Conformation, enzymic activity, and immunochemistry of a lysozyme derivative modified at tryptophan 123 by reaction with 2,3-dioxo-5-indolinesulfonic acid.

Reaction of hen egg-white lysozyme with 2,3-dioxo-5-indolinesulfonic acid (DISA) yielded a homogeneous derivative which was modified at a single tryptophan residue. The modification was located at Trp-123. The absorption spectrum of the derivative showed a new peak in the visible range with lambdamax at 365 nm. In addition, the absorption maximum in the ultraviolet which appears in lysozyme at 280 nm was shifted to 270 nm in the derivative and appreciably enhanced. In ORD measurements, the rotatory behaviors of lysozyme and its derivative were identical at the 233 nm negative minimum and the 199 nm positive extremum. CD measurements gave equal [theta] values for lysozyme and derivative at the two negative ellipticity bands at 208 and 220 nm. Although no conformational differences between lysozyme and derivative were observed by ORD and CD measurements, some changes were detectable by chemical methods. Accessibility to tryptic hydrolysis and susceptibility of the disulfide bonds to reduction were increased in the derivative relative to lysozyme. The lytic activity of the derivative, which retained the same pH optimum as native lysozyme, was greatly (50%) decreased, probably as a result of the slight conformational change. With several antisera to lysozyme, the native protein and its derivative had equal antigenic reactivities. The findings were instrumental in further delineation of an antigenic reactive site in lysozyme.     

Antigenic structure of human hemoglobin: Delineation of the antigenic site (site 2) within region 41–65 of the alpha chain by immunochemistry of synthetic peptides

A comprehensive synthetic approach consisting of a series of consecutive, uniform overlapping peptides encompassing the entire protein chain was recently used to determine the full antigenic profile of the α-chain of human hemoglobin (Hb). The peptides synthesized enabled the localization of five major “continuous” antigenic regions within the α chain. The present findings describe the delineation of an antigenic site (site 2) residing within the region 41–65. Ten peptides representing the α-chain regions 41–55, 51–65, 45–54, 45–56, 45–58, 45–60, 48–56, 49–56, 50–56, and 51–56 were synthesized and purified. Quantitative radioimmunoadsorbent titrations were used to determine binding to peptide adsorbents of radioiodinated anti-Hb antibodies that were raised in rabbit, goat, and outbred mouse. In one set of peptides, the N-terminal was fixed while the C-terminal end was increased by increments of two residues from Gln-54 to Lys-60 (i.e., peptides 45–54, 45–45, 45–58, and 45–60). Binding studies revealed that maximum antibody activity resided in peptide 45–45, indicating that Lys-56 marks the C-terminal boundary of the site. In the second set of peptides, the C-terminal was fixed at Lys-56 while the peptides were elongated at their N-terminal by one-residue increments from Gly-51 to Leu-48. Antibody-binding studies with these peptides indicated that Ser-49 defines the N-terminal boundary of the site. Therefore, the antigenic site within region 41–65 of the α chain comprises residues 49–56. The relevance of these findings to the immune recognition of Hb and other proteins is discussed.     

LDL-apheresis depletes apoE-HDL and pre-β1-HDL in familial hypercholesterolemia: relevance to atheroprotection

Subnormal HDL-cholesterol (HDL-C) and apolipoprotein (apo)AI levels are characteristic of familial hypercholesterolemia (FH), reflecting perturbed intravascular metabolism with compositional anomalies in HDL particles, including apoE enrichment. Does LDL-apheresis, which reduces HDL-cholesterol, apoAI, and apoE by adsorption, induce selective changes in HDL subpopulations, with relevance to atheroprotection? Five HDL subpopulations were fractionated from pre- and post-LDL-apheresis plasmas of normotriglyceridemic FH subjects (n = 11) on regular LDL-apheresis (>2 years). Apheresis lowered both plasma apoE (−62%) and apoAI (−16%) levels, with preferential, genotype-independent reduction in apoE. The mass ratio of HDL2:HDL3 was lowered from ∼1:1 to 0.72:1 by apheresis, reflecting selective removal of HDL2 mass (80% of total HDL adsorbed). Pre-LDL-apheresis, HDL2 subpopulations were markedly enriched in apoE, consistent with ∼1 copy of apoE per 4 HDL particles. Large amounts (50-66%) of apoE-HDL were removed by apheresis, preferentially in the HDL2b subfraction (−50%); minor absolute amounts of apoE-HDL were removed from HDL3 subfractions. Furthermore, pre-β1-HDL particle levels were subnormal following removal (−53%) upon apheresis, suggesting that cellular cholesterol efflux may be defective in the immediate postapheresis period. In LDL-receptor (LDL-R) deficiency, LDL-apheresis may enhance flux through the reverse cholesterol transport pathway and equally attenuate potential biglycan-mediated deposition of apoE-HDL in the arterial matrix.     

