Comparative studies on the interaction of proteins with a polydimethylsiloxane elastomer. I. Monolayer protein capture capacity (PCC) as a function of protein pl,buffer pH and buffer ionic strength

Polydimethylsiloxane (PEP) is widely used in medical prostheses and therefore is in contact with plasma and secretory proteins. Two pair of globular proteins, lactoferrin (Lf) and transferrin (Trf), and bovine IgG1 and IgG2a, which differ substantially between pair members in their pl, were used to study the interaction of a PEP widely used in breast implants and soluble protein. Studies were done using iodinated proteins over a concentration range that resulted in an apparent protein monolayer. Secondary incubations with dilute protein solutions were needed to form the monolayer on PEP, possibly as a consequence of micro air bubbles trapped on its highly textured surface as shown by atomic force microscopy. Immunoassay quality polystyrene microtiter wells were used as controls. Adsorption studies were routinely performed at pH 4, 7 and 10 and at ionic strengths corresponding to 0.95, 9.5 and 90.0 mS. The protein capture capacity (PCC) of PEP for Lf and Trf was optimal at physiological pH and ionic strength and comparable under these conditions to that of Immulon 2 (Imm 2) microtiter wells. While increasing the ionic strength and pH further increases the PCC of Imm 2 for Lf and Trf, this markedly lowered the PCC of PEP for these proteins suggesting that initial polar interactions may precede subsequent hydrophobic bonding to PEP. This was tested using a hydrophilic variant of PEP, which when tested in a 90.0 mS buffer, showed a >five-fold lower PCC at neutral and alkaline pH. The greatly reduced PCC of the hydrophilic variant might also suggest that hydrophilic variants of silicone would be more biocompatible than those currently used. The PCC of PEP for the IgGs was less than that of Imm 2 but still optimal at physiological conditions. Consistent with the data on Lf/Trf, PCC progressively decreased with increasing ionic strength at alkaline pH. Differences in pl between the protein pairs had only a marginal effect on the PCC of PEP. Monolayer adsorption on both PEP and Imm 2 was slowly reversible and greater in the presence of free ligand (<2% in 16 h) suggesting that the process follows Mass Law principles. However, even in the presence of non-ionic detergent and free ligand, 85–90% remained bound on either surface. Thus, desorption of proteins in the monolayer should not complicate subsequent immunochemical studies conducted on adsorbed monolayers. © 1997 John Wiley & Sons, Ltd.     

Effect of succinylation of antibodies on their conformation and interaction with the antigen

Using succinic anhydride, a succinylated derivative of anti-urease IgG having 49 ± 6% modification was prepared and its physicochemical and immunological properties were studied. IgG undergoes substantial changes in its native conformation on succinylation, which was mainly attributed to electrostatic destabilization of the native protein conformation. The modified IgG exhibited a decrease in its cross-reactivity with urease. This decrease is attributed to the conformational change in IgG upon succinylation and/or is due to the disruption of the lysine residues in the antigen-binding site of IgG upon succinylation, which may be involved in binding the antigen. IgG was able to bind to the specific antigen although its conformation was partially modified. Therefore, partial modification of the conformation of the antigen-binding site of IgG is permissible in order to bind to the antigen. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 12, pp. 1642–1647.     

Biochemical and ultrastructural studies on the interaction of basic proteins with mitochondria: a primary effect on membrane configuration

