ABSTRACT
The polyclonal antibodies purified by affine chromatography against tyrosyl-tRNA synthetase (TyrRS) immobilized on the column with affigel-sepharose have been obtained from the bovine liver. The immunospecificity of these antibodies and their influence on enzymatic activity of TyrRS from the bovine liver have been investigated. We have stated that the polyclonal antibodies inhibited TyrRS enzymatic activity in aminoacylation of homologous tRNA(Tyr) by 47%. As it has been shown by immunoblotting the antibodies reacted both with the native enzyme (M(r) 2.59 kDa) and with the proteolytic cleaved functionally active form of the enzyme (M 2.39 kDa).
Subject(s)
Antibodies/isolation & purification , Isoenzymes/immunology , Liver/immunology , Tyrosine-tRNA Ligase/immunology , Animals , Cattle , Chromatography, Affinity , Enzymes, Immobilized , Immunochemistry , Liver/enzymologyABSTRACT
The interaction of the cow mammary gland tRNA(IAGLeu), having a long variable loop, with the cognate aminoacyl-tRNA synthetase has been studied by the alkylation with ethylnitrosourea. It was shown that leucyl-tRNA synthetase protects from alkylation 3'-phosphates of the nucleotides 12-13 in D-loop, 23-24 in D-stem and 37-43 in the anticodon arm of tRNA(IAGLeu). All regions of interaction with the aminoacyl-tRNA synthetase are located in the same plane of tRNA whereas the long variable loop is in another plane.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Mammary Glands, Animal/metabolism , RNA, Transfer, Leu/genetics , Alkylation , Animals , Autoradiography , Base Sequence , Cattle , Electrophoresis, Polyacrylamide Gel , Female , Molecular Sequence Data , Nucleic Acid Conformation , RNA, Transfer, Leu/metabolismABSTRACT
The structural accessibility of tryptophan residues in leucyl-tRNA synthetase from cow mammary gland has been studied using chemical modifications by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide. The modifications were monitored by UV absorbance and intrinsic fluorescence of the enzyme's tryptophan residues. Under native conditions, at pH 7,8, only two exposed tryptophan residues are modified in each subunit of the dimeric enzyme. Under denaturing conditions, in 6 M guanidine hydrochloride solution, internal tryptophan residues are also modified as a consequence of unfolding of the native tertiary structure of the enzyme. Modifications of tryptophan residues resulted in inactivation of leucyl-tRNA synthetase both in aminoacylation and ATP-PPi exchange reactions. In the specific complex of leucyl-tRNA synthetase with the cognate tRNALeu one of exposed tryptophan residues is protected by tRNALeu and is not modified by the above reagents.
Subject(s)
2-Hydroxy-5-nitrobenzyl Bromide , Amino Acyl-tRNA Synthetases , Bromosuccinimide , Leucine-tRNA Ligase , Nitrophenols , Succinimides , Tryptophan/analysis , Amino Acyl-tRNA Synthetases/metabolism , Animals , Cattle , Chemical Phenomena , Chemistry , Female , Fluorescence , In Vitro Techniques , Indicators and Reagents , Leucine-tRNA Ligase/metabolism , Mammary Glands, Animal/enzymology , Protein Denaturation , Spectrophotometry, UltravioletABSTRACT
Definite separation of somatotropin and prolactin using sodium dodecylsulphate polyacrylamide gel electrophoresis takes place in continuous systems with pH 7.0-7.2 containing acetate or chloride monovalet anions; substitution of the ions by polyvalent phosphate or borate anions interfered with the separation. The hormones precipitated by trichloracetic acid may be separated and quantitatively determined after dissolving in the weak alkaline solutions. The hormones are distinctly separated from serum proteins and may be quantitatively determined in media containing up to 15% of serum.
Subject(s)
Electrophoresis, Polyacrylamide Gel/methods , Growth Hormone/analysis , Prolactin/analysis , Sodium Dodecyl Sulfate , Animals , Cattle , Female , Hydrogen-Ion Concentration , Indicators and Reagents , Pituitary Gland, Anterior/analysis , RatsABSTRACT
The somatotropin-releasing activity of rat hypothalamus was studied in the experiments in vitro incubating of adenohypophysis with the hypothalamic extracts or the whole hypothalamus and serotonin. The studied activity is shown to be increased in the thyroidectomized and hyperthyroid rats. Exogenic somatotropin prevents this increase. A conclusion is drawn that a link of hypothalamic adenohypophysotropic hormones of the regulatory system of the somatotropic function is not an area of the specific disturbances with hypo- and hyperthyroidism which might lead to hyposomatotropism.
