Natalia Kulikova

A polarized localization of amino acid/carnitine transporter B0,+ (ATB0,+) in the blood–brain barrier

Brain capillary endothelial cells control the uptake and efflux from the brain of many hydrophilic compounds due to highly specialized transporters often localized in a polarized way. Localization of Na(+)- and Cl(-)-dependent amino acid and carnitine transporter B(0,+) (ATB(0,+)) was studied in a co-culture of bovine brain capillary endothelial cells (BBCEC) grown on filters above astrocytes (an in vitro blood-brain barrier model). Immunoblotting and three-dimensional immunocytochemistry analysis with anti-B(0,+)antibodies demonstrated the presence of this transporter and its prevalent co-localization with P-glycoprotein i.e. at the apical side. The sensitivity of leucine uptake through the apical membrane to 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH), D-serine as well as sodium and chloride replacement confirm the functioning of ATB(0,+) and suggests an important physiological role of ATB(0,+) in controlling the delivery of amino acids and carnitine to the brain.

Palmitoylcarnitine regulates estrification of lipids and promotes palmitoylation of GAP‐43

Palmitoylcarnitine was previously shown to promote differentiation of neuroblastoma NB‐2a cells. It was also observed to increase palmitoylation of several proteins and to diminish incorporation of palmitic acid to phospholipids, as well as to affect growth associated protein GAP‐43 by decreasing its phosphorylation and interaction with protein kinase C. The present study was focused on influence of palmitoylcarnitine on palmitoylation of GAP‐43 and lipid metabolism. Althought palmitoylcarnitine did not significantly affect the total phospholipids and fatty acid content, it increased incorporation of palmitate moiety to triacylglicerides and cholesterol esters, with a decrease of free cholesterol content. The presence of palmitoylcarnitine significantly increased the amount of covalently bound palmitate to GAP‐43, which can regulate the signal transduction pathways.

Does Calponin Interact with Caldesmon

The roles of calponin and caldesmon and their interaction in regulation of smooth muscle contraction are controversial. Recently, strong binding between these two proteins has been reported (Graceffa, P., Adam, L. P., and Morgan, K. G. (1996) J. Biol. Chem. 271, 30336–30339). Results in this paper fail to confirm their data and are consistent with the concept of independent functions for calponin and caldesmon. To examine the ability of duck gizzard caldesmon to interact with calponin, three caldesmon derivatives, each containing a different sulfhydryl-specific reporter probe (6-acryloyl-2-dimethylaminonaphtalene,N-(1-pyrenyl)iodoacetamide, andN-iodoacetyl-N′-(5-sulfo-1-naphtylo)ethylenediamine) attached to a single cysteine located in the C-terminal domain, were synthesized. Addition of calponin to labeled caldesmon at both low and physiological salt concentrations did not induce any changes in fluorescence intensity or maximum shift. Under the same conditions, calmodulin and tropomyosin (known to bind to the C terminus of caldesmon) produced substantial changes in these spectral parameters. Gel filtration of an equimolar caldesmon-calponin mixture on a fast protein liquid chromatography Superose-12 column revealed two base-line-separated peaks, the first containing only caldesmon and the second only calponin, thus confirming the lack of any interaction between these two proteins. Also, the addition of calponin did not change the fluorescence parameters of labeled caldesmon in complexes with F-actin and F-actin-tropomyosin.

The effect of Ca2+ on the structure of synthetic filaments of smooth muscle myosin.

Using electron microscopy and negative staining we have studied the effect of Ca2+ on the structure of synthetic filaments of chicken gizzard smooth muscle myosin under conditions applied by Frado and Craig (1989) for demonstration of the influence of Ca2+ on the structure of synthetic filaments of scallop striated muscle myosin. The results show that Ca2+ induces the transition of compact, ordered structure of filaments with a 14.5 nm axial repeat of the myosin heads close to the filament backbone (characteristic of the relaxing conditions) to a disordered structure with randomly arranged myosin heads together with subfragments-2 (S-2) seen at a distance of up to 50 nm from the filament backbone. This order/disorder transition is much more pronounced in filaments formed of unphosphorylated myosin, since a substantial fraction of phosphorylated filaments in the relaxing solution is already disordered due to phosphorylation. Under rigor conditions some of the filaments of unphosphorylated and phosphorylated myosin retain a certain degree of order resembling those under relaxing conditions, while most of them have a substantially disordered appearance. The results indicate that Ca2+-induced movement of myosin heads away from the filament backbone is an inherent property of smooth muscle myosin, like molluscan muscle myosin regulated exclusively by Ca2+ binding, and can play a modulatory role in smooth muscle contraction.

