Dynamic Behavior of Vitronectin at the Cell-Material Interface.

Considering that vitronectin (VN) can promote both cell adhesion and matrix degradation, it is likely to play a dual role at the cell-biomaterial interface. In this paper we therefore describe details of the dynamic interplay between matrix adhesion and pericellular proteolysis in endothelial cells adhered to glass model substratum. Initially we show that coating concentration determines protein organization at the surface. When the protein coating density approached saturation (63 ng cm-2), VN spontaneously organized itself in multimeric aggregates at the surface (30-50 nm in diameter). At subsaturation protein density (17 ng cm-2) VN molecules were present predominantly as single entities, indicating that a minimum coating density was required for VN multimerization. By fluorescent visualization of surface-associated VN in different ways, we provide the first evidence of significant proteolytic remodelling of VN by endothelial cells (HUVECs) at the sites of αv integrin clusters. The degree of proteolysis was estimated using a novel approach relying on dequenching of FITC-labeled VN upon proteolytic activity, showing that about one-third of the surface-associated VN was proteolytically altered by adhering HUVECs. In addition, we demonstrate that HUVECs can internalize surface-associated VN and deposit it in a linear pattern along longitudinal actin filaments. Deposited VN was partly colocalized with urokinase receptors. Taken altogether, we elucidate the complex and dynamic behavior of VN during initial cell-biomaterials interactions, the equilibrium if which could have a significant impact on the biocompatibility of any blood contacting implants.

Plastocyanin microheterogeneity in Scenedesmus acutus MT8.

Two total plastocyanin (PC) fractions - loosely bound (lPC) and strongly bound (sPC) were extracted (84% and 16%, respectively) from the homogenate of Scenedesmus acutus MT8. Two-fold isolation-purification procedure including DE-52 chromatography separated IPC into a smaller oxidized [IPC (II)] and a larger reduced [IPC(I)] fractions, in contrast to sPC, where sPC(ll) greatly dominated over sPC(I). Analytical isoelectric focusing (IEF) separated IPC(II) into two main fractions only in the presence of 8 M urea, implying microheterogeneity. Preparative IEF in immobiline pH-gradient of 3.2-4.1 separated IPC(II) into two blue fractions - a more alkaline IPC(II) and a more acidic IPC"(II), which were probably stereoisomers. Their UV-Vis spectra exhibited rarely observed tryptophane (291.5 nm) and some differences at 270 and 287 nm. The exact molecular masses of apo-/holo-lPC (10131 Da/10194 Da) were determined by mass spectrometry. The number of -SH groups was determined from the mass difference between alkylated with 4-vinylpyridine (4-VP) and non-alkylated protein. Additionally, a simple procedure for simultaneous separation of both primary structure and stereoisomers of PC was developed.