Hydrophobic Surface Regions Research Articles

Cellular Responses on a Wettability Gradient Surface with Continuous Variations in Surface Compositions of Carbonate and Hydroxyl Groups

This article reports adhesion, spreading, migration, and proliferation responses of bovine endothelial cells (ECs) on the surface with a unidirectional wettability gradient. The surface chemical gradient, in which molar fractions of cyclic carbonate and hydroxyl groups were inversely varied, was prepared by continuous immersion of a poly(vinylene carbonate) (PVCa) film into an aqueous solution of sodium hydroxide at a constant speed. Advancing water contact angles of the surface, which ranged from 60° for an untreated PVCa surface to around 20° for a well-treated surface, gradually decreased with the distance from the untreated end of the film. Quantitative analyses of cellular characteristics of adhered cells on the gradient surface, which included the initial adhesion rate and the morphological indices of adhered cells such as spreading area, peripheral length, and circular coefficient, showed that these cells gave the highest values on the untreated PVCa surface and rapidly decreased on the surface regions with shorter periods of hydrolysis, followed by a slower decrease on surface regions with longer periods ofhydrolysis with increasing distance from the untreated end. Little adhesion occurred in a well-hydrolyzed, very hydrophilic surface region. The highest migration rate of adhered cells occurred on the slightly hydrophobic surface region (advancing contact angle, 35°) where adhered cells tended to delaminate as tissue formation proceeded. These results indicate that each cellular potential of ECs is dependent on the wettability of the gradient surface, producing polymorphorous states of adhered cells. Thus, the wettability gradient surface served as an excellent substrate to simultaneously study cellular response dependence on surface chemical composition and wettability.

X-ray crystal structure of the two site-specific mutants His35Gln and His35Leu of azurin from Pseudomonas aeruginosa

The three-dimensional structures of two site-specific mutants of the blue copper protein azurin from Pseudomonas aeruginosa have been solved by a combination of isomorphous replacement and Patterson search techniques, and refined by energy-restrained least-squares methods. The mutations introduced by recombinant DNA techniques involve residue His35, which was exchanged for glutamine and leucine, to probe for its suggested role in electron transfer. The two mutants, His35Gln (H35Q) and His35Leu (H35L), crystallize non-isomorphously in the orthorhombic space group P2 12 12 1 with unit cell dimensions of a = 109·74 A ̊ , b = 99·15 A ̊ , c = 47·82 A ̊ for H35Q, a = 57·82 A ̊ , b = 81·06 A ̊ , c = 110·03 A ̊ for H35L. In each crystal form, there are four molecules in the asymmetric unit. They are arranged as a dimer of dimers in the H35Q case and are distorted from ideal C2 symmetry in H35L. The final crystallographic R-value is 16·3% for 20,747 reflections to a resolution of 2·1 Å for H35Q and 17·0% for 32,548 reflections to 1·9 Å for H35L. The crystal structures reported here represent the first crystallographically refined structures for azurin from P. aeruginosa. The structure is very similar to that of azurin from Alcaligenes denitrificans. The copper atom is located about 7 Å below a hydrophobic surface region and is ligated by five donor groups in a distorted trigonal bipyramidal fashion. The implications for electron transfer properties of the protein are discussed in terms of the mutation site and the packing of the molecules within the tetramer.

Prothoracicotropic hormone has an insulin-like tertiary structure

A three-dimensional model of PTTH-II has been constructed using interactive computer graphics and energy minimisation techniques, assuming homology with porcine insulin, the structure of which has been determined by X-ray analysis. The model shows that PTTH-II can assume an insulin-like tertiary structure, which is compact with the exception of the sequence variable NH 2-terminal amino acids of the B chain. Most of the hydrophobic core residues including A2 Ile, A6 Cys, A11 Cys, A16 Leu, A20 Cys, B11 Leu, B15 Leu and B19 Cys are identical in PTTH-II and insulins. The glycines at A1, B8 and B23 allow the chain to assume the characteristic tertiary interactions of the insulin fold and although polypeptide chains are shorter at the COOH-termini of the A and B chains and extended at the NH 2-terminus of the B chain, the insulin-like tertiary structure can still be assumed. It is unlikely that PTTH-II forms either dimers or hexamers, characteristic of porcine and human insulin, and the model is consistent with the inability of PTTH-II to bind anti-insulin antibodies or insulin receptors. A hydrophobic surface region of PTTH-II may be involved in intermolecular actions of physiological relevance. We discuss the implications of our model for evolution of this family of hormones and growth factors.

