Purification of soluble tetrodotoxin-sensitive protein from the cytoplasmic fraction of bovine brain tissue

Highly purified protein preparations promoting TTX-dependent sodium permeability in the liposomal membrane were separated from the cytoplasmic fraction of bovine brain tissue. The protein was purified using anion-exchange and separation chromatography and gel filtration. The separated protein was found to be a high-molecular acidic glycoprotein forming 55 kD subunits during denaturation under reducing conditions. It was thought to contribute to the formation of voltage-dependent sodium channels in the cell membrane.A. A. Bogomolets Institute of Physiology and A. V. Palladin Institute of Biochemistry, Academy of Sciences of the USSR, Kiev. Translated from Neirofiziologiya, Vol. 19, No. 2, pp. 202–209, March–April, 1987.     

Ultrafine organization of Leptomonas peterhoffi flagellates cultured in broth and solid nutrient media

The morphology of in vitro grown lower trypanosomatids L. peterhoffi was studied by means of electron microscopy. The flagellates from both liquid and solid culture media are represented by uninucleate cells of two structural types. Type I flagellates are characterized by dense cytoplasm enriched with numerous ribosomes. Type II flagellates are most abundant in the cultures; they display a less dense cytoplasm and fewer ribosomes. The flagella of L. peterhoffi of both types form enlargements, which are most expressed at the outlet of the flagellar pocket. The nuclei of some cells contain twisted threads about 10 nm in diameter. L. peterhoffi from the liquid media usually possess long, narrow and curved flagellar pockets. On the solid medium, amoeboid and hemispherical colonies composed of both uninucleate and giant multinucleate cells are formed. In these cells the flagellar pockets are usually short and straight. Outside the flagellar pocket, the axoneme often becomes looped in the flagellar enlargements of the colonial uninucleate cells.     

Study of the interaction of Na+ and K+-ATPase of erythrocytes with ouabain. Effect of acetyl phosphate and p-nitrophenyl phosphate]

Effects of ATP, acetyl phosphate (AcP) and p-nitrophenyl phosphate (p-NPP) on the inhibition of the Na+, K+-ATPase activity were studied. ATP, AcP and p-NPP were found to facilitate the ouabain-induced inhibition of the enzyme activity only after the injection of these phosphorylyzing agents into the erythrocyte ghosts. Inside the ghosts Na+ ions enhanced the effects of the phosphorylyzing agents. K+ ions in the environment removed the stimulating effects of ATP, AcP and p-NPP on the ouabain-induced inhibition of Na+, K+-ATPase activity. It is concluded that the sites of AcP and p-NPP hydrolysis as well as the active center for ATP are localized on the inner surface of the cell membrane.     

Age Peculiarities of the Calpain/Calpastatin Cerebral System in Rats: Relation to the Hypothesis of Brain Aging

We studied age-related changes in the activity of calpain, those in the activity of its endogenous inhibitor calpastatin, and the ratio of these indices in the brain of rats of four age groups (2-3 weeks, 2-3, 5-6, and 24 months). The activity of calpain was estimated using FITC casein as the substrate. In a soluble fraction of the brain homogenate, the enzyme activity in general increased with age. In mature rats (5 to 6 months old), this index exceeded 3.65 times the corresponding index in juvenile (2 to 3 weeks old) animals, while in old animals this index somewhat decreased. In the fraction obtained after separation of calpain from other components using DEAE-cellulose chromatography, the age-related trend toward an increase in the activity of calpain was preserved, but it was much more moderate. The activity of calpastatin demonstrated an opposite direction of age changes: it was the maximum in 2-3-week-old animals and gradually decreased (by 27%) in old rats. We also found that the efficacy of inhibitory action of calpastatin in the cerebral tissue with respect to the activity of calpain was, as a rule, redundant. In this case, the ratio of inhibitor/enzyme activities decreased with age; this index was 1.65, 1.33, 1.1, and 1.0 or less in 2-3-week-old, 2-month-old, mature, and old animals. Therefore, we found that the intensity of calpain-mediated proteolysis in the rat brain increases from the juvenile period to the mature age and somewhat decreases in old individuals. Such alterations are developed at the expense of both an increase in the activity of the enzyme and weakening of the action of its inhibitor (calpastatin).     

Membranotropic properties of latrotoxin-like protein: Studies on liposomes

Fusion and changes in permeability of artificial phospholipid vesicles (liposomes) caused by the influence of α-latrotoxin-like protein (L protein) from the gray matter of the bovine brain were studied using a hydrophobic fluorescent probe, R18. It was shown that L protein stimulates fusion of negatively charged liposomes. This effect becomes stronger in acidified media. The influence of L protein on the permeability of phosphatidylcholine liposome membrane is also a pH-dependent process.