The cytochrome P-450_K containing monooxygenase system of rat kidney cortex microsomes catalyzes the hydroxylation of various saturated fatty acids of medium chain length to the corresponding ω- and (ω-1)-hydroxy derivatives. The hydroxylation activity, as well as the ratio between the two hydroxylated products, vary with the carbon chain length of the fatty acid. Optimal hydroxylation activity is observed with myristic acid which yields the 13- and 14-hydroxylated products at a ratio of about 1. The ω/(ω-1)-hydroxylation ratio decreases with increasing carbon chain length of the fatty acid. On the other hand, with lauric acid as a substrate the ratio between ω- and (ω-1)-hydroxylation does not change significantly with varying time of incubation or substrate concentration, or incubation in a medium containing D₂O or after induction of enhanced hydroxylation activity by starvation of the animals. Furthermore, 12-hydroxylauric acid and capric acid—which is almost exclusively ω-hydroxylated by rat kidney cortex microsomes—inhibit both 11- and 12-hydroxylation of lauric acid to a similar extent whereas 11-hydroxylauric acid does not seem to inhibit either 11- or 12-hydroxylation.C₁₀-C₁₆ fatty acids produce the type I spectral change upon addition to rat kidney cortex microsomes and seem to interact with similar amounts of the cytochrome P-450_K present in these particles. In agreement with the metabolic studies, 12-hydroxylauric acid interacts with cytochrome P-450_K giving rise to a reverse type I spectral change, whereas 11-hydroxylauric acid does not produce an observable spectral change. Finally, results of binding experiments with a series of derivatives of dodecane suggest that type I binding to cytochrome P-450_K requires, besides a proper chain length, the presence of a carbonyl group together with an electron pair on a neighboring atom at the end of the carbon chain. A chain length of 14 carbon atoms seems to be optimal and it is suggested that this chain length may correspond to the distance between a possible binding site and the catalytic site of cytochrome P-450_K     

The interaction of proteins with hydroxyapatite: II. Role of acidic and basic groups

The elution behavior from hydroxyapatite columns of the modification products of seven basic and three acidic proteins has been investigated. Three classes of NH₂ derivatives were prepared. These consisted of (1) replacement by a guanidyl group with no change in charge; (2) blocking with loss of charge; and (3) replacement of positive charges by negative ones. Two types of COOH derivatives were prepared: (1) blocking with loss of charge; and (2) replacement of COOH by SO₃H with no change in charge. The elution behavior of the derivatives in PO₄, F^?, Cl^?, ClO₄^?, and Ca²⁺ ion eluants showed that (1) the elution patterns are determined by the isoelectric points of the proteins, there being no symmetry between the binding or elution behavior of acidic and basic proteins; (2) the binding of basic proteins requires the presence of a high density of positively charged groups; (3) the binding of all proteins to hydroxyapatite equilibrated with phosphate buffer is enhanced by a decrease in the number of their negative charges; and (4) calcium ions affect the binding of proteins to hydroxyapatite at the level of carboxyls, since clusters of carboxyls strengthen both the interaction with Ca²⁺ and the binding to hydroxyapatite.     

Studies on the Fc gamma receptor of the murine macrophage-like cell line P388D1. II. Binding of human IgG subclass proteins and their proteolytic fragments.

Binding studies of human IgG proteins to murine P388D1 cells indicated that they bind to an apparently homogeneous Fc receptor population. The association constant was 0.89 x 10(6)M-1 at 22 degrees C and was comparable to the binding affinities of homologous murine IgG2a and IgG2b. The number of receptor sites was found to be approximately 6 x 10(5)/cell. Fc gamma 1 and Fc gamma 3 fragments bound with an affinity comparable to that of the parent proteins. The P388D1 receptors could discriminate between the human IgG subclasses; the relative cytophilic activity was IgG3 greater than IgG1 greater than IgG4 and IgG2 was devoid of binding activity. Fragments corresponding to the C gamma 2 and C gamma 3 domains of human IgG1 were both unable to bind to the P388D1 receptors either alone or in equimolar combination. This suggests that the cytophilic site may be formed cooperatively by interaction between the two domains. The integrity of the hinge region appeared to be essential for full expression of cytophilic activity since reduction of the hinge-region disulfides in both human IgG1 and its Fc fragment markedly decreased their binding affinity. In addition, a mutant IgG1 molecule lacking the hinge region was significantly less cytophilic than its normal counterpart.     

Comparative studies on the interaction of benzpyrene with cells derived from poikilothermic and homeothermic vertebrates. II. Effect of temperature on benzpyrene metabolism and cell multiplication

The effect of incubation temperature on cell multiplication and on the efficiency of benzpyrene (BP) metabolism to water-soluble derivatives was compared in cell cultures derived from three poikilothermic and three homeo-thermic vertebrate species. The fish cells grew optimally at about 20°C and the amphibian and reptilian cells at about 30°C, and in general, these cells multiplied over broader ranges of temperature than the mouse, hamster or chick cells. In each cell system, the maximum temperature supporting efficient BP metabolism exceeded the maximum temperature supporting cell growth by 4 to 8°, but the range of temperatures supporting near-maximal BP metabolism was also considerably broader in the poikilothermic than in the homeothermic vertebrate cultures.     

Structural studies on metal-serum albumin. IV. The interaction of Zn(II), Cd(II) and Hg(II) with HSA and BSA.

There have been no detailed and reliable studies on the environment and configuration of Zn(II), Cd(II) and Hg(II) in the metal centers of human serum albumin and bovine serum albumin to date. In this paper the authentic evidence for the involvement of the cystinyl sulfur atoms in the ligation to the zinc group ions has been obtained from the X-ray photoelectron spectra. The belief that each of the zinc group ions possesses several similar binding sites in human- and bovine serum albumin and is bound to the deprotonated thiol group (-RS-) of the cysteinyl residues to form tetrahedral and linear metal centers has been further confirmed by the treatment of ligand to metal charge transfer data with Jorgensen's method. According to these results, we have inferred that these binding sites may be located at the seventeen disulfide bridges, most likely at the seven pairs of adjacent disulfide bridges between positions 75 and 567, in the serum albumins.     

Fluorescence studies on the interaction of inhibitor 2 and okadaic acid with the catalytic subunit of type 1 phosphoprotein phosphatases.

Phosphatase inhibitor 2 was mutagenized and expressed in Escherichia coli to produce a protein with a single cysteinyl residue at position 129. The newly introduced sulfhydryl group was labeled with a maleimide derivative of coumarin (CPM). The resulting fluorescent inhibitor 2 molecule (CPM-I2) retains biological activity and binds to the catalytic subunit of type 1 phosphatase (PP1-C) with a Kd similar to the Ki of native I2 (2-3 nM). Fluorescence anisotropy data indicate that kinase FA (glycogen synthase kinase 3) does not dissociate the CPM-I2.PP1-C complex but rather causes a conformational change in the I2 molecule that is retained even after the CPM-I2 is displaced by an excess of native I2. The fluorescence data presented here also indicate that okadaic acid and I2 are competitive for binding to PP1-C, even after kinase FA treatment of the CPM-I2.PP1-C complex.     

Further studies on the interaction of actin with heavy meromyosin and subfragment 1 in the presence of ATP.

It has been postulated that, during the hydrolysis of ATP, both normal and SH1-blocked heavy meromyosin undergo a rate-limiting transition from a refractory state which cannot bind to actin to a nonrefractory state which can bind to actin. This model leads to several predictions which were studied in the present work. First, the fraction of heavy meromysin or subfragment 1 which remains unbound to actin when the ATPase equals Vmax should have the same properties as the original protein. In the present study it was determined that the unbound protein has normal ATPase activity which suggests that it is unbound to actin for a kinetic reason rather than because it is a permanently altered form of the myosin. Second, if the heavy meromyosin heads act independently half as much subfragment 1 as heavy meromyosin should bind to actin. Experiments in the ultracentrifuge demonstrate that about half as much subfragment 1 as heavy meromyosin sediments with the actin at Vmax. Third, the ATP turnover rate per actin monomer at infinite heavy meromyosin concentration should be much higher than the ATP turnover rate per heavy meromyosin head at infinite actin concentration. This was found to be the case for SH1-blocked heavy meromyosin since, even at very high concentrations of SH1-blocked heavy meromyosin, in the presence of a fixed actin concentration, the actin-activated ATPase rate remained proportional to the SH1-blocked heavy meromyosin concentration. All of these results tend to confirm the refractory state model for both SH1-blocked heavy meromyosin and unmodified heavy meromyosin and subfragment 1. However, the nature of the small amount of heavy meromyosin which does bind to actin in the presence of ATP at high actin concentration remains unclear.     

The primary structure of rat ribosomal proteins P0, P1, and P2 and a proposal for a uniform nomenclature for mammalian and yeast ribosomal proteins.

The covalent structures of rat ribosomal proteins P0, P1, and P2 were deduced from the sequences of nucleotides in recombinant cDNAs. P0 contains 316 amino acids and has a molecular weight of 34,178; P1 has 114 residues and a molecular weight of 11,490: and P2 has 115 amino acids and a molecular weight of 11,684. The rat P-proteins have a near identical (16 of 17 residues) sequence of amino acids at their carboxyl termini and are related to analogous proteins in other eukaryotic species. A proposal is made for a uniform nomenclature for rat and yeast ribosomal proteins.     

Identification of the protein-protein contact site and interaction mode of human VDAC1 with Bcl-2 family proteins

Bcl-2 family of proteins plays differential roles in regulation of mitochondria-mediated apoptosis, by either promoting or inhibiting the release of apoptogenic molecules from mitochondria to cytosol. Bcl-2 family proteins modulate the mitochondrial permeability through interaction with adenine nucleotide translocator (ANT), voltage-dependent anion channel (VDAC), ADP/ATP exchange, or oxidative phosphorylation during apoptosis. Although the mitochondrial homeostasis is affected by the relative ratio of pro- and anti-apoptotic Bcl-2 family members, the molecular mechanism underlying the release of mitochondrial intermembrane proteins remains elusive. Here we reported the biochemical evidence that both pro-apoptotic Bax and anti-apoptotic Bcl-X(L) might simultaneously contact the putative loop regions of human VDAC1, and the existence of VDAC1-Bax-Bcl-X(L) tertiary complex in vitro suggested that VDAC1 channel conformation and mitochondrial permeability could be determined by the delicate balance between Bax and Bcl-X(L).     

Spin label and 2H-NMR studies on the interaction of melanotropic peptides with lipid bilayers

The interaction of the cationic tridecapeptide -melanocyte stimulating hormone (-MSH) and the biologically more active analog [Nle⁴, DPhe⁷]--MSH with lipid membranes was investigated by means of ESR of spin probes incorporated in the bilayer, and NMR of deuterated lipids. All spin labels used here, stearic acid and phospholipid derivatives labeled at the 5^th and 12^th position of the hydrocarbon chain, and the cholestane label, incorporated into anionic vesicles of DMPG (1,2-dimyristoyl-sn-glycero-3-phosphoglycerol) in the liquid-crystalline phase, indicated that both peptides decrease the motional freedom of the acyl chains. No peptide effect was detected with neutral lipid bilayers. Changes in the -deuteron quadrupolar splittings and spin lattice relaxation time of DMPG deuterated at the glycerol headgroup paralleled the results obtained with ESR, showing that the peptides cause a better packing both at the headgroup and at the acyl chain bilayer regions. The stronger effect caused by the more potent analog in the membrane structure, when compared to the native hormone, is discussed in terms of its larger lipid association constant and/or its deeper penetration into the bilayer.     

Comparative studies on the diagnosis of hepatitis E virus antibodies with ORF-2 and ORF-3 proteins expressed in insect cells

Hepatitis E is a worldwide health problem, especially in developing countries. The virus genome contains three different open reading frames (ORFs): ORF-1, which is believed to encode nonstructural proteins, and ORF-2 and ORF-3, which are believed to encode structural proteins. Presently, serologic tests for the detection of human antibodies to hepatitis E virus (HEV) infection are primarily based on the ORF-2 structural protein expressed inEscherichia coli, insect cells or synthetic peptides. We report here the comparative studies on the diagnosis of HEV infection with full-length ORF-2 and ORF-3 proteins expressed in insect cells. We found that 31 of 74 (42%) sera were positive for IgM antibody to HEV (anti-HEV) using the ORF-2 protein as an antigen, as compared to 6 of 74 sera (8%) using the ORF-3 protein as an antigen (p<0.001). Similarly, 49 of 74 sera (66%) were positive for IgG anti-HEV utilizing the ORF-2 protein versus 12 of 74 sera (16%) when the ORF-3 protein was used (p<0.001). These results suggest that the recombinant ORF-2 protein is more sensitive as a diagnostic antigen for detecting antibodies to HEV in both acute-phase and convalescent-phase sera than ORF-3 protein.     

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

ABSTRACT

Prion diseases involve the conversion of the endogenous cellular prion protein, PrP^C, into a misfolded infectious isoform, PrP^Sc. Several functions have been attributed to PrP^C, and its role has also been investigated in the olfactory system. PrP^C is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp^?/? mice showed impaired behavior in olfactory tests. Given the high PrP^C expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrP^C-binding partners. Ten different putative PrP^C ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrP^C with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrP^C-Stub1 interaction are under investigation. The PrP^C-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrP^C is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein.