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ATP activity of actomyosin in dilated cardiomyopathy (DCMP) was studied together with impact on the above activity of tropomyosin-troponin regulatory complexes recovered from the normal myocardium and cardiac muscle of a DCMP patient. Recordable in DCMP was a striking decline (1.5-fold) in ATP activity of actomyosin. But no significant difference was to be seen in sensitivity to Ca2+ ions of actomyosins obtained from the normal myocardium and cardiac muscle of the DCMP patient. The cardial tropomyosin-troponin regulatory complex from the DCMP patient's myocardium was shown to be endowed with somewhat more manifest activity compared to the analogous complex recovered from the normal myocardium.

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).

A specific chemical modification of histidyl residues in tyrosyl-tRNA synthetase by diethyl pyrocarbonate was performed. It is shown that five of sixteen histidyl residues can react with diethyl pyrocarbonate in the native conditions. Modification of two histidyl residues per dimer results in the inactivation of tyrosyl-tRNA synthetase in both steps of the tRNATyr aminoacylation. All substrates protect tyrosyl-tRNA synthetase against inactivation with diethyl pyrocarbonate, the most effective protector being combination of ATP and tyrosine. Histidyl residues of tyrosyl-tRNA synthetase are suggested to be involved in the catalytic mechanism of aminoacylation of tRNATyr.

Functionally active proteolytic modified form of tyrosyl-tRNA-synthetase has been isolated in a homogeneous form from the bovine liver under incomplete blocking of endogenous proteolysis. The isolation scheme is described. From the data of gel electrophoresis under denaturing conditions the molecular weight of this form is 39 +/- 1.5 kDa and from the data of gel filtration under native conditions -84 kDa. Thus, this form as well as the native enzyme is a dimer of the alpha 2-type. As compared to the native enzyme (Mm 2 x 59 kDa) a proteolytically modified form has a fragment of the polypeptide chain about 20 kDa long split out (this fragment is not essential for catalytic activity). The values of catalytic characteristics of the modified form in tRNA(Tyr) aminoacylation reaction (Km = 1.19 microM and kcat = 2.99 min-1) are close to those obtained for the main form of the enzyme (0.69 microM and 2.97 min-1, respectively). Amino acid composition of the low-molecular form of tyrosyl-tRNA-synthetase has been determined. It was found that the fragment split out in limited proteolysis was characterized by very high content of positively charged lysine residues (46 residues). A proteolytically modified form of tyrosyl-tRNA-synthetase possesses, like the main form, the affinity to high-molecular rRNA but it is eluted from the column filled with rRNA-sepharose at lower salt concentration (50 mM KCl) as compared to the main form of the enzyme (100 mM KCl).

The affinity of eukaryotic tyrosyl-tRNA synthetases from bovine liver and from yeast for E. coli ribosomal RNA and synthetic polyribonucleotides has been studied by protein binding on the rRNA-Sepharose column and enzyme inhibition by high molecular weight RNAs. Tyrosyl-tRNA synthetase from bovine liver (Mr 2.59 kDa) was fully retained on the rRNA-Sepharose and eluted by buffer with 100 mM KCl. The functionally active modified form of bovine liver tyrosyl-tRNA synthetase obtained by endogenous limited proteolysis (Mr 2.38 kDa) partially maintains the affinity for rRNA and is eluted by 50 mM KCl. The highest rRNA-binding ability was revealed for yeast tyrosyl-tRNA synthetase eluted by 200 mM KCl. The E. coli tyrosyl-tRNA synthetase was not retained on rRNA-Sepharose. The aminoacylation activities of both bovine liver and yeast tyrosyl-tRNA synthetases were efficiently inhibited by rRNA and the inhibition was partially competitive in respect to tRNA(Tyr). At the same time the activities of proteolytically modified bovine tyrosyl-tRNA synthetase and E. coli tyrosyl-tRNA synthetase were not influenced by the addition of rRNA. Synthetic single- and double-stranded polyribonucleotides specifically inhibited the activity of bovine tyrosyl-tRNA synthetase to different extent. The inhibition degree of bovine liver tyrosyl-tRNA synthetase decreased in the order: poly (G) greater than poly (I) greater than poly (I).poly (C) greater than poly (G).poly (C) greater than poly (C) greater than poly (A). Poly (U) did not inhibit the activity of bovine liver tyrosyl-tRNA synthetase.

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.

Genes rplJ, coding for ribosomal protein L10 of Salmonella typhimurium and Klebsiella pneumoniae, have been cloned on pUC plasmid. The resultant multicopy recombinant plasmids were detrimental for the growth of normal JM101 E. coli host cells and harmless for the mutant JF3029 host. This negative effect is the evidence for the ability of heterologous L10 proteins to regulate expression of rplJL genes in E. coli. Nucleotide sequence was determined completely for S. typhimurium rplJL' DNA portion and partially for rplJL' genes of K. pneumoniae. According to the nucleotide sequence data obtained three amino acid substitutions differ L10 proteins of S. typhimurium and E. coli and the long range, providing for the coupled translations of L10 and L7/L12 cistrons in E. coli mRNA is also valid for S. typhimurium and K. pneumoniae.

A method for isolating tyrosyl-tRNA synthetase from Thermus thermophilus is described, including ammonium sulfate fractionation, chromatography on DEAE-sepharose, hydroxyapatite, heparin-sepharose and hydrophobic chromatography on Toyopearl HW-65. The yield of the purified enzyme was 1.6 mg per 1 kg of T. thermophilus cells. The enzyme is a dimer protein of the alpha 2 type with molecular weight of 100 kDa.

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.

The nucleosides of tRNA(IAGLeu) (with a long variable loop) from the cow mammary gland included in formation of the three-dimensional structure have been analysed by the chemical modification methods. Exposed guanosine and cytidine residues were detected by means of dimethylsulfate, whereas diethylpyrocarbonate was used to detect exposed adenosine residues. The low level of the modification was characteristic of guanosine residues in positions 10 (m2G), 13, 15, 23, 24, 29, 30, 47 H, 51, 52, 53, 57; of cytidine residues in positions 48 (m5C), 56 and those involved in Watson--Crick pairing; of adenosine residues in positions 14, 22, 31, 42, 59, 64. Most bases of tRNA(IAGLeu) thus detected are similarly located in the yeast tRNA(Phe) molecule, which suggests a common role of these bases in the formation of the spacial structure of both tRNAs.

tRNA2Leu from cow mammary gland has been degraded with pancreatic ribonuclease, and the fragments obtained were separated by DEAE-cellulose micro-column chromatography in 7 M urea at pH 7,5. Rechromatography was performed on a DEAE-cellulose micro-column at pH 3,7 and also on Dowex 1 X 2 in a formiate system. Nucleotide analysis was carried out with the aid of T2-RNase hydrolysis followed by chromatography on anion-exchanger AG 1 X 8. Nucleosides were separated on Aminex A-6 at pH 9,8. 15 minor components were shown to be present: T, 2 psi, 2Um, 2D, m5C, ac4C, m1G, 2m2G, m22G, m1A and N, the N is not identified so far. The structure of oligonucleotides was established by terminal analysis, hydrolysis with T1-RNase and also using incomplete hydrolysis by the snake venom phosphodiesterase.

Tyrosyl-tRNA synthetase of beef liver has been isolated and its properties have been studied. Tyrosyl-tRNA synthetase is a structural dimer of alpha 2 type. Mr of the enzyme subunit is about 59 kDa. Km values for substrates have been determined and compared with kinetic properties of tyrosyl-tRNA synthetases from different sources. The polymorphism of tyrosyl-tRNA synthetase was studied. The enzyme was separated into two different forms by chromatography on phosphocellulose P 11. P1-form is active only in the amino acid activation reaction. This form is not due to the phosphorylation of the enzyme. The low molecular weight form (38 kDa) was also isolated. This form appeared due to the limited endogenic proteolysis of the main form and retained full activity in the aminoacylation reaction. Tyrosyl-tRNA synthetase from beef liver has non-specific affinity to rRNA-sepharose.

The oligonucleotides obtained by digestion of tRNA2Leu from cow mammary gland with T1 RNase were separated by micro-column chromatography on DEAE-cellulose in 7 M urea at pH 7,5 and 3,7, and in addition on Dowex 1 x 2. The digest consisted of 18 individual components, the larger being a tridecanucleotide. Micro-column chromatography of nucleotides on anion-exchanger AG 1 x 8 and nucleosides on Aminex A-6 was used to determine the base composition of the oligonucleotides. The oligonucleotide structure was established using terminal analysis, hydrolysis by pancreatic and U2-RNases and incomplete hydrolysis by snake venom phosphodiesterases. The total primary structure of tRNA2Leu was derived from overlapping fragments isolated after its complete hydrolysis with pancreatic and T1 RNase and using data obtained on S1-nuclease digestion of tRNA. The methods of rapid gel-sequencing were also employed for checking the nucleotide sequence of tRNA2Leu from cow mammary gland.

P-chloromercuribenzoate (PCMB) inhibition of the leucyl-t-RNA-synthetase (LeuRS) activity was studied both in the tRNA-aminoacylation and ATP-[32P]-pyrophosphate exchange reactions. It is established that the enzyme contained sulphydryl (SH) groups essential for catalytic activity. No protection of substrates from the PCMB action was observed in both reactions. It is shown that SH-groups essential for tRNA-aminoacylation were of no use for leucine activation. The PCMB modified enzyme was reactivated (80%) after the treatment with dithiothreitol. The quantity of cysteine residues in the native and denatured forms of LeuRS was determined.

The primary structure of tRNA(1Ser) from the bovine liver has been studied. pG- A-C-G-A-G-G-U-G-G-C-ac4C-G-A-G-D-Gm-G-D-D-A-A-G-G- C-m2(2)-G-A-psi-G-G-A-m3C-U-G-C-U-A*-A-psi-C-C-A-U-Um-G-psi- G-C-U-m3C-U-G-C-A-C-G-m5C-G-U-G-G-G-T-psi-C-G-m1A-A- U-C-C-C-A-U-C-C-U-C-G-U-C-G-C-C-AOH. A comparison of the nucleotide sequence of tRNA(1Ser) from the bovine liver with already known sequences of serine tRNA revealed a number of common nucleotides, some of them, probably, participated in specific interaction with seryl-tRNA synthetase.

tRNA-Leu 1, tRNA-Leu 2 and tRNA-Leu 4 were isolated from a lactating cow mammary gland by combination of several chromatographic methods. Chromatography on a Sepharose 4B column with a reverse salt gradient was used as the first step. Individual tRNAsLeu were further purified by RPC-5 column chromatography at pH 4.5 and 7.5. For isolation of tRNA-Leu 2 a RPC-5 column was additionally used at pH 3.3 in the presence of 7 M urea. Using micro-column chromatography of Ti-RNAases digests, it was demonstrated that tRNA-Leu 1 and tRNA-Leu 2+ are similar in their primary structure and differ essentially from tRNA-Leu 4.

The phosphates of the tRNA-Leu IAG from cow mammary gland (tRNA which has a long variable loop) participating in the formation of three-dimensional structure were studied by alkylation with ethylnitrosourea and methylnitrosourea. A low degree of modification was observed for the phosphates of the following nucleotides: 7, 8, 9, 10 (at the bend site between the acceptor and D-stem); 18, 19, 20A and 21 in the D-loop; 47H and 49 at the joint of variable and T-stem; 57, 58 and 59 in the T-loop.

The primary structure of the tRNA2Leu from a lactating cow mammary gland was established T1 and pancreatic ribonuclease digestion were determined by an ultramicrospectrophotometric method using the equipment for microcolumn chromatography. tRNA2Leu is composed of 85 nucleotide residues, including 15 modified nucleotides. The anticodon region of tRNA2Leu is respresented by the sequence CAG. The primary structures of tRNA2Leu from the cow mammary gland and leucine tRNAs from E. coli and yeast were compared.

Interactions of leucyl-tRNA synthetase with substrates were studied by fluorescence spectroscopy. The formation of enzyme-substrate complexes results in the quenching of protein fluorescence. The equilibrium binding constants were determined for L-leucine, ATP, tRNAleu and leucyladenylate. It is shown that the interaction of the enzyme with ATP or tRNAleu leads to 10-30-fold increase in the binding constants for subsequent interaction of the second substrate. The data obtained indicate to the cooperative interaction between ATP and tRNA binding sites.

The nucleotide sequence of the tRNALeuIAG from a lactating cow mammary gland was determined by ultramicrospectrophotometrical method and rapid gel sequencing procedure. The chain length of this tRNA is 85 nucleotides, 15 of them including 6 psi, are modified nucleotides. The primary structure of tRNALeuIAG is absolutely identical to major species of leucyl tRNAIAG from bovine liver and differs in 21 positions from cow mammary gland tRNALeuCAG.