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F Karush

Publications and source records attributed to F Karush.

15 recordsLinked to original sources
PubMed1990

Probing the transglutaminase-mediated, posttranslational modification of proteins during development.

Sphaerechinus granularis eggs were fertilized in seawater in the presence of 0.2 mM dansylcadaverine, and development was allowed to take place with this compound in the medium. gamma-Glutamyldansylcadaverine, indicative of the utilization of the amine tracer by intrinsic transglutaminase, was isolated from the embryonic proteins, and identity of the product with the chemically synthesized gamma-glutamyl derivative of dansylcadaverine was confirmed. Covalent labeling of proteins occurring during development was examined by means of electrophoresis in NaDodSO4, followed by immunoblotting with an antibody that specifically recognized the dansyl hapten. There was an increase in the total uptake of the tracer at an essentially constant rate with each cell division, from 2- to 8- and 64-cell stages. Moreover, multiple protein labeling was evident in all specimens. The described concept of studying posttranslational modifications in vivo by transglutaminase through detection of the haptenic or specific ligand recognizable group of an incorporated small amine substrate will undoubtedly be of general utility for probing the functions of this family of enzymes in other cell types as well.

Animals↗
PubMed1990

Bacterial expression of immunoglobulin VH proteins.

A bacterial expression system in Escherichia coli has been developed that produces as much as 10 mg/l of culture of the VH protein associated with monoclonal antibodies specific for the 5-dimethylaminonaphthalene-2-sulfonyl (Dns) group. This system has been applied to the expression of the VH genes derived from a low-affinity, IgM-producing hybridoma and from a high-affinity, IgG-producing cell line. The plasmid vectors (contributed by Dr William F. Studier) utilize a T7 expression cassette whose activity is initiated by infection with a lambda phage derivative carrying the T7 RNA polymerase gene. The VH proteins were extracted from the bacterial pellet in 8 M urea and purified by chromatography in 8 M urea. Recombinants with the homologous light (L) chains were prepared to yield VHL molecules. These were used to measure intrinsic affinity for Dns-lysine by resonance energy transfer. The association constants were 7 x 10(6) M-1 and 7 x 10(9) M-1 for the low- and high-affinity systems, respectively. These values are not significantly different from those observed with monoclonal antibodies secreted from the corresponding cell lines. This system lends itself to the quantitative evaluation of the binding properties of the VH protein itself as well as the modulation of affinity by site-directed mutagenesis.

Escherichia coli↗
PubMed1975

Antibody affinity. VIII. Measurement of affinity of anti-lactose antibody by fluorescence quenching with a DNP-containing ligand.

The method of fluorescence quenching by bound ligand for the measurement of antibody affinity has been extended to anti-lactose antibodies. The basis for this extension is the use of a lactosyl ligand covalently linked to the 2,4-dinitrophenyl (DNP) group. This group serves as a sensor of the bound state by causing fluorescence quenching similar to that observed with anti-DNP antibody. The synthesis and characterization of this new ligand, N-(Nalpha-acetyl,Nepsilon-DNP-L-lysyl)-rho-aminophenyl-beta-lactoside, are described. The affinity of its interaction with three rabbit IgG and one equine IgM antilactose antibody preparations has been measured. The Qmax values range from 24 to 47% and the calculated association constants from 2.5 times 10-5 M-1 to 7.2 times 10-5 M-1. For one IgG preparation, measurement of the association constant by equilibrium dialysis with the tritiated ligand gave a value of 6.75 times 10-5 M-1 compared to the value of 7.2 times 10-5 M-1 obtained by fluorescence quenching. It is evident that with monoclonal anti-lactose antibody the accurate determination of Qmax, which is possible in this system, provides another structurally dependent clonal characterization of the variable region of the antibody. This optical probe along with others can be used for the identification of individual clones of antibody-producing cells through time and inheritance.

Aminoglycosides↗
PubMed1975

Equine anti-hapten antibody. IX. IgM anti-lactose antibodies.

The immune response to a bacterial vaccine of Streptococcus faecalis (strain N) was characterized in all of the seven horses studied by the sustained production of about 90% IgM anti-lactose antibody over a period of 44 weeks with maximum values of the total antibody ranging from 4 mg/ml of serum to 12 mg/ml of serum. With respect to the binding of a lactose-containing ligand the association constants of the antibodies purified from sera obtained between 5 and 44 weeks fell in the range of 1 times 10-5 M-1 to 2 times 10-5 M-1. Not only was there no significant indication of maturation of a-finity in this period but there was a selective limitation of affinity compared to that of 7S antibodies. It was inferred that the synthesis of IgM antibody involves the selective utilization of V-H and/or V-L genes.

Adsorption↗
PubMedDate not supplied

Primary B-cell response to neuropeptide Y and bovine pancreatic polypeptide.

An analysis of the murine primary response to protein epitopes has been made with two small highly structured proteins, neuropeptide Y (NPY) and bovine pancreatic polypeptide (BPP), both of 36-amino acid residue length and containing helical structures. A group of cell lines producing monoclonal IgM antibody have been prepared consisting of six anti-NPY and two anti-BPP. The VH nucleotide sequences have been determined and characterized as germ-line either by identity to established germ-line sequences or by inference from the germ-line character of the D and JH segments. The intrinsic association constants for the homologous ligands have been estimated to range from 10(4) to 10(7) M-1 based on competitive ELISA. No severe restriction in the utilization of VH families, D segments or JH segments appears to be involved in this response. Among the eight cell lines, three VH families were represented as well as all three families of D segments and all of the JH segments, although some preference for JH3 was indicated. The length of the N(D)N sequences was also not subject to restriction, ranging from 9 to 29. Two unusual features of the CRD3s were noted, one involving the utilization of an uncommon DSP2 segment and the other the apparent occurrence of a D-D fusion.

Amino Acid Sequence↗