Rapid purification of calcium-activated protease by calcium-dependent hydrophobic-interaction chromatography

Both low Ca 2+- and high Ca 2+-requiring forms of Ca 2+-activated protease (calpains I and II) were found to bind to phenyl-Sepharose in a calcium-dependent manner, suggesting that both enzymes expose a hydrophobic surface region in the presence of Ca 2+. Inclusion of leupeptin in column buffers prevented the loss of activity during hydrophobic-interaction and substrate-affinity chromatography. Under these conditions calpain II (high calcium-requiring form) was rapidly purified from bovine brain and rabbit skeletal muscle using successive phenyl-Sepharose and casein-Sepharose columns.

Aggregation of porcine pancreatic phospholipase A2 and Its zymogen induced by submicellar concentrations of negatively charged detergents.

The interaction of two sodium n-alkyl sulfates (C10 and C12) with porcine pancreatic phospholipase A2 and its zymogen was studied by various spectroscopic techniques, equilibrium gel filtration, calorimetry, and photochemically induced dynamic nuclear polarization 1H NMR. At very low concentrations of n-alkyl sulfate [up to 0.07 X critical micelle concentration (cmc)] the enzyme (Mr 14 000) is able to build up a complex with the detergent molecules having a molecular weight of about 90 000. This complex consists of 6 enzyme molecules and about 40 n-alkyl sulfate monomers. The formation of the detergent-protein aggregate occurs in a two-step process: First, two detergent molecules strongly bind to a hydrophobic surface region of the protein, previously called interface recognition site [Pieterson, W. A., Vidal, J. C., Volwerk, J. J., & de Haas, G. H. (1974) Biochemistry 13, 1455-1460]. Subsequently, at higher detergent concentrations suddenly 6 enzyme molecules aggregate, probably including about 30 additional detergent monomers. Although the zymogen of the pancreatic phospholipase A2 seems to form comparable high molecular weight aggregates with these detergents, there are spectroscopic differences, and higher detergent concentrations are required. Moreover, as will be shown in the following paper [Hille, J. D. R., Egmond, M. R., Dijkman, R., van Oort, M. G., Sauve, P., & de Haas, G. H. (1983) Biochemistry (following paper in this issue)], only the phospholipase A2 becomes superactivated in these complexes.

Escherichia coli phosphoenolpyruvate dependent phosphotransferase system. Complete purification of enzyme I by hydrophobic interaction chromatography

We have observed that EI possesses extremely hydrophobic surface regions. In light of this property, a high yield, rapid procedure has been developed for the complete purification of EI using solely hydrophobic interaction chromatography on commercially available resins. The entire procedure can be completed in 4-5 days with a 55-65% recovery of EI activity and an 870-fold purification.

Identification and extent of fluid bilayer regions in membranous cytochrome oxidase

Membranous cytochrome oxidase isolated from mitochondria provides a useful model membrane for studies of hydrophobic lipid association with a fairly well characterized functional protein complex. In order to examine the lipid environments, isotropic and oriented samples of cytochrome oxidase of varying phospholipid content were spin labeled with 5-doxylstearic acid (the 4′,4′-dimethyloxazolidine- N-oxyl derivative of 5-ketostearic acid), and examined by ESR spectroscopy. The isotropic samples exhibit two spectral components. Based on the relative intensity of the two components, the mobility, and the response to hydration, the two components are interpreted as arising from two lipid environments: (1) a layer of lipid bound to the hydrophobic protein surface (boundary lipid) and (2) fluid phospholipid bilayer regions. The extent of the bilayer regions is estimated from integration of the two components of the ESR spectra for samples with different phospholipid/protein ratios. The anisotropy of macroscopically oriented membranous cytochrome oxidase samples containing 0.33 and 0.49 mg phospholipid/mg protein confirms the presence of phospholipd bilayer regions. A spectral analysis reveals a remarkable similarity between the phospholipid bilayer regions in the cytochrome oxidase model membranes and bilayers formed by the isolated lipids, whereas the boundary lipid component has no counterpart in lipid bilayers.