Myosin : Subunits and their interactions

There is fairly general agreement that myosin isolated from rabbit skeletal muscle has a molecular weight of about 500,000. The higher values that have been reported apparently reflect protein aggregation related to the method of preparation. On the basis of present evidence, the myosin molecule has an elongate helical core of two f subunits (average weight about 215,000) that extend into a globular head region containing three g subunits (average weight about 20,000). Myosin may be dissociated into subunits by a number of methods. In 5 M guanidine, the myosin molecule is dissociated into f and g subunits, while at pH above 10, the g subunits are dissociated from the intact fibrous core of myosin. The dissociation of g subunits at pH 10 is accompanied by the loss of both ATPase activity and actin-binding capacity; however, the exact biological significance of the g subunits is presently uncertain. In preliminary studies, the f subunits appear to contain the sulfhydryl residues currently implicated in myosin ATPase, and there is some indication of allosteric regulation of enzymic activity.     

Study of the antigenic relationship between animal and human immunoglobulins using antisera to human immunoglobulins]

Cross-reactivity of monospecific antisera to human immunoglobulins with animal sera of 10 species was studied by immunoelectrophoresis and radial immunodiffusion. Antisera to IgG were shown to reveal IgG of all the species studied, antisera to IgM and especially to IgA cross reacted less extensively. The greatest number of cross reactions were given by the antisera obtained as a result of hyperimmunization. Hyperimmune monospecific antisera to human IgG, IgA, and IgM can be used for the identification of animal immunoglobulins during their isolation from the sera and for their quantitation by radial immunodiffusion.     

Degeneracy of antibody specificity

Evidence was obtained, by direct binding assays (radioimmunoassays) and by inhibition of binding assays, that after immunization some of the antibody molecules produced are degenerate in that they bind not only the immunizing antigen, but also unrelated ligands. It can be concluded that the exquisite specificity of the immune response is not necessarily a property of any given antibody molecule but is, at least to some extent, due to the summation of specificities held in common by the population of antibody molecules produced during the response.     

Purification of homogeneous murine immunoglobulins with anti-fructofuranan specificity.

Homogenous IgAK immunoglobulins ABPC-47 new, EPC-109, UPC-61 (anti-2,1 fructofuranan specificity) and homogeneous IgAK ABPC-48 and IgGK UPC-10 (anti-2,6 fructofuranan specificity) were purified by affinity chromatography by using inulin- and grass levan-Sepharose columns, respectively.     

Monoclonal antibody with specificity to mitotic chromosomes of primates

Fusion of a cell in mitosis with a cell in interphase results in the condensation of chromatin in the interphase nucleus into chromosomes. Premature chromosome condensation is caused by certain proteins, called mitotic factors, that are present in the mitotic cell and are localized on chromosomes. Extracts from mitotic cells were used to immunize mice to produce monoclonal antibodies specific for cells in mitosis. Among the antibodies obtained, the MPM-4 antibody defines a 125-kD polypeptide antigen located on mitotic chromosomes by indirect immunofluorescence. Although the polypeptide antigen is present in approximately equal concentrations in extracts of interphase cells and mitotic cells, as revealed by immunoblots, it cannot be detected cytologically in the former. Cell fractionation experiments showed that the 125-kD antigen is found in the cytoplasm of interphase cells and metaphase cells, but is concentrated in fractions containing metaphase chromosomes, although not detectable in interphase nuclei. Even though the antigen is apparently primate-specific, it binds to mitotic chromosomes and prematurely condensed chromosomes in human-rodent cell hybrids without regard to the species of origin of the mitotic inducer. The presence of the antigen in the cytoplasm of interphase cells and the chromosomes of mitotic cells suggests a relationship between the presence of the antigen on chromosomes and the process of chromosome condensation and decondensation.     

Structure and specificity of antibody molecules.

The structure of the Fab' fragment of a human myeloma protein (IgG1 (lambda) New) has been determined by X-ray crystallographic analysis to a nominal resolution of 0.2 nm. Each of the structure subunits corresponding to the variable and to the constant homology regions of the light and heavy polypeptide chains contains two irregular beta-sheets which are roughly parallel to each other and surround a tighly packed interior of hydrophobic side chains. The regions of the hypervariable sequences in the light and heavy chains occur in close spatial proximity at one end of the molecule, defining the active site of IgG New. The role of these hypervariable regions in defining the size and shape of the active site of different immunoglobulins is discussed on the basis of the three-dimensional model of Fab' New. Several ligands that bind to the active centre of IgG New have been used to obtain crystalline ligand-Fab' New complexes which were investigated by difference Fourier maps. These studies are analysed in terms of the biological function and specificity of antibodies.     

Evolutionary pattern of specificity regions in light chains of immunoglobulins

An examination of the distribution of changes of single amino acids in the specificity (S) regions of light chains of immunoglobulins G shows that the changes, in terms of minimum base changes at each site, correspond quite well with the Poisson distribution, if it is assumed that approximately ten sites are invariant and five in the hinge region are hypervariable. The findings are concordant with other evidence that the S regions of light chains are evolving in a manner similar to that in other series of homologous proteins, such as the cytochromes c and hemoglobins, by the incorporation of randomly occurring adaptive or neutral point mutations into the genome and the discarding of deleterious point mutations. It is suggested that mutations in S regions are predominantly adaptive and that those in C regions are usually deleterious, thus accounting for the variability of S and the constancy of C sequences.This work was supported by Grant NsG 479 from the National Aeronautics and Space Administration to the University of California, Berkeley.     

Anti-nitroxide immunoglobulin G: analysis of antibody specificity and their application as probes for spin-labeled proteins

Antibodies have been elicited to the nitroxide spin-label 4-maleimido-2,2,6,6-tetramethyl-piperidinyl-1-oxy conjugated, via protein sulfhydryl groups, to bovine serum albumin. Antibody-hapten cross-reactivity was demonstrated by double immunodiffusion and by a broadening of the nitroxide electron paramagnetic resonance spectrum. The specificity of the antibodies with respect to hapten structure was examined by means of a simple filter binding assay. Under these conditions, antibodies were shown to distinguish between the nitroxide and hydroxylamine derivatives and between spin-labels comprising either five- or six-membered ring structures. In addition, protein-bound nitroxide spin-labels were detected at the nanogram level by immunoblotting. By use of this method, the specificity of the antibody-hapten reaction predicted by the filter binding assay procedure was utilized to differentially detect various types of bound spin-label. Finally, antibodies were used to identify protein-bound nitroxide spin-label of protein fractionated by gel electrophoresis.     

Transformation of specific antibodies to polyreactive immunoglobulins and their binding to blood vessel walls

It was shown that 30-50% ethanol or 40-70% dimetilsulfoxide could efficiently induce in vitro transformation of specific monoclonal antibodies (mAbs) into non-specific polyreactive immunoglobulins (PRIG). Intravenous injection 0.4 ml of FeSO4-EDTA mixture (60 and 30 mkM respectively) could induce increase of PRIG reactivity in the blood-stream. Intramuscle injection of either 0.1 ml of 40% ethanol, or 0.1 ml of FeSO4-EDTA mixture into muscle of hind limb of C57B1 mice leads to the substantial binding of circulated immunoglobulins to the blood vessels of the muscle. The similar effect could also be induced by ischemia/reperfusion of mice hind limb. In the case of intravenous injection of specific to ovalbumin biotinilated mAbs, the subsequent intramuscle injection of 0.1 ml of 40% ethanol induces apparent transformation of these mAbs into PRIG and their binding to the blood vessels. Intramuscle injection of 0.1 ml of FeSO4-EDTA mixture induces less than ethanol though noticeable effect. The obtained data have shown that cord-blood circulating specific antibodies could be transformed into PRIG at some conditions in vivo. If so, this process might play an important role in the organism defence against infections but could, probably, facilitate the development of atherosclerosis, cardiac infarct, cerebral stroke or tumors.     

Computer simulation of antibody binding specificity

A Monte Carlo algorithm that searches for the optimal docking configuration of hen egg white lysozyme to an antibody is developed. Both the lysozyme and the antibody are kept rigid. Unlike the work of other authors, our algorithm does not attempt to explicitly maximize surface contact, but minimizes the energy computed using coarse-grained pair potentials. The final refinement of our best solutions using all-atom OPLS potentials (Jorgensen and Tirado-Rives⁸) consistently yields the native conformation as the preferred solution for three different antibodies. We find that the use of an exponential distance-dependent dielectric function is an improvement over the more commonly used linear form. © 1993 Wiley-Liss, Inc.