Visualization of caldesmon binding to synthetic filaments of smooth muscle myosin.

We have used synthetic filaments of unphosphorylated chicken gizzard myosin with a compact, highly ordered structure under relaxing conditions (in the absence of Ca2+ and in the presence of ATP) to visualize the mode of caldesmon binding to myosin filaments by negative staining and immunogold electron microscopy. We demonstrate that the addition of caldesmon to preformed myosin filaments leads to the appearance of numerous smooth projections curving out from the filament surface. The addition of caldesmon or its N-terminal fragment resulted in the partial masking of myosin filament periodicity. However, it did not change the inner structure of the filaments. It is demonstrated that most caldesmon molecules bind to myosin filaments through the N-terminal part, while the C-terminal parts protrude from the filament surface, as confirmed by immunoelectron microscopy visualization. Together with the available biochemical data on caldesmon binding to both actin and myosin and electron microscopic observations on the mode of caldesmon attachment to actin filaments with the C-termini of the molecules curving out from the filaments, the visualization of caldesmon attachment to myosin filaments completes the scenario of actin to myosin tethering by caldesmon.

Polymerization of actin induced by actin-binding fragments of caldesmon

Our earlier studies revealed that caldesmon causes assembly of G-actin into polymers morphologically indistinguishable from those formed in the presence of salt (Gałazkiewicz, B., Belagyi, J. and Dabrowska, R. (1989) Eur. J. Biochem. 181, 607-614). In this work we have investigated the effect of actin-binding fragments of caldesmon on actin polymerization process followed by measurements of the changes in fluorescence of pyrenyl conjugated with G-actin and ATP hydrolysis. The results indicate that C-terminal 34 kDa fragment of caldesmon containing two actin-binding sites and 19 kDa containing high-affinity binding site have similar capability to polymerize actin to that of intact molecule. Binding of each of these fragments to G-actin causes bypassing of nucleation phase. The 11.5 kDa fragment comprising low affinity actin-binding site has much lower potency to polymerize actin. Conformation of actin monomers in filaments formed upon 19 kDa fragment and that formed upon 11.5 kDa fragment differs. The former fragment seems to resemble more conformation of monomers in filaments formed upon intact caldesmon than the latter one.

Caldesmon inhibits the rotation of smooth actin subdomain-1 and alters its mobility during the ATP hydrolysis cycle.

Smooth muscle thin filaments have been reconstituted in muscle ghost fibers by incorporation of smooth muscle actin, tropomyosin and caldesmon. For the first time, rotation of subdomain-1 and changes of its mobility in IAEDANS-labeled actin during the ATP hydrolysis cycle simulated using nucleotides and non-hydrolysable ATP analogs have been demonstrated directly. Binding of caldesmon altered the mobility and inhibited the rotation of actin subdomain-1 during the transition from AM * *.ADP.Pi to AM state, resulting in inhibition of both strong and weak-binding intermediate states. These new results imply that regulation of actomyosin interaction by caldesmon during the ATPase cycle is fulfilled via the inhibition of actin subdomain-1 rotation toward the periphery of the thin filament, which decreases the area of the specific binding between actin and myosin molecules and is likely to underlie at least in part the mechanism of caldesmon-induced contractility suppression.

Disturbance of smooth muscle regulatory function by Eisenia foetida toxin lysenin: insight into the mechanism of smooth muscle contraction.

Lysenin, a toxin present in the coelomic fluid of the earthworm Eisenia foetida, is known to cause a long-lasting contraction of rat aorta smooth muscle strips. We addressed the mechanisms underlying its action on smooth muscle cells and present the first report demonstrating a completely new property of lysenin unrelated to its basic sphingomyelin-binding ability. Here we report lysenin enhancement effect on smooth muscle actomyosin ATPase activity and the ability of networking the actin filaments. The maximum enhancement of the ATPase activity of actomyosin at 120 mM KCl was observed at a molar ratio of lysenin to actin of about 1:10(5), while at 70 mM KCl at the ratio of about 1:10(6). The effect of lysenin became most pronounced only when both smooth muscle regulatory proteins, tropomyosin and caldesmon, were present. Co-sedimentation experiments indicated that lysenin did not displace neither tropomyosin nor caldesmon from the thin filament. Thus, the lysenin-dependent abolishment of the inhibitory effect of caldesmon on the ATPase activity was related rather to the modification of the filament structure. The ability of the toxin to exert its stimulatory effect at extremely low concentrations (as low as one molecule of lysenin per 10(6) actin molecules) may result from the long-range cooperative transitions in the entire thin filament with an involvement of smooth muscle tropomyosin, while the role of caldesmon may be limited exclusively to the inhibition of ATPase activity.