Synthesis of a novel series of cytotoxic bisindole alkaloids

Original cytotoxic bisindole alkaloids with a 1,2,3,4-tetrahydroquinoline bridge were synthesized by reductive amination with various anilines. The most cytotoxic compounds display a high and dose-dependent cell cycle effect with accumulation in the G1 phase. Influence of substitution of the starting aniline on the reaction and on cytotoxicity of produced dimers was pointed out.     

Recognition of inter-transmembrane regions of acetylcholine receptor alpha subunit by antibodies, T cells and neurotoxins. Implications for membrane-subunit organization

Three regions of the alpha chain of Torpedo californica acetylcholine receptor (AChR), corresponding to residues alpha 262-276, alpha 388, 408 and alpha 427-437 were synthesized, purified and characterized. The first two peptides have been proposed to occupy inter-transmembrane regions while the third represented the C-terminal segment, proposed by various models to be either extracellular or intracellular. Peptide alpha 388-408 stimulated a good response in the AChR-primed T cells of H-2s haplotype mice, a low response in the H-2q haplotype and no response in the H-2b haplotype. Peptide alpha 427-437 stimulated AChR-primed T cells of the H-2s haplotype, but caused no response in the q and b haplotypes. Peptide alpha 262-276 evoked no in vitro stimulation in any of the s, q or b haplotypes. In antibody binding studies, peptide alpha 388-408 bound antibodies raised against free AChR or against membrane-bound AChR. The other two peptides showed little or no binding activity. Further, peptide alpha 388-408 bound specifically both 125I-labelled bungarotoxin and cobratoxin, while the other two peptides had no binding activity. These results were consistent with only one of the models for subunit organization within the membrane.     

Effects of amino acid substitutions outside an antigenic site on protein binding to monoclonal antibodies of predetermined specificity obtained by peptide immunization: demonstration with region 56-62 (antigenic site 2) of myoglobin.

This work was carried out in order to study the effects of substitutions outside antigenic site 2 of sperm whale myoglobin (SpMb) on the reactivity of protein variants with antisite 2 monoclonal antibodies (mAbs). A synthetic peptide corresponding to region 56–62 (site 2) of SpMb was used as an immunogen in mice in its free form (i.e., without coupling to any carrier) to prepare a panel of mAbs whose predetermined specificity is directed, by design, against this region. The binding of three of these mAbs to eight Mbs from different species was studied. Myoglobins of Pacific common dolphin, finback whale, and horse, which have no substitutions within region 56–62 relative to SpMb, showed remarkable differences in their cross-reactivities and relative affinities with each of the mAbs. Myoglobins of badger, chicken, and dog, although they have an identical substitution within the site (Ala-57 to Gly), exhibited cross-reactivities with a given mAb that were affected differently. Echidna Mb, which has one replacement (Glu-59 to Ala) within region 56–62, displayed greatly reduced cross-reactivities and relative binding affinities. The results, especially those from Mbs that have no substitutions within the boundaries of site 2, clearly indicate that substitutions outside site 2 of Mb can exert drastic effects on the binding of the Mb variants with mAbs whose specificity was predesigned to be against the site. These indirect effects and their impact on site reactivity will completely explain previous findings on cross-reactivities of Mb variants with mAbs of unknown specificity and will rule out the postulations of discontinuous sites in Mb, which were based on the assumption that every substitution affecting reactivity is directly involved in binding to antibody.