary wastes had a low count of harmful bacteria. Storage of the material at room temperature resulted in moulds and odours after a week, indicating that the material deteriorated quite rapidly. The addition of organic acids or formaldehyde kept the material quite stable for several weeks.     

Electrophoretic separation of molecular species associated with the thermal transition of chymotrypsinogen A

Solutions of thermally unfolded chymotrypsinogen A were rapidly cooled to 0 °C and subjected to gel electrophoresis. In addition to the native protein, two other electrophoretic species were observed. Both appear to result from reversible interaction with the transition process. Of the three electrophoretic species revealed, that which represents the reversibly unfolded state is the intermediate as gauged by its mobility.