Whole sheets of plasma membrane, each with their attached flagellum, were purified from Trypanosoma brucei. The method devised for their isolation included a new technique of cell breakage that used a combination of osmotic stress followed by mechanical sheer and avoided the problem of extreme vesiculation as well as the trapping of organelles in cell 'ghosts'. The purified membranes all contained the pellicular microtubular array. The antigenic surface coat was completely released from the plasma membrane during the isolation procedure. The membranes had a very high cholesterol/phospholipid ratio (1.54). A large proportion (42%) of the cellular DNA was recovered in the plasma-membrane fraction unless a step involving deoxyribonuclease treatment, which decreased the DNA content to less than 13%, was included before secrose-density gradient centrifugation. This step also aided the separation of plasma membranes from other cellular components. The ouabain-sensitive Na+ + K+-stimulated adenosine triphosphatase and adenylate cyclase co-purified with the plasma membranes. Although 5'-nucleotidase was thought to be a plasma-membrane component, it was easily detached from the membrane. The purified membranes were essentially free of L-alanine-alpha-oxoglutarate aminotransferase, L-asparte-alpha-oxoglutarate aminotransferase, malate dehydrogenase, oligomycin-sensitive adenosine triphosphatase, glucose 6-phosphatase, Mg2+-stimulated p-nitrophenyl phosphatase and catalase.

No abstract

A rapid method for preparing Leydig cells from rat testes is described. An interstitial cell suspension, prepared by collagenase treatment of decapsulated testes, was centrifugal for 10 min over a cushion of 60% (v/v) Percoll to remove red blood cells, and then centrifuged for 20 min in a 0-60% linear density gradient of Percoll. Seventy-four per cent of the cells present in that fraction of the gradient comprising 35-50% Percoll were Leydig cells; the yield from each testis was about 1.5 x 10(6) cells. The Leydig cells appeared viable, excluded Trypan blue, possessed high-affinity binding sites for human chorionic gonadotrophin (hCG) and synthesized increased quantities of testosterone in response to hCG. The cells could be stored overnight in 20% (v/v) glycerol at -20 degrees C, with only minimal effect on the specific activities of a number of enzymes used as markers of subcellular components. Testosterone production in vitro by the cells after storage for 20 h was greater than that of hCG-stimulated fresh cells and was not further increased by hCG.

Abstract— The loss of at least two different cell types in the basal ganglia of the choreic brain led us to examine the activity of enzymes involved in the metabolism of glucose. Cellular ATPase, HK, G6‐PDH, PFK., LDH, GDH were measured. Post mortem stability studies indicated that these enzymes were more unstable in human brain than mouse brain. The most stable enzyme was GDH. HK activity appeared to increase after freezing, suggesting release from another compartment. PFK and G6‐PDH activity decreased by 70% over the usual time and temperature period for autopsy. In the autopsied brain tissue we were still able to measure significant activities that allowed us to determine the distribution of these enzymes and the similar post mortem handling of control and choreic brain allowed us to compare these two groups. The activity of HK and G6‐PDH was higher in the frontal cortex than in the basal ganglia. Ouabain insensitive ATPase and PFK were higher in the basal ganglia than the frontal cortex. GDH activity, an enzyme that is very active in glial cells, was increased in the choreic globus pallidus, an area with a very high glial to neuronal cell ratio. Although there was a wide variation in PFK activity that appeared to be related to the pre‐mortem clinical state there was a significant decrease in PFK activity in the putamen of choreic post mortem brain when compared to controls. These findings do not indicate an absolute defect in any of the enzymes studied in choreic brain but further studies might prove worthwhile.

No abstract