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Detection of new ligand-defective mutations of apolipoprotein B (apoB) will enable identification of sequences involved in binding to the LDL receptor. Genomic DNA from patients attending a lipid clinic was screened by singlestrand conformation polymorphism analysis for novel mutations in the putative LDL receptor-binding domain of apoB-100. A 46-yr-old woman of Celtic and Native American ancestry with primary hypercholesterolemia (total cholesterol [TC] 343 mg/dl; LDL cholesterol [LDL-C] 241 mg/ dl) and pronounced peripheral vascular disease was found to be heterozygous for a novel Arg3531-Cys mutation, caused by a C-T transition at nucleotide 10800. One unrelated 59-yr-old man of Italian ancestry was found with the same mutation after screening 1,560 individuals. He had coronary heart disease, a TC of 310 mg/dl, and an LDL-C of 212 mg/ dl. A total of eight individuals were found with the defect in the families of the two patients. They had an age-and sex-adjusted TC of 240±14 mg/dl and LDL-C of 169±+10 mg/dl. This compares with eight unaffected family members with age-and sex-adjusted TC of 185±12 mg/dl and LDL-C of 124±+12 mg/dl. In a dual-label fibroblast binding assay, LDL from the eight subjects with the mutation had an affinity for the LDL receptor that was 63% that of control LDL. LDL from eight unaffected family members had an affinity of 91%. By way of comparison, LDL from six patients heterozygous for the Arg3500-Gln mutation had an affinity of 36%. The percentage mass ratio of the defective Cys3531 LDL to normal LDL was 59:41, as determined using the mAb MB19 and dynamic laser light scattering. Thus, the defective LDL had accumulated in the plasma of these patients. Using this mass ratio, it was calculated that the defective Cys353, LDL particles bound with 27% of normal affinity.Deduced haplotypes using 10 apoB gene markers showed J. E. Chatterton's present address is

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We propose a new model for the segmental flexibility of immunoglobulin G (IgG). The flexibility of native and mildly reduced anti-5-(dimethylamino)naphthalene-1-sulfonyl (anti-dansyl) antibody was reexamined by nanosecond fluorescence spectroscopy using deconvolution and lamp-shift corrections. The rabbit antibodies used for this study were purified of dimers and other aggregates. The original results indicated that the decay of fluorescence anisotropy involved two rotational correlation times. It was suggested that the short rotational correlation time, phi s, represented a flexible Fab arm motion over a restricted angle and that the long correlation time, phi L, represented global tumbling of the molecule [Yguerabide, J., Epstein, H. F., & Stryer, L. (1970) J. Mol. Biol. 51, 573--590]. Our new data indicate that the long correlation time primarily represents motions of the Fab segments and not global tumbling of the entire molecule. This interpretation implies a more flexible model for IgG. Thus, in solution the antibody arms appear to move over a wide angle and are not restricted to 33 degrees as was suggested in the earlier model. Simple diffusion calculations and other evidence suggest that phi s may represent V-module flexibility about the switch peptides or Fab twisting around its long axis, whereas phi L may represent wagging or wobbling motions of the Fab arms about the hinge region. The faster motions appear to occur over small angles whereas the slower wagging or wobbling motions responsible for most of the decay of anisotropy appear to be much less restricted. The biological function of IgG and anisotropy changes resulting from hinge disulfide cleavage are interpreted in terms of the proposed model. We also demonstrate a useful method for comparison of time-dependent and steady-state fluorescence polarization data.

Normal IgG and myeloma proteins of the IgG1, 2, 3,and 4 subclasses were mixed with human Clq and studied in the analytical ultracentrifuge for complex formation. Binding of IgG to Clq is apparent both from the enlargement of area and from the increase in sedimentation rate of the well-separated schlieren peak of Clq. The accurate determination of binding parameters requires that sedimentation rates be corrected for hydrodynamic interaction, and area measurements corrected for the Johnston-Ogston effect. At the highest immunoglobulin concentrations employed in these studies more than ten IgG molecules are bound to each Clq. If we assume that the number of binding sites must be an integral multiple of 6, then the data best support a 12 binding site model, although an 18 site model cannot be rule out. Myeloma IgG proteins of all subclasses bind to Clq, with affinities decreasing in the order G3 greater than G1 greater than G2 greater than G4. No binding of IgA to Clq could be detected.