Electron microscopy of human factor VIII/Von Willebrand glycoprotein: effect of reducing reagents on structure and function

The structure of native and progressively reduced human factor VIII/von Willebrand factor (FVIII/vWF) was examined by electron microscopy and SDS gel electrophoresis and then correlated with its biological activities. Highly resolved electron micrographs of well-spaced, rotary- shadowed FVIII/vWF molecules showed their structure to consist of a very flexible filament that contains irregularly spaced small nodules. Filaments ranged from 50 to 1,150 nm with a mean length of 478 nm and lacked fixed, large globular domains as seen in fibrinogen and IgM. A population of multimeric FVIII/vWF species ranging in molecular weight from 1 to 5 million daltons and differing in size alternately by one and two subunits was observed on SDS-2% polyacrylamide-0.5% agarose gel electrophoresis. With progressive reduction of disulfide bonds by dithiothreitol (DTT), the electron microscopic size of FVIII/vWF decreased in parallel with increased electrophoretic mobility on SDS- agarose gels; between 0.1 and 0.5 mM DTT its structure changed from predominantly fibrillar species to large nodular forms. A 50% loss of vWF specific activity and FVIII procoagulant activity occurred at 0.4 mM DTT and 1 mM DTT, respectively, corresponding to the reduction of 4 and 12 disulfide bonds of the 62 disulfides per 200,000-dalton subunit. We conclude that reduction of a few critical disulfide bonds results in a major structural change by electron microscopy and a concomitant loss of approximately 50% of the vWF function.     

Immunochemical characteristics of hepatitis B e antigen subspecificities, HBeAg/1 and HBeAg/2

The molecular interrelation between hepatitis B e Ag 1 (HBeAg/1) and HBeAg/2 was examined immunochemically. Major polypeptides with m.w. around 17,000 (P17) and one minor polypeptide with m.w. 18,000 (P18) that had HBeAg activity were consistently detected in 12 different serum samples by immunoblotting analysis. To examine the polypeptide composition of HBeAg/1 and HBeAg/2, precipitin lines of HBeAg/1-anti-HBeAg/1 and HBeAg/2-anti-HBeAg/2 were separately cut from the agarose gel and analyzed by immunoblotting. HBeAg/1 and HBeAg/2 showed similar P17 and P18 compositions. Monomeric forms of P17 and P18 in a solution of 0.1% SDS showed HBeAg/1 activity, whereas polymeric forms of these polypeptides showed HBeAg/2 activity. The stability of HBeAg subspecificities to SDS and 2-ME was examined. When HBeAg was incubated in a buffer containing 0.1% SDS and 0.1% 2-ME, only the HBeAg/2 precipitin line disappeared in agarose gel concurrently with the conversion of HBeAg molecules to a monomeric from a polymeric form. In contrast, neither monomerization nor the disappearance of HBeAg/2 was seen when SDS or 2-ME was used by itself. These results indicate that hydrophobic forces and disulfide bonds may be involved in the association of P17 and P18 in serum and that HBeAg/2 activity depends upon association of HBeAg-polypeptides but that HBeAg/1 activity does not. The possibility that the epitopes of HBeAg/2 are specific for the conformation of polymerized molecules is discussed.     

Association of terminal complement proteins in solution and modulation by suramin.

The association of terminal complement proteins was investigated by analytical ultracentrifugation and multi-angle laser light scattering. Native C8 and C9 formed a heterodimer in solution of physiological ionic strength with a free-energy change DeltaG degrees of -8.3 kcal/mol and a dissociation constant Kd of 0.6 microM (at 20 degrees C) that was ionic strength- and temperature-dependent. A van't Hoff plot of the change in Kd was linear between 10 and 37 degrees C and yielded values of DeltaH degrees = -12.9 kcal/mol and DeltaS degrees = -15.9 cal mol-1 deg-1, suggesting that electrostatic forces play a prominent role in the interaction of C8 with C9. Native C8 also formed a heterodimer with C5, and low concentrations of polyionic ligands such as protamine and suramin inhibited the interaction. Suramin induced high-affinity trimerization of C8 (Kd = 0.10 microM at 20 degrees C) and dimerization of C9 (Kd = 0.86 microM at 20 degrees C). Suramin-induced C8 oligomerization may be the primary reason for the drug's ability to prevent complement-mediated hemolysis. Analysis of sedimentation equilibria and also of the fluorescence enhancement of suramin when bound to protein provided evidence for two suramin-binding sites on each C9 and three on each C8 in the oligomers. Oligomerization could be reversed by high suramin concentrations, but 8-aminonaphthalene-1,3,6- trisulfonate (ANTS2- ), which mimics half a suramin molecule, could not compete with suramin binding and oligomerization suggesting that the drug also binds nonionically to the proteins.     

