Purified sarcoplasmic reticulum (SR) vesicles from dog heart were used as an antigen to produce monoclonal antibodies (mAbs) to the Ca2'-ATPase. Nine of twelve clones of hybridoma cells produce mAbs which crossreact with seven SR preparation isolated from cardiac and skeletal muscles of various species. Three mAbs of IgM type interact with the 45-kDa tryptic fragment of rabbit skeletal muscle Ca2 +-ATPase and markedly inhibit Ca2+ uptake (by 95%) and ATPase activity (by 80%) and decrease (by 30-50%) the steady-state level of the Ca2+-ATPase phosphoenzyme. The ATPase activity could be completely blocked by one of these mAbs if the incubation medium was supplemented with 2 pM orthovanadate. On the other hand, when SR vesicles were treated with increasing concentrations of a nonioinic detergent CI2Es, the inhibiting effect of mAb 4B4 is diminished. It is concluded that the mAbs inhibit the Ca2+-ATPase only if the enzyme exists in an oligomeric form. The inhibition of the SR activities is due to an effect of the mAbs on the whole active center of the enzyme, rather than on a single partial reaction.

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Mouse hybridoma antibody E5D2 reacting with murine mono- and polyclonal IgG1 has been produced. MonAb E5D2 recognizes the antigenic determinant (epitope) buried in intact IgG1 and expressed upon mild reduction of interchain S-S bridges. Neither H nor L chains alone maintain epitope E5D2. Reassociation of gamma 1 chains (H chains of IgG1) with L chains results in complete restoration of this antigenic determinant. The data strongly suggest that epitope E5D2 depends on the quaternary structure of IgG1. The epitope is also expressed by reduced F(ab)2 fragment of IgG1 but is not connected with its antigen binding site. The likely localization of the epitope E5D2 is the interface between CH and CL domains. The second produced monAb F6C2 reacts with CH1-CL region of reduced mouse IgG2. Small-angle X-ray scattering experiments have demonstrated pronounced decrease of the radius of gyration of reduced IgG1 as compared to the intact one. This indicates general conformational changes of IgG1 molecule following mild reduction of Fab region S-S groups. Epitope E5D2 is the first quaternary antigenic subclass specific determinant described for C the region of mouse IgG. Thus, serologic expression of epitope E5D2 reveals precise conformational perturbations of small area near reduced S-S bridges while small-angle scattering demonstrates accompanying general transformation of IgG structure.

The immunoglobulin G (IgG) fraction of the antiserum from rabbits immunized with homogeneous beef pancreas tryptophanyl-tRNA synthetase inhibits the enzyme activity in the reactions of both tRNATrp aminoacylation and tryptophan activation. Fab fragments of IgG act in a similar way. Common antigenic determinants have been detected in tryptophanyl-tRNA synthetases from beef, pig, chicken and rat livers using pure antibodies against beef pancreas tryptophanyl-tRNA synthetase. This observation indicates the evolutional stability of certain structural features of tryptophanyl-tRNA synthetases.The interaction of antibodies with the fragments of beef tryptophanyl-tRNA synthetase produced by endogenous and tryptic proteolysis of the enzyme has been studied. One third of the antiserum antibodies interacting with the C-terminal fragment of the enzyme ( M , = 40000) inhibits its activity whereas the antibodies to the N-terminal fragment ( M , z 20000) have no effect on the enzyme activity.The immunochemical identity of the two synthetase fragments differing in their enzymatic activity supports the assumption that the loss of enzymatic activity of the tryptic fragment is caused by lack of a small peptide which is retained in case of endogenous proteolysis; probably the amino acid residues of this peptide participate in formation of active centre of tryptophanyl-tRNA synthetase. A radioimmunochemical method is described for determining the number of antigenic determinants. One molecule of tryptophanyl-tRNA synthetase was found to bind 9( * 1) molecules of Fab fragments. Antibodies against tryptophanyl-tRNA synthetase from beef pancreas do not inhibit noticeably the activity of reverse transcriptase from avian myeloblastosis virus. No antigenic determinants in common have been detected in reverse transcriptase and tryptophanyl-tRNA synthetase by radioimmunochemical assays.The fidelity in reproducing the genetically predetermined structure of proteins in the course of their biosynthesis depends on the operation of two coupled Abhrevmtions. Albumin, human serum albumin. Proteolytic fragment, fragment of tryptophanyl-tRNA synthetase with a molecular weight of about 2 x 40000, which is produced upon endogenous proteolysis of the enzyme. Tryptic fragment, fragment of tryptophanyl-tRNA synthetase with a molecular weight of about 2 x 40000. which is produced upon limited hydrolysis of the enzyme with trypsin.Enzyme. Tryptophanyl-tRNA synthetase, or L-tryptophan : tRNA ligase (AMP-forming) (EC 6.1.1.2). systems, i.e. tRNA aminoacylation and the ribosomal steps of synthesis. The crucial role in the specificity of tRNA aminoacylation is played by aminoacyltRNA synthetases. That is why studies on the structure and functions of these enzymes are among the most important problems of molecular biology and biochemistry [l, 21. The immunochemical approach used in the studies of enzymes has proved to be very helpful in furnishing valuable information about their structure and functions [3]. We employed this approach (a) in order to