Effects of cadmium on manganese peroxidase competitive inhibition of MnII oxidation and thermal stabilization of the enzyme.

Inhibition of manganese peroxidase by cadmium was studied under steady-state and transient-state kinetic conditions. CdII is a reversible competitive inhibitor of MnII in the steady state with Ki approximately 10 microM. CdII also inhibits enzyme-generated MnIII-chelate-mediated oxidation of 2,6-dimethoxyphenol with Ki approximately 4 microM. CdII does not inhibit direct oxidation of phenols such as 2,6-dimethoxyphenol or guaiacol (2-methoxyphenol) in the absence of MnII. CdII alters the heme Soret on binding manganese peroxidase and exhibits a Kd approximately 8 microM, similar to Mn (Kd approximately 10 microM). Under transient-state conditions, CdII inhibits reduction of compound I and compound II by MnII at pH 4.5. However, CdII does not inhibit formation of compound I nor does it inhibit reduction of the enzyme intermediates by phenols in the absence of MnII. Kinetic analysis suggests that CdII binds at the MnII-binding site, preventing oxidation of MnII, but does not impair oxidation of substrates, such as phenols, which do not bind at the MnII-binding site. Finally, at pH 4.5 and 55 degrees C, MnII and CdII both protect manganese peroxidase from thermal denaturation more efficiently than CaII, extending the half-life of the enzyme by more than twofold. Furthermore, the combination of half MnII and half CdII nearly quadruples the enzyme half-life over either metal alone or either metal in combination with CaII.     

Free fatty acids activate a high-affinity saturable pathway for degradation of low-density lipoproteins in fibroblasts from a subject homozygous for familial hypercholesterolemia.

This paper describes a mechanism for degradation of low-density lipoprotein (LDL) in fibroblasts unable to synthesize the LDL receptor. In this cell line, long-chain free fatty acids (FFA) activated 125I-LDL uptake; unsaturated FFA were the most efficient. The first step of this pathway was the binding of LDL apoB to a single class of sites on the plasma membrane and was reversible in the presence of greater than or equal to 10 mM suramin. Binding equilibrium was achieved after a 60-90-min incubation at 37 degrees C with 1 mM oleate; under these conditions, the apparent Kd for 125I-LDL binding was 12.3 micrograms/mL. Both cholesterol-rich (LDL and beta-VLDL) and triglyceride-rich (VLDL) lipoproteins, but not apoE-free HDL, efficiently competed with 125I-LDL for this FFA-induced binding site. After LDL bound to the cell surface, they were internalized and delivered to lysosomes; chloroquine inhibited subsequent proteolysis of LDL and thereby increased the cellular content of the particles. A physiological oleate to albumin molar ratio, i.e., 1:1 (25 microM oleate and 2 mg/mL albumin), was sufficient to significantly (p less than 0.01) activate all three steps of this alternate pathway: for example, 644 +/- 217 (25 microM oleate) versus 33 +/- 57 (no oleate) ng of LDL/mg of cell protein was degraded after incubation (2 h, 37 degrees C) with 50 micrograms/mL 125I-LDL. We speculate that this pathway could contribute to the clearance of both chylomicron remnants and LDL.     

Retention of a bean phaseolin/maize gamma-Zein fusion in the endoplasmic reticulum depends on disulfide bond formation

Most seed storage proteins of the prolamin class accumulate in the endoplasmic reticulum (ER) as large insoluble polymers termed protein bodies (PBs), through mechanisms that are still poorly understood. We previously showed that a fusion between the Phaseolus vulgaris vacuolar storage protein phaseolin and the N-terminal half of the Zea mays prolamin gamma-zein forms ER-located PBs. Zeolin has 6 Cys residues and, like gamma-zein with 15 residues, is insoluble unless reduced. The contribution of disulfide bonds to zeolin destiny was determined by studying in vivo the effects of 2-mercaptoethanol (2-ME) and by zeolin mutagenesis. We show that in tobacco (Nicotiana tabacum) protoplasts, 2-ME enhances interactions of newly synthesized proteins with the ER chaperone BiP and inhibits the secretory traffic of soluble proteins with or without disulfide bonds. In spite of this general inhibition, 2-ME enhances the solubility of zeolin and relieves its retention in the ER, resulting in increased zeolin traffic. Consistently, mutated zeolin unable to form disulfide bonds is soluble and efficiently enters the secretory traffic without 2-ME treatment. We conclude that disulfide bonds that lead to insolubilization are a determinant for PB-mediated protein accumulation in the ER.     

Native disulfide bonds in plasma retinol-binding protein are not essential for all-trans-retinol-binding activity

A human plasma retinol-binding protein (RBP) mutant, named RBP-S, has been designed and produced in which the six native cysteine residues, involved in the formation of three disulfide bonds, have been replaced with serine. A hexa-histidine tag was also added to the C-terminus of RBP for ease of purification. The removal of the disulfide bonds led to a decrease in the affinity of RBP for all trans-retinol. Data indicates all-trans-retinol binds RBP and RBP-S with Kd = 4 x 10(-8) M and 1 x 10(-7) M, respectively, at approximately 20 degrees C. RBP-S has reduced stability as compared to natural RBP below pH 8.0 and at room temperature. Circular dichroism in the far-UV shows that there is a relaxation of the RBP structure upon the removal of its disulfide bonds. Circular dichroism in the near-UV shows that in the absence of the disulfide bonds, the optical activity of RBP is higher in the 310-330 nm than in the 280-290 nm range. This work suggests that the three native disulfide bonds aid in the folding of RBP but are not essential to produce a soluble, active protein.     

Calmodulin regulation of Ca2+ transport in human erythrocytes.

Inside-out vesicles of human erythrocytes took up Ca2+ against an electrochemical gradient. This Ca2+ uptake was dependent on ATP and was stimulated by calmodulin. Treatment of vesicles with 1 mM-EDTA exposed an apparent low-CA2+-affinity Ca2+-transport component with Kd of about 100 microM-Ca2+ or more. This was converted into a single high-Ca2+-affinity transport activity of Kd about 2.5 microM-Ca2+ in the presence of 2 micrograms of calmodulin/ml, showing that the decrease in transport activity after EDTA treatment was reversible. Vesicles not extracted with EDTA showed mainly apparent high-Ca2+-affinity kinetics even in the absence of added calmodulin. Trifluoperazine (30 microM) and calmodulin-binding protein (20 micrograms/ml) inhibited about 50% of the high-affinity Ca2+ uptake and (Ca2+ + Mg2+)-ATPase (Ca2+-activated, Mg2+-dependent ATPase) activity of these vesicles, indicating that the vesicles isolated by the procedure used retained some calmodulin from the erythrocytes. Comparison of Ca2+ transport and (Ca2+ + Mg2+)-ATPase activities in inside-out vesicles yielded a variable Ca2+/P1 stoichiometric ratio. At low free Ca2+ concentrations (below 20 micro-Ca2+), a Ca2+/P1 ration of about 2 was found, whereas at higher Ca2+ concentrations the stoichiometry was approx. 1. The stoichiometry was not significantly altered by calmodulin.     

Inhibition of microbial growth by ajoene, a sulfur-containing compound derived from garlic.

Ajoene, a garlic-derived sulfur-containing compound that prevents platelet aggregation, exhibited broad-spectrum antimicrobial activity. Growth of gram-positive bacteria, such as Bacillus cereus, Bacillus subtilis, Mycobacterium smegmatis, and Streptomyces griseus, was inhibited at 5 micrograms of ajoene per ml. Staphylococcus aureus and Lactobacillus plantarum also were inhibited below 20 micrograms of ajoene per ml. For gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, and Xanthomonas maltophilia, MICs were between 100 and 160 micrograms/ml. Ajoene also inhibited yeast growth at concentrations below 20 micrograms/ml. The microbicidal effect of ajoene on growing cells was observed at slightly higher concentrations than the corresponding MICs. B. cereus and Saccharomyces cerevisiae were killed at 30 micrograms of ajoene per ml after 24 h of cultivation when cultivation was started at 10(5) cells per ml. However, the minimal microbicidal concentrations for resting cells were at 10 to 100 times higher concentrations than the corresponding MICs. The disulfide bond in ajoene appears to be necessary for the antimicrobial activity of ajoene, since reduction by cysteine, which reacts with disulfide bonds, abolished its antimicrobial activity.     

Allosteric effect of ATP on Na(+),K(+)-ATPase conformational kinetics

The kinetics of the E2 --> E1 conformational change of unphosphorylated Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 degrees C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize the E2 conformation. When rabbit enzyme was mixed with 130 mM NaCl alone or with 130 mM NaCl and varying concentrations of Na2ATP simultaneously, a fluorescence decrease was observed. In the absence of ATP, the fluorescence decrease followed a biexponential time course, but at ATP concentrations after mixing of >or=50 microM, the fluorescence transient could be adequately fitted by a single exponential. On the basis of the agreement between theoretical simulations and experimental traces, we propose that in the absence of bound ATP the conformational transition occurs as a two step reversible process within a protein dimer, E2:E2 --> E2:E1 --> E1:E1. In the presence of 130 mM NaCl, the sum of the forward and backward rate constants for the E2:E2 --> E2:E1 and E2:E1 --> E1:E1 transitions were found to be 10.4 (+/-1.0) and 0.49 (+/-0.02) s-1, respectively. At saturating concentrations of ATP, however, the transition occurs in a single reversible step with the sum of its forward and backward rate constants equal to 35.2 (+/-0.3) s-1. It was found that ATP acting at a high affinity site (Kd approximately 0.25 microM), stimulated the reverse reaction, E1ATP --> E2ATP, in addition to its known allosteric low affinity (Kd approximately 71 microM) stimulation of the forward reaction, E2ATP --> E1ATP.     

Stability and peptide binding affinity of an SH3 domain from the Caenorhabditis elegans signaling protein Sem-5.

We have determined the thermodynamic stability and peptide binding affinity of the carboxy-terminal Src homology 3 (SH3) domain from the Caenorhabditis elegans signal-transduction protein Sem-5. Despite its small size (62 residues) and lack of disulfide bonds, this domain is highly stable to thermal denaturation--at pH 7.3, the protein has a Tm of 73.1 degrees C. Interestingly, the protein is not maximally stable at neutral pH, but reaches a maximum at around pH 4.7 (Tm approximately equal to 80 degrees C). Increasing ionic strength also stabilizes the protein, suggesting that 1 or more carboxylate ions are involved in a destabilizing electrostatic interaction. By guanidine hydrochloride denaturation, the protein is calculated to have a free energy of unfolding of 4.1 kcal/mol at 25 degrees C. We have also characterized binding of the domain to 2 different length proline-rich peptides from the guanine nucleotide exchange factor, Sos, one of Sem-5's likely physiological ligands in cytoplasmic signal transduction. Upon binding, these peptides cause about a 2-fold increase in fluorescence intensity. Both bind with only modest affinities (Kd approximately equal to 30 microM), lower than some previous estimates for SH3 domains. By fluorescence, the domain also appears to associate with the homopolymer poly-L-proline in a similar fashion.     

Tyrosine kinase inhibitors and the contractile action of epidermal growth factor-urogastrone and other agonists in gastric smooth muscle.

We examined the effects of the tyrosine kinase (TK) inhibitors, genistein, and tyrphostin (RG-50864) on the contractile action of epidermal growth factor - urogastrone (EGF-URO), transforming growth factor-alpha (TGF-alpha), and other agonists in two smooth muscle bioassay systems (guinea pig gastric longitudinal muscle, LM, and circular muscle, CM). We also studied the inhibition by tyrphostin of EGF-URO stimulated protein phosphorylation in identical smooth muscle strips. The selective inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha induced contraction, but not of carbachol- and bradykinin-mediated contraction, occurred at much lower concentrations (genistein, less than 7.4 microM (2 micrograms/mL); tyrphostin, less than 20 microM (4 micrograms/mL)) than those used in previously published studies with these TK inhibitors. In LM tissue, the IC50 values were for genistein 1.1 +/- 0.1 microM (0.30 micrograms/mL; mean +/- SEM) and 3.6 +/- 0.5 microM (0.74 micrograms/mL) for tyrphostin, yielding a molar potency ratio (GS: TP) of 1:3 in the longitudinal preparation. In CM tissue, the IC50 values were 3.0 +/- 0.3 microM (0.81 micrograms/mL) for genistein and 2.4 +/- 0.2 microM (0.49 micrograms/mL) for tyrphostin, yielding a molar potency ratio (GS:TP) of 1.0:0.8 in the circular strips. The inhibition by genistein and tyrphostin of EGF-URO and TGF-alpha mediated contraction was rapid (beginning within minutes) and was reversible upon washing the preparations free from the enzyme inhibitors. In intact tissue strips studied under bioassay conditions, tyrphostin (40 microM) also blocked EGF-URO triggered phosphorylation of substrates detected on Western blots using monoclonal antiphosphotyrosine antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitor stabilization of human immunodeficiency virus type-2 proteinase dimer formation

We report the first direct observation of the subunit self-association behavior of highly purified recombinant human immunodeficiency virus type-2 (HIV-2) proteinase. Multiple samples of enzyme were subjected to sedimentation equilibrium analytical ultracentrifugation sequentially at 8.8 degrees C and two pH values in the presence and absence of a C2 symmetric, peptidomimetic inhibitor. At both pH values the enzyme exhibited sedimentation equilibrium behavior which fit a monomer-dimer-tetramer model. In the absence of inhibitor, the apparent Kd for dimer formation was less than approximately 100 microM and the apparent Kd for the weaker dimer-tetramer association was greater than approximately 100 microM. In the presence of inhibitor, at either pH, dimer formation was more strongly favored as indicated by a approximately 5-14-fold decrease in the apparent Kd for dimer formation and a approximately 1.2-4-fold increase in the apparent Kd for tetramer formation. The enhanced formation of dimer and decrease in higher order self-associated forms in the presence of an inhibitor is consistent with inhibitor stabilization of an active dimer. The inhibitor-induced stabilization of the dimeric species is consistent with a model for substrate-induced formation of active proteinase dimers in virion assembly.     

Effects of disulfide bonds on compactness of protein molecules revealed by volume, compressibility, and expansibility changes during reduction

To elucidate the effects of disulfide bonds on the compactness of protein molecules, the partial specific volume (v(o)) and coefficients of adiabatic compressibility (beta(s)(o)) and thermal expansibility (alpha) of five globular proteins (ovalbumin, beta-lactoglobulin, lysozyme, ribonuclease A, and bovine serum albumin) were measured in aqueous solutions with pH values of 7 and 2 at 25 degrees C when their disulfide bonds were totally reduced by carboxamidomethylation. Circular dichroism and fluorescence spectra show that the secondary and tertiary structures are partly disrupted by reduction, depending on the number of disulfide bonds in the proteins and the pH of the medium. The conformational changes are accompanied by decreases in v(o) and beta(s)(o) and by an increase in alpha, indicating that reduction decreases the internal cavity and increases surface hydration. The beta(s)(o) values of native or oxidized proteins decrease, and the effects of reduction on the volumetric parameters become more significant as the number of disulfide bonds increases and as they are formed over a larger distance in the primary structure. These results demonstrate that disulfide bonds play an important role, mainly via entropic forces, in the three-dimensional structure and compactness of protein molecules.     

Inhibition of the chymotrypsin-like activity of the pituitary multicatalytic proteinase complex.

The multicatalytic proteinase complex (MPC), also referred to as proteasome, is a large molecular mass intracellular particle (approximately 700 kDa), which exhibits three distinct proteolytic activities designated as chymotrypsin-like, trypsin-like, and peptidylglutamyl-peptide hydrolyzing (PGPH), all sensitive to inhibition by 3,4-dichloroisocoumarin (DCI). The presence of a component resistant to inhibition by DCI with an apparent preference toward bonds on the carboxyl side of branched-chain amino acids has also been recently established. Peptide aldehydes and peptide alpha-keto esters containing a hydrophobic residue in the P1 position have been tested as potential inhibitors of the chymotrypsin-like activity. Three peptide aldehydes (benzyloxycarbonyl)-Leu-Leu-phenylalaninal (Z-LLF-CHO), N-acetyl-Leu-Leu-norleucinal (Ac-LLnL-CHO), and N-acetyl-Leu-Leu-methioninal (Ac-LLM-CHO) were found to be slow-binding reversible inhibitors with Ki values of 0.46, 5.7, and 33 microM, respectively. The simplest kinetic model for inhibition is consistent with a mechanism involving a slow and reversible association of the enzyme with the inhibitor to form a EI complex. The aldehyde inhibitors also inhibited the trypsin-like and PGPH activities of the complex albeit with much higher Ki values than those for chymotrypsin-like activity. Z-LLF-CHO, the most selective of the three aldehydes, did not inhibit the PGPH activity at concentrations of up to 200 microM and inhibited the trypsin-like activity with a Ki approximately 2 orders of magnitude higher than that for the chymotrypsin-like activity. The activity of the DCI-resistant component was not affected by Z-LLF-CHO.(ABSTRACT TRUNCATED AT 250 WORDS)     

One-pot conversion of RAFT-generated multifunctional block copolymers of HPMA to doxorubicin conjugated acid- and reductant-sensitive crosslinked micelles

N-(2-Hydroxypropyl)methacrylamide (HPMA) containing polymers that are widely used as anticancer drug carriers. We have synthesized new amphiphilic block copolymers of HPMA with a functional monomer 2-(2-pyridyldisulfide)ethylmethacrylate (PDSM) via reversible addition-fragmentation chain transfer (RAFT) polymerization. In a one-pot reaction, the versatility of PDS groups on poly(PDSM)- b-poly(HPMA) was used to conjugate an anticancer drug, doxorubicin (DOX), and also simultaneously crosslink the micellar assemblies via acid-cleavable hydrazone bonds and reducible disulfide bonds. DOX-conjugated crosslinked micelles with an average diameter of approximately 60 nm were observed to be formed in aqueous medium. Disintegration of the micelles into unimers in the presence of a disulfide reducing agent confirmed the crosslinking via disulfide bonds. While the release of DOX from the crosslinked micelles at pH 5.0 was faster compared to the release at pH 7.4, a high proportion of released DOX was found to retain the original active structure. Overall results demonstrate the simplicity and the versatility of the poly(PDSM)- b-poly(HPMA) system, which are potentially important in the design of new generation of polymer therapeutics.     

A new spectroscopic approach to examining the role of disulfide bonds in the structure and unfolding of soybean trypsin inhibitor

Although it is well-known that disulfide bonds stabilize the secondary structure of many proteins, it is difficult to directly probe both disulfide bond formation/breakage and the resulting secondary structural changes during the course of the protein folding/unfolding process. In this work, we have used a new, real-time spectroscopic approach to examine how the reduction of two disulfide bonds affects the secondary structure of soybean trypsin inhibitor (STI). The disulfide bonds are reduced with tris(2-carboxyethyl)phosphine (TCEP) at 40 degrees C, and the reduction process is probed in real-time using sulfur X-ray absorption spectroscopy. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies are used concurrently to determine the structural changes caused by reduction of the disulfide bonds. Results demonstrate a noncooperative reduction of the two disulfide bonds within 5 min, likely because they are located on the surface of the protein. The unfolding of STI lags behind; dramatic changes are not observed until 60-90 min after the reduction was initiated. The CD and FTIR spectra indicate a decrease in the amount of extended (hydrated) coil, suggesting that the STI structure slowly collapses after the disulfide bonds are reduced. Thus, although the disulfide bonds are not located near the active site of STI, they play a crucial role in stabilizing the protein structure, which is necessary to sustain enzymatic activity.     

Conformational domains and structural transitions of human von Willebrand protein

The conformational states of human von Willebrand protein (vWF) were studied by using ultraviolet (UV) difference, circular dichroism, and fluorescence spectrophotometric techniques in order to gain insight into the forces that maintain its asymmetric, flexible shape. vWF has 24% alpha-helix and 18% beta-pleated sheet structure in the native state. Disulfide bond reduction and carboxamidation reduced the beta-pleated sheet content by 50% without affecting the content of alpha-helix. In addition, the quantum yield of intrinsic (tryptophan/tyrosine) fluorescence decreased by 33% after reduction and alkylation, and the affinity of the hydrophobic fluorescent probes 8-anilino-1-naphthalenesulfonate and 6-(p-toluidino)-2-naphthalenesulfonate for vWF was reduced 2.5-fold. In contrast, intrinsic fluorescence quenching by acrylamide and the UV difference spectrum did not change following reduction. An analysis of changes in the intrinsic fluorescence polarization and the emission maximum shift induced by thermal and guanidine hydrochloride denaturation revealed single, smooth transitions for both native and reduced vWF, suggesting the existence of an ordered structure in both species. This study shows that (1) disulfide reduction and carboxamidation cause significant conformational changes in vWF, (2) vWF may contain discrete, ordered, conformational domains linked by regions of random polypeptide chain, and (3) specific tertiary structural domains within vWF are not affected by disulfide reduction and carboxamidation. This structural model would explain both the asymmetry and flexibility of the molecule.     

Dimer model for the microfibrillar protein fibulin-2 and identification of the connecting disulfide bridge.

Fibulin-2 is a novel extracellular matrix protein frequently found in close association with microfibrils containing either fibronectin or fibrillin. The entire protein and its predicted domains were obtained as recombinant products and examined by ultracentrifugation and electron microscopy. This demonstrated a disulfide-linked homodimer of 175 kDa subunits. Partial reduction to monomers identified specifically an odd Cys574 residue responsible for dimer formation in one of three anaphylatoxin-like modules that constitute the central globular domain I (13 kDa) of fibulin-2. Furthermore, a Cys574-Ser mutation abolished disulfide connection but not non-covalent dimerization of fibulin-2. The C-terminal region (85 kDa) was shown to represent a 35-nm-long rod consisting of 11 calcium-binding EGF-like modules (domain II) and a small terminal globe (domain III). The unique N-terminal domain N (55 kDa) was also rod-shaped (approximately 38 nm) and rich in galactosamine indicating extensive O-glycosylation. A dimer model is proposed indicating mainly a rod-like shape of 80 nm length based on an anti-parallel association of two subunits through their domains I. This model also implies alignment of domains II and N between different subunits. This was demonstrated by surface plasmon resonance assay which showed a distinct interaction between domains N and II with a Kd of approximately 0.7 microM.     

Mechanism of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes: III. Serum-bound and oxidized 2-mercaptoethanol are available for the augmentation

The fetal calf serum (FCS) that was incubated with 2-mercaptoethanol (2-ME) followed by the removal of free 2-ME could support the antibody response to sheep erythrocytes in vitro as effectively as native FCS plus 2-ME. The supporting activity of 2-ME-pulsed FCS was reversibly abrogated by the treatment with dithiothreitol followed by dialysis. In addition, iodoacetamidetreated FCS did not acquire the supportiveness by 2-ME pulsing. These observations suggest that the activity of 2-ME-pulsed FCS would be due to the mixed disulfide between 2-ME and FCS components. On the other hand, the disulfide form of 2-ME (2-ME_ox) could also augment the antibody response as effectively as fresh 2-ME (the reduced form). These derivatized forms of 2-ME as well as fresh 2-ME was found to stimulate the transport of [³⁵S]cystine into murine lymphocytes when the uptake was examined by the long-term experiments (24 hr). These stimulations were thought to be mediated by the formation of the mixed disulfide between 2-ME and cysteine because the lymphocytes promoted the reaction of [³⁵S]cystine with 2-ME_ox- or 2-ME-pulsed FCS to produce the mixed disulfide that had been shown to be taken up by the lymphocytes four to five times more rapidly than cystine. Therefore, it was suggested that 2-ME_ox and 2-ME-pulsed FCS could augment the antibody response in a similar fashion to 2-ME by stimulating the uptake of cystine, an essential